The human cytomegalovirus

Pharmacology & Therapeutics 98 (2003) 269 – 297 www.elsevier.com/locate/pharmthera Associate editor: S. Pestka The human cytomegalovirus Santo Landolfoa,*, Marisa Garigliob, Giorgio Gribaudoa, David Lemboa a Department of Public Health and Microbiology, University of Turin, Via Santena 9, 10126 Turin, Italy b Department of Medical Sciences, University of Eastern Piedmont, Novara, Italy Abstract Human cytomegalovirus (HCMV), a betaherpesvirus, represents the major infectious cause of birth defects, as well as an important pathogen for immunocompromised individuals. The viral nucleocapsid containing a linear double-stranded DNA of 230 kb is surrounded by a proteinaceous tegument, which is itself enclosed by a loosely applied lipid bilayer. Expression of the HCMV genome is controlled by a cascade of transcriptional events that leads to the synthesis of three categories of viral proteins designated as immediate-early, early, and late. Clinical manifestations can be seen following primary infection, reinfection, or reactivation. About 10% of infants are infected by the age of 6 months following transmission from their mothers via the placenta, during delivery, or by breastfeeding. HCMV is a significant post-allograft pathogen and contributes to graft loss independently from graft rejection. Histopathologic examination of necropsy tissues demonstrates that the virus enters via the epithelium of the upper alimentary, respiratory, or genitourinary tracts. Hematogenous spreading is typically followed by infection of ductal epithelial cells. Infections are kept under control by the immune system. However, total HCMV clearance is rarely achieved, and the viral genome remains at selected sites in a latent state. Virological and molecular detection of HCMV, as well as serological demonstration of a specific immune response, are used for diagnosis. Treatment of HCMV infections is difficult because there are few options. The presently available drugs produced a significant clinical improvement, but suffer from poor oral bioavailability, low potency, development of resistance in clinical practice, and dose-limiting toxicities. D 2003 Elsevier Science Inc. All rights reserved. Keywords: HCMV; Gene expression; Epidemiology; Transplant; Diagnosis; Antiviral therapy Abbreviations: ab, antibody; AIDS, acquired immunodeficiency syndrome; AP, assembly protein; AP-1, activator protein-1; ATF, activating transcription factor; BMT, bone marrow transplant; CDV, cidofovir; CMV, cytomegalovirus; CPE, cytopathic effect; CREB, cyclic AMP-responsive element binding protein; crs, cis-repression signals; CTL, cytotoxic T lymphocyte; DB, dense bodies; E, early; ER, endoplasmic reticulum; GCV, ganciclovir; GPCR, Gprotein-coupled receptor; HCMV, human cytomegalovirus; HIV, human immunodeficiency virus; IE, immediate-early; Ig, immunoglobulin; L, late; mCP, minor capsid protein; MCP, major capsid protein; MHC, major histocompatibility complex; MIE, major immediate-early; NF, nuclear factor; NK, natural killer; ORF, open reading frame; PBL, peripheral blood leukocytes; PCR, polymerase chain reaction; PFA, foscarnet; p.i., post-infection; Rb, Retinoblastoma; TAP, transporter associated with antigen processing; UL, unique long; US, unique short. Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The human cytomegalovirus . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Virus structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Virus genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Virus growth cycle and viral gene expression . . . . . . . . . . . . . 2.3.1. Range of cell permissivity and in vitro growth . . . . . . . . 2.3.2. Virus binding and penetration . . . . . . . . . . . . . . . . 2.3.3. Regulation of viral gene expression . . . . . . . . . . . . . 2.3.4. Characteristics and functions of the immediate-early proteins 2.3.5. Characteristics and functions of the early proteins . . . . . . 2.3.6. Viral DNA replication . . . . . . . . . . . . . . . . . . . . * Corresponding author. Tel.: +39-11-6706604; fax: +39-11-6636436. E-mail address: [email protected] (S. Landolfo). 0163-7258/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0163-7258(03)00034-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 270 270 272 273 273 273 274 274 276 277 270 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 2.3.7. Characteristics and functions of the late proteins . . 2.3.8. Virion assembly, maturation, and egress . . . . . . 3. Epidemiology of human cytomegalovirus infection . . . . . . . . . 4. Infection routes . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Pathogenesis and pathology . . . . . . . . . . . . . . . . . . . . . 6. Host defences . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Cell-mediated immunity . . . . . . . . . . . . . . . . . . . 6.2. Humoral immunity. . . . . . . . . . . . . . . . . . . . . . 6.3. Immune evasion by human cytomegalovirus . . . . . . . . 6.4. Persistence and release from the host . . . . . . . . . . . . 7. Clinical features associated with human cytomegalovirus infection. 7.1. Infection in normal hosts . . . . . . . . . . . . . . . . . . 7.2. Congenital infection . . . . . . . . . . . . . . . . . . . . . 7.3. Cytomegalovirus infection in the immunocompromised host 8. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Virus detection. . . . . . . . . . . . . . . . . . . . . . . . 8.1.1. Viremia . . . . . . . . . . . . . . . . . . . . . . . 8.1.2. Antigenemia . . . . . . . . . . . . . . . . . . . . 8.1.3. DNAemia . . . . . . . . . . . . . . . . . . . . . . 8.1.4. RNAemia . . . . . . . . . . . . . . . . . . . . . . 8.2. Detection of the immune response. . . . . . . . . . . . . . 9. Prevention of human cytomegalovirus infection and disease . . . . 9.1. Human cytomegalovirus vaccines . . . . . . . . . . . . . . 10. Antiviral treatment . . . . . . . . . . . . . . . . . . . . . . . . . 10.1. Currently available drugs . . . . . . . . . . . . . . . . . . 10.2. Therapeutic approaches. . . . . . . . . . . . . . . . . . . 10.2.1. Prophylaxis . . . . . . . . . . . . . . . . . . . . 10.2.2. Pre-emptive treatment . . . . . . . . . . . . . . . 10.2.3. Treatment of an established disease . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction 2. The human cytomegalovirus The human cytomegalovirus (HCMV) is a widespread pathogen responsible for generally asymptomatic and persistent infections in healthy people. It may, however, cause severe disease in the absence of an effective immune response, as in immunologically immature and immunocompromised individuals. Its impact, therefore, has increased in recent decades due to the rise in organ allografting, immunosuppressive treatment, and human immunodeficiency virus (HIV)-infected patients. Furthermore, it is the leading infectious agent causing birth defects (Griffiths, 2000; Pass, 2001). HCMV is a member of the betaherpesvirinae subfamily, whose virion structure, kinetics of viral gene expression, and persistence for the lifetime of their host are typical of other herpesviruses. However, its strict species specificity, salivary gland tropism, and slow growth in cell cultures differentiate it as the prototype betaherpesvirus (Mocarski & Courcelle, 2001). This paper provides an overview of the general characteristics of HCMV structure, growth, and replication, as well as the pathogenesis, epidemiology, diagnosis, and management of the infections it causes. 2.1. Virus structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 278 278 279 280 281 281 282 282 283 284 284 284 284 285 285 285 285 286 286 286 286 286 287 287 288 288 288 288 289 289 The virion of HCMV consists of a 100-nm diameter icosahedral nucleocapsid containing a 230-kbp, doublestranded linear DNA genome surrounded by a proteinaceous layer defined as the tegument or matrix, which, in turn, is enclosed by a lipid bilayer containing a large number of viral glycoproteins. The mature virion particle is 150 – 200 nm in diameter. HCMV-infected cell cultures produce the infectious virions and another two types of morphological particles: noninfectious enveloped particles and dense bodies (DB). Noninfectious enveloped particles are defective viral particles composed of enveloped immature capsids (type B) that lack DNA, but contain the viral scaffolding/assembly protein (AP) normally absent from fully mature nucleocapsids (C-capsids). DB are enveloped particles that lack an assembled nucleocapsid and viral DNA, but contain several tegument proteins of which ppUL83 (pp65 or lower matrix protein) is the most abundant. The relative amounts of the three viral forms depends on the number of passages in cell culture and the viral strain (Mocarski & Courcelle, 2001). 271 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 Table 1 HCMV proteins discussed in this review1 Table 1 (continued) Gene Kinetic class Protein name(s) Function(s) UL36 UL37 UL122 IE IE IE UL123 IE UL123 IE IRS1 IE pUL36 vMIA IE1491aa, IE1 – 72 IE2579aa, IE2 – 86 IE2425aa, IE2 – 55 pIRS1 US3 IE gpUS3 TRS1 IE pTRS1 UL4 UL40 UL44 UL46 E E-L E-L E-L gpUL4, gp48 gpUL40 ppUL44, p52 mC-BP UL54 E UL55 E DNA pol, pUL54 gB Inhibition of apoptosis Inhibition of apoptosis Regulation of viral and host gene expression Regulation of viral and host gene expression Regulation of viral and host gene expression Transactivator of viral gene expression Down-modulation of MHC Class I expression Transactivator of viral gene expression Minor envelope glycoprotein Immune evasion DNA processivity factor Minor capsid-binding protein, capsid structure DNA polymerase UL57 E UL69 E-L UL70 E-L UL78 E Helicaseprimase pUL78 UL84 E-L ppUL84 UL85 UL97 E-L E-L mCP pUL97 UL102 E UL105 E UL112 E UL113 E UL114 US2 E E Helicaseprimase Helicaseprimase Early pp family Early pp family pUL114 gpUS2 US6 E-L gpUS6 US11 E gpUS11 US27 E pUS27 US28 E pUS28 UL32 L UL33 L pp150, ppUL32 gpUL33 pUL57, ssDNA BP ppUL69 Major envelope glycoprotein, constituent of gCI Single-stranded DNA-binding protein Transactivator, dysregulation of host cell cycle Subunit of the helicaseprimase complex Similar to glucocorticoid receptors Initiation of oriLyt-specific DNA replication mCP, capsid structure phosphotransferase, GCV-activating enzyme Subunit of the helicaseprimase complex Subunit of the helicaseprimase complex Organization of viral DNA replication centers Organization of viral DNA replication centers Uracil DNA glycosylase Down modulation of MHC Class I expression Inhibition of TAP-mediated peptide translocation Down modulation of MHC Class I expression Similar to glucocorticoid receptors C-C chemokine receptor, immune evasion Major tegument component, basic phosphoprotein Similar to glucocorticoid receptors, minor envelope gp Gene Kinetic class Protein name(s) Function(s) UL48.5 L SCP UL73 L gN UL74 L gO UL75 L gH UL80 L UL80.5 L Assemblin precursor AP UL82 L pp71, ppUL82 UL83 L UL86 UL94 UL99 UL100 L L L L pp65, ppUL83 MCP pUL94 pp28 gM UL115 L gL UL123 L gIE2338aa Smallest capsid protein, capsid structure Envelope glycoprotein, constituent of gCII Envelope glycoprotein, constituent of gCIII Envelope glycoprotein, constituent of gCIII Assemblin protease (pUL80a), capsid assembly AP scaffolding protein, capsid assembly Transactivator, dysregulation of host cell cycle Major tegument component, lower matrix protein MCP, capsid structure Virion protein? Tegument protein Envelope glycoprotein, constituent of gCII Envelope glycoprotein, constituent of gCIII Regulation of viral and host gene expression 1 See Mocarski and Courcelle (2001) for a complete list of all HCMV genes, their products, and their functions. Of the more than 30 viral proteins found in the complete infectious virion, four constitute the capsid; namely, pUL46, pUL48.5, the minor capsid protein (mCP), and the major capsid protein (MCP) encoded by UL85 and UL86, respectively (Table 1). Three APs encoded by UL80 associate with capsid and play roles in maturation. The 162 capsomer shell of HCMV is composed of hexameric and pentameric units of the MCP located at the vertices of a T = 16 icosahedral lattice, where adjacent capsomers are joined by surface structures produced by the association of pUL48.5 and pUL46. The phospholipid envelope contains 6 virus encoded glycoproteins, including gpUL55 (gB), gpUL73 (gN), gp UL74 (gO), gpUL75 (gH), UL100 (gM), and gpUL115 (gL). These glycoproteins play essential roles in virus entry into host cells, cell-to-cell spread, and virion maturation (Britt & Mach, 1996). Mutational analysis has revealed that disruption of gB, gH, gL, and gM open reading frames (ORFs) results in the failure to produce infectious progeny, and underscores the essential role of these proteins for productive replication. Only gO is dispensable for viral growth in cultured fibroblasts (Hobom et al., 2000). Two other glycoproteins, gpUL4 (gp48) and the seven-spanning transmembrane gpUL33, are found in the virion and are also probably located in the envelope. The protein products of the six major glycoprotein-encoding genes associate to form three complexes that are highly conserved within herpesvi- 272 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 ruses and previously have been designated as gCI, gCII, and gCIII (Gretch et al., 1988). At least two are required for viral entry: gCI, composed of homodimeric gB molecules, and gCIII, a heteroligomeric complex composed of gH, gL, and gO (Theiler & Compton, 2001). gB is an essential glycoprotein that plays a crucial role in virus binding, since it is the major cell surface, heparan sulfate proteoglycan-binding glycoprotein. It also participates in viral entry, cell-to-cell spread, and cell fusion. By virtue of its abundance and ability to elicit neutralizing antibodies (abs), it has been proposed as a molecule of choice for a recombinant subunit vaccine (Gonczol & Plotkin, 2001). gCIII is necessary for the final stage of virus entry via pH-independent fusion between the viral envelope and the cell membrane (Huber & Compton, 1998). gCII results from the association of gM and gN (Kari et al., 1994). All three complexes induce neutralizing abs. Because of their interaction with the immune system, the genes encoding gB, gH, and gN have been detected as highly polymorphic loci that can be clustered in different genotypes in clinical isolates. The occurrence of ab-escaping mutant viruses selected by the pressure of the humoral immune response may be related to cell tropism and virulence, and is thought to contribute to the pathogenicity of HCMV. The remaining 20 – 25 structural virion proteins probably are located in the still poorly characterized amorphous layer between the nucleocapsid and the envelope (Baldick & Shenk, 1996). The tegument proteins may be involved in the maturation of progeny virions, or may influence viral and cellular events in the early stages of infection, such as release of viral DNA from disassembling virus particles or the regulation of viral and cellular promoters. Most tegument proteins are phosphorylated and are highly immunogenic. The most abundant are ppUL32 (pp150 or basic phosphoprotein) and ppUL83. Due to its large amounts, pp65 is the target antigen in antigenemia assays for rapid diagnosis of HCMV-substained clinical infections. Tegument proteins such as ppUL69 and ppUL82 (pp71) may play important regulatory roles in both viral and cellular gene expression. They are, in fact, transactivators of viral gene expression, and deeply impact on host cell physiology by dysregulating cycle progression. UL69 stimulates expression from the ie1/ie2 enhancer-promoter. It is required for efficient viral replication, and arrests cells in G1 by an unknown mechanism (Lu & Shenk, 1999; Hayashi et al., 2000). UL82 is a transcriptional activator of activating transcription factor (ATF)/cyclic AMP-response element binding protein (CREB) or the activator protein-1 (AP-1)containing promoter and, thus, stimulates the activity of the ie1/ie2 enhancer-promoter. It also increases the infectivity of transfected viral DNA, is required for viral replication at low multiplicities of infection, and accelerates the G1 to S transition of quiescent cells (Liu & Stinski, 1992; Baldick et al., 1997; Bresnahan & Shenk, 2000b). Two more transactivator proteins (pIRS1 and pTRS1) are associated with virions and DB (Romanowski et al., 1997). Five viral RNAs (designated as virion RNAs) have been detected in preparations of highly purified, infectious HCMV particles. These HCMV transcripts probably are located in the tegument and are delivered to the host cell on infection. However, the functional significance of their presence in the virions and that of their protein products remains to be defined (Bresnahan & Shenk, 2000a). 2.2. Virus genome The HCMV genome is the largest of all herpesviruses and has a high G + C content. Like that of herpes simplex virus-1, it contains an arrangement of unique long (UL), unique short (US), and repeat regions. Since each long and short region can be oriented in either direction, four genome isomers are produced in viral progeny (Class E structure) (Fig. 1). In contrast, the genomes of animal CMV, as well as those of other betaherpesviruses, are linear without repeat regions (Class F genomes). Inversion of UL and US regions is mediated by direct repeat sequences (a, b, c) at the genome termini and by inverted repeat elements at the UL-US junction (a0, b0, c0). The repeated a sequence that occurs as a direct element at the termini and in the inverted orientation at the UL/US junction promotes genome isomerization, since it contains the cis-acting pac (packaging) elements needed for DNA cleavage (Mocarski & Courcelle, 2001). The AD169 laboratory strain is the only completely sequenced HCMV. Analysis of its 230-kbp genome has revealed that it encodes 225 ORFs of  100 or more amino acids (Chee et al., 1990a; Novotny et al., 2001). These ORFs are designated sequentially according to their location within the unique and repeated regions. Additional ORFs have been identified in the Towne and Toledo laboratory strains. In the latter, as well as in clinical isolates, the inverted b0 repeat is deleted and replaced by an additional UL region of  15 kbp, containing 19 additional ORFs that are absent in the AD169 genome (Cha et al., 1996). The unique ORF UL1 – 154 and US1 – 36 blocks are separated by duplicated IRL1 –14 and J1I genes and the partially repeated IRS1 gene. The UL region is flanked at the 50 end by the duplicated TRL1 – 14 and J1L (identical to IRL1 – 14 Fig. 1. Structure of the four HCMV genome isomers. S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 and J1I), whereas the US gene block is flanked at its 30 end by the TRS1 gene and by the third copy of a J1 gene (J1S). Comparison of the AD169 amino acid sequences with those of other herpesvirus genomes has revealed that the protein products of more than 40 ORFs share high similarity to proteins encoded by alpha- and gammaherpesviruses (Chee et al., 1990a; Karlin et al., 1994), and provided further evidence of a common origin of the three subfamilies. Of the herpesvirus-conserved ORFs,  25% appear to encode functions related to viral DNA metabolism and replication, whereas the remaining 75% are thought to be involved in the maturation and structural organization of virions. The UL ORF-encoding functions involved in DNA replication and repair, nucleotide metabolism, or virion structure are grouped in seven conserved gene blocks (A – G) also found in other herpesviruses, such as herpes simplex virus-1 and Epstein-Barr virus, although in a different order in the alpha- and gammaherpesviruses. The occurrence of these conserved blocks in a characteristic order specific to each subfamily suggests that the functions encoded are probably conserved in all herpesviruses. The US ORF and those located within the repeated regions of the HCMV genome are less well conserved than in the other herpesviruses (Chee et al., 1990a). Sequence homology searches and experimental biochemical and/or genetic studies have assigned functional roles to only some of the more than 200 HCMV ORFs (Novotny et al., 2001). However, analysis of the phenotypes of spontaneous deletion mutants of the AD169 strain, as well as those of virus-bearing deletions or inactivation at specific loci, has indicated that the products of more than 50 HCMV ORFs are dispensable for productive replication in fibroblast cultures. These findings, along with observations that the proteins responsible for functions common to all herpesviruses, such as basic DNA replication, virion organization, and maturation, do not account for all the ORFs, indicate that many ORFs still await functional characterization. It is likely, therefore, that much of the coding capability has evolved to optimize infection by influencing dissemination, growth in target tissues and pathogenesis, and in counteracting host immune reactivity (Mocarski & Courcelle, 2001). 273 macrophage-granulocyte progenitors in the bone marrow and in peripheral monocytes (Kondo et al., 1994; Soderberg-Naucler et al., 1997). In contrast, in vitro, the only cells fully permissive for replication of laboratory strains are human skin or lung fibroblasts, whereas clinical isolates replicate preferentially on endothelial cell cultures. Some transformed cell lines derived from glioblastomas, such as U373MG, as well as primary arterial smooth muscle cells, support productive infection, although at lower levels than in fibroblasts (Sinzger et al., 1995; Plachter et al., 1996). In fibroblasts, the replication cycle is slow, with a typical cytopathic effect (CPE) characterized by cell rounding and enlargement with both intra- and perinuclear inclusions (Fig. 2). Only laboratory strains, such as AD169 or Towne, produce a generalized CPE, with lysis of the entire cell monolayer, and release high titers of virions starting from 3 to 4 days post-infection (p.i.). Recently, isolated strains mainly spread on fibroblast monolayers by cell-to-cell contact and produce a limited CPE, with dispersed foci and poor virion yields (Pass, 2001). 2.3.2. Virus binding and penetration Virus attachment and penetration are rapid and efficient in both permissive and nonpermissive cell types. However, since productive replication is observed in a very restricted range of human cells, a post-penetration block to viral gene expression is thought to restrict replication in nonpermissive cells (Sinzger et al., 2000). The poorly characterized receptor(s) for HCMV is widely distributed among host cell types, and contributes to the broad viral tropism observed during natural infections. Viral entry is the result of a cascade of interactions between viral and cellular proteins that culminate in fusion of the virion envelope with the cellular plasma membrane by a pH-independent mechanism. During the initial virus-cell interactions, as observed with other herpesviruses, HCMV attaches to the cell surface by low-affinity binding of gB to heparan sulfate proteoglycans (Compton et al., 1993). The subsequent interaction of gB with its nonheparin receptor then turns the weak adhesion of the viral particle into a more stable binding or docking state. 2.3. Virus growth cycle and viral gene expression 2.3.1. Range of cell permissivity and in vitro growth Autopsy specimens show that HCMV infects a wide range of epithelial tissues. The ductal epithelial cell is most commonly infected, and develops a typical cytopathology (Pass, 2001; Bissinger et al., 2002). However, during natural infection, it is thought that HCMV replicates productively in epithelial cells, endothelial cells, smooth muscle cells, mesenchymal cells, hepatocytes, granulocytes, and monocyte-derived macrophages (Plachter et al., 1996; Sinzger & Jahn, 1996; Sinzger et al., 1996; Kahl et al., 2000; Bissinger et al., 2002). Latent viral DNA, thus, can be detected in Fig. 2. HCMV cytopathic effect on a human fibroblasts monolayer. 274 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 However, final fusion of the viral envelope with the cell membrane to allow viral penetration is thought to require a further priming event mediated by the heteroligomeric gHgL-gO complex with as yet unidentified receptors (Theiler & Compton, 2001). Fusion of the virus and cell membranes is followed by entry into the host cytoplasm of the nucleocapsid and tegument proteins, and their rapid translocation into the nucleus, where pp65 is detected < 1 hr p.i. Interaction of HCMV glycoproteins with their receptors is enough to generate an intracellular signal transduction pathway, leading to the alteration of cellular gene expression. Most changes in the profiles of host gene activity resemble those induced by binding of interferons to their receptors (Zhu et al., 1997, 1998; Browne et al., 2001). The specific viral ligand triggering this response is gB, and its interaction with its as yet unidentified receptor is thought to be the main mechanism by which HCMV modifies host cell gene expression in the very early phases of infection (Boyle et al., 1999; Simmen et al., 2001). In addition, engagement of gB and gH with their receptors is sufficient to induce the activity of the cellular transcription factors nuclear factor-kB (NF-kB) and Sp1 (Yurochko et al., 1997). 2.3.3. Regulation of viral gene expression During productive infection, the HCMV genome is expressed in a temporally coordinated and regulated cascade of transcriptional events that lead to the synthesis of three categories of viral proteins described as immediate-early (IE or a), early (E or b), and late (L or g) (Fig. 3). Failure in the expression of early gene and subsequent viral DNA replication rather than attachment and/or penetration may be the restricting event in nonpermissive cells. HCMV genes are transcribed in the infected cell nucleus by RNA polymerase II and the associated basal transcription machinery, with the intervention of host-encoded transcription factors whose activity may be stimulated by viral transactivators (Fortunato & Spector, 1999; Mocarski & Courcelle, 2001). 2.3.4. Characteristics and functions of the immediate-early proteins HCMV gene expression initiates from a few IE proteins within l hr p.i. without de novo protein synthesis. The IE genes include the major IE (MIE) UL122/123 genes (IE1 and IE2) and auxiliary genes, such as UL36 – UL38, UL115 –UL119, IRS1/TRS1, and US3 (Table 1). The MIE proteins, alone or in synergism, are required for subsequent expression by acting as transactivators and autostimulators of viral genes. In addition, these proteins have a deep impact on host cell physiology since they regulate the expression of a large number of host cell genes (Fortunato & Spector, 1999). MIE proteins are encoded by the ie1/ie2 genes (UL122/ UL123), whose expression is regulated by a complex enhancer-modulator element that functions in a tissue- and cell-type-specific manner (Nelson et al., 1990; Meier & Stinski, 1996), and exerts its strong transcriptional activity through interactions with several host transcription factors whose binding sites are closely distributed within the regulatory element. An  500-bp segment upstream from the TATA box of the ie1/ie2 genes contains several repeat elements with binding sites for NF-kB, AP-1, Sp1, and CREB/ATF (Boshart et al., 1985). Since their cognate DNA-binding activities are rapidly activated by HCMV infection, their binding to the corresponding sites is thought to contribute to the very strong activity of the MIE enhancer. The CREB/ATF and AP-1 sites are also responsive to the tegument transactivator ppUL82 (Liu & Stinski, 1992). In addition, NF-1, serum response factor, Elk-1, CCAAT/ enhancer-binding protein, and YY1 sites have been identified within the enhancer segment. The cell type- and differentiation state-specific enhancer activity is related to the availability of appropriate transcription factors in a specific cell type, and is thought to depend on the modulator region. This element spans  500 bp upstream from the core of the MIE enhancer. In transient transfection assays, Fig. 3. HCMV gene expression and viral gene product functions during productive infection. S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 the modulator region represses the transcriptional activity of the enhancer in undifferentiated cells lines, although these findings could not be reproduced with mutagenized viruses in which the modulator element was removed (Nelson et al., 1990; Meier & Stinski, 1997). The MIE region produces a set of transcripts synthesized from different start sites and differentially spliced and polyadenylated to give rise to four protein products. The most abundant in IE times of infection are IE1491aa and IE2579aa. These share the same amino terminal 85 amino acid long segment, but their differentially spliced exons are 4 (UL123) and 5 (UL122), respectively. In addition, a less abundant IE2 isoform (IE2425aa) is encoded by a spliced transcript bearing an in-frame deletion within UL122. The fourth IE protein expressed from the MIE region is a true late 40-kDa polypeptide (gIE2338aa) encoded by a polyadenylated transcript that originates late in infection from a start site within UL122. Both IE1491aa and IE2579aa are expressed throughout replication, albeit with slightly different kinetic profiles. Their ability to regulate transcription of several viral and cellular genes has been studied extensively (Fortunato & Spector, 1999; Mocarski & Courcelle, 2001). IE1479aa is a nuclear phosphoprotein of  72 kDa (IE172), whose functions are not needed for viral growth in fibroblast cultures infected at high multiplicity of infection, but are required after a low multiplicity of infection. The presence of IE1-72, thus, is crucial when infection starts from a single virion (Mocarski et al., 1996). Generation of mutants lacking UL123 and, hence, IE1-72 expression has confirmed in infected cells its functions, as predicted by transfection assays. The first role of IE1-72 is consistent with its ability to positively autoregulate expression of the ie1/ie2 and US3 genes by activating the corresponding enhancer elements through NF-kB-binding sites (Mocarski et al., 1996). In addition, IE1-72 cooperates with IE2579aa to regulate the expression of viral genes belonging to the subsequent categories, as predicted by transient transfection assays in which co-expression of the two IE proteins were found to be required for maximal activity of the promoters of E (UL44 and UL54), as well as L (UL83), genes (Fortunato & Spector, 1999). IE1-72 is a weaker and less promiscuous transactivator than IE2579aa. In these assays, however, it stimulates the activity of several cellular promoters, including those of dihydrofolate reductase, DNA polymerase a, c-fos, c-myc, NF-kB p65 subunit, and thymidylate synthase genes (Hagemeier et al., 1992a; Wade et al., 1992; Hayhurst et al., 1995; Yurochko et al., 1995; Gribaudo et al., 2002). In addition, IE1 may affect the host cell cycle regulatory pathway by targeting the Retinoblastoma (Rb) protein family and E2F-mediated transcriptional mechanisms since it interacts with cellular p107, a member of the Rb family, and activates an E2F-responsive promoter, such as that of the DHFR gene, by alleviating p107mediated repression (Poma et al., 1996; Johnson et al., 1999). Moreover, it interacts physically with E2F1 and transactivates the DHFR promoter only if its E2F sites are 275 retained (Margolis et al., 1995). Lastly, IE1-72 is endowed with a kinase activity that autophosphorylates and phosphorylates the p107 and p130, as well as E2F1, -2, and -3. The putative catalytic domain of IE1-72 is also required for the activation of E2F-dependent transcription (Pajovic et al., 1997). IE2579aa is an 86-kDa (IE2-86) nuclear phosphorylated polypeptide whose activities are critical for viral replication. A bacterial artificial chromosome clone of HCMV with a deletion of exon 5 unique to IE2-86 (the UL122 ORF), in fact, did not activate E gene expression nor yield progeny virus upon transfection in permissive cells (Marchini et al., 2001). Since it is difficult to generate mutant viruses lacking IE2-86 and complementing long-term cell lines expressing fully functional wild-type IE2-86, most studies have relied on transient transfection experiments, many of which have indeed established that IE2-86 behaves as a strong transcriptional regulator by either stimulating or repressing both HCMV and cellular genes (Fortunato & Spector, 1999; Mocarski & Courcelle, 2001; Song & Stinski, 2002). During productive infection, IE2-86 is the key protein regulating the transition from the IE to the E, and very likely to the L phases of HCMV gene expression. Characterization of its function in transient transfection assays, in fact, has demonstrated that alone or in combination with IE1, it transactivates several E and L promoters (Fortunato & Spector, 1999), and IE2-86-binding sites have been identified just upstream from the TATA box of three E promoters. These transactivating properties probably are attributable to the interaction of IE2-86 with factors of the basal-transcription machinery and a variety of promoter-specific transcription factors. Recombinant IE2-86 produced in bacteria, in fact, interacts with the TATA-binding protein, TFIIB, TAFII-130, and TFIID (Hagemeier et al., 1992b; Caswell et al., 1993; Jupp et al., 1993; Lukac et al., 1997). It also interacts in vitro with other transcription factors, such as histone acetyltransferase, CREB, Sp1, c-Jun, and ATF-2, as well as with cell-cycle regulatory factors, including p53, pRb, and p21 (Speir et al., 1993; Hagemeier et al., 1994; Sommer et al., 1994; Fortunato et al., 1997). These interactions indicate that IE2-86 may act as an adapter-like protein by stabilizing basal and specific transcription factors on the promoter region (Mocarski & Courcelle, 2001). Consistent with this model, the occurrence of cis-acting regulatory elements; namely, AP-1, CREB/ATF, and Sp1, upstream from some E promoters contributes to the overall promoter responsiveness to IE2-86-mediated transactivation (Fortunato & Spector, 1999). In addition to their regulation of the expression of E and L genes, IE2-86 and gIE2338aa repress expression of ie1/ie2 and US3 (Fortunato & Spector, 1999; Mocarski & Courcelle, 2001). This down-regulation is mediated by IE2 binding to cis-repression signals (crs) sequences located between the TATA box and the transcription initiation sites of these genes (Pizzorno & Hayward, 1990; Cherrington et al., 1991). By down-regulating transcription from its own 276 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 promoter, therefore, IE2-86 mediates autoregulation of its own expression and contributes to the reduction of IE gene expression in the late stages of infection. In vitro transfection assays have established that binding of IE2-86 to crs confers the IE2-dependent repression of homologous and heterologous promoters, provided that the sequence element is located near the transcription start site. Moreover, purified bacterial IE2-86 binds to crs through its C-terminus and alters RNA polymerase pre-initiation complex formation in the in vitro repression of transcription assays. IE2-86 also modulates several cell processes, including cycle control and apoptosis (Zhu et al., 1995; Kalejta & Shenk, 2002). In fact, expression of IE2-86 by either transient transfection or replicative-defective adenovirus arrests cells in an early S phase by mechanisms that are not known yet (Wiebush & Hagemeier, 1999, 2001; Murphy et al., 2000). Their limited DNA synthesis and high levels of cyclin E-dependent kinase activity show that these cells are blocked one step further than the G1 restriction point of the cycle. IE2-86 stimulates quiescent, serum-starved cells into the early S phase, but prevents them from progressing through this phase, and cell division does not occur. Its ability to arrest the cycle thus greatly contributes to the late G1/early S phase synchronization and host DNA synthesis inhibition observed in HCMV-infected cells (Jault et al., 1995; Lu & Shenk, 1996; Bresnahan et al., 1996; Dittmer & Mocarski, 1997; Salvant et al., 1998). These modifications lead to a more favorable environment for viral replication, since the precursors for DNA synthesis produced by the host are available, but are not utilized by the DNA replication machinery of the cell (Fortunato et al., 2000; Kalejta & Shenk, 2002). The functions of the other IE genes are heterogeneous. TRS1 and IRS cooperate with IE1-72 and IE2-86 in the transactivation of E promoters. The glycoproteins encoded by the US3 gene are involved in the establishment of immune evasion in infected cells, since these endoplasmic reticulum (ER)-located proteins prevent the transport of the assembled major histocompatibility complex (MHC) Class I from ER to the Golgi and, thus, down-modulate MHC Class I antigen presentation (Stasiak & Mocarski, 1992; Jones et al., 1996). In contrast, the UL36 –38 region encodes proteins such as UL36 and UL37 that are endowed with antiapoptotic properties and that act at different steps in the caspase cascade (Skaletskaya et al., 2001; Goldmacher, 2002). UL36, UL37, US3, and IRS1 are all dispensable for replication in cell culture. 2.3.5. Characteristics and functions of the early proteins Expression of E or b genes depends on the presence of functional IE proteins and is unaffected by inhibitors of viral DNA replication (Fortunato & Spector, 1999). They are divided into two subclasses: b1 (E) and b2 (E-L) according to their time of expression. b1 genes are transcribed within 4 –8 hr p.i., b2 transcription 8– 24 hr p.i.. The functional data indicate that E genes encode mostly non-structural proteins, including viral DNA replication factors, repair enzymes, and proteins involved in immune evasion (Mocarski & Courcelle, 2001). The expression profiles of microarrays of viral DNA recently have provided a temporal map of IE, E, and L genes in the entire viral genome (Chambers et al., 1999). Hybridization of such microarrays to cDNAs prepared from HCMV-infected cells treated with ganciclovir (GCV) to block viral DNA replication has revealed that 36% of the more than 150 ORFs scored positive for expression were unaffected by GCV and, therefore, classified as E. These E genes are dispersed throughout the HCMV genome. Unlike the genes in the UL region, most US genes show E class expression properties. However, several E genes, such as UL4, UL44, UL54, and UL112/113, are also transcribed late in infection through several mechanisms, including activation of a promoter different and independent from that transcriptionally active in the E times, initiation of transcription from a new start site, and alteration of the splicing pattern as infection proceeds (Fortunato & Spector, 1999; Mocarski & Courcelle, 2001). It is believed that transcription of E genes is stimulated by IE2-86 alone or in cooperation with IE1-72 through transactivation of the corresponding promoters in a TATA boxdependent manner that requires both the host basal transcription initiation complex and sequence-specific transcription factors, such as CREB/ATF and Sp1 (Fortunato & Spector, 1999). Both E and L transcripts may have a polycistronic structure due to the relatively few polyadenylation signals in the genome that generate families of 30 co-terminal transcripts. In addition, expression of several E genes studied in some detail is regulated by both transcriptional and post-transcriptional mechanisms (Mocarski & Courcelle, 2001). Functional roles in viral DNA replication have been identified for the UL112/113 family of DNA-binding proteins that contribute to organization of the so-called replication centers within the nucleus of infected cells for the viral DNA polymerase encoded by the UL54 ORF and the UL44 gene product that acts as a polymerase processivity factor (Mocarski & Courcelle, 2001). Several other E proteins, however, are involved in establishment of immune evasion in the productively infected cell, such as the glycoproteins encoded by US2 and US11, which bind the MHC Class I heavy chains and transport them in a retrograde fashion from the ER into the cytosol, where they are degraded by the proteasome (Shamu et al., 1999; Story et al., 1999). The E-expressed US27 and US28 ORFs have homology to the CC chemokine receptors (Chee et al., 1990b). However, US28 alone is a receptor for the CC chemokines RANTES and monocyte chemoattractant peptide-1. It sequesters them from the extracellular environment by internalization and, thus, prevents elimination of HCMV-infected cells by chemokine-activated immune cells (Gao & Murphy, 1994; Bodaghi et al., 1998). In addition, the E gene UL4 encodes an early structural glycoprotein (gp48). This is a non- S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 essential component of the viral envelope, since a mutant virus with disruption of the UL4 gene produces virus progeny without impaired replication kinetics (Hobom et al., 2000) (Table 1). 2.3.6. Viral DNA replication HCMV genome replication, inversion, and packaging occur in the nucleus of the infected cells. Viral DNA synthesis begins later than 16 hr p.i. It requires the activities of essential and specific viral proteins and the active contribution of several cellular proteins (Mocarski & Courcelle, 2001). Examination of CMV genome sequences has shown that, unlike other herpesviruses, they do not encode deoxyribonucleotide biosynthetic enzymes, such as thymidine kinase, dihydrofolate reductase, thymidylate synthase, and an active form of ribonucleotide reductase (Chee et al., 1990a; Rawlinson et al., 1996). Thus, the virus must depend on the host cell metabolism to ensure a sufficient supply of dNTPs for its DNA replication. As a result, it does not shut off host macromolecular synthesis, but stimulates cellular transcription and translation. CMV evidently has developed strategies to stimulate the biochemical pathways involved in the biosynthesis of DNA precursors. Several reports have demonstrated that the early consequences of CMV infection are similar to those observed in serum-deprived cells exposed to growth factors (Fortunato et al., 2000). These include cell cycle functions, such as nuclear translocation of Cdk2; induction of cyclin E and B; pRb hyperphosphorylation; activation of E2F-dependent transcription; and activation of c-myc, c-jun, and c-fos proto-oncogenes (Jault et al., 1995; Margolis et al., 1995; Bresnahan et al., 1996, 1997; Salvant et al., 1998). Moreover, there is a substantial increase in the expression of cellular enzymes involved in nucleotide metabolism, including thymidine kinase (Estes & Huang, 1977), ornithine decarboxylase (Isom, 1979) and topoisomerase II (Benson & Huang, 1990), dihydrofolate reductase (Wade et al., 1992; Lembo et al., 1999; Song & Stinski, 2002), folylpolyglutamate synthetase (Cavallo et al., 2001), thymidylate synthase (Gribaudo et al., 2000, 2002; Song & Stinski, 2002), deoxycytidilate deaminase (Gribaudo et al., manuscript in preparation), and ribonucleotide reductase (Lembo et al., 2000; Song & Stinski, 2002). Despite the induction of an S phase-like state, CMV-infected cells, as discussed in Section 2.3.4, fail to undergo cellular DNA replication and division as a result of blocks in cell cycle progression that prevent the host DNA replication machinery from competing with the virus for access to DNA precursors (Fortunato et al., 2000; Kalejta & Shenk, 2002). The ability of HCMV to stimulate the expression of cellular enzymes for DNA precursor synthesis is crucial for its productive replication in the quiescent or terminally differentiated non-dividing cells it encounters during natural infection, since their expression is stringently repressed in these cells. Six herpesvirus-conserved ORFs in the HCMV genome provide the core replication proteins for viral DNA replica- 277 tion. Among them, the single-stranded DNA-binding protein ppUL57 prevents the reannealing of DNA strands following unwinding by the helicase-primase complex. This, in turn, is composed of three subunits encoded by UL70, UL102, and UL105, the DNA polymerase encoded by UL54 and the DNA polymerase processivity factor UL44 that prevents dissociation of UL54 from the template (Griffiths, 2000; Mocarski & Courcelle, 2001). Replication also requires other viral proteins to maximize DNA replication, such as UL84, UL112/113, and UL114. UL84 encodes a 75-kDa phosphoprotein, which stably interacts with IE286, functions as an origin-specific initiator factor, and stimulates the viral origin (oriLyt)-dependent DNA synthesis (Sarisky & Hayward, 1996). The phosphoproteins encoded by the UL112/113 region regulate the establishment of the so-called replication centers corresponding to subnuclear sites of HCMV DNA synthesis. UL112/113 localize to small intranuclear globular sites representing the early precursors of the replication centers and recruit the core replication proteins and enzymes (Penfold & Mocarski, 1997; Ahn et al., 1999). Finally, the protein encoded by UL114 expresses a functional uracil DNA glycosylase activity that appears to be required for efficient viral DNA replication in post-mitotic cells, since a mutant virus with a substitution mutation in UL114 showed a defect in transition to high-level, late-phase DNA replication (Courcelle et al., 2001). IE proteins, such as the transactivators encoded by the ie1/ie2 and TRS1/IRS1 genes and those expressed by the UL36-38 region, are also required for transient complementation of oriLyt-dependent DNA synthesis, although their roles in this process are not known. HCMV DNA replication proceeds through initial circularization of the input genome within 4 hr p.i., followed by DNA synthesis via a bidirectional q mechanism from a single origin (oriLyt) of replication that undergoes a switch to a late-phase rolling circle form of DNA replication (Mocarski & Courcelle, 2001). The latter is responsible for most of the viral DNA produced during the late stages of infection in the form of large concatemeric replicating units lacking terminal fragments that are subsequently cleaved into lengths that can be encapsulated. The oriLyt of HCMV maps within the UL region close to UL57 (Masse et al., 1992). It spans  2000 bp, containing repeated nucleotide sequences, as well as transcription factor-binding sites, and encodes a number of short transcripts. The integrity of this region is needed for efficient replication, since mutations targeting different repeated sequence elements dramatically reduce the initiation of viral DNA synthesis (Mocarski & Courcelle, 2001). During the late stages of viral DNA replication, newly synthesized genomes mature through their inversion, cleavage, and packaging (McVoy & Adler, 1994). Inversion occurs in concatemeric units and leads to the generation of progeny genomes as a pool of four isomers that only differ in the orientation of their L and S components (Fig. 1). The mechanism leading to inversion is thought to require 278 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 a recombination that needs the repeated a sequences at the termini and the UL/US junction. Packaging of the genome into preformed B capsids then follows its cleavage at the essential cleavage/packaging signals pac1 and pac2. These sequences are highly conserved among the herpesvirus genomes and are contained within a 220-bp element located in the S component of the HCMV DNA near the repeated a sequence (Mocarski & Courcelle, 2001). 2.3.7. Characteristics and functions of the late proteins The L proteins are the last class of gene products expressed during HCMV replication (Table 1). Their transcription begins more than 24 hr p.i. and requires prior viral DNA replication (Fortunato & Spector, 1999). Late or g gene expression leads to the synthesis of two subclasses of L proteins (g1 and g2) in accordance with their time of expression and sensitivity to viral DNA replication inhibitors. g1 (leaky late) transcription occurs 24– 36 hr p.i., and is reduced by such inhibitors. g2 (true late) transcription occurs 24 – 48 hr p.i., and is strictly dependent on DNA replication. The L proteins have mainly structural roles and primarily contribute to the assembly and morphogenesis of the virion (Mocarski & Courcelle, 2001). Most HCMV genes belong to the L class. Examination of the expression profiles of DNA microarrays from the whole HCMV genome has shown that at 72 hr p.i., in the absence of a viral DNA synthesis inhibitor, 26% and 32% of the transcriptionally active genes are g1 and g2, respectively (Chambers et al., 1999). Little is known about the transcriptional regulation of L genes and the requirement of viral and/or cellular factors for their expression. Of the L genes studied in detail, the g1 UL83 gene promoter is transactivated by the combination of IE2-86 and IE1-72, whereas deletion analysis of the g2 UL94 and UL99 promoters revealed that they require a minimal promoter element containing little more then the TATA box for full gene activation in late infection (Fortunato & Spector, 1999; Mocarski & Courcelle, 2001). 2.3.8. Virion assembly, maturation, and egress Formation of HCMV capsids and packaging of viral DNA occur in the nucleus. Subsequently, nucleocapsids acquire a primary envelopment by budding at the nuclear membrane, and further mature through a de-envelopment/reenvelopment process in the cytoplasm before leaving the cell via an exocytotic-like pathway (Mocarski & Courcelle, 2001; Mettenleiter, 2002). Nucleocapsid particles accumulate in inclusions that confer the typical ‘‘owl’s eye’’ appearance of the infected cell nucleus. The initial step is the interaction of pUL86 with the scaffolding AP pUL80.5 (the AP precursor) in the cytoplasm and subsequent translocation into the nucleus (Gibson, 1996), where oligomerization of these complexes catalyzed by AP leads to the formation of hexons and pentons that interact with pUL85-pUL46 complexes to form the B capsid precursor shell. The subsequent association of capsid intermediates with pUL48.5 completes the formation of B capsids that are now ready to package viral DNA and, after insertion of viral genomes, remove AP (Butcher et al., 1998). During capsid formation, a series of proteolytic cleavages catalyzed by the assemblin protein (pUL80a) leads to maturation of the UL80 precursor to assemblin/ APs and the dissociation of UL86 from AP (Gibson, 1996). Capsids are initially enwrapped through budding at the nuclear membrane, where they acquire a primary envelope derived from its inner leaflet (Gibson, 1996). They then cross the lumen, fuse with the outer leaflet of the nuclear membrane or the ER membrane with which it is contiguous, lose their primary envelope, and move into the cytoplasm. Here, HCMV virion particles further mature by acquiring their tegument. The tegumented capsids then receive their definitive envelope by budding into vesicles of the Golgi apparatus (Sanchez et al., 2000). Both tegumentation and reenvelopment are driven by multiple specific protein-protein interactions to secure the integrity of the viral particle (Mettenleiter, 2002). These mature particles are retained within the vesicles and transported to the cell surface via the Golgi network, which is enlarged due to the accumulation of nucleocapsids and DB. The Golgi alterations during the late replication stages create inclusions around the nucleus that result in its characteristic kidney-like appearance (Pass, 2001). Progeny virus accumulates in the cytoplasm, and infectious virus is released into the extracellular compartment beginning at 72 hr p.i. In the very late stages, however, a substantial number of viral particles are still associated with the cell. 3. Epidemiology of human cytomegalovirus infection HCMV is one of the most successful parasites. It is found in both the developed industrial societies and in isolated aboriginal groups (Griffiths, 2000; Pass, 2001). Following infection, it is excreted in body fluids (urine, saliva, tears, semen, milk, and cervical secretions) for months to years. Infection is usually mild and subclinical. The unsuspecting host is thus able to spread the virus both vertically and horizontally. Virus can appear following primary infection, reinfection, or reactivation. About 10% of infants are infected by the age of 6 months, following transmission from their mothers via the placenta, during delivery, and by breast feeding (Pass, 1985; Stagno et al., 1986). Transplacental infection can occur both in women infected for the first time during pregnancy and those infected long before conception (recurrent infection). Primary infection during pregnancy as a source of fetal infection was suggested by the observation that transplacental transmission ranges from 20% to 40% when primary infection varies from 0.7% to 4.1% (Pass & Boppana, 1999). In the case of recurrent infections, high rates of congenital infections are observed in populations with higher rates of maternal seropositivity, suggesting that most congenital infections are caused by S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 reactivation of latent virus (Stagno et al., 1973; Yeager & Martin, 1977). Stagno et al. (1977, 1982), for example, found an incidence of  2% of congenital infections among births from mothers seropositive long before conception (as documented by virus shedding and serum abs). Infection during delivery is the outcome of shedding from the vagina or cervix, followed by ingestion of infected secretions by the offspring. Shedding close to the time of delivery has been noted in from 2% to 28% of the mothers (Reynolds et al., 1973). About 50% of the newborns may be infected in this way, and they usually begin to shed the virus themselves when they are 6 weeks old. Breast-feeding is the most common route of transmission. Transmission is a function of its duration and the relatively low virus load in the milk (Dworsky et al., 1983). It has been shown, for example, that infants nursed for < 1 month do not become infected compared with almost 40% of those nursed longer. Moreover, 69% of infants are infected when the virus can be isolated from the milk, whereas only 10% are infected when the mother is seropositive, but negative for virus in the milk. Polymerase chain reaction (PCR) studies have also demonstrated a strong relationship between the presence of viral DNA in milk and transmission to the infant (Vochem et al., 1998). The precise route of infection throughout childhood is not known, although close contact is required. Infection increases when people live in crowded, unhygienic conditions, and hence is most common in socially disadvantaged countries. Most children from populations with low socioeconomic backgrounds, in fact, are infected at the onset of puberty, whereas < 40% of adolescents from the industrial countries are infected, followed by an increase of  1% per year (Griffiths, 2000; Pass, 2001). Infection is life long. Latent CMV may reactivate and produce infectious virions that are shed in the saliva, urine, and other body fluids. Reactivation is usually asymptomatic, and enables the virus to spread horizontally and vertically. The incidence of congenital infection is highest in poor communities, since most women are infected before puberty. Reactivation is thus a more frequent cause of congenital infection than primary maternal infection, although the latter presents a greater risk to the fetus than recurrent infection. An epidemiological distinction is often drawn between reinfection and reactivation as opposed to latent infection. In clinical practice, however, the term ‘‘recurrent’’ is used to indicate both possibilities. 4. Infection routes Primary infection does not usually result in a clinical illness, and cannot be identified in pregnant women. Its status as a risk factor for fetus infection is thus uncertain. Intrauterine infection occurs in only one-third of pregnant women with primary infection. The ways in which the fetus escapes infection are unknown, although macrophages in the placenta may constitute a barrier (Burton & Watson, 1997). 279 Ingestion of infected maternal in genital secretions or breast milk is the main perinatal route of infection. ‘‘Copious’’ quantities of genital secretions containing high virus titers as a result of recurrent maternal infection surround the fetus during delivery, and upon ingestion, may result in virus transmission to the newborn (Stagno et al., 1982). Virus titers are usually low in breast milk, but long-term feeding results in the build-up of an effective inoculum. Newborns from women with infected breast milk who fed with formula milk are not infected. Once ingested, the virus infects the mucosa of the oropharynx, esophagus, or the upper airways (Stagno et al., 1982). The absence of symptoms following postnatal infection makes it impossible to determine the transmission routes. Molecular analysis of CMV isolates has greatly increased our understanding of CMV epidemiology. PCR amplification of selected segments of the genome and hybridization of the variable junction region of the CMV genome require only small amounts of virus for diagnosis and allow faster sequencing of viral DNA. Chou and Dennison (1991) compared envelope glycoprotein gene sequences and found that clinical and laboratory strains can be grouped into four gB genotypes, although epidemiologic and clinical significance of the gB genotype distribution in immunocompromised patients remains unclear. Primary infection with HCMV via blood transfusions was noted in the mid-1960s, although epidemiologic studies have demonstrated that this is an uncommon route. Attempts to culture HCMV from fresh donor blood have rarely been successful. It is thus assumed that the virus is latent in the blood cells of healthy donors and is reactivated following transfusion when they encounter an allogeneic stimulus. The nature of the leukocytes carrying latent virus is unknown, although attention is being increasingly focused on the monocytes/macrophages (Plachter et al., 1996; SoderbergNaucler et al., 1997, 2001). HCMV is a significant post-allograft pathogen. Several studies have shown that seronegative recipients of an organ from a seropositive donor are at risk of acquiring a primary infection and that  60% – 80% develop a more severe disease than seropositive recipients of seropositive organs, suggesting that acquired immunity modulates infection (Peterson et al., 1980; Pollard, 1988). The contribution of immunity to resistance to HCMV disease, however, decreases as the level of immunosuppression increases. Variables that contribute to post-transplant infection include donor and recipient serological status, type of immunosuppression, source of allograft, HLA matching of donor and recipient, and type and amount of blood products used (Rubin et al., 1985; Miller et al., 1991). Since the main determinant of transmission via blood is the presence of leukocytes, reduction of the cell contamination of blood products has dramatically decreased the incidence of transfusion-acquired infection in allograft recipients (Miller et al., 1991). The effects of infection extend well beyond the immediate post-transplant period. It appears that HCMV 280 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 contributes to graft loss independently of graft rejection. Following kidney transplantation, a histologically distinct glomerulopathy may result from infection of the graft and may significantly alter its survival (Richardson et al., 1981; Herrera et al., 1986). Following liver transplantation, a distinct bile duct sclerosis syndrome has been related to active HCMV infection. HCMV may perhaps increase the expression of MHC antigens in the graft and may favor its rejection (Donaldson et al., 1987; Vierling & Fennel, 1985), although the way in which this could take place has not been defined. The role of HCMV infection in rejection is most convincing in the case of cardiac allografts. Accelerated development of coronary atherosclerosis associated with post-transplant HCMV infection has been reported and found to be independent of reports of age, lipid levels, and immunosuppression regimen (Loebe et al., 1990). Interactions between HCMV IE proteins and p53 in vessel smooth muscle cells have also been noted, and the virus may thus contribute to the formation of atherosclerotic plaques in the heart vessels (Streblow et al., 2001). The incidence of HCMV infection following allogenic bone marrow transplant (BMT) ranges from 32% to 70%, with an average of 50%, regardless of the prior serological status of the recipient and donor. Typing of strains has shown that infection is due to a virus derived from the recipient (Ruutu et al., 1990; Rubie et al., 1993; Winston et al., 1993). Its incidence in seronegative recipients receiving a seropositive marrow is lower than in seropositive recipients receiving a seropositive marrow, suggesting a transfer of adoptive immunity from donor to recipient (Griffiths, 2000). Thus, the most critical event is reactivation of a latent virus in seropositive BMT recipients, whereas in seronegative recipients, transmission mostly occurs through the larger quantities of blood products that they receive compared with solid organ transplant recipients. Increasing evidence indicates that apart from the serological status of donor and recipient, administration of blood products carries a significant risk of exposure to HCMV infection following BMT. Another major risk factor is the immunosuppressive regimen. Reactivation of infection ranges between 20% with cyclosporine and 60% with OKT3 administration. Hematologic, hepatic, and gastrointestinal abnormalities are relatively common. Uncertainty, however, persists with regard to both the role of virus replication in the onset of graft versus host disease and the toxicity of the pretransplant therapy. The most significant HCMV-associated syndrome in BMT is viral pneumonia, with an incidence ranging between 10 and 15%. Its pathogenesis is unknown, although several mechanisms, such as uncontrolled replication leading to organ damage and respiratory failure or immunologic destruction of the lungs triggered by HCMV, have been suggested. HCMV interaction during the induction and progression of HIV infection has not been definitively established. Even so, HCMV is a leading opportunistic agent in acquired immunodeficiency syndrome (AIDS). Autopsies have shown that 90% of patients develop active HCMV infection and 40% display sight- and/or life-threatening diseases. The increased survival of HIV-infected individuals following more effective prophylaxis and treatment of bacterial and protozoal infections, accompanied by virtual inactivity of their immune system, has augmented the importance of HCMV infection in these patients. Invasive HCMV infection, in fact, increases rapidly as CD4+ lymphocyte counts fall (Gallant et al., 1992; Webster et al., 1992; Leach et al., 1993). Three major organ systems are clinically affected: the CNS, the lungs, and the gastrointestinal tract. Retinitis is the most frequent CNS infection directly attributable to HCMV replication, and the most sight-threatening (Vinters et al., 1989; Sison et al., 1991; Pertel et al., 1992; Faber et al., 1992). Autoptic immunohistochemical analysis has shown HCMV presence in all layers of the retina, without underlying choroidal involvement. Absence of virus in the endothelium of the retinal microvasculature indicates that its hematogenous spread does not follow replication in these cells, but that perhaps the virus breaks through the capillary endothelium. 5. Pathogenesis and pathology Histopathologic and immunohistochemical examination of necropsy tissues has indicated that the virus initially enters via the epithelium of the upper alimentary, respiratory, or genitourinary tracts. However, since infection is readily established by transfusion and transplantation, initial infection of epithelial cells does not seem essential (Sinzger & Jahn, 1996). Cytotrophoblasts form a barrier between the maternal and fetal circulation, but readily allow HCMV replication in vitro, suggesting that the fetus is infected hematogenously (Halwachs-Baumann et al., 1998; Hemmings et al., 1998). Leukocytes and vascular endothelial cells aid the spread of HCMV. During acute infection, HCMV can be isolated from buffy coat preparations of leukocytes, but not from the plasma, and is presumably carried by one or more cell subsets. Viral antigens are found mainly in neutrophils and monocytes in acute infection (Sinzger & Jahn, 1996) and transmission through blood products from seropositive donors can be prevented by removal of leukocytes (Pass, 2001). Moreover, immunohistochemical double-labeling has shown that macrophages infiltrating organ tissues support virus replication, whereas granulocytes show no signs of viral late gene expression (Sinzger & Jahn, 1996). Lastly, virus encoded chemokines may facilitate dissemination of CMV from the initial replication site by attracting neutrophils and monocytes (Penfold et al., 1999). Saederup et al. (1999), in fact, have demonstrated that mouse CMV-encoded b-chemokine promotes monocyte-associated viremia, whereas viruses with deletions in the gene for this b-chemokine have significantly reduced levels of viremia. Endothelial cells detached from S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 the basal membrane as a result of HCMV infection are also involved in hematogenous spreading of the virus. However, the molecular mechanisms responsible for the interaction between these cell populations and the microvascular system, and subsequent transmission of the virus to the target organs, have not been determined. Hematogenous spreading is typically followed by infection of ductal epithelial cells. Fibroblasts are rarely involved, despite the fact that the virus can replicate in such cells in vitro. CMV is recognized histologically by its characteristic ‘‘owl’s eye’’ intranuclear inclusions with a surrounding halo and marginated chromatin. Typical cytomegalic cells are found in the salivary gland, bile duct, bronchial and renal tubular epithelium, islet cells, epithelial cells of the inner ear, the capillary endothelium, astrocytes, and neurons (Griffiths, 2000; Pass, 2001). In cases of severe disseminated disease, evidence of HCMV involvement can be found in all organs. The present review, however, will be confined to those most commonly involved (for further details, see Ho, 1991 and Becroft, 1981). Salivary gland involvement is probably chronic and probably the outcome of subclinical congenital and perinatal infection, since it is more frequent in infants and young children and diminishes with age. In contrast, viruria is a constant feature in all age groups as the result of replication in the genitourinary tract (Plachter et al., 1996). In the kidneys, infected cells are found in the proximal tubules, Henle’s loop, and the collecting tubules, but seldom in the glomeruli. As already mentioned in Section 4, infection is directly involved in renal transplant dysfunction and rejection, whereas it rarely leads to dysfunction in normal individuals. Elevated liver enzyme levels indicative of subclinical hepatitis are frequently associated with HCMV infection in immunocompetent individuals. Cytomegalic cells are usually found in the bile duct epithelium, less frequently in the capillary endothelium, and rarely in the parenchyma. Together with cytomegalic cells, areas of inflammatory infiltrates are a dominant feature in histological sections, possibly suggesting that liver dysfunction might result directly from virus cytopathogenicity or indirectly from the inflammatory reaction to HCMV infection (Phillips et al., 1977). HCMV infection in HIV patients often affects other parts, or even the entire digestive system (Rene et al., 1988). The colon is the most common replication site, followed by the esophagus, rectum, and small intestine. The direct effects of infection range from punctate, superficial ulcers to deep ulcerations, with extensive necrotizing involvement and gut perforation. HCMV pneumonia is infrequent in immunocompetent individuals, but often is severe in immunosuppressed patients, especially BMT and heart-lung recipients. Infected cells are mainly observed in the alveolar and bronchial epithelium, along with inflammatory infiltrates (Apperley & Goldman, 1988; Wreghitt et al., 1988). The range of clinical presentation, from severe pulmonary dysfunction and limited focal infection, together with similar findings in 281 animal models, suggests that dysfunction may be due to virus-associated dysregulation of the immune response. CNS damage is a frequent feature of congenital infection (Fowler et al., 1992). Symptoms include mental retardation, seizures, hypotonia, and hearing loss due to various alterations in the form of many small, necrotic foci surrounded by an inflammatory infiltrate. In histological sections, inclusion-bearing cells positive for viral antigens have been found in neurons, glia, ependyma, choroid plexus, meninges, and vascular endothelium. These cells are also widespread in the semicircular canals, vestibular membrane, and cochleae. CNS involvement was once regarded as infrequent, except in congenital infection. Today, however, CNS tissues are the major target organs in AIDS patients, where HCMV replicates efficiently in various brain structures and in specialized organs, such as the retina and cochlea. Histopathologic alterations include microglial nodules, focal parenchymal necrosis, necrotizing ventriculitis, hemorrhages, astrocytic proliferation, and perivascular inflammation. Acute retinal infection frequently leads to blindness, probably as the outcome of direct lysis of the retinal pigment epithelial cells during virus replication. In conclusion, organ dysfunction seems to coincide with HCMV replication, although the frequent association of an inflammatory reaction indicates that other pathogenetic mechanisms are also operative. 6. Host defences HCMV infections are kept under control by the immune system. Total HCMV clearance, however, is rarely achieved, and the viral genome remains at selected sites in a latent state (Reddehase et al., 1994). Initiation of productive replication results in transient virus shedding and recrudescent disease (Mocarski, 1996). Since severe infection is usually restricted to individuals with impaired cell-mediated immunity, it is evident that this arm of the immune response provides the most protection. Even so, the supportive role of the humoral system in keeping CMV loads below critical thresholds must not be overlooked. 6.1. Cell-mediated immunity Mouse models have been employed to determine the role of specific components of the immune response and their recognition of viral proteins (Koszinowski et al., 1990). Early studies demonstrated that a CMV-specific cytotoxic T lymphocyte (CTL) response was required for recovery from CMV infection. Suppression of CTLs caused reactivation and dissemination of natural infection. Further studies showed that both natural killer (NK) cells and CD8+ cytotoxic T-cells are of primary importance in the prevention of recurrence (Jonjic et al., 1990; Polic et al., 1996; Hengel et al., 1998). Adoptive transfer of CD8+ cells 282 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 protected mice from lethal challenge independent of CD4+ T helper cells. CD8+ T-cell clones specific for both structural and nonstructural proteins were identified. However, CD8+ depletion with abs or in mouse mutants deficient in MHC Class I expression revealed additional antiviral activities mediated by CD4+-derived cytokines not seen when CD8+ cells are present. Lastly, experiments with mice deficient for perforin and granzyme- or Fas-mediated cytotoxicity have shown that replication is controlled by NK and CD8+ cells via the perforin and granzyme-mediated pathway, whereas the Fas-Fas ligand system is not critical (Riera et al., 2000, 2001). As already mentioned in Section 4, since infection (apart from congenital forms) is most severe in patients with dramatically impaired cell-mediated immunity, such as BMT recipients, and those with AIDS, it is evident that this arm of the immune response provides the most protection (Pass et al., 1983; Zanghellini et al., 1999). Its investigation in studies of lymphocyte proliferation in response to CMV antigens has shown that in most seropositive immunocompetent adults, this usually takes the form of CD4+ cell proliferation in response to envelope glycoproteins gB and gH, the lower matrix protein pp65, and IE proteins, in addition to other proteins (Liu et al., 1993; Davignon et al., 1995; He et al., 1995; Beninga et al., 1995). The function of proliferating CD4+ cells in resistance to HCMV infection is not clear. Studies in mice point to several antiviral effector functions, such as the production of cytokines and CD4+mediated cytotoxicity. The virus-encoded proteins that are targets of CTLs include a collection of structural and nonstructural forms (Boppana & Britt, 1996; Kern et al., 1999). Comparison of CTL precursor frequencies for envelope gB or the 72-kDa IE1 protein revealed a low frequency of gBspecific CTLs ( < 6%), whereas 18 – 58% of CTLs recognized IE1-expressing target cells. The high frequencies for pp65 and IE1 proteins found in subsequent studies show that they are the chief targets of the CTL-mediated immune response (Pass, 2001). Although a direct role for virus-specific CTL responses in the resolution of infection has yet to be defined, in vivo evidence of the control of HCMV infection by CTLs has been obtained through the adoptive transfer of virus-specific clones. Passive administration of autologous HCMVspecific CD8+ CTLs at set intervals after BMT to seronegative recipients of marrow from seropositive donors generated a vigorous CMV-specific CTL response without onset of viremia or CMV disease (Walter et al., 1995). These studies provided the first evidence for the role of CTLs in controlling human CMV infection, and opened new therapeutic perspectives for CMV treatment in organ transplantation. 6.2. Humoral immunity During primary infection, immunocompetent individuals produce anti-HCMV immunoglobulin (Ig)M class abs that persist for 3 – 4 months, followed a few weeks later by IgG class abs that persist for life. Experimental and clinical findings show that the humoral response is beneficial. Mice immunized against murine CMV gB were protected against a lethal challenge (Rapp et al., 1992), and immunization of pregnant guinea pigs against guinea pig CMV envelope glycoprotein protected their fetuses in the same way (Harrison et al., 1995). Intrauterine infection is less severe when transmitted by means of recurrent rather than primary maternal infection (Griffiths, 2000). During primary infection, women who transmit the virus in utero have high levels of total IgG characterized by low avidity and neutralizing activity. Correlation of an enhanced ab response with a poor fetal prognosis suggests that fetal ab is responsible for immunopathology. Primary infection is more frequent and more severe when the renal transplant recipient is seronegative and the donor seropositive. Alleviation of this severity by preimmunization with the attenuated Towne strain (Plotkin et al., 1984) or administration of high-titer anti-CMV Igs (Snydman et al., 1987) also shows that humoral immunity is beneficial. Many CMV proteins are recognized by the humoral immune system (Landini & Michelson, 1988; Landini et al., 1988). The envelope glycoproteins (mainly gB and gH) are the targets of virus-neutralizing abs in both human and mouse models (Pass, 2001). The predominance of gB as a target is best explained by its dominant immunogenicity and abundance compared with other components of the envelope. Anti-gH abs have a potent, complement-independent, but minor neutralizing activity. Viral tegument components, including pp28 (UL99), pp65 (UL83), and pp150 (UL32), trigger an intense and long-lasting ab response that provides an indirect measure of viral replication and correlates with the clinical outcome. However, these abs are unable to react with the surface of virions and infected cells, and are of limited importance in a protective response. 6.3. Immune evasion by human cytomegalovirus A characteristic feature of infection in the normal host is persistence of the viral genome in a nonproductive form at specific anatomical sites for months or even years (Hengel et al., 1998). This ability to avoid elimination by the immune system is the result of (1) induction of a latent state of infection, (2) exploitation of immunologically privileged tissues for replication (i.e., epithelial cells of the salivary glands expressing an insufficient number of MHC Class I molecules to trigger clearance by CD8+ cell, and (3) expression of genes that interfere with the immune response (Mocarski, 2002). Table 2 summarizes the main mechanisms exploited by HCMV to evade the immune response. Escape from CD8+ cells is mediated by several mechanisms, all of which block the expression of MHC Class I molecules complexed with a potentially important CTL target, the 72-kDa IE1 phosphoprotein, or which S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 Table 2 HCMV genes associated with immune evasion Immune function Virus genes Mechanisms NK cell activity UL 40 MHC Class I antigen presentation/ processing US 2 Up-regulation of HLA expression: binding of inhibitory NK cell receptors Export of MHC Class I heavy chains from the ER VIA Sec61 Retention of MHC Class I complexes in the ER Inhibition of TAP-mediated peptide translocation into the ER Dislocation of MHC Class I heavy chains into cytosol Inhibition of 72-kDa IE1 antigen presentation to CD+ T-cells Degradation of MHC Class II antigen by down-regulating antigen presentation to CD4+ T-cells b-Chemokine-binding proteins Putative GPCR homologue Putative transferrin receptor homologue US 3 US 6 US 11 UL 83 MHC Class II antigen presentation US 2 Modulation of chemokine activity US 27/28 UL 78 UL 144 degrade MHC Class II molecule proteins, and, thus, prevent presentation of viral antigen to CD4+ lymphocytes. HCMV deletion mutants have been used to map two gene regions whose products are associated with the downregulation of MHC Class I complex formation; namely, US11 and that spanning US2 and US5 (Machold et al., 1997). Both US2- and US11-encoded proteins lead to the accumulation of MHC Class I heavy chains in the cytosol of infected cells, where they are degraded by the proteosome, thus shortening their half-life and preventing their expression on the cell surface. Binding of peptides to MHC Class I molecules depends on transport across the ER membrane by a specific transporter complex designated TAP 1/2 (transporter associated with antigen processing) (Sadasivan et al., 1996). Only after their introduction into the ER can peptides form heterodimers with MHC Class I molecules and b2-microglobulin and be recognized by CD8+ CTLs. Despite a significant increase in the expression of TAP molecules in infected cells, the HCMV glycoprotein encoded by the US6 gene inactivates the TAP system by interfering with formation of the TAP1TAP2-MHC Class I-b2-microglobulin complex. The finding that lysis of HCMV-infected fibroblasts by IE-specific CTL is usually low, but increases in fibroblasts infected with a mutant HCMV lacking the UL83 encoding the matrix pp65, suggests that phosphorylation of the 72-kDa IE1 protein by pp65 interferes with its antigenic processing and presentation to CD8+ cells (Wiertz et al., 1996; Gilbert et al., 1996; Jones & Sun, 1997). Experiments have demonstrated that NK cells are involved in the control of CMV infection. Beige mice (genetically deficient in functional NK cells) are more susceptible to murine CMV (Shellam et al., 1981). In 283 humans, MHC Class I HLA – E molecules protect target cells from NK cells by binding to inhibitory receptors on their surface. A convincing mechanism for escape from NK cell attack stems from the observation that the HCMV UL40 gene encodes for a glycoprotein containing a nine amino acid sequence homologous to the MHC Class I sequence that up-regulates the expression of Class I HLA-E molecules (Tomasec et al., 2000). Lastly, mechanisms interfering with chemokine-driven inflammation are exploited by HCMV to evade the immune response. HCMV-infected cells express viral G-proteincoupled receptor (GPCR) homologues encoded by US27, US28, UL33, and UL78, which are needed for virus replication in vitro (Chee et al., 1990b; Bodaghi et al., 1998). The US28-encoded GPCR binds to and sequesters several b-chemokines and, thus, enables the virus to evade the immune response. 6.4. Persistence and release from the host Cells in bone marrow and peripheral blood are the chief reservoirs for latent CMV infection. CMV DNA is found in a small percentage of peripheral blood monocytes, and gene expression is limited to the E genes. These findings and the demonstration that tissue macrophages differentiated from circulating monocytes also express E and L genes have led to the formulation of the following model: bone marrow precursors of blood monocytes are the site of CMV latency and provide a means of dissemination upon differentiation into circulating monocytes (Pass, 2001). Differentiation of latently infected monocytes into macrophages leads to reactivation and productive infection. Support for this model is provided by the reactivation of CMV by allogeneic stimulation in vitro of peripheral blood mononuclear cells from seropositive healthy donors (Soderberg-Naucler et al., 1997). Moreover, it has been found that a small percentage (close to 0.01%) of CD33+/CD14+ or CD33+/CD15+ bone marrow mononuclear cells from seropositive human donors express transcripts associated with latency. In contrast, T lymphocytes, B lymphocytes, and CD33- mature granulocytes and macrophages are negative (Hahn et al., 1998). Recurrent infection can be defined as indefinite, but intermittent, excretion of the virus from single or multiple sites. It should be distinguished from the prolonged excretion typical of primary infection and also infection in the immunocompromised host (Pass, 2001), where productive infection, as measured by viral excretion, is markedly increased. In allograft recipients and AIDS patients, the incidence of virus excretion from multiple sites approaches 100%. During primary, recurrent, or persistent infection, CMV is shed in multiple body fluids, such as saliva, urine, tears, cervicovaginal fluid, breast milk, and semen, probably due to virus replication in glandular epithelial cells, accompanied by virus release into excretions. 284 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 7. Clinical features associated with human cytomegalovirus infection 7.1. Infection in normal hosts HCMV infection in normal immunocompetent hosts is generally subclinical, but nonetheless, is thought to account for 8% of all the cases of mononucleosis (Nesmith & Pass, 1995). Clinical manifestations of mononucleosis due to HCMV are very similar to that induced by the more common Epstein-Barr virus. Persistent fever, myalgia, headache, cervical lymphadenopathy, splenomegaly, and nonspecific constitutional symptoms are common, and may persist for weeks. Rash is also present in  30% of patients. Uncommon complications include pneumonia, myocarditis, hemolytic anemia, retinitis, gastrointestinal ulceration, hepatitis, CNS involvement (Guillain-Barré syndrome), and peripheral neuropathy. Common laboratory findings include a negative heterophil ab response (Paul-Bunnel test), atypical lymphocytosis, and elevated liver aminotransferases. Slight elevation of serum bilirubin and jaundice are occasional findings. 7.2. Congenital infection From 5% to 10% of congenitally infected newborns display cytomegalic inclusion disease, whose symptoms include intrauterine growth retardation, jaundice, hepatosplenomegaly, thrombocytopenia, petechiae, chorioretinitis, and hepatitis, along with CNS involvement in the form of microcephaly, encephalitis, seizures, and focal neurological signs. Most of the non-CNS (liver and blood-forming organs) manifestations are self-limiting and resolve without therapy in the vast majority of cases. In contrast, the neurological damage is permanent and accounts for the long-term morbidity and poor prognosis of cytomegalovirus inclusion disease (Boppana et al., 1992). Long-term prospective studies indicate that 80% of infants with symptomatic congenital infection will display serious life-long neurological abnormalities. In 11 –20%, damage caused by the virus leads to such a severe life-threatening organ dysfunction that the patient dies during infancy. Long-term follow-up shows that  15% of the asymptomatic infected infants at birth (90 – 95% are asymptomatic) may develop hearing defects or impaired intellectual performance. Differences in the reported incidence of hearing defects are probably attributable to the use of different evaluation methods. 7.3. Cytomegalovirus infection in the immunocompromised host HCMV is a significant opportunistic pathogen in immunocompromised patients. Primary infection, reactivation of latent virus, and reinfection are possible and are often clinically silent. The onset of infection is marked by spiking pyrexia, which may resolve in a few days (Pass, 2001). Its severity is roughly parallel with the level of immunosuppression, and is greatest in BMT recipients and AIDS patients with low CD4+ T-cell counts. Solid organ transplant recipients, patients receiving immunosuppressive chemotherapy, and subjects with congenital immunodeficiencies may also be symptomatic. Table 3 summarizes the major clinical diseases related to the type of immunocompromised host. Some patients may develop a CMV syndrome associated with pneumonitis. This has a poor prognosis (up to 90% mortality). Retinitis may be present alone following virus dissemination. Replication of CMV in the gut may be asymptomatic or associated with extensive ulceration due to erosion of the neighboring blood vessels and hemorrhages. Replication in the CNS of AIDS patients produces some of the symptoms observed in congenital infection, and is often followed by encephalopathy and/or peripheral polyradiculopathy. It is not certain whether CMV infection and disease are simply markers of the immune dysfunction that follows HIV replication or whether CMV infection itself promotes HIV progression, although it is clear that highly active antiretroviral therapy has reduced HIV-1 viral load, along with a reduction in CMV viremia and disease (Gerna et al., 1998a; O’Sullivan et al., 1999). Clinical signs of HCMV infection in transplant recipients may be absent or severe, although severe infection is now less frequent as a result of better prophylaxis. HCMV is initially localized in the transplanted organ, i.e., hepatitis occurs generally in liver transplant recipients, pancreatitis in pancreas transplant recipients, but then spreads throughout the gastrointestinal tract and to the retina, skin, endometrium, lungs, and CNS (Rubin, 1994). HCMV disease is more difficult to treat in BMT compared with solid organ transplant recipients, and HCMV pneumonia has a high mortality rate, despite the recent introduction of specific antiviral drugs (Reed et al., 1988). Lastly, an immunosuppressive syndrome often related to HCMV infection in the late post-transplant period is characterized by superinfection with bacteria, fungi, and protozoa, perhaps due to disturbance of both the humoral and cellular immune response by HCMV. Table 3 Clinical syndromes associated with HCMV infection in the immunocompromised patient Syndromes AIDS Solid transplant BMT Esophagitis Gastritis Enterocolitis Hepatitis Pancreatitis Pneumonitis Retinitis Encephalopathy Polyradiculopathy + + + +   ++ + + + + + ++ ++ + +   + + + +  ++ +   S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 285 8. Diagnosis Virological and molecular detection of HCMV and serological demonstration of a specific immune response are used for diagnosis, although acute infection is usually diagnosed through detection of the virus in body fluids. 8.1. Virus detection HCMV can be isolated from several body fluids, such as urine, saliva, vaginal secretions, amniotic fluid, and blood, following primary or recurrent infections (Gerna et al., 1990; Revello et al., 1999). Moreover, HCMV-infected cells can be identified in biopsy samples (e.g., liver, lungs), owing to ‘‘owl’s eye’’ inclusions or, more efficiently, by immunohistochemistry, in situ PCR, or nucleic acid hybridization. Electron microscopy is employed to detect the virus in the urine from congenitally or perinatally infected infants since, unlike other human herpesviruses, the HCMV virions are present in high titers. However, it is generally agreed that acute HCMV infection is best diagnosed by detection of the virus in the blood since the level of viremia correlates with the clinical picture and the prognosis. The most widely used assays for detection and quantitation of HCMV load in blood include tests for viremia, antigenemia, DNAemia, and IE and L mRNAemia. 8.1.1. Viremia Viremia is measured by an assay that allows the detection and the quantification of infectious viral particles in the blood. Conventional methods are the inoculation of low passage ( < 25) human embryonic lung or foreskin fibroblasts with peripheral blood leukocytes (PBL) and identification of the characteristic focal CPE (Fig. 2). This usually develops very slowly, and the cultures must be carefully observed for at least 21 days before being reported as negative. This expensive and time-consuming Fig. 4. HCMV viremia assay. This picture was kindly provided by Rossana Cavallo. Fig. 5. HCMV antigenemia assay. This picture was kindly provided by Rossana Cavallo. technique has been replaced by the ‘‘shell vials’’ assay, which retains the specificity and sensitivity of virus isolation, but provides results within 24 hr (Gleaves et al., 1984; Griffiths et al., 1984). A human fibroblast monolayer is inoculated with 2  105 PBL and fixed 18 hr later. The nuclei of the infected fibroblasts are stained by immunofluorescence using a monoclonal ab reactive with the MIE protein p72 (Fig. 4). The assay is based on the assumption that each stained cell was infected by a single PBL carrying an infectious virus. Several studies indicate that transplanted patients with HCMV viremia in the range of 10– 100 infected fibroblasts following inoculation with 2  105 PBL have a high risk of developing symptomatic HCMV diseases (Gerna et al., 1995; Grossi et al., 1995) and should be treated with antiviral drugs (Gerna et al., 1991). Moreover, viremia is a rapid way to monitor the efficacy of an antiviral treatment since it decreases sharply 1 or 2 days after the beginning of an effective regimen (Gerna et al., 1993), whereas persistent or increasing viremia levels predict the emergence of a drug-resistant HCMV strain (Gerna et al., 1992a). 8.1.2. Antigenemia Antigenemia is measured by the quantitation of leukocyte nuclei positive, in an immunofluorescence assay, for the HCMV lower matrix phosphoprotein pp65 in a cytospin preparation of 2  105 PBL (van der Bij et al., 1988a, 1988b; Revello et al., 1989; Gerna et al., 1992b) (Fig. 5). This rapid assay provides a result in a few hours. Antigenemia is an indirect marker of disseminated infection, since pp65 is transferred to uninfected leukocytes by infected cells after transient fusion of plasma membranes and exchange of cytoplasmic material (Gerna et al., 2000b). By comparison with the test for infectious virus, the test for viral antigen gives a positive result earlier after the onset of infection and becomes negative later in the late phase of a systemic infection. Since high levels of 286 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 antigen are frequently found in patients with HCMV disease and low levels correlate with asymptomatic infections, the test for antigenemia is sensitive enough to monitor infection and antiviral treatment in immunocompromised patients (van den Berg et al., 1989; Grossi et al., 1995). 8.1.3. DNAemia HCMV DNA in clinical samples is currently detected and quantitated by qualitative or quantitative PCR. Qualitative PCR detects viral DNA in the amniotic fluid (Revello et al., 1995) and the aqueous humor of AIDS patients with HCMV retinits (Gerna et al., 1994a), whereas quantitative PCR provides more clinically useful information concerning the virus load in blood. PCR measurement of DNA levels produces good prognostic information in both transplant recipients and HIV-positive individuals (Kidd et al., 1993; Gerna et al., 1994b, 1998b; Bowen et al., 1997). 8.1.4. RNAemia This assay is a good marker for monitoring active HCMV replication and disease, since it detects mRNAs transcribed from IE or L genes in PBL or whole blood (Gozlan et al., 1996; Lam et al., 1998). Reverse transcription-PCR was the first technique employed to detect viral transcripts. Its major drawbacks, namely, instability of the RNA and the difficulty of discriminating PCR products from a DNA or RNA template when working with unspliced viral transcripts, cause both false negatives and false positives. However, these difficulties have now been circumvented by nucleic acid sequence-based amplification, which allows specific amplification of unspliced viral mRNAs in a DNA background, and is a highly promising technique for monitoring HCMV infection in transplant recipients (Blok et al., 1998; Aono et al., 1998; Gerna et al., 1999, 2000a) and for the early diagnosis of both pregnant women with primary infection and congenitally infected newborns (Revello et al., 2001). 8.2. Detection of the immune response Serological determination of a past or recent HCMV infection in immunocompetent individuals rests on detection of virus-specific IgG or IgM abs. Occurrence of a primary infection is conventionally deduced from seroconversion from IgG ab negative to IgG ab positive in the interval between two serological assays. A specific IgM response may be serologic evidence of a recent primary infection. Detection of IgM abs, however, is not reliable because false positives may be induced by rheumatoid factor, antinuclear abs, and other cross-reactive factors not yet identified. Moreover, true-positive tests detect IgM persistence in patients with a past HCMV infection. Interpretation of a positive IgM assay in pregnant women is assisted by determination of the avidity of HCMV-specific IgG, since low-avidity IgG is produced early in infection and highavidity IgG is a marker of past or recurrent infection (Blackburn et al., 1991; Grangeot-Keros et al., 1997; Bodeus et al., 1998; Lazzarotto et al., 1999). Detection of anti-HCMV IgG abs is a useful way of documenting a past infection, and they are also a marker of potential infectivity, since reactivation of a latent infection may occur. Serologic assays are thus of great clinical importance in donor selection, since HCMV can be transmitted by blood donation and organ transplantation. 9. Prevention of human cytomegalovirus infection and disease Steps to prevent infection in groups at risk have been devised in light of what is known about the transmission routes for HCMV. In solid organ transplant recipients, for example, the risk of a primary infection could be reduced by matching seronegative donors and recipients, although this approach is hampered by the scarcity of donor organs (Stratta et al., 1990). Prophylaxis with antiviral agents and pre-emptive therapy (see Section 10) have proved useful in transplant patients. Transmission to immunocompromised individuals, pregnant women, and premature newborns through donor blood can be avoided by using HCMVseronegative, filtered, or leukocyte-deprived blood products. This method places a greater burden on blood transfusion centers, but should be used whenever possible, since it significantly reduces HCMV transmission (Gilbert et al., 1989; Sayers et al., 1992). Administration of an HCMV vaccine to secure pre-exposure immunization would be another way of protecting groups at risk. 9.1. Human cytomegalovirus vaccines Development of a vaccine to prevent congenital HCMV infection is a priority concern, since the virus is a leading cause of CNS damage in children (Pass, 1996). The potential benefit of vaccine-induced immunity has been estimated at 40-fold reduction with respect to intrauterine infection and 25- to 30-fold reduction with respect to decrease in CNS damage (Britt, 1996). However, no vaccine is available and live-virus vaccines have not obtained official approval (Elek & Stern, 1974; Plotkin et al., 1994). Both humoral and cellmediated immunity are needed to prevent HCMV disease. Many studies indicate that gB and pp65 are absolutely necessary vaccine components to secure the induction of neutralizing abs and CTL responses (Britt et al., 1990; Gonczol et al., 1991; Riddell et al., 1991; Marshall et al., 1992; Wills et al., 1996). Moreover, gH and gN for neutralizing abs and IE1 and pp150 for CTL responses could increase the protective immune response (Kern et al., 1999; Gyulai et al., 2000). There are a variety of options for the elaboration of a safe and effective vaccine: attenuated live virus vaccines (Towne strain), recombinant live vac- S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 cines using chimeric viruses comprising genomic portions of the virulent strain Toledo and the attenuated strain Towne, canarypox-HCMV recombinants expressing gB and pp65, vaccination with HCMV DB, subunit gB vaccine; pp65 peptide-based vaccine, and DNA vaccines encoding the gB and pp65 proteins. The attenuated Towne strain, the recombinant gB, and the canarypox gB and pp65 vaccines already have been tested in clinical trials. The attenuated Towne vaccine has been tested both in healthy volunteers and in transplant recipients (Plotkin et al., 1975, 1976, 1989, 1991; Adler et al., 1995, 1998). It induced neutralizing abs and an CTL response and reduced HCMV disease in seronegative renal transplant recipients, but did not prevent infection. However, there was no evidence of its efficacy in a placebo-controlled trial in seronegative mothers of HCMV-infected children. In conclusion, it is immunogenic, but the protection it offers is less effective than that seen after natural infection. In addition, a live virus is a potential risk factor in transplant recipients, and all live attenuated herpesvirus vaccines are overshadowed by their possible oncogenicity. To circumvent these risks, poxviruses with limited potential for replication in humans (canarypox) have been tested as vehicles for the expression of recombinant gB and pp65 (Adler et al., 1999; Berencsi et al., 2001). A subunit vaccine composed of a modified gB protein was evaluated after combination with a new powerful adjuvant, MF59, based on an oil-in-water emulsion of squalene. Results of a trial of 46 seronegative adults established safety, immunogenicity, and optimal antigen dose (Pass et al., 1999). Whether the neutralizing abs elicited by this vaccine will be sufficient to prevent recurrence or primary HCMV infection is still an open question (Drulak et al., 2000). Other promising preclinical approaches include vaccination with HCMV DB (Pepperl et al., 2000) and administration of DNA vaccine encoding viral immunogenic proteins to elicit a humoral and a CTL response (Endresz et al., 1999, 2001). 10. Antiviral treatment 10.1. Currently available drugs Treatment of HCMV infections is difficult because there are few options. To date, the nucleoside analog GCV, the nucleotide analog cidofovir [CDV; (S)-1-(3-hydroxy-2phosphonylmethoxypropyl) cytosine], and the pyrophosphate analog foscarnet (PFA) have been licensed for serious or life-threatening HCMV infections in immunocompromised individuals. These drugs have produced clinical improvement in many patients, but suffer from poor oral bioavailability, low potency, development of resistance in clinical practice, and dose-limiting toxicities, and hospitalization is sometimes required. GCV is a competitive inhibitor of viral DNA polymerase (UL54), and its antiviral activity requires monophosphor- 287 ylation in the infected cell by the phosphotransferase encoded by the UL97 gene of HCMV (Littler et al., 1992), followed by diphosphorylation by cellular kinases. It is administered by intravenous infusion, and is usually the front-line drug for the treatment of HCMV infections. An oral formulation that is effective for prophylaxis in immunocompromised patients has also been approved (Spector et al., 1996). However, its side-effects include potentially dose-limiting neutropenia and the emergence of GCVresistant strains due to UL97 and UL54 gene mutations, mainly in AIDS patients during prolonged maintenance therapy (Erice et al., 1989; Lurain et al., 1994; Chou et al., 1995; Baldanti et al., 1995, 1998), and less frequently in solid organ transplant recipients (Limaye et al., 2000, 2002). PFA is a noncompetitive inhibitor of the pyrophosphatebinding site of HCMV DNA polymerase, which does not require prior activation by a virally encoded enzyme. It is administered intravenously as an alternative to GCV in the event of GCV resistance or severe side-effects, although its application is limited by nephrotoxicity and disturbance of the electrolyte balance (Vogel et al., 1998; Chrisp & Clissold, 1991). Long-term exposure to PFA may lead to the emergence of resistant strains due to UL54 mutations (Knox et al., 1991; Baldanti et al., 1996). CDV is a competitive inhibitor of the HCMV DNA polymerase, and has been approved for the treatment of HCMV retinitis (De Clercq, 1998). One of its advantages compared with GCV and PFA is its long intracellular halflife (Ho et al., 1992; Aduma et al., 1995). Its intermittent intravenous administration is also possible. Toxicology evaluations in animals have demonstrated that nephrotoxicity is its major side-effect, although this can be reduced by its less frequent administration, coupled with a high probenecid dose and saline hydration (Cundy et al., 1994). Cross-resistance between GCV and CDV could become a concern since they share the same target, the product of the UL54 gene. The emergence of GCV-resistant strains to GCV through UL54 mutations in vitro or in AIDS patients is well documented (Smith et al., 1997; Cherrington et al., 1998). Double resistance to GCV and PFA in strains from AIDS patients has been reported (Baldanti et al., 1996), and high-level GCV-resistant strains with both UL97 and UL54 mutations were found to be cross-resistant to CDV (Smith et al., 1997). At present, however, CDV is effective against most of the GCV-resistant isolates studied, as well as PFAresistant viruses generated in vitro. An antisense oligonucleotide against the HCMV IE2 mRNA (ISIS2922, fomivirsen) has also been developed (Azad et al., 1993; Anderson et al., 1996) for intravitreal application in patients with retinitis (De Smet et al., 1999) who do not respond to conventional management. A promising new class of anti-HCMV compounds is formed of benzimidazole ribosides, which do not inhibit the viral DNA polymerase. The benzimidazole analog 1263W94 has many characteristics that make it an attractive candidate for development; namely, high potency in vitro, 288 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 selectivity, good oral bioavailability, and lower toxicity than current therapies. Initial clinical trials have provided encouraging results, including good tolerability and linear pharmacokinetics over a wide dose range (Chulay et al., 1999). 10.2. Therapeutic approaches The antiviral agents described in the previous section are used to treat and prevent HCMV infections in immunocompromised patients. In the immunocompetent host, infections normally resolve spontaneously and antiviral therapy is not indicated. Three major therapeutic approaches are currently employed to manage HCMV infections and diseases: prophylaxis, pre-emptive therapy, and treatment of an established disease. 10.2.1. Prophylaxis In prophylaxis, the antiviral drug is administered before active HCMV infection is detected to prevent its occurrence. The therapeutic value of an antiviral prophylaxis in AIDS patients at high risk for HCMV disease is controversial. A randomized clinical trial of oral GCV versus placebo found that prophylaxis significantly reduced disseminated disease and the incidence of retinitis (Spector et al., 1996). In contrast, Brosgart et al. (1998) found no significant reduction in the occurrence of HCMV disease. Moreover, it is still not clear whether oral GCV prophylaxis improves survival and sight preservation compared with pre-emptive therapy (Masur et al., 1996). GCV prophylaxis after allogenic BMT in seropositive recipients was significantly protective in two studies (Goodrich et al., 1993; Boeckh & Bowden, 1995), but failed in a third (Winston et al., 1993) and did not reduce overall mortality (Goodrich et al., 1993; Winston et al., 1993; Boeckh & Bowden, 1995), probably because drug-induced neutropenia led to fatal bacterial and fungal infections. In one study of heart transplant recipients, GCV prophylaxis exhibited a protective effect in seropositive patients, but not in primary infection (Merigan et al., 1992), but the opposite result was obtained in a different study (Macdonald et al., 1995). For renal transplant recipients, prophylaxis is recommended when the donor or recipient is seropositive and if the recipient is subjected to an immunosuppressive regimen, including an antilymphocyte treatment, or for a seronegative recipient of a graft from a seropositive donor (Jassal et al., 1998). In conclusion, antiviral prophylaxis may be useful in some cases, but it is a high-cost strategy that exposes all patients to drug toxicity (marrow suppression and renal toxicity), the selection of antiviral resistance, and the risk of late-onset HCMV infection and disease (Emery, 2001). 10.2.2. Pre-emptive treatment Pre-emptive treatment was first described in the early 1990s (Rubin, 1991) as a means of reducing the incidence of HCMV disease by withholding antiviral drugs until they would be maximally effective. Antiviral treatment is initiated when HCMV positivity is detected in the blood or broncoalveolar lavage fluid using sensitive methods, such as PCR, nucleic acid sequence-based amplification, and tests for viral antigen. The advantages for the management of HCMV infection include the targeting of antiviral therapy to those most at risk for future disease, reducing the number of patients exposed to antiviral toxicity, lowering the risk of drug resistance, and maximizing the cost:benefit ratio. In the BMT setting, pre-emptive GCV administration reduced HCMV disease, and was literally life saving (Goodrich et al., 1991; Einsele et al., 1995) in contrast to its lack of effect on survival when used prophylactically (Goodrich et al., 1993; Winston et al., 1993; Boeckh & Bowden, 1995). The pre-emptive approach also prevented HCMV diseases in liver transplant recipients (Singh et al., 1994). A prospective study of the predictive value of PCR for CMV in asymptomatic kidney transplant recipients indicated that while a substantial number of recipients become positive for HCMV-PCR after transplantation, only a minority would develop CMV disease. Negative CMV-PCR assay is an accurate negative predictor for CMV disease, but the value of HCMV-PCR as a guide for pre-emptive anti-CMV therapy in kidney transplant recipients is limited (Benedetti et al., 1998). 10.2.3. Treatment of an established disease It is generally agreed that it is better to prevent HCMV diseases with pre-emptive approaches than to treat an established disease, since an ongoing virus infection may induce pathological phenomena unresponsive to antiviral drugs and extensive tissue damage that may result in target organ failure. Clinical trials on AIDS patients with retinitis showed that intravenous GCV, PFA, and CDV slow the progression of the disease and lead to remission (Palestine et al., 1991; Spector et al., 1993; Polis et al., 1995; Chavez-de la Paz et al., 1997; Lalezari et al., 1998). In one study, PFA showed a higher survival benefit than GCV (Anonymous, 1992). Retinitis can also be treated with intravitreal injections of antiviral compounds or by inserting an intraocular implant that slowly releases the drug into the vitreous fluid (Masur et al., 1996; Jacobson, 1997 –1998). All antiviral compounds suppress active replication of the virus, but do not eliminate it. Unless immune reconstitution occurs as a consequence of highly active antiretroviral therapy, chronic suppressive maintenance anti-HCMV management is required, and will be equally effective when administered intravenously, per os, or locally (Drew et al., 1995). However, while oral and local administration avoid the inconveniences and risks associated with the intravenous route, they are less effective in preventing disease in the contralateral eye (Jacobson, 1997– 1998; Cheung & Teich, 1999). Maintenance therapy may result in the appearance of resistant and refractory strains (Drew et al., 1991; Cherrington et al., 1996; Jabs et al., 1998a, 1998b), S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 whose estimated emergence rate is  25 – 33% after 9 months. These rates are similar for GCV, PFA, and CDV (Jabs et al., 1998a, 1998b). The efficacy of the antisense oligonucleotide fomivirsen in treating HCMV retinitis in AIDS patients has been demonstrated recently (The Vitravene Study Group, 2002a, 2002b). GCV or PFA regimens have enjoyed some success in the treatment of symptomatic gastrointestinal (esophagitis, enterocolitis), respiratory (pneumonia), and neurologic (encephalitis, peripheral neuropathy, polyradiculoneuropathy) HCMV diseases in AIDS (Dieterich et al., 1993; Blanshard et al., 1995; Cheung & Teich, 1999). No antiviral agents have been approved as yet for the treatment of congenital HCMV infections. Acknowledgments The authors’ research is supported by grants from MURST-CNR Biotechnology program L. 95/95, MURST (40% and 60%), from the AIDS Research Project, and by grants from the Ricerca Sanitaria Finalizzata (Regione Piemonte). References Adler, S. P., Starr, S. E., Plotkin, S. A., Hempfling, S. H., Buis, J., Manning, M. L., & Best, A. M. (1995). Immunity induced by primary human cytomegalovirus infection protects against secondary infection among women of childbearing age. J Infect Dis 171, 26 – 32. Adler, S. P., Hempfling, S. H., Starr, S. E., Plotkin, S. A., & Riddell, S. (1998). Safety and immunogenicity of the Towne strain cytomegalovirus vaccine. Pediatr Infect Dis J 17, 200 – 206. Adler, S. P., Plotkin, S. A., Gonczol, E., Cadoz, M., Meric, C., Wang, J. B., Dellamonica, P., Best, A. M., Zahradnik, J., Pincus, S., Berencsi, K., Cox, W. I., & Gyulai, Z. (1999). A canarypox vector expressing cytomegalovirus (CMV) glycoprotein B primes for antibody responses to a live attenuated CMV vaccine (Towne). J Infect Dis 180, 843 – 846. Aduma, P., Connelly, M. C., Srinivas, M. C., & Fridland, A. (1995). Metabolic diversity and antiviral activities of acyclic nucleoside phosphonates. Mol Pharmacol 47, 816 – 822. Ahn, J. H., Jang, W. J., & Hayward, G. S. (1999). The human cytomegalovirus IE2 and UL112-113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein associated nuclear bodies (PODs or ND10). J Virol 73, 10471 – 10548. Anderson, K. P., Fox, M. C., Brown-Driver, V., Martin, M. J., & Azad, R. F. (1996). Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediateearly RNA. Antimicrob Agents Chemother 40, 2004 – 2011. Anonymous (1992). Mortality in patients with the acquired immunodeficiency syndrome treated with either foscarnet or ganciclovir for cytomegalovirus retinitis. Studies of Ocular Complications of AIDS Research Group, in collaboration with the AIDS Clinical Trials Group. N Engl J Med 326, 213 – 220. Erratum: N Engl J Med 326, 1172 (1992). Aono, T., Kondo, K., Miyoshi, H., Tanaka-Taya, K., Kondo, M., Osugi, Y., Hara, J., Okada, S., & Yamanishi, K. (1998). Monitoring of human cytomegalovirus infections in pediatric bone marrow transplant recipients by nucleic acid sequence-based amplification. J Infect Dis 178, 1244 – 1249. 289 Apperley, J. F., & Goldman, J. M. (1988). Cytomegalovirus: biology, clinical features and methods for diagnosis. Bone Marrow Transplant 3, 253 – 264. Azad, R. F., Driver, V. B., Tanaka, K., Crooke, R. M., & Anderson, K. P. (1993). Antiviral activity of a phosphorothioate oligonucleotide complementary to RNA of the human cytomegalovirus major immediateearly region. Antimicrobiol Agents Chemother 37, 1945 – 1954. Baldanti, F., Silini, E., Sarasini, A., Talarico, C. L., Stanat, S. C., Biron, K. K., Furione, M., Bono, F., Palu, G., & Gerna, G. (1995). A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate. J Virol 69, 796 – 800. Baldanti, F., Underwood, M. R., Stanat, S. C., Biron, K. K., Chou, S., Sarasini, A., Silini, E., & Gerna, G. (1996). Single amino acid changes in the DNA polymerase confer foscarnet resistance and slow-growth phenotype, while mutations in the UL97-encoded phosphotransferase confer ganciclovir resistance in three double-resistant human cytomegalovirus strains recovered from patients with AIDS. J Virol 70, 1390 – 1395. Baldanti, F., Simoncini, L., Talarico, C. L., Sarasini, A., Biron, K. K., & Gerna, G. (1998). Emergence of a ganciclovir-resistant human cytomegalovirus strain with a new UL97 mutation in an AIDS patient. AIDS 12, 816 – 818. Baldick, C. J., & Shenk, T. (1996). Proteins associated with purified human cytomegalovirus particles. J Virol 70, 6097 – 6105. Baldick, C. J., Marchini, A., Patterson, C. E., & Shenk, T. (1997). Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle. J Virol 71, 4400 – 4408. Becroft, D. M. O. (1981). Prenatal cytomegalovirus infection: epidemiology, pathology, and pathogenesis. In H. S. Rosenberg, & J. Bernstein (Eds.), Perspective in Pediatric Pathology ( pp. 203 – 241). New York: Masson Press. Benedetti, E., Mihalov, M., Asolati, M., Kirby, J., Dunn, T., Raofi, V., Fontaine, M., & Pollak, R. (1998). A prospective study of the predictive value of polymerase chain reaction assay for cytomegalovirus in asymptomatic kidney transplant recipients. Clin Transplant 12, 391 – 395. Beninga, J., Kropff, B., & Mach, M. (1995). Comparative analysis of fourteen individual human cytomegalovirus proteins for helper T cell response. J Gen Virol 76, 153 – 160. Benson, J. D., & Huang, E.-S. (1990). Human cytomegalovirus induces expression of cellular topoisomerase II. J Virol 64, 9 – 15. Berencsi, K., Gyulai, Z., Gonczol, E., Pincus, S., Cox, W. I., Michelson, S., Kari, L., Meric, C., Cadoz, M., Zahradnik, J., Starr, S., & Plotkin, S. (2001). A canarypox vector-expressing cytomegalovirus (CMV) phosphoprotein 65 induces long-lasting cytotoxic T cell responses in human CMV-seronegative subjects. J Infect Dis 183, 1171 – 1179. Bissinger, A. L., Singzer, C., Kaiserling, E., & Jahn, G. (2002). Human cytomegalovirus as a direct pathogen: correlation of multiorgan involvement and cell distribution with clinical and pathological findings in a case of congenital inclusion disease. J Med Virol 67, 200 – 206. Blackburn, N. K., Besselaar, T. G., Schoub, B. D., & O’Connell, K. F. (1991). Differentiation of primary cytomegalovirus infection from reactivation using the urea denaturation test for measuring antibody avidity. J Med Virol 33, 6 – 9. Blanshard, C., Benhamou, Y., Dohin, E., Lernestedt, J. O., Gazzard, B. G., & Katlama, C. (1995). Treatment of AIDS-associated gastrointestinal cytomegalovirus infection with foscarnet and ganciclovir: a randomized comparison. J Infect Dis 172, 622 – 628. Blok, M. J., Goossens, V. J., Vanherle, S. J., Top, B., Tacken, N., Middeldorp, J. M., Christiaans, M. H., van Hooff, J. P., & Bruggeman, C. A. (1998). Diagnostic value of monitoring human cytomegalovirus late pp67 mRNA expression in renal-allograft recipients by nucleic acid sequence-based amplification. J Clin Microbiol 36, 1341 – 1346. Bodaghi, B., Jones, T. R., Zipeto, D., Vita, C., Sun, L., Laurent, L., Arenzana-Seisdedos, F., Virelizier, J. L., & Michelson, S. (1998). Chemokine sequestration by viral chemoreceptors as a novel viral 290 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells. J Exp Med 188, 855 – 866. Bodeus, M., Feyder, S., & Goubau, P. (1998). Avidity of IgG antibodies distinguishes primary from non-primary cytomegalovirus infection in pregnant women. Clin Diagn Virol 9, 9 – 16. Boeckh, M., & Bowden, R. (1995). Cytomegalovirus infection in marrow transplantation. Cancer Treat Res 76, 97 – 136. Boppana, S. B., & Britt, W. J. (1996). Recognition of human cytomegalovirus gene products by HCMV-specific cytotoxic T cells. Virology 222, 293 – 296. Boppana, S. B., Pass, R. F., Britt, W. J., Stagno, S., & Alford, C. A. (1992). Symptomatic congenital cytomegalovirus infection: neonatal morbidity and mortality. Pediatr Infect Dis 11, 93 – 99. Boshart, M., Weber, F., Jahn, G., Dorsch-Hasler, K., Fleckenstein, B., & Schaffner, W. (1985). A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41, 521 – 530. Bowen, E. F., Sabin, C. A., Wilson, P., Griffiths, P. D., Davey, C. C., Johnson, M. A., & Emery, V. C. (1997). Cytomegalovirus (CMV) viraemia detected by polymerase chain reaction identifies a group of HIVpositive patients at high risk of CMV disease. AIDS 11, 889 – 893. Boyle, K. A., Pietropaolo, R. L., & Compton, T. (1999). Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol Cell Biol 19, 3607 – 3613. Bresnahan, W. A., & Shenk, T. (2000a). A subset of viral transcripts packaged within human cytomegalovirus particles. Science 288, 2373 – 2376. Bresnahan, W. A., & Shenk, T. (2000b). UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirusinfected cells. Proc Natl Acad Sci USA 97, 14506 – 14511. Bresnahan, W. A., Boldogh, I., Thompson, E. A., & Albrecht, T. (1996). Human cytomegalovirus inhibits cellular DNA synthesis and arrests productively infected cells in late G1. Virology 224, 150 – 160. Bresnahan, W. A., Thompson, E. A., & Albrecht, T. (1997). Human cytomegalovirus infection results in altered Cdk2 subcellular localization. J Gen Virol 78, 1993 – 1997. Britt, W. J. (1996). Vaccines against human cytomegalovirus: time to test. Trends Microbiol 4, 34 – 38. Britt, W. J., & Mach, M. (1996). Human cytomegalovirus glycoproteins. Intervirology 39, 401 – 412. Britt, W. J., Vugler, L., Butfiloski, E. J., & Stephens, E. B. (1990). Cell surface expression of human cytomegalovirus (HCMV) gp55-116 (gB): use of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody response. J Virol 64, 1079 – 1085. Brosgart, C. L., Louis, T. A., Hillman, D. W., Craig, C. P., Alston, B., Fisher, E., Abrams, D. I., Luskin-Hawk, R. L., Sampson, J. H., Ward, D. J., Thompson, M. A., & Torres, R. A. (1998). A randomized, placebo-controlled trial of the safety and efficacy of oral ganciclovir for prophylaxis of cytomegalovirus disease in HIV-infected individuals. Terry Beirn Community Programs for Clinical Research on AIDS. AIDS 12, 269 – 277. Browne, E. P., Wing, B., Coleman, D., & Shenk, T. (2001). Altered cellular mRNA levels in human cytomegalovirus infected fibroblasts: viral block to the accumulation of antiviral mRNAs. J Virol 75, 12319 – 12330. Burton, G. J., & Watson, A. L. (1997). The structure of the human placenta: implications for initiating and defending against virus infections. Rev Med Virol 7, 219 – 228. Butcher, S. J., Aitken, J., Mitchell, J., Gowen, B., & Dargan, D. J. (1998). Structure of the human cytomegalovirus B capsid by electron microscopy and image reconstruction. J Struct Biol 124, 70 – 76. Caswell, R., Hagemeier, C., Chiou, C.-J., Hayward, G., Kouzarides, T., & Sinclair, J. H. (1993). The human cytomegalovirus 86K immediate early (IE2) protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via region of IE2 required for transcriptional regulation. J Gen Virol 74, 2691 – 2698. Cavallo, R., Lembo, D., Gribaudo, G., & Landolfo, S. (2001). Murine cytomegalovirus infection induces cellular folylpolyglutamate synthetase activity in quiescent cells. Intervirology 44, 224 – 226. Cha, T. A., Tom, E., Kemble, G. W., Duke, G. M., Mocarski, E. S., & Spaete, R. R. (1996). Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol 70, 78 – 83. Chambers, J., Angulo, A., Amaratunga, D., Guo, H., Jiang, Y., Wan, J. S., Bittner, A., Frueh, K., Jackson, M. R., Peterson, P. A., Erlander, M. G., & Ghazal, P. (1999). DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression. J Virol 73, 5757 – 5766. Chavez-de la Paz, E., Arevalo, J. F., Kirsch, L. S., Munguia, D., Rahhal, F. M., De Clercq, E., & Freeman, W. R. (1997). Anterior nongranulomatous uveitis after intravitreal HPMPC (cidofovir) for the treatment of cytomegalovirus retinitis. Analysis and prevention. Ophthalmology 104, 539 – 544. Erratum: Ophthalmology 104, 1063 (1997). Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M., Cerny, R., Horsnell, T., Hutchison III, C. A., Kouzarides, T., Martignetti, J. A., Preddie, E., Satchwell, S. C., Tomlinson, P., Weston, K. M., & Barrell, B. G. (1990a). Analysis of the protein-coding content of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154, 125 – 169. Chee, M. S., Satchwell, S. C., Preddie, E., Weston, K. M., & Barrell, B. G. (1990b). Human cytomegalovirus encodes three G protein-coupled receptor homologues. Nature 344, 774 – 777. Cherrington, J. M., Khoury, E. L., & Mocarski, E. S. (1991). Human cytomegalovirus IE2 negatively regulates a gene expression via a short target sequence near the transcription start site. J Virol 65, 887 – 896. Cherrington, J. M., Miner, R., Hitchcock, M. J., Lalezari, J. P., & Drew, W. L. (1996). Susceptibility of human cytomegalovirus to cidofovir is unchanged after limited in vivo exposure to various regimens of drug. J Infect Dis 173, 987 – 992. Cherrington, J. M., Allen, A. S. J. W., Mulato, S., Miner, R., Drew, W. L., & Chen, M. S. (1998). Sensitivities of human cytomegalovirus (HCMV) clinical isolates to cidofovir. Antiviral Res 26, A319. Cheung, T. W., & Teich, S. A. (1999). Cytomegalovirus infection in patients with HIV infection. Mt Sinai J Med 66, 113 – 124. Chou, S. W., & Dennison, K. M. (1991). Analysis of interstrain variation in cytomegalovirus glycoprotein B sequences encoding neutralizationrelated epitopes. J Infect Dis 163, 1229 – 1234. Chou, S., Guentzel, S., Michels, K. R., Miner, R. C., & Drew, W. L. (1995). Frequency of UL97 phosphotransferase mutations related to ganciclovir resistance in clinical cytomegalovirus isolates. J Infect Dis 172, 239 – 242. Chrisp, P., & Clissold, S. P. (1991). Foscarnet: a review of its antiviral activity, pharmacokinetic properties and therapeutic use in immunocompromised patients with cytomegalovirus retinitis. Drugs 41, 104 – 129. Chulay, J., Biron, K., Wang, L., Underwood, M., Chamberlain, S., Frick, L., Good, S., Davis, M., Harvey, R., Townsend, L., Drach, J., & Koszalka, G. (1999). Development of novel benzimidazole riboside compounds for treatment of cytomegalovirus disease. Adv Exp Med Biol 458, 129 – 134. Compton, T., Nowlin, D. M., & Cooper, N. R. (1993). Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. Virology 193, 834 – 841. Courcelle, C. T., Courcelle, J., Prichard, M. N., & Mocarski, E. S. (2001). Requirement for uracil-DNA glycosylase during the transition to latephase cytomegalovirus DNA replication. J Virol 75, 7592 – 7601. Cundy, K. C., Li, Z. H., & Lee, A. (1994). Effect of concomitant probenecid on the tissue distribution and urinary excretion of HPMPC in preclinical models. Pharm Res 11, S449. Davignon, J. L., Clement, D., Alriquet, J., Michelson, S., & Davrinche, C. (1995). Analysis of the proliferative T cell response to human cytomegalovirus major immediate-early protein (IE1): phenotype, frequency and variability. Scand J Immunol 41, 247 – 255. De Clercq, E. (1998). Antiviral agents that are active against CMV: potential of cidofovir for the treatment of CMV and other virus infections. In M. Scholz, H. F. Rabenau, H. W. Doerr, & J. Cinatl, Jr. (Eds), CMV-Related Immunopathology, Monographs in Virology, Vol. 21 (pp. 193 – 214). Basel: Karger. De Smet, M. D., Meenken, C. J., & van den Horn, G. J. (1999). Fomivirsen S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 a phosphorothioate oligonucleotide for the treatment of CMV retinitis. Ocul Immunol Inflamm 7, 189 – 198. Dieterich, D. T., Kotler, D. P., Busch, D. F., Crumpacker, C., Du Mond, C., Dearmand, B., & Buhles, W. (1993). Ganciclovir treatment of cytomegalovirus colitis in AIDS: a randomized, double-blind, placebo-controlled multicenter study. J Infect Dis 167, 278 – 282. Dittmer, D., & Mocarski, E. S. (1997). Human cytomegalovirus infection inhibits G1/S transition. J Virol 71, 1629 – 1634. Donaldson, P. T., Alexander, G. J., O’Grady, J., Neuberger, J., Portmann, B., Thick, M., Davis, H., Calne, R. Y., & Williams, R. (1987). Evidence for an immune response to HLA class I antigens in the vanishing-bile duct syndrome after liver transplantation. Lancet 1, 945 – 948. Drew, W. L., Miner, R. C., Busch, D. F., Follansbee, S. E., Gullett, J., Mehalko, S. G., Gordon, S. M., Owen, W. F., Jr., Matthews, T. R., & Buhles, W. C. (1991). Prevalence of resistance in patients receiving ganciclovir for serious cytomegalovirus infection. J Infect Dis 163, 716 – 719. Drew, W. L., Ives, D., Lalezari, J. P., Crumpacker, C., Follansbee, S. E., Spector, S. A., Benson, C. A., Friedberg, D. N., Hubbard, L., Stempien, M. J., Shadman, A., & Buhles, W. (1995). Oral ganciclovir as maintenance treatment for cytomegalovirus retinitis in patients with AIDS. N Engl J Med 333, 615 – 620. Drulak, M. W., Malinoski, F. J., Fuller, S. A., Stewart, S. S., Hoskin, S., Duliege, A. M., Sekulovich, R., Burke, R., & Winston, S. (2000). Vaccination of seropositive subjects with CHIRON CMV gB subunit vaccine combined with MF59 adjuvant for production of CMV immune globulin. Viral Immunol 13, 49 – 56. Dworsky, M., Yow, M., Stagno, S., Pass, R. F., & Alford, C. (1983). Cytomegalovirus infection of breast milk and transmission in infancy. Pediatrics 72, 295 – 299. Einsele, H., Ehninger, G., Hebart, H., Wittkowski, K. M., Schuler, U., Jahn, G., Mackes, P., Herter, M., Klingebiel, T., & Loffler, J. (1995). Polymerase chain reaction monitoring reduces the incidence of cytomegalovirus disease and the duration and side effects of antiviral therapy after bone marrow transplantation. Blood 86, 2815 – 2820. Elek, S. D., & Stern, H. (1974). Development of a vaccine against mental retardation caused by cytomegalovirus infection in utero. Lancet i, 1 – 5. Emery, V. C. (2001). Prophylaxis for CMV should not now replace preemptive therapy in solid organ transplantation. Rev Med Virol 11, 83 – 86. Endresz, V., Kari, L., Berencsi, K., Kari, C., Gyulai, Z., Jeney, C., Pincus, S., Rodeck, U., Meric, C., Plotkin, S. A., & Gonczol, E. (1999). Induction of human cytomegalovirus (HCMV)-glycoprotein B (gB)-specific neutralizing antibody and phosphoprotein 65 (pp65)-specific cytotoxic T lymphocyte responses by naked DNA immunization. Vaccine 17, 50 – 58. Endresz, V., Burian, K., Berencsi, K., Gyulai, Z., Kari, L., Horton, H., Virok, D., Meric, C., Plotkin, S. A., & Gonczol, E. (2001). Optimization of DNA immunization against human cytomegalovirus. Vaccine 19, 3972 – 3980. Erice, A., Chou, S., Biron, K. K., Stanat, S. C., Balfour, H. H., Jr., & Jordan, M. C. (1989). Progressive disease due to ganciclovir-resistant cytomegalovirus in immunocompromised patients. N Engl J Med 320, 289 – 93. Estes, J. E., & Huang, E. -S. (1977). Stimulation of cellular thymidine kinase by human cytomegalovirus. J Virol 24, 13 – 21. Faber, D. W., Wiley, C. A., Lynn, G. B., Gross, J. G., & Freeman, W. R. (1992). Role of HIV and CMV in the pathogenesis of retinitis and retinal vasculopathy in AIDS patients. Invest Ophthalmol Vis Sci 33, 2345 – 2353. Fortunato, E. A., & Spector, D. H. (1999). Regulation of human cytomegalovirus gene expression. Adv Virus Res 54, 61 – 128. Fortunato, E. A., Sommer, M. H., Yoder, K., & Spector, D. H. (1997). Identification of domains within the human cytomegalovirus major immediate-early 86-kilodalton protein and the retinoblastoma protein required for physical and functional interaction with each other. J Virol 71, 8176 – 8185. Fortunato, E. A., McElroy, A. K., Sanchez, V., & Spector, D. H. (2000). Exploitation of cellular signalling and regulatory pathways by human cytomegalovirus. Trends Microbiol 8, 111 – 119. 291 Fowler, K. B., Stagno, S., Pass, R. F., Britt, W. J., Boll, T. J., & Alford, C. A. (1992). The outcome of congenital cytomegalovirus infection in relation to maternal antibody status. N Engl J Med 326, 663 – 667. Gallant, J. E., Moore, R. D., Richman, D. D., Keruly, J., & Chaisson, R. E. (1992). Incidence and natural history of cytomegalovirus disease in patients with advanced human immunodeficiency virus disease treated with zidovudine. J Infect Dis 166, 1223 – 1227. Gao, J. L., & Murphy, P. M. (1994). Human cytomegalovirus open reading frame US28 encodes a functional b chemokine receptor. J Biol Chem 269, 28539 – 28542. Gerna, G., Revello, M. G., Percivalle, E., Zavattoni, M., Marea, M., & Battaglia, M. (1990). Quantification of human Cytomegalovirus viremia by using monoclonal antibodies to different viral proteins. J Clin Mirobiol 28, 2681 – 2688. Gerna, G., Zipeto, D., Parea, M., Revello, M. G., Silini, E., Percivalle, E., Zavattoni, M., Grossi, P., & Milanesi, G. (1991). Monitoring of human cytomegalovirus infections and ganciclovir treatment in heart transplant recipients by determination of viremia, antigenemia, and DNAemia. J Infect Dis 164, 488 – 498. Gerna, G., Baldanti, F., Zavattoni, M., Sarasini, A., Percivalle, E., & Revello, M. G. (1992a). Monitoring of ganciclovir sensitivity of multiple human cytomegalovirus strains coinfecting blood of an AIDS patient by an immediate-early antigen plaque assay. Antiviral Res 1, 333 – 345. Gerna, G., Revello, M. G., Percivalle, E., & Morini, F. (1992b). Comparison of different immunostaining techniques and monoclonal antibodies to the lower matrix phosphoprotein (pp65) for optimal quantitation of human cytomegalovirus antigenemia. J Clin Microbiol 30, 1232 – 1237. Gerna, G., Percivalle, E., Revello, M. G., & Morini, F. (1993). Correlation of quantitative human cytomegalovirus pp65- p72- and p150-antigenemia, viremia and circulating endothelial giant cells with clinical symptoms and antiviral treatment in immunocompromised patients. Clin Diagn Virol 1, 47 – 59. Gerna, G., Baldanti, F., Sarasini, A., Furione, M., Percivalle, E., Revello, M. G., Zipeto, D., & Zella, D. (1994a). Effect of foscarnet induction treatment on quantitation of human cytomegalovirus (HCMV) DNA in peripheral blood polymorphonuclear leukocytes and aqueous humor of AIDS patients with HCMV retinitis. Antimicrob Agents Chemother 38, 38 – 44. Gerna, G., Furione, M., Baldanti, F., & Sarasini, A. (1994b). Comparative quantitation of human cytomegalovirus DNA in blood leukocytes and plasma of transplant and AIDS patients. J Clin Microbiol 32, 709 – 717. Gerna, G., Furione, M., Baldanti, F., Percivalle, E., Comoli, P., & Locatelli, F. (1995). Quantitation of human cytomegalovirus DNA in bone marrow transplant recipients. Br J Haematol 91, 674 – 683. Gerna, G., d’Arminio Monforte, A., Zavattoni, M., Sarasini, A., Testa, L., Revello, M. G., & Moroni, M. (1998a). Sharp drop in the prevalence of human cytomegalovirus leuko-DNAemia in HIV-infected patients following highly active antiretroviral therapy. AIDS 12, 118 – 120. Gerna, G., Zavattoni, M., Baldanti, F., Sarasini, A., Chezzi, L., Grossi, P., & Revello, M. G. (1998b). Human cytomegalovirus (HCMV) leukodnaemia correlates more closely with clinical symptoms than antigenemia and viremia in heart and heart-lung transplant recipients with primary HCMV infection. Transplantation 65, 1378 – 1385. Gerna, G., Baldanti, F., Middeldorp, J. M., Furione, M., Zavattoni, M., Lilleri, D., & Revello, M. G. (1999). Clinical significance of expression of human cytomegalovirus pp67 late transcript in heart, lung, and bone marrow transplant recipients as determined by nucleic acid sequencebased amplification. J Clin Microbiol 37, 902 – 911. Gerna, G., Baldanti, F., Lilleri, D., Parea, M., Alessandrino, E., Pagani, A., Locatelli, F., Middeldorp, J., & Revello, M. G. (2000a). Human cytomegalovirus immediate-early mRNA detection by nucleic acid sequencebased amplification as a new parameter for preemptive therapy in bone marrow transplant recipients. J Clin Microbiol 38, 1845 – 1853. Gerna, G., Percivalle, E., Baldanti, F., Sozzani, S., Lanzarini, P., Genini, E., Lilleri, D., & Revello, M. G. (2000b). Human cytomegalovirus replicates abortively in polymorphonuclear leukocytes after transfer from 292 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 infected endothelial cells via transient microfusion events. J Virol 74, 5629 – 5638. Gibson, W. (1996). Structure and assembly of the virion. Intervirology 39, 389 – 400. Gilbert, G. L., Hayes, K., Hudson, I. L., & James, J. (1989). Prevention of transfusion-acquired cytomegalovirus infection in infants by blood filtration to remove leucocytes. Neonatal Cytomegalovirus Infection Study Group. Lancet i, 1228 – 1231. Gilbert, M. J., Riddell, S. R., Plachter, B., & Greenberg, P. D. (1996). Cytomegalovirus selectively blocks antigen processing and presentation of its immediate-early gene product. Nature 383, 720 – 722. Gleaves, C. A., Smith, T. F., Shuster, E. A., & Pearson, G. R. (1984). Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. J Clin Microbiol 19, 917 – 919. Goldmacher, V. S. (2002). vMIA, a viral inhibitor of apoptosis targeting mitochondria. Biochimie 84, 177 – 185. Gonczol, E., & Plotkin, S. (2001). Development of a cytomegalovirus vaccine: lessons from recent clinical trials. Expert Opin Biol Ther 1, 401 – 412. Gonczol, E., deTaisne, C., Hirka, G., Berencsi, K., Lin, W. C., Paoletti, E., & Plotkin, S. (1991). High expression of human cytomegalovirus (HCMV)gB protein in cells infected with a vaccinia-gB recombinant: the importance of the gB protein in HCMV immunity. Vaccine 9, 631 – 637. Goodrich, J. M., Mori, M., Gleaves, C. A., Du Mond, C., Cays, M., Ebeling, D. F., Buhles, W. C., DeArmond, B., & Meyers, J. D. (1991). Early treatment with ganciclovir to prevent cytomegalovirus disease after allogeneic bone marrow transplantation. N Engl J Med 325, 1601 – 1607. Goodrich, J. M., Bowden, R. A., Fisher, L., Keller, C., Schoch, G., & Meyers, J. D. (1993). Ganciclovir prophylaxis to prevent cytomegalovirus disease after allogeneic marrow transplant. Ann Intern Med 118, 173 – 178. Gozlan, J., Laporte, J. P., Lesage, S., Labopin, M., Najman, A., Gorin, N. C., & Petit, J. C. (1996). Monitoring of cytomegalovirus infection and disease in bone marrow recipients by reverse transcription-PCR and comparison with PCR and blood and urine cultures. J Clin Microbiol 34, 2085 – 2088. Grangeot-Keros, L., Mayaux, M. J., Lebon, P., Freymuth, F., Eugene, G., Stricker, R., & Dussaix, E. (1997). Value of cytomegalovirus (CMV) IgG avidity index for the diagnosis of primary CMV infection in pregnant women. J Infect Dis 175, 944 – 946. Gretch, D. R., Kari, B., Rasmussen, L., Gehrz, R. C., & Stinski, M. F. (1988). Identification and characterization of three distinct families of glycoprotein complexes in the envelopes of human cytomegalovirus. J Virol 62, 875 – 881. Gribaudo, G., Riera, L., Lembo, D., De Andrea, M., Gariglio, M., Rudge, T. L., Johnson, L. F., & Landolfo, S. (2000). Murine cytomegalovirus stimulates cellular thymidylate synthase gene expression in quiescent cells and requires the enzyme for replication. J Virol 74, 4979 – 4987. Gribaudo, G., Riera, L., Rudge, T. L., Caposio, P., Johnson, L. F., & Landolfo, S. (2002). Human cytomegalovirus infection induces cellular thymidylate synthase gene expression in quiescent fibroblasts. J Gen Virol 83, 2983 – 2993. Griffiths, P. D. (2000). Cytomegalovirus. In A. J. Zuckerman, J. E. Banatvala, & J. R. Pattison (Eds.), Principles and Practice of Clinical Virology ( pp. 79 – 116). London: John Wiley and Sons. Griffiths, P. D., Panjwani, D. D., Stirk, P. R., Ball, M. G., Ganczakowski, M., Blacklock, H. A., & Prentice, H. G. (1984). Rapid diagnosis of cytomegalovirus infection in immunocompromised patients by detection of early antigen fluorescent foci. Lancet ii, 1242 – 1245. Grossi, P., Minoli, L., Percivalle, E., Irish, W., Vigano, M., & Gerna, G. (1995). Clinical and virological monitoring of human cytomegalovirus infection in 294 heart transplant recipients. Transplantation 59, 847 – 851. Gyulai, Z., Endresz, V., Burian, K., Pincus, S., Toldy, J., Cox, W. I., Meric, C., Plotkin, S., Gonczol, E., & Berencsi, K. (2000). Cytotoxic T lymphocyte (CTL) responses to human cytomegalovirus pp65, IE1-Exon4, gB, pp150, and pp28 in healthy individuals: reevaluation of prevalence of IE1-specific CTLs. J Infect Dis 181, 1537 – 1546. Hagemeier, C., Walker, S., Caswell, R., Kouzarides, T., & Sinclair, J. H. (1992a). The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoter via basal promoter elements. J Gen Virol 73, 2385 – 2393. Hagemeier, C., Walker, S., Caswell, R., Kouzarides, T., & Sinclair, J. H. (1992b). The human cytomegalovirus 80-kilodalton but not the 72-kilodalton immediate-early protein transactivates heterologous promoters in a TATA box-dependent mechanism and interacts directly with TFIID. J Virol 66, 4452 – 4456. Hagemeier, C., Caswell, R., Hayhurst, G., Sinclair, J. H., & Kouzarides, T. (1994). Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein. EMBO J 13, 2897 – 2903. Hahn, G., Jores, R., & Mocarski, E. (1998). Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc Natl Acad Sci USA 95, 3937 – 3942. Halwachs-Baumann, G., Wilders-Trucshnig, M., Desoye, G., Hahn, T., Kiesel, L., Klingel, K., Rieger, P., Jahn, G., & Sinzger, C. (1998). Human trophoblast cells are permissive to the complete replicative cycle of human cytomegalovirus. J Virol 72, 7598 – 7602. Harrison, C. J., Britt, W. J., Chapan, N. M., Mullican, J., & Tracy, S. (1995). Reduced congenital cytomegalovirus (CMV) infection after maternal immunization with a guinea pig CMV glycoprotein before gestational primary CMV infection in the guinea pig model. J Infect Dis 172, 1212 – 1220. Hayashi, M. L., Blankenship, C., & Shenk, T. (2000). Human cytomegalovirus UL69 protein is required for efficient accumulation of infected cells in the G1 phase of the cell cycle. Proc Natl Acad Sci USA 97, 2692 – 2696. Hayhurst, G. P., Bryant, L. A., Caswell, R. C., Walker, S. M., & Sinclair, J. H. (1995). CCAAT box-dependent activation of the TATA-less human DNA polymerase a promoter by the human cytomegalovirus 72kilodalton major immediate-early protein. J Virol 69, 182 – 188. He, H., Rinaldo, C. R., Jr., & Morel, P. A. (1995). T cell proliferative responses for five human cytomegalovirus proteins in healthy seropositive individuals: implications for vaccine development. J Gen Virol 76, 1603 – 1610. Hemmings, D. G., Kilani, R., Nyukiforuk, C., Preiksaitis, J., & Guibert, L. J. (1998). Permissive cytomegalovirus infection of primary villous term and first trimester trophoblasts. J Virol 72, 4970 – 4979. Hengel, H., Brune, W., & Koszinowski, U. H. (1998). Immune evasion by cytomegalovirus-survival strategies of a highly adapted opportunist. Trends Microbiol 6, 190 – 197. Herrera, G. A., Alexander, R. W., Cooly, C. F., Luke, R. G., Kelly, D. R., Curtis, J. J., & Gockerman, J. P. (1986). Cytomegalovirus glomerulopathy: a controversial lesion. Kidney Int 29, 725 – 733. Ho, M. (1991). Cytomegalovirus. In G. L. Mandell, J. E. Bennet, & R. Dolin (Eds.), Principles and Practices of Infectious Diseases ( pp. 1351 – 1364). New York: Churchill Livingstone. Ho, H.-T., Woods, K. L., Bronson, J. J., De Boeck, H., Martin, J. C., & Hitchcock, M. J. (1992). Intracellular metabolism of the antiherpes agent (S)-1-(3-hydroxy-2-(phosphonylmethoxy)propyl)-cytosine. Mol Pharmacol 41, 97 – 202. Hobom, U., Brune, W., Messerle, M., Hahn, G., & Koszinowski, U. H. (2000). Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: mutational analysis of human cytomegalovirus envelope glycoproteins genes. J Virol 74, 7720 – 7729. Huber, M. T., & Compton, T. (1998). The human cytomegalovirus UL74 gene encodes the third component of the glycoprotein H-glycoprotein L-containing envelope complex. J Virol 72, 8191 – 8197. Isom, H. C. (1979). Stimulation of ornithine decarboxylase by human cytomegalovirus. J Gen Virol 42, 265 – 278. Jabs, D. A., Enger, C., Dunn, J. P., & Forman, M., for the Cytomegalovirus Retinitis and Viral Resistance Study Group (1998a). Cytomegalovirus S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 retinitis and viral resistance: 4. Ganciclovir resistance. J Infect Dis 177, 770 – 773. Jabs, D. A., Enger, C., Forman, M., & Dunn, J. P., for the Cytomegalovirus Retinitis and Viral Resistance Study Group (1998b). Incidence of foscarnet resistance and cidofovir resistance in in patients treated for cytomegalovirus retinitis. Antimicrob Agents Chemother 42, 2240 – 2244. Jacobson, M. A. (1997 – 1998). Cytomegalovirus retinitis: new developments in prophylaxis and therapy. AIDS Clin Rev, 249 – 269. Jassal, S. V., Roscoe, J. M., Zaltzman, J. S., Mazzulli, T., Krajden, M., Gadawski, M., Cattran, D. C., Cardella, C. J., Albert, S. E., & Cole, E. H. (1998). Clinical practice guidelines: prevention of cytomegalovirus disease after renal transplantation. J Am Soc Nephrol 9, 1697 – 1708. Jault, F. M., Jault, J. M., Ruchti, F., Fortunato, E. A., Clark, C., Corbeil, J., Richman, D. D., & Spector, D. H. (1995). Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb, and p53, leading to cell cycle arrest. J Virol 69, 6697 – 6704. Johnson, R. A., Yurochko, A. D., Poma, E. E., Zhu, L., & Huang, E.-S. (1999). Domain mapping of the human cytomegalovirus IE1-72 and cellular p107 protein-protein interaction and the possible functional consequences. J Gen Virol 80, 1293 – 1303. Jones, T. R., & Sun, L. (1997). Human cytomegalovirus US2 destabilizes major histocompatibility complex class I heavy chains. J Virol 71, 2970 – 2979. Jones, T. R., Wiertz, E. J., Sun, L., Fish, K. N., Nelson, J. A., & Ploegh, H. L. (1996). Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains. Proc Natl Acad Sci USA 93, 11327 – 11333. Jonjic, S., Pavic, I., Lucin, P., Rukavina, D., & Koszinowski, U. H. (1990). Efficacious control of cytomegalovirus infection after long-term depletion of CD8+ lymphocytes. J Virol 64, 5457 – 5464. Jupp, R., Hoffmann, S., Stenberg, R. M., Nelson, J. A., & Ghazal, P. (1993). Human cytomegalovirus IE86 protein interacts with promoterbound TATA-binding protein via a specific region distinct from the autorepression domain. J Virol 67, 7539 – 7546. Kahl, M., Siegel-Axel, D., Stenglein, S., Jahn, G., & Sinzger, C. (2000). Efficient lytic infection of human arterial endothelial cells by human cytomegalovirus strains. J Virol 74, 7628 – 7635. Kalejta, R. F., & Shenk, T. (2002). Manipulation of the cell cycle by human cytomegalovirus. Front Biosc 7, 295 – 306. Kari, B., Li, W., Cooper, J., Goertz, R., & Radeke, B. (1994). The human cytomegalovirus UL100 gene encodes the gC-II glycoproteins recognized by group 2 monoclonal antibodies. J Gen Virol 75, 3081 – 3086. Karlin, S., Mocarski, E. S., & Schachtel, G. A. (1994). Molecular evolution of herpesviruses: genomic and protein sequence comparisons. J Virol 68, 1886 – 1902. Kern, F., Surel, I. P., Faulhaber, N., Frommel, C., Schneider-Mergener, J., Schonemann, C., Reinke, P., & Volk, H. D. (1999). Target structures of the CD8 + -T-cell response to human cytomegalovirus: the 72-kilodalton major immediate-early protein revisited. J Virol 73, 8179 – 8184. Kidd, I. M., Fox, J. C., Pillay, D., Charman, H., Griffiths, P. D., & Emery, V. C. (1993). Provision of prognostic information in immunocompromised patients by routine application of the polymerase chain reaction for cytomegalovirus. Transplantation 56, 867 – 871. Knox, K. K., Drobyski, W. R., & Carrigan, D. R. (1991). Cytomegalovirus isolate resistant to ganciclovir and foscarnet from a marrow transplant patient. Lancet ii, 1292 – 1293. Kondo, K., Kaneshima, H., & Mocarski, E. S. (1994). Human cytomegalovirus latent infection of granulocyte-macrophage progenitors. Proc Natl Acad Sci USA 91, 11879 – 11883. Koszinowski, U. H., Del Val, M., & Reddehase, M. J. (1990). Cellular and molecular basis of the protective immune response to cytomegalovirus infection. Curr Top Microbiol Immunol 154, 189 – 215. Lalezari, J. P., Holland, G. N., Kramer, F., McKinley, G. F., Kemper, C. A., Ives, D. V., Nelson, R., Hardy, W. D., Kuppermann, B. D., Northfelt, D. W., Youle, M., Johnson, M., Lewis, R. A., Weinberg, D. V., Simon, G. L., Wolitz, R. A., Ruby, A. E., Stagg, R. J., & Jaffe, H. S. (1998). 293 Randomized, controlled study of the safety and efficacy of intravenous cidofovir for the treatment of relapsing cytomegalovirus retinitis in patients with AIDS. J Acquir Immune Def Syndr Hum Retrovirol 17, 339 – 344. Lam, K. M., Oldenburg, N., Khan, M. A., Gaylore, V., Mikhail, G. W., Strouhal, P. D., Middeldorp, J. M., Banner, N., & Yacoub, M. (1998). Significance of reverse transcription polymerase chain reaction in the detection of human cytomegalovirus gene transcripts in thoracic organ transplant recipients. J Heart Lung Transplant 17, 555 – 565. Landini, M. P., & Michelson, S. (1988). Human cytomegalovirus proteins. In J. L. Melnick (Ed.), Progress in Medical Virology ( pp. 152 – 185). Basel: Karger. Landini, M. P., Rossier, E., & Schmitz, H. (1988). Antibodies to human cytomegalovirus structural polypeptides during primary infection. J Virol Methods 22, 309 – 317. Lazzarotto, T., Spezzacatena, P., Varani, S., Gabrielli, L., Pradelli, P., Guerra, B., & Landini, M. P. (1999). Anticytomegalovirus (anti-CMV) immunoglobulin G avidity in identification of pregnant women at risk of transmitting congenital CMV infection. Clin Diagn Lab Immunol 6, 127 – 129. Leach, C. T., Cherry, J. D., English, P. A., Hennessey, K., Giorgi, J. V., Visscher, B. R., Dudley, J. P., & Detels, R. (1993). The relationship between T-cells and CMV infection in asymptomatic HIV-1 antibodypositive homosexual men. J Acquir Immune Def Syndr Hum Retrovirol 6, 407 – 413. Lembo, D., Gribaudo, G., Cavallo, R., Riera, L., Angeretti, A., Hertel, L., & Landolfo, S. (1999). Human cytomegalovirus stimulates cellular dihydrofolate reductase activity in quiescent cells. Intervirology 42, 30 – 36. Lembo, D., Gribaudo, G., Hofer, A., Riera, L., Cornaglia, M., Mondo, A., Angeretti, A., Gariglio, M., Thelander, L., & Landolfo, S. (2000). Expression of an altered ribonucleotide reductase activity associated with the replication of murine cytomegalovirus in quiescent fibroblasts. J Virol 74, 11557 – 11565. Limaye, A. P., Corey, L., Koelle, D. M., Davis, C. L., & Boeckh, M. (2000). Emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants. Lancet 356, 645 – 649. Limaye, A. P., Raghu, G., Koelle, D. M., Ferrenberg, J., Huang, M. L., & Boeckh, M. (2002). High incidence of ganciclovir-resistant cytomegalovirus infection among lung transplant recipients receiving preemptive therapy. J Infect Dis 185, 20 – 27. Littler, E., Stuart, A. D., & Chee, M. S. (1992). Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir. Nature 358, 160 – 162. Liu, B., & Stinski, M. F. (1992). Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements. J Virol 66, 4434 – 4444. Liu, Y. N., Curtsinger, J., Donahue, P. R., Klaus, A., Optiz, G., Cooper, J., Karr, R. W., Bachs, F. H., & Gehrz, R. C. (1993). Molecular analysis of the immune response to human cytomegalovirus glycoprotein B. I. Mapping of HLA-restricted helper T cell epitopes on gp93. J Gen Virol 74, 2207 – 2214. Loebe, M., Schuler, S., Zais, O., Warnecke, H., Fleck, E., & Hetzer, R. (1990). Role of cytomegalovirus infection in the development of coronary disease in the transplanted hearth. J Heart Transplant 9, 707 – 711. Lu, M., & Shenk, T. (1996). Human cytomegalovirus infection inhibits cell cycle progression at multiple points, including the transition from G1 to S. J Virol 70, 8850 – 8857. Lu, M., & Shenk, T. (1999). Human cytomegalovirus UL69 protein induces cells to accumulate in G1 phase of the cell cycle. J Virol 73, 676 – 683. Lukac, D. M., Harel, N. Y., Tanese, N., & Alwine, J. C. (1997). TAF-like functions of human cytomegalovirus immediate-early proteins. J Virol 71, 7227 – 7239. Lurain, N. S., Spafford, L. E., & Thompson, K. D. (1994). Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir. J Virol 68, 4427 – 4431. Macdonald, P. S., Keogh, A. M., Marshman, D., Richens, D., Harvison, A., Kaan, A. M., & Spratt, P. M. (1995). A double-blind placebo-controlled 294 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 trial of low-dose ganciclovir to prevent cytomegalovirus disease after heart transplantation. J Heart Lung Transplant 14, 32 – 38. Machold, R. P., Wiertz, F. J., Jones, T. R., & Ploegh, H. L. (1997). The HCMV gene products US11 and US2 differ in their ability to attack allelic forms of murine major histocompatibility complex (MHC) class I heavy chains. J Exp Med 185, 363 – 366. Marchini, A., Liu, H., & Zhu, H. (2001). Human cytomegalovirus with IE2 (UL122) deleted fails to express early lytic genes. J Virol 75, 1870 – 1878. Margolis, M. J., Pajovic, S., Wong, E. L., Wade, M., Jupp, R., Nelson, J. A., & Clifford Azizkhan, J. (1995). Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2F-dependent activation of dihydrofolate reductase transcription. J Virol 69, 7759 – 7767. Marshall, G. S., Rabalais, G. P., Stout, G. G., & Waldeyer, S. L. (1992). Antibodies to recombinant-derived glycoprotein B after natural human cytomegalovirus infection correlate with neutralizing activity. J Infect Dis 165, 381 – 384. Masse, M. J., Karlin, S., Schachtel, G. A., & Mocarski, E. S. (1992). Human cytomegalovirus origin of DNA replication (oriLyt) resides within a highly complex repetitive region. Proc Natl Acad Sci USA 89, 5246 – 5250. Masur, H., Whitcup, S. M., Cartwright, C., Polis, M., & Nussenblatt, R. (1996). Advances in the management of AIDS-related cytomegalovirus retinitis. Ann Intern Med 125, 126 – 136. McVoy, M. A., & Adler, S. P. (1994). Human cytomegalovirus DNA replicates after early circularization by concatemer formation, and inversion occurs within concatemer. J Virol 68, 1040 – 1051. Meier, J. L., & Stinski, M. F. (1996). Regulation of human cytomegalovirus immediate-early gene expression. Intervirology 39, 331 – 342. Meier, J. L., & Stinski, M. F. (1997). Effect of a modulator deletion on transcription of the human cytomegalovirus major immediate-early genes in infected undifferentiated and differentiated cells. J Virol 71, 1246 – 1255. Merigan, T. C., Renlund, D. G., Keay, S., Bristow, M. R., Starnes, V., O’Connell, J. B., Resta, S., Dunn, D., Gamberg, P., & Ratkovec, R. M. (1992). A controlled trial of ganciclovir to prevent cytomegalovirus disease after heart transplantation. N Engl J Med 326, 1182 – 1186. Mettenleiter, T. C. (2002). Herpesvirus assembly and egress. J Virol 76, 1537 – 1547. Miller, W. J., McCullough, J., Balfour, H. H., Haake, R. J., Ramsay, N. K., Goldman, A., Bowman, R., & Kersey, J. (1991). Prevention of cytomegalovirus infection following bone marrow transplantation: a randomized trial of blood product screening. Bone Marrow Transplant 7, 227 – 234. Mocarski, E. S. (1996). Cytomegalovirus and their replication. In B. N. Fields, D. M. Knipe, & P. M. Howley (Eds.), Virology ( pp. 2447 – 2492). Philadelphia: Lippincott-Raven. Mocarski, E. S. (2002). Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion. Trends Microbiol 10, 332 – 339. Mocarski, E. S., & Courcelle, C. T. (2001). Cytomegalovirus and their replication. In D. Knipe, & P. Howley (Eds.), Fields Virology ( pp. 2629 – 2673). Philadelphia: Lippincott, Williams and Wilkins. Mocarski, E. S., Kemble, G. W., Lyle, J. M., & Greaves, R. F. (1996). A deletion mutant in the human cytomegalovirus gene encoding IE1 (491 aa) is replication defective due to a failure in autoregulation. Proc Natl Acad Sci USA 94, 11321 – 11326. Murphy, E. A., Streblow, D. N., Nelson, J. A., & Stinski, M. F. (2000). The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells. J Virol 74, 7108 – 7118. Nelson, J. A., Gnann, J. W., & Ghazal, P. (1990). Regulation and tissuespecific expression of human cytomegalovirus. Curr Top Microbiol Immunol 154, 75 – 100. Nesmith, J. D., & Pass, R. F. (1995). Cytomegalovirus infection in adolescent. In G. D. Overturf, & R. F. Jacobs (Eds.), Adolescent Medicine: State of the Art Reviews. Viral Infections in Adolescents ( pp. 79 – 90). Philadelphia: Hanley and Belfus. Novotny, J., Rigoutsos, I., Coleman, D., & Shenk, T. (2001). In silico structural and functional analysis of the human cytomegalovirus (HHV5) genome. J Mol Biol 310, 1151 – 1166. O’Sullivan, C. E., Drew, W. L., McMullen, D. J., Miner, R., Lee, J. Y., Kaslow, R. A., Lazar, J. G., & Saag, M. S. (1999). Decrease of cytomegalovirus replication in human immunodeficiency virus-infected patients after treatment with highly active antiretroviral therapy. J Infect Dis 180, 847 – 849. Pajovic, S., Wong, E. L., Black, A. R., & Azizkhan, C. J. (1997). Identification of a viral kinase that phosphorylates specific E2Fs and pocket proteins. Mol Cell Biol 17, 6459 – 6464. Palestine, A. G., Polis, M. A., De Smet, M. D., Baird, B. F., Falloon, J., Kovacs, J. A., Davey, R. T., Zurlo, J. J., Zunich, K. M., Davis, M., et al. (1991). A randomized, controlled trial of foscarnet in the treatment of cytomegalovirus retinitis in patients with AIDS. Ann Intern Med 115, 665 – 673. Pass, R. F. (1985). Epidemiology and transmission of cytomegalovirus infection. J Infect Dis 152, 243 – 256. Pass, R. F. (1996). Immunization strategy for prevention of congenital cytomegalovirus infection. Infect Agents Dis 5, 240 – 244. Pass, R. F. (2001). Cytomegalovirus. In D. Knipe, & P. Howley (Eds.), Fields Virology ( pp. 2675 – 2705). Philadelphia: Lippincott, Williams and Wilkins. Pass, R. F., & Boppana, S. (1999). Cytomegalovirus. In D. J. Jeffries, & C. N. Hudson (Eds.), Viral Infections in Obstetrics and Gynaecology ( pp. 35 – 56). New York: Arnold. Pass, R. F., Stagno, S., & Britt, W. J. (1983). Specific cell-mediated immunity and the natural history of congenital infection with cytomegalovirus. J Infect Dis 148, 953 – 961. Pass, R. F., Duliege, A. M., Boppana, S., Sekulovich, R., Percell, S., Britt, W., & Burke, R. L. (1999). A subunit cytomegalovirus vaccine based on recombinant envelope glycoprotein B and a new adjuvant. J Infect Dis 180, 970 – 975. Penfold, M. E., & Mocarski, E. S. (1997). Formation of cytomegalovirus DNA replication compartments defined by localization of viral proteins and DNA synthesis. Virology 239, 46 – 61. Penfold, M. E., Dairaghi, D. J., Duke, G. M., Saedurup, N., Mocarski, E. S., Kemble, G. W., & Schall, T. J. (1999). Cytomegalovirus encodes a potent alpha chemokine. Proc Natl Acad Sci USA 96, 9839 – 9844. Pepperl, S., Munster, J., Mach, M., Harris, J. R., & Plachter, B. (2000). Dense bodies of human cytomegalovirus induce both humoral and cellular immune responses in the absence of viral gene expression. J Virol 74, 6132 – 6146. Pertel, P., Hirschtick, R., Phair, J., Chmiel, J., Poggensee, L., & Murphy, R. (1992). Risk of developing cytomegalovirus retinitis in persons infected with the human immunodeficiency virus. J Acquir Immune Def Syndr Hum Retrovirol 5, 1069 – 1074. Peterson, P. K., Balfour Jr., H. H., Marker, S. C., Fryd, D. S., Howard, R. J., & Simmons, R. L. (1980). Cytomegalovirus disease in renal allograft recipients: a prospective study of the clinical features, risk factors and impact on renal transplantation. Medicine 59, 283 – 300. Phillips, C. A., Fanning, W. L., & Gump, D. W. (1977). Cytomegalovirus in immunologically normal adults. JAMA 238, 2299 – 2300. Pizzorno, M. C., & Hayward, G. S. (1990). The IE2 gene products of human cytomegalovirus specifically down-regulate expression from the major immediate-early promoter through a target sequence located near the cap site. J Virol 64, 6154 – 6165. Plachter, B., Sinzger, C., & Jahn, G. (1996). Cell types involved in replication and distribution of human cytomegalovirus. Adv Virus Res 46, 195 – 261. Plotkin, S. A., Furukawa, T., Zygraich, N., & Huygelen, C. (1975). Candidate cytomegalovirus strain for human vaccination. Infect Immun 12, 521 – 527. Plotkin, S. A., Farquhar, J., & Horberger, E. (1976). Clinical trials of immunization with the Towne 125 strain of human cytomegalovirus. J Infect Dis 134, 470 – 475. S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 Plotkin, S. A., Smiley, M. L., Friedman, H. M., Starr, S. E., Fleisher, G. R., Wlodaver, C., Dafoe, D. C., Friedman, A. D., Grossman, R. A., & Barker, C. F. (1984). Towne-vaccine induced prevention of cytomegalovirus disease after renal transplants. Lancet i, 528 – 530. Plotkin, S. A., Starr, S. E., Friedman, H. M., Gonczol, E., & Weibel, R. E. (1989). Protective effects of Towne cytomegalovirus vaccine against low-passage cytomegalovirus administered as a challenge. J Infect Dis 159, 860 – 865. Plotkin, S. A., Starr, S. E., Friedman, H. M., Brayman, K., Harris, S., Jackson, S., Tustin, N. B., Grossman, R., Dafoe, D., & Barker, C. (1991). Effect of Towne live virus vaccine on cytomegalovirus disease after renal transplant. A controlled trial. Ann Intern Med 114, 525 – 531. Plotkin, S. A., Higgins, R., Kurtz, J. B., Morris, P. J., Campbell, D. A., Jr., Shope, T. C., Spector, S. A., & Dankner, W. M. (1994). Multicenter trial of Towne strain attenuated virus vaccine in seronegative renal transplant recipients. Transplantation 58, 1176 – 1178. Polic, B., Jonjic, S., Pavic, I., Crnkovic, I., Zorica, I., Hengel, H., Lucin, P., & Koszinowski, U. H. (1996). Lack of MHC class I complex expression has no effect on spread and control of cytomegalovirus infection in vivo. J Gen Virol 77, 217 – 225. Polis, M. A., Spooner, K. M., Baird, B. F., Manischewitz, J. F., Jaffe, H. S., Fisher, P. E., Falloon, J., Davey, R. T. J., Kovacs, J. A., Walker, R. E., Whitcup, S. M., Nussenblatt, R. B., Lane, H. C., & Masur, H. (1995). Anticytomegaloviral activity and safety of cidofovir in patients with human immunodeficiency virus infection and cytomegalovirus viruria. Antimicrob Agents Chemother 39, 882 – 886. Pollard, A. B. (1988). Cytomegalovirus infections in renal, heart, heart-lung and liver transplantation. Pediatr Infect Dis J 7, 97 – 102. Poma, E. E., Kowalik, T. F., Zhu, L., Sinclair, J. H., & Huang, E.-S. (1996). The human cytomegalovirus IE1-72 protein interacts with the cellular p107 protein and relieves p107-mediated transcriptional repression of an E2F-responsive promoter. J Virol 70, 7867 – 7877. Rapp, M., Messerle, M., Buhler, B., Tannheimer, M., Keil, G. M., & Koszinowski, U. R. (1992). Identification of the murine cytomegalovirus glycoprotein B gene and its expression by recombinant vaccinia virus. J Virol 66, 4399 – 4406. Rawlinson, W. D., Farrell, H. E., & Barrell, B. G. (1996). Analysis of the complete DNA sequence of murine cytomegalovirus. J Virol 70, 8833 – 8849. Reddehase, M. J., Balthesen, M., Rapp, M., Jonjic, S., Pavic, I., & Koszinowski, U. H. (1994). The conditions of primary infection define the load of latent viral genome in organs and the risk of recurrent cytomegalovirus disease. J Exp Med 179, 185 – 193. Reed, E. C., Bowden, R. A., Dandliker, P. S., Lilleby, K. E., & Meyers, J. D. (1988). Treatment of cytomegalovirus pneumonia with ganciclovir and intravenous cytomegalovirus immunoglobulin in patients with bone marrow transplants. Ann Intern Med 109, 783 – 788. Rene, E., Marche, C., Chevalier, T., Rouzioux, C., Regnier, B., Saimot, A. G., Negesse, Y., Matheron, S., Leport, C., & Wolff, B. (1988). Cytomegalovirus colitis in patients with acquired immunodeficiency syndrome. Dig Dis Sci 33, 741 – 750. Revello, M. G., Percivalle, E., Zavattoni, M., Parea, M., Grossi, P., & Gerna, G. (1989). Detection of human cytomegalovirus immediate early antigen in leukocytes as a marker of viremia in immunocompromised patients. J Med Virol 29, 88 – 93. Revello, M. G., Baldanti, F., Furione, M., Sarasini, A., Percivalle, E., Zavattoni, M., & Gerna, G. (1995). Polymerase chain reaction for prenatal diagnosis of congenital human cytomegalovirus infection. J Med Virol 47, 462 – 466. Revello, M. G., Zavattoni, M., Furione, M., Baldanti, F., & Gerna, G. (1999). Quantification of human cytomegalovirus DNA in amniotic fluid of mothers of congenitally infected fetuses. J Clin Mirobiol 37, 3350 – 3352. Revello, M. G., Lilleri, D., Zavattoni, M., Stronati, M., Bollani, L., Middeldorp, J. M., & Gerna, G. (2001). Human cytomegalovirus immediate-early messenger RNA in blood of pregnant women with primary 295 infection and of congenitally infected newborns. J Infect Dis 184, 1078 – 1081. Reynolds, D. W., Stagno, S., Hosty, T. S., Tiller, M., & Alford, C. A., Jr. (1973). Maternal cytomegalovirus excretion and perinatal infection. N Engl J Med 289, 1 – 5. Richardson, W. P., Colvin, R. B., Cheeseman, S. H., Tolkoff-Rubin, N. E., Herrin, J. T., Cosimi, A. B., Collins, A. B., Hirsch, M. S., McCluskey, R. T., Russell, P. S., & Rubin, R. H. (1981). Glomerulopathy associated with cytomegalovirus viremia in renal allografts. N Engl J Med 305, 57 – 63. Riddell, S. R., Rabin, M., Geballe, A. P., Britt, W. J., & Greenberg, P. D. (1991). Class I MHC-restricted cytotoxic T lymphocyte recognition of cells infected with human cytomegalovirus does not require endogenous viral gene expression. J Immunol 146, 2795 – 2804. Riera, L., Gariglio, M., Valente, G., Mullbacher, A., Museteanu, C., Landolfo, S., & Simon, M. M. (2000). Murine cytomegalovirus replication in salivary glands is controlled by both perforin and granzymes during acute infection. Eur J Immunol 30, 1350 – 1355. Riera, L., Gariglio, M., Pagano, M., Gaiola, O., Simon, M. M., & Landolfo, S. (2001). Control of murine cytomegalovirus replication in salivary glands during acute infection is independent of the Fas ligand/Fas system. Microbiologica 24, 231 – 238. Romanowski, M. J., Garrido-Guerrero, E., & Shenk, T. (1997). pIRS1 and pTRS1 are present in human cytomegalovirus virions. J Virol 71, 5703 – 5705. Rubie, H., Attal, M., Campardou, A. M., Gayet-Mengelle, C., Payen, C., Sanguignol, F., Calot, J. P., Charlet, J. P., Robert, A., & Huguet, F. (1993). Risk factors for cytomegalovirus infection in BMT recipients transfused exclusively with seronegative blood products. Bone Marrow Transplant 11, 209 – 214. Rubin, R. H. (1991). Preemptive therapy in immunocompromised hosts. N Engl J Med 324, 1057 – 1059. Rubin, R. H. (1994). Infection in the organ transplant recipient. In R. H. Rubin, & L. S. Young (Eds.), Clinical Approach to Infection in the Compromised Host ( pp. 629 – 705). New York: Plenum Medical Book Co. Rubin, R. H., Tolkoff-Rubin, N. E., Oliver, D., Rota, T. R., Hamilton, J., Betts, R. F., Pass, R. F., Hillis, W., Szmuness, W., & Farrell, M. L. (1985). Multicenter seroepidemiologic study of the impact of cytomegalovirus infection on renal transplantation. Transplantation 40, 243 – 249. Ruutu, P., Ruutu, T., Volin, L., Tukiainen, P., Ukkonen, P., & Hovi, T. (1990). Cytomegalovirus is frequently isolated in bronchoalveolar lavage fluid of bone marrow transplant recipients without pneumonia. Ann Intern Med 112, 913 – 916. Sadasivan, B., Lehner, P. J., Ortmann, B., Spies, T., & Cresswell, P. (1996). Role for clareticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP. Immunity 5, 103 – 114. Saederup, N., Lin, Y. C., Dairaghi, D. J., Schall, T. J., & Mocarski, E. S. (1999). Cytomegalovirus-encoded b chemokine promotes monocyte-associated viremia in the host. Proc Natl Acad Sci USA 96, 10881 – 10886. Salvant, B. S., Fortunato, E. A., & Spector, D. H. (1998). Cell cycle dysregulation by human cytomegalovirus: influence of the cell cycle phase at the time of infection and effects on cyclin transcription. J Virol 72, 3729 – 3741. Sanchez, V., Sztul, E., & Britt, W. (2000). Accumulation of virion tegument and envelope proteins in a stable cytoplasmatic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly. J Virol 74, 975 – 986. Sarisky, R. T., & Hayward, G. S. (1996). Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLytdependent DNA replication and formation of replication compartments in cotransfection assays. J Virol 70, 7398 – 7413. Sayers, M. H., Anderson, K. C., Goodnough, L. T., Kurtz, S. R., Lane, T. A., Pisciotto, P., & Silberstein, L. E. (1992). Reducing the risk for transfusion-transmitted cytomegalovirus infection. Ann Intern Med 116, 55 – 62. 296 S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 Shamu, C. E., Story, C. M., Rapoport, T. A., & Ploegh, H. L. (1999). The pathway of US11-dependent degradation of MHC class I heavy chains involves a ubiquitin-conjugated intermediate. J Cell Biol 147, 45 – 58. Shellam, G. R., Allen, J. E., Papdimitriou, J. M., & Bancroft, J. M. (1981). Increased susceptibility to cytomegalovirus infection in beige mutant mice. Proc Natl Acad Sci USA 78, 5104 – 5108. Simmen, K. A., Singh, J., Luukkonen, B. G. M., Lopper, M., Bittner, A., Miller, N. E., Jackson, M. R., Compton, T., & Fruh, K. (2001). Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B. Proc Natl Acad Sci USA 98, 7140 – 7145. Singh, N., Yu, V. L., Mieles, L., Wagener, M. M., Miner, R. C., & Gayowski, T. (1994). High-dose acyclovir compared with short-course preemptive ganciclovir therapy to prevent cytomegalovirus disease in liver transplant recipients. A randomized trial. Ann Intern Med 120, 375 – 381. Sinzger, C., & Jahn, G. (1996). Human cytomegalovirus cell tropism and pathogenesis. Intervirology 39, 302 – 319. Sinzger, C., Grefte, A., Plachter, B., Gouw, A. S., The, T. H., & Jahn, G. (1995). Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues. J Gen Virol 76, 741 – 750. Sinzger, C., Plachter, B., Grefte, A., The, T. H., & Jahn, G. (1996). Tissue macrophages are infected by human cytomegalovirus. J Infect Dis 173, 240 – 245. Sinzger, C., Kahl, M., Laib, K., Klingel, K., Rieger, P., Plachter, B., & Jahn, G. (2000). Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus. J Gen Virol 81, 3021 – 3035. Sison, R. F., Holland, G. N., MacArthur, L. J., Wheeler, N. C., & Gottlieb, M. S. (1991). Cytomegalovirus retinopathy as the initial manifestation of the acquired immunodeficiency syndrome. Am J Ophthalmol 112, 243 – 249. Skaletskaya, A., Bartle, L. M., Chittenden, T., McCormick, A. L., Mocarski, E. S., & Goldmacher, V. S. (2001). A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation. Proc Natl Acad Sci USA 98, 7829 – 7834. Smith, I. L., Cherrington, J. M., Jiles, R. E., Fuller, M. D., Freeman, W. R., & Spector, S. A. (1997). High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes. J Infect Dis 176, 69 – 77. Snydman, D. R., Werner, B. G., Heinze-Lacey, B., Berardi, V. P., Tilney, N. L., Kirkman, R. L., Milford, E. L., Cho, S. I., Bush Jr., H. L., & Levey, A. S. (1987). Use of cytomegalovirus immune globulin to prevent cytomegalovirus disease in renal transplant recipients. N Engl J Med 317, 1049 – 1054. Soderberg-Naucler, C., Fish, K. N., & Nelson, J. A. (1997). Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell 91, 119 – 126. Soderberg-Naucler, C., Streblow, D. N., Fish, K. N., Allan-Yorke, J., Smith, P. P., & Nelson, J. A. (2001). Reactivation of latent human cytomegalovirus in CD14 + monocytes is differentiation dependent. J Virol 75, 7543 – 7554. Sommer, M. H., Scully, A. L., & Spector, D. H. (1994). Transactivation by the human cytomegalovirus IE2 86-kilodalton protein requires a domain that binds to both the TATA box-binding protein and the retinoblastoma protein. J Virol 68, 6223 – 6231. Song, Y., & Stinski, M. F. (2002). Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes: a DNA microarray analysis. Proc Natl Acad Sci USA 99, 2836 – 2841. Spector, S. A., Weingeist, T., Pollard, R. B., Dieterich, D. T., Samo, T., Benson, C. A., Busch, D. F., Freeman, W. R., Montague, P., Kaplan, H. J., Keller, L., Crager, M., De Armond, B., Buhles, W., & Feinberg, J. (1993). A randomized, controlled study of intravenous ganciclovir therapy for cytomegalovirus peripheral retinitis in patients with AIDS. AIDS Clinical Trials Group and Cytomegalovirus Cooperative Study Group. J Infect Dis 168, 557 – 563. Spector, S. A., McKinley, G. F., Lalezari, J. P., Samo, T., Andruczk, R., Follansbee, S., Sparti, P. D., Havlir, D. V., Simpson, G., Buhles, W., Wong, R., & Stempien, M. (1996). Oral ganciclovir for the prevention of cytomegalovirus disease in persons with AIDS. Roche Cooperative Oral Ganciclovir Study Group. N Engl J Med 334, 1491 – 1497. Speir, E., Modali, R., Huang, E.-S., Leon, M. B., Shawl, F., Finkel, T., & Epstein, S. E. (1993). Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science 265, 391 – 394. Stagno, S., Reynolds, D. W., Lakeman, A., Charamella, L. J., & Alford, C. A. (1973). Congenital cytomegalovirus infection: consecutive occurrence due to viruses with similar antigenic compositions. Pediatrics 52, 788 – 794. Stagno, S., Reynolds, D. W., Huang, E. S., Thames, S. D., Smith, R. J., & Alford, C. A. (1977). Congenital cytomegalovirus infection: occurrence in an immune population. N Engl J Med 296, 1254 – 1258. Stagno, S., Pass, R. F., Dworsky, M. E., & Alford, C. A., Jr. (1982). Maternal cytomegalovirus infection and perinatal transmission. Clin Obstet Gynecol 25, 563 – 576. Stagno, S., Pass, R. F., Cloud, G., Britt, W. J., Henderson, R. E., Walton, P. D., Veren, D. A., Page, F., & Alford, C. A. (1986). Primary cytomegalovirus infection in pregnancy. Incidence, transmission to fetus, and clinical outcome. JAMA 256, 1904 – 1908. Stasiak, P. C., & Mocarski, E. S. (1992). Transactivation of the human cytomegalovirus ICP36 gene promoter requires the a gene product TRS1 in addition to the IE1 and IE2. J Virol 66, 1050 – 1058. Story, C. M., Furman, M. H., & Ploegh, H. L. (1999). The cytosolic tail of class I MHC heavy chain is required for its dislocation by the human cytomegalovirus US2 and US11 gene products. Proc Natl Acad Sci USA 96, 8516 – 8521. Stratta, R. J., Wood, R. P., Langnas, A. N., Duckworth, R. M., Shaefer, M. S., Marujo, W., Pillen, T. J., Markin, R. S., & Shaw, B. W., Jr. (1990). Donor selection for orthotopic liver transplantation: lack of an effect of gender or cytomegalovirus (CMV) status. Transplant Proc 22, 410 – 413. Streblow, D. N., Orloff, S. L., & Nelson, J. A. (2001). Do pathogens accelerate atherosclerosis? J Nutr 13, 2798S – 2804S. Theiler, R. N., & Compton, T. (2001). Characterization of the signal peptide processing and membrane association of human cytomegalovirus glycoprotein O. J Biol Chem 276, 39226 – 39231. The Vitravene Study Group (2002a). A randomized controlled clinical trial of intravitreous fomivirsen for treatment of newly diagnosed peripheral cytomegalovirus retinitis in patients with AIDS. Am J Ophthalmol 133, 467 – 474. The Vitravene Study Group (2002b). Randomized dose-comparison studies of intravitreous fomivirsen for treatment of cytomegalovirus retinitis that has reactivated or is persistently active despite other therapies in patients with AIDS. Am J Ophthalmol 133, 475 – 483. Tomasec, P., Braud, V. M., Rickards, C., Powell, M. B., McSharry, B. P., Gadola, S., Cerundolo, V., Borysiewicz, L. K., McMichael, A. J., & Wilkinson, G. W. (2000). Surface expression of HLA E an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science 287, 1031 – 1033. van den Berg, A. P., van der Bij, W., van Son, W. J., Anema, J., van der Giessen, M., Schirm, J., Tegzess, A. M., & The, T. H. (1989). Cytomegalovirus antigenemia as a useful marker of symptomatic cytomegalovirus infection after renal transplantation—a report of 130 consecutive patients. Transplantation 48, 991 – 995. van der Bij, W., Schirm, J., Torensma, R., van Son, W. J., Tegzess, A. M., & The, T. H. (1988a). Comparison between viremia and antigenemia for detection of cytomegalovirus in blood. J Clin Microbiol 26, 2531 – 2535. van der Bij, W., Torensma, R., van Son, W. J., Anema, J., Schirm, J., Tegzess, A. M., & The, T. H. (1988b). Rapid immunodiagnosis of active cytomegalovirus infection by monoclonal antibody staining of blood leucocytes. J Med Virol 25, 179 – 188. Vierling, J. M., & Fennel, R. H. (1985). Histopathology of early and late human hepatic allograft rejection: evidence of progressive destruction of interlobular bile ducts. Hepatology 5, 1076 – 1082. Vinters, H. V., Kwok, M. K., Ho, H. W., Anders, K. H., Tomiyasu, U., S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 Wolfson, W. L., & Robert, F. (1989). Cytomegalovirus in the nervous system of patients with the acquired immune deficiency syndrome. Brain 112, 245 – 268. Vochem, M., Hamprecht, K., Jahn, G., & Speer, C. P. (1998). Transmission of cytomegalovirus to preterm infants through breast milk. Pediatr Infect Dis J 17, 53 – 58. Vogel, J.-U., Scholz, M., & Cinatl, J., Jr. (1998). Treatment of CMV diseases. Intervirology 40, 357 – 367. Wade, M., Kowalik, T. F., Mudryj, M., Huang, E.-S., & Azizkhan, J. C. (1992). E2F mediates dihydrofolate reducatse promoter activation and multiprotein complex formation in human cytomegalovirus infection. Mol Cell Biol 12, 4364 – 4374. Walter, E. A., Greenberg, P. D., Gilbert, M. J., Finch, R. J., Watanabe, K. S., Thomas, E. D., & Riddell, S. R. (1995). Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med 333, 1038 – 1044. Webster, A., Philips, A. N., Lee, C. A., Janossy, G., Kernoff, P. B., & Griffiths, P. D. (1992). Cytomegalovirus (CMV) infection, CD4+ lymphocyte counts and the development of AIDS in HIV-1 infected haemophiliac patients. Clin Exp Immunol 88, 6 – 9. Wiebush, L., & Hagemeier, C. (1999). Human cytomegalovirus 86-kilodalton IE2 protein blocks cell cycle progression in G1. J Virol 73, 9274 – 9283. Wiebush, L., & Hagemeier, C. (2001). The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclindependent kinase activation. EMBO J 20, 1086 – 1098. Wiertz, E. J., Jones, T. R., Sun, L., Bogyo, M., Geuze, H. J., & Ploegh, H. L. (1996). The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84, 769 – 779. Wills, M. R., Carmichael, A. J., Mynard, K., Jin, X., Weekes, M. P., Plachter, B., & Sissons, J. G. (1996). The human cytotoxic T-lympho- 297 cyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol 70, 7569 – 7579. Winston, D. J., Ho, W. G., Bartoni, K., Du Mond, C., Ebeling, D. F., Buhles, W. C., & Champlin, R. E. (1993). Ganciclovir prophylaxis of cytomegalovirus infection and disease in allogeneic bone marrow transplant recipients. Results of a placebo-controlled, double-blind trial. Ann Intern Med 118, 179 – 184. Wreghitt, T. G., Hakim, M., Gray, J. J., Kucia, S., Wallwork, J., & English, T. A. (1988). Cytomegalovirus infections in heart and heart and lung transplantation. J Clin Pathol 41, 660 – 667. Yeager, A. S., & Martin, H. P. (1977). Congenital cytomegalovirus infection: outcome for the subsequent sibling. Clin Pediatr 16, 455 – 458. Yurochko, A. D., Kowalik, T. F., Huong, S. M., & Huang, E.-S. (1995). Human cytomegalovirus upregulates NF-kB activity by transactivating the NF-kB p105/p50 and p65 promoter. J Virol 69, 5391 – 5400. Yurochko, A. D., Mayo, M. W., Poma, E. E., Baldwin Jr., A. S., & Huang, E.-S. (1997). Induction of the transcription factor Sp1 during human cytomegalovirus infection mediates upregulation of the p65 and p105/ p50 NF-kB promoters. J Virol 71, 4638 – 4648. Zanghellini, F., Boppana, S. B., Emery, V. C., Griffiths, P. D., & Pass, R. F. (1999). Asymptomatic primary cytomegalovirus infection: virologic and immunologic features. J Infect Dis 180, 702 – 707. Zhu, H., Shen, Y., & Shenk, T. (1995). Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J Virol 69, 7960 – 7970. Zhu, H., Cong, J. P., & Shenk, T. (1997). Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: induction of the interferon-responsive RNAs. Proc Natl Acad Sci USA 94, 13985 – 13990. Zhu, H., Cong, J. P., Mamtora, G., Gingeras, T., & Shenk, T. (1998). Cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays. Proc Natl Acad Sci USA 95, 14470 – 14475.