Papers by Wolfgang Hammerschmidt
Cell, Nov 1, 1988
We have identified a cis-acting element of Epstein-Barr virus (EBV) that mediates viral DNA repli... more We have identified a cis-acting element of Epstein-Barr virus (EBV) that mediates viral DNA replication during the lytic phase of this virus's life cycle. This lytic origin of DNA replication, termed oriLyt, is complex in structure in that it contains multiple regions that are required for replication and additional DNA sequences that increase replication. One of the required regions of oriLyt can be functionally substituted by a transcriptional enhancing element. DNA replication mediated by oriLyt depends on EBV DNA polymerase and yields a concatemeric molecule. A vector, which contains both oriP (the EBV plasmid origin of replication) and oriLyt, can be maintained as a plasmid in latently EBV-infected cells and can be amplified 100- to 1000-fold in cells in which the lytic phase of the viral life cycle is induced.
PubMed, Feb 1, 1990
We have two goals in this review: the first is to relate what has been learned about DNA replicat... more We have two goals in this review: the first is to relate what has been learned about DNA replication from the study primarily of herpes simplex virus type 1 (HSV-1); the second is to note briefly facets of this virus's mode of DNA replication that might serve as points of intervention for novel chemotherapeutic approaches in order to deal with primary and recurrent herpesvirus infections in man and animals. For the first goal we shall both summarize what has been learned and attempt to identify directions that may be pursued in order to further our understanding of DNA replication by herpesviruses. For the second goal we shall propose two schemes for the screening for drugs that might interfere uniquely with the DNA replication of this family of viruses.
Journal of Virology, 1995
Journal of Virology, Jun 1, 1997
The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the immortalizin... more The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the immortalizing activity of EBV in primary, human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it influences signaling pathways of infected cells. LMP-1 has been found to associate with members of the tumor necrosis factor receptor-associated factor (TRAF) family of proteins. As with LMP-1, the TRAF molecules have been shown to participate in cell signaling pathways. We have characterized and mapped in detail a region of LMP-1 that associates with TRAF1, TRAF2, and TRAF3. TRAF3 alone associates with LMP-1 in a yeast two-hybrid assay, whereas all three TRAF molecules associate with LMP-1 under various conditions when they are assayed in extracts of human cells. TRAF1, TRAF2, and TRAF3 appear to associate independently with LMP-1 but bind an overlapping target site. TRAF3 associates with LMP-1 most avidly and can compete with TRAF1 and TRAF2 for binding to LMP-1. TRAF2 associates with truncated derivatives of the carboxy terminus of LMP-1 more efficiently than with the intact terminus, indicating that LMP-1's conformation may regulate its association with TRAF2. Finally, point mutations that decrease LMP-1's association with the three TRAF molecules to 3 to 20% of wild-type levels do not detectably affect otherwise intact LMP-1's induction of NF-B activity. Therefore, these associations are not necessary for the majority of intact LMP-1's induction of this signaling pathway.
Springer eBooks, 1989
Epstein-Barr virus (EBV) is a human herpesvirus that is causally associated with several malignan... more Epstein-Barr virus (EBV) is a human herpesvirus that is causally associated with several malignancies including nasopharyngeal carcinoma, African Burkitt’s lymphoma, and lymphoproliferative disorders in immunosuppressed people (reviewed in Miller 1985). In vitro EBV infects human B cells and immortalizes between 10%-100% of them (Sugden and Mark 1977; reviewed in zur Hausen 1981). It seems likely that EBV’s capacity to immortalize cells in culture is related to its tumorigenicity in vivo. This likelihood has led researchers to study the mechanism of immortalization of B cells by this virus. The EBV genes required to initiate and/or to maintain the immortalized state in vitro have not been identified. However, at least eight viral genes are found to be transcribed consistently in B cells immortalized in vitro, and circumstantial evidence exists that at least several of these gene products will be required for this process (reviewed in Knutson and Sugden 1988). Until recently, there was no evidence that causally linked any one of these eight viral genes with changes in the growth control of EBV-infected cells. It has now been shown that one viral gene, BNLF-1 (or LMP), when expressed from either of two heterologous promoters and introduced into either of two established rodent cell lines, Rat-1 (Wang et al. 1985) or BALB/3T3 cells (Baichwal and Sugden 1988), induces these cells to grow in an anchorage-independent fashion in agarose.
Oncogene, Mar 18, 2013
Epstein-Barr virus (EBV) has evolved exquisite controls over its host cells, human B lymphocytes,... more Epstein-Barr virus (EBV) has evolved exquisite controls over its host cells, human B lymphocytes, not only directing these cells during latency to proliferate and thereby expand the pool of infected cells, but also to survive and thereby persist for the lifetime of the infected individual. Although these activities ensure the virus is successful, they also make the virus oncogenic, particularly when infected people are immunosuppressed. Here we show, strikingly, that one set of EBV's miRNAs both sustain BL (Burkitt's lymphoma) cells in the absence of other viral oncogenes and promote the transformation of primary B lymphocytes. Burkitt's Lymphoma cells were engineered to lose EBV and found to die by apoptosis and could be rescued by constitutively expressing viral miRNAs in them. Two of these EBV miRNAs were found to target Caspase 3 to inhibit apoptosis at physiological concentrations.
Cold Spring Harbor Monograph Archive, 2006
Epstein-Barr virus (EBV) is fascinating and informative both as a human pathogen and as a replico... more Epstein-Barr virus (EBV) is fascinating and informative both as a human pathogen and as a replicon. EBV has become a paradigm for human tumor viruses: It is the first virus recognized to cause cancer in people; it causes both lymphomas and carcinomas, yet these tumors arise infrequently although most people in the world are infected with the virus. EBV is maintained as an extrachromosomal replicon in infected normal and tumor cells. These viral plasmids replicate once per cell cycle, are licensed, require a single viral protein for their synthesis and segregation, but can use either of two functionally distinct origins of DNA synthesis for their replication. Viral progeny are not synthesized under these conditions, and the cells are said to be latently infected. This plasmid replication is consistent with survival of EBV’s host cells, and viral genes are required for proliferation and/or survival of the infected cells. EBV also has an additional mode of replication that is inconsistent with cell survival. Rare cells in an infected population change to support EBV’s lytic cycle in which viral DNA replicates 100-fold or more without cell division. Viral progeny are synthesized, and host cells die. This lytic mode of replication uses a third kind of viral origin of DNA synthesis and many viral proteins to carry out that synthesis. Here, we describe the three modes of EBV’s replication as a function of the viral origins used and the viral and cellular proteins that mediate initiation and elongation of DNA synthesis from these origins.
MBio, Dec 21, 2021
The innate immune system serves as frontline defense against pathogens, such as bacteria and viru... more The innate immune system serves as frontline defense against pathogens, such as bacteria and viruses. Natural killer (NK) cells are a part of innate immunity and can both secrete cytokines and directly target cells for lysis. NK cells express several cell surface receptors, including NKG2D, which bind multiple ligands. People with deficiencies in NK cells are often susceptible to uncontrolled infection by herpesviruses, such as Epstein-Barr virus (EBV). Infection with EBV stimulates both innate and adaptive immunity, yet the virus establishes lifelong latent infection in memory B cells. We show that the EBV oncogene EBNA1, previously known to be necessary for maintaining EBV genomes in latently infected cells, also plays an important role in suppressing NK cell responses and cell death in newly infected cells. EBNA1 does so by downregulating the NKG2D ligands ULBP1 and ULBP5 and modulating expression of c-Myc. B cells infected with a derivative of EBV that lacks EBNA1 are more susceptible to NK cell-mediated killing and show increased levels of apoptosis. Thus, EBNA1 performs a previously unappreciated role in reducing immune response and programmed cell death after EBV infection, helping infected cells avoid immune surveillance and apoptosis and thus persist for the lifetime of the host.
mBio, 2021
Epstein-Barr virus (EBV) is a ubiquitous human pathogen, infecting up to 95% of the world’s adult... more Epstein-Barr virus (EBV) is a ubiquitous human pathogen, infecting up to 95% of the world’s adult population. Initial infection with EBV can cause infectious mononucleosis.
eLife, 2021
Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the p... more Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing, and fork directionality profiles obtained by RNA-seq, Repli-seq, and OK-seq. Both, the origin recognition complex (ORC) and the minichromosome maintenance complex (MCM) are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating regions, and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late ori...
Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the p... more Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing and fork directionality profiles obtained by RNA-seq, Repli-seq and OK-seq. ORC and MCM are strongly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early-than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late replicating initiation zones and gene deserts of stochastic r...
Journal of Virology, 1995
We have assembled derivatives of Epstein-Barr Virus (EBV) that include 71 kbp of noncontiguous DN... more We have assembled derivatives of Epstein-Barr Virus (EBV) that include 71 kbp of noncontiguous DNA sequences cloned into a prokaryotic F-factor plasmid. These mini-EBVs, when introduced into an EBV-containing lymphoblastoid cell, can be packaged by the endogenous helper virus. One such mini-EBV was found to have a single C residue deleted from its EBNA3a open reading frame. When packaged, this mini-EBV initiates proliferation of infected primary human B lymphocytes only in conjunction with a complementing helper virus. Proliferation of the infected cells, however, was maintained either alone by the mini-EBV containing the mutated EBNA3a open reading frame or alone by its derivative in which the EBNA3a open reading frame had been healed of its lesion by recombination with the helper virus. The mini-EBV with a wild-type EBNA3a open reading frame when packaged alone can both initiate and maintain proliferation upon infection of primary human B lymphocytes. These findings identify 41% o...
Journal of Virology, 1989
A previously unrecognized activity has been associated with the product of the BNLF-1 gene of Eps... more A previously unrecognized activity has been associated with the product of the BNLF-1 gene of Epstein-Barr virus. This gene encodes the latent membrane protein of Epstein-Barr virus. When the gene was expressed at high levels, it was toxic to all cell lines tested, which included six human B-lymphoid lines as well as BALB/3T3, 143/EBNA-1, and HEp-2 cells. The BNLF-1 gene was previously shown to induce anchorage-independent and tumorigenic growth in Rat-1 and BALB/3T3 cells. We demonstrate here that only those mutations in the BNLF-1 gene that score positively in the anchorage-independent growth assay were cytotoxic when expressed at high levels. It is therefore possible that the same activities of the latent membrane protein that are necessary to induce anchorage-independent growth of some rodent cell lines also confer toxicity to many cell lines when expressed at high levels.
Eukaryotic replication initiates during S phase from origins that have been licensed in the prece... more Eukaryotic replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with replication initiation events obtained by Okazaki fragment sequencing. We demonstrate that MCM is displaced from early replicating, actively transcribed gene bodies, while ORC is mainly enriched at active TSS. Late replicating, H4K20me3 containing initiation zones display enhanced ORC and MCM levels. Furthermore, we find early RTDs being primarily enriched in ORC, compared to MCM, indicating that ORC levels are involved in organizing the temporal order of DNA replication. The organizational connection between active transcription and replication competence directly links changes in the transcriptional program to flexible replication patterns, which ensures the cell’s flexibility to respond to environmental cues.
Proceedings of the National Academy of Sciences, 2016
Significance Most humans are infected for their lifetime with Epstein–Barr virus (EBV), which can... more Significance Most humans are infected for their lifetime with Epstein–Barr virus (EBV), which can cause cancer and other EBV-associated diseases. Infected individuals develop strong immune responses to this virus, in particular cytotoxic CD8 + T cells, but viral infection is never cleared nor is EBV eliminated from the body. This suggests that certain viral molecules might prevent effective elimination of EBV-infected cells by CD8 + T cells. EBV is rich in genes coding for microRNAs, many with unknown function. We show that viral microRNAs interfere with recognition and killing of EBV-infected cells by CD8 + T cells. Multiple mechanisms and molecules are targeted by microRNAs to achieve this immune evasion. Therefore, targeting of viral microRNAs may improve antiviral immunity and therapy.
Journal of Experimental Medicine, 2016
Epstein-Barr virus (EBV) is a tumor virus that establishes lifelong infection in most of humanity... more Epstein-Barr virus (EBV) is a tumor virus that establishes lifelong infection in most of humanity, despite eliciting strong and stable virus-specific immune responses. EBV encodes at least 44 miRNAs, most of them with unknown function. Here, we show that multiple EBV miRNAs modulate immune recognition of recently infected primary B cells, EBV's natural target cells. EBV miRNAs collectively and specifically suppress release of proinflammatory cytokines such as IL-12, repress differentiation of naive CD4+ T cells to Th1 cells, interfere with peptide processing and presentation on HLA class II, and thus reduce activation of cytotoxic EBV-specific CD4+ effector T cells and killing of infected B cells. Our findings identify a previously unknown viral strategy of immune evasion. By rapidly expressing multiple miRNAs, which are themselves nonimmunogenic, EBV counteracts recognition by CD4+ T cells and establishes a program of reduced immunogenicity in recently infected B cells, allowin...
Trends in Molecular Medicine, 2004
Two proteins of Epstein-Barr Virus make formerly unrecognized contributions to maintaining the tu... more Two proteins of Epstein-Barr Virus make formerly unrecognized contributions to maintaining the tumors of Burkitt's lymphomas and Hodgkin's disease. The Epstein-Barr nuclear antigen 1 (EBNA1) protein can support the synthesis and maintenance of the viral genome. New data show that inhibiting EBNA1 in Burkitt's lymphoma cells induces cell death by apoptosis. Therefore, EBNA1 inhibits apoptosis and, according to recent findings, does so independently of other viral genes. The latent membrane protein 2a (LMP2a) binds to signaling molecules that are engaged by the B-cell receptor and inhibits the signaling that is mediated by antigen binding. New findings have revealed how LMP2a overcomes the apoptosis that normally results from the absence of functional B-cell receptors, and explain how Hodgkin's disease tumor cells, which are B cells, survive but lack functional antibodies.
Cold Spring Harbor perspectives in biology, 2013
Epstein-Barr virus (EBV) is a paradigm for human tumor viruses: it is the first virus recognized ... more Epstein-Barr virus (EBV) is a paradigm for human tumor viruses: it is the first virus recognized to cause cancer in people; it causes both lymphomas and carcinomas; yet these tumors arise infrequently given that most people in the world are infected with the virus. EBV is maintained extrachromosomally in infected normal and tumor cells. Eighty-four percent of these viral plasmids replicate each S phase, are licensed, require a single viral protein for their synthesis, and can use two functionally distinct origins of DNA replication, oriP, and Raji ori. Eighty-eight percent of newly synthesized plasmids are segregated faithfully to the daughter cells. Infectious viral particles are not synthesized under these conditions of latent infection. This plasmid replication is consistent with survival of EBV's host cells. Rare cells in an infected population either spontaneously or following exogenous induction support EBV's lytic cycle, which is lethal for the cell. In this case, the...
Virology, 1996
oriLyt, the cis-acting element of the lytic origin of DNA replication of Epstein-Barr virus, is a... more oriLyt, the cis-acting element of the lytic origin of DNA replication of Epstein-Barr virus, is activated by the viral transactivator BZLF1 which belongs to the extended bZIP class of transcription factors. Seven binding sites for BZLF1, so-called ZRE sites, are located within oriLyt. By mutational analysis of individual ZRE sites, we found that lytic DNA replication is dependent on only four of these sites which colocate with the promoter of the BHLF1 gene. The remaining three ZRE sites distal to the BHLF1 promoter were dispensable for DNA replication and did not contribute to long-range transcriptional activation of this promoter by BZLF1. This finding indicated that a similar set of ZRE sites is involved in DNA replication and transcriptional activation. To determine the function of BZLF1 in DNA replication, BZLF1 mutants with successive deletions in the transactivation domain were analyzed in replication assays. Unexpectedly, most BZLF1 mutants which failed to support DNA replication were found to be equally defective in transcriptional activation. Therefore, similar transacting domains of BZLF1 are involved both in replication and in transcription.
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Papers by Wolfgang Hammerschmidt