Despite advances in hepatitis C treatment, substantial clinical hurdles remain to achieve univers... more Despite advances in hepatitis C treatment, substantial clinical hurdles remain to achieve universal cure and global control of infection. Saeed et al. identified SEC14L2 as a host factor permitting replication of clinical HCV isolates in cell culture, providing a novel system to model infection of patient-derived viruses.
Hepatitis C virus (HCV) remains a major global health burden, with more than 130 million individu... more Hepatitis C virus (HCV) remains a major global health burden, with more than 130 million individuals chronically infected and at risk for the development of hepatocellular carcinoma (HCC). The recent clinical licensing of direct-acting antivirals enables viral cure. However, limited access to therapy and treatment failure in patient subgroups warrants a continuing effort to develop complementary antiviral strategies. Furthermore, once fibrosis is established, curing HCV infection does not eliminate the risk for HCC. High-throughput approaches and screens have enabled the investigation of virus-host interactions on a genome-wide scale. Gain- and loss-of-function screens have identified essential host-dependency factors in the HCV viral life cycle, such as host cell entry factors or regulatory factors for viral replication and assembly. Network analyses of systems-scale data sets provided a comprehensive view of the cellular state following HCV infection, thus improving our understand...
Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a con... more Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a ...
Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcino... more Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus (HBV) and hepatitis D virus (HDV) use the sodium taurocholate co-transporting polypeptide (NTCP, a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high-throughput infectious cell culture model enabling functional genomics of HDV entry and infection. Using a targeted RNAi entry screen we identified glypican 5 (GPC5) as a common host cell entry factor for HBV and HDV. These findings advance our understanding of virus cell entry and open new avenues for curative therapies. Since glypicans have been shown to play a role in the control of cell division and growth regulation, virus-GPC5 interactions may also play a role in the pathogenesis of virus-induced liver disease and cancer. This article is protected by copyright. All rights reserved.
Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcino... more Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus (HBV) and hepatitis D virus (HDV) use the sodium taurocholate co-transporting polypeptide (NTCP, a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high-throughput infectious cell culture model enabling functional genomics of HDV entry and infection. Using a targeted RNAi entry screen we identified glypican 5 (GPC5) as a common host cell entry factor for HBV and HDV. These findings advance our understanding of virus cell entry and open new avenues for curative therapies. Since glypicans have been shown to play a role in the control of cell division and growth regulation, virus-GPC5 interactions may also play a role in the pathogenesis of virus-induced liver disease and cancer. This article is protected by copyright. All rights reserved.
Hepatitis C virus (HCV) causes persistent infection in the majority of infected individuals. The ... more Hepatitis C virus (HCV) causes persistent infection in the majority of infected individuals. The mechanisms of persistence and clearance are only partially understood. Antibodies against host cell entry receptors have been shown to inhibit HCV infection in cell culture and animal models. In this study we aimed to investigate whether anti-receptor antibodies are induced during infection in humans in vivo and whether their presence is associated with outcome of infection. We established an ELISA using a recombinant CD81- claudin-1 (CLDN1) fusion protein to detect and quantify antibodies directed against extracellular epitopes of the HCV CD81-CLDN1 co-receptor complex. The presence of anti-receptor antibodies was studied in serum of patients from a well-defined cohort of a single-source HCV outbreak of pregnant women and several control groups including uninfected pregnant women, patients with chronic hepatitis B and D virus (HBV/HDV) infection and healthy individuals. The virus-neutralizing activity of antibodies was determined using recombinant cell culture-derived HCV (HCVcc). Our results demonstrate that HCV infected patients have statistically significantly higher anti-CD81/CLDN1 antibody titers during the early phase of infection than controls, the titers being significantly higher in resolvers compared to persisters. Functional studies using immunoadsorption and HCV cell culture models demonstrate that HCV-neutralizing anti-receptor antibodies are induced in early phase of HCV infection but not in control groups. The virus-neutralizing properties of these antibodies suggest a role for control of viral infection in conjunction with anti-viral responses. Characterization of these anti-receptor antibodies opens new avenues to prevent and treat HCV infection. This article is protected by copyright. All rights reserved.
Hepatitis C virus (HCV) is a positive strand RNA virus that primarily infects human hepatocytes. ... more Hepatitis C virus (HCV) is a positive strand RNA virus that primarily infects human hepatocytes. HCV infections constitute a global health problem with 80 million people currently chronically infected. Recent studies reported that cholesterol 25-hydroxylase (CH25H) is expressed as an interferon-stimulated gene (ISG) and mediates antiviral activities against different enveloped viruses through the production of 25-hydroxycholesterol (25HC). However, the intrinsic regulation of human CH25H (hCH25H) expression within the liver as well as its mechanistic effects on HCV infectivity remains elusive. In this study, we characterized the expression of hCH25H using liver biopsies and primary human hepatocytes (PHH). In addition, the antiviral properties of this protein and its enzymatic product, 25HC, were further characterized against HCV in tissue culture. hCH25H mRNA levels were significantly upregulated both in HCV-positive liver biopsies and HCV-infected PHH. The expression of hCH25H in PHH was primarily and transiently induced by type-I interferon (IFN). Transient expression of hCH25H in human hepatoma cells restricted HCV infection in a genotype-independent manner. This inhibition required the enzymatic activity of CH25H. We observed an inhibition of viral membrane fusion during the entry process by 25HC, which was not due to a virucidal effect. Yet the primary effect by 25HC on HCV was at the level of RNA replication which was observed by using subgenomic replicons of two different genotypes. Further analysis using electron microscopy revealed that 25HC inhibited formation of the membranous web (MW), the HCV replication factory, independent of RNA replication. In conclusion, HCV infection causes the upregulation of interferon-inducible CH25H in vivo. Its product 25HC restricts HCV primarily at the level of RNA replication by preventing the formation of viral replication factory. This article is protected by copyright. All rights reserved.
Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current... more Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current antiviral therapy fails to clear infection in a substantial proportion of cases. Drug development is focused on nonstructural proteins required for RNA replication. Individuals undergoing orthotopic liver transplantation face rapid, universal reinfection of the graft. Therefore, antiviral strategies targeting the early stages of infection are urgently needed for the prevention of HCV infection. In this study, we identified the polyphenol, epigallocatechin-3-gallate (EGCG), as an inhibitor of HCV entry. Green tea catechins, such as EGCG and its derivatives, epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), have been previously found to exert antiviral and antioncogenic properties. EGCG had no effect on HCV RNA replication, assembly, or release of progeny virions. However, it potently inhibited Cell-culture-derived HCV (HCVcc) entry into hepatoma cell lines as well as primary human hepatocytes. The effect was independent of the HCV genotype, and both infection of cells by extracellular virions and cell-to-cell spread were blocked. Pretreatment of cells with EGCG before HCV inoculation did not reduce HCV infection, whereas the application of EGCG during inoculation strongly inhibited HCV infectivity. Moreover, treatment with EGCG directly during inoculation strongly inhibited HCV infectivity. Expression levels of all known HCV (co-)receptors were unaltered by EGCG. Finally, we showed that EGCG inhibits viral attachment to the cell, thus disrupting the initial step of HCV cell entry. Conclusion: The green tea molecule, EGCG, potently inhibits HCV entry and could be part of an antiviral strategy aimed at the prevention of HCV reinfection after liver transplantation. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (HA 4393/2-1; to T.V.H.) and (Ci 171/2-1; to S.C.) and from the CIHR and BWF (to L.M.S.). T.
Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of second... more Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of secondary metabolites in bacteria, fungi and plants. Some of these metabolites have played important roles during the early evolution of land plants by providing protection from various environmental assaults including UV irradiation. The genome of the moss, Physcomitrella patens, contains at least 17 putative CHS superfamily genes. Three of these genes (PpCHS2b, PpCHS3 and PpCHS5) exist in multiple copies and all have corresponding ESTs. PpCHS11 and probably also PpCHS9 encode non-CHS enzymes, while PpCHS10 appears to be an ortholog of plant genes encoding anther-specific CHS-like enzymes. It was inferred from the genomic locations of genes comprising it that the moss CHS superfamily expanded through tandem and segmental duplication events. Inferred exon-intron architectures and results from phylogenetic analysis of representative CHS superfamily genes of P. patens and other plants showed tha...
Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with va... more Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide a-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain-and v-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated a-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.
Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of second... more Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of secondary metabolites in bacteria, fungi and plants. Some of these metabolites have played important roles during the early evolution of land plants by providing protection from various environmental assaults including UV irradiation. The genome of the moss, Physcomitrella patens, contains at least 17 putative CHS superfamily genes. Three of these genes (PpCHS2b, PpCHS3 and PpCHS5) exist in multiple copies and all have corresponding ESTs. PpCHS11 and probably also PpCHS9 encode non-CHS enzymes, while PpCHS10 appears to be an ortholog of plant genes encoding anther-specific CHS-like enzymes. It was inferred from the genomic locations of genes comprising it that the moss CHS superfamily expanded through tandem and segmental duplication events. Inferred exon-intron architectures and results from phylogenetic analysis of representative CHS superfamily genes of P. patens and other plants showed that intron gain and loss occurred several times during evolution of this gene superfamily. A high proportion of P. patens CHS genes (7 of 14 genes for which the full sequence is known and probably 3 additional genes) are intronless, prompting speculation that CHS gene duplication via retrotransposition has occurred at least twice in the moss lineage. Analyses of sequence similarities, catalytic motifs and EST data indicated that a surprisingly large number (as many as 13) of the moss CHS superfamily genes probably encode active CHS. EST distribution data and different light responsiveness observed with selected genes provide evidence for their differential regulation. Observed diversity within the moss CHS superfamily and amenability to gene manipulation make Physcomitrella a highly suitable model system for studying expansion and functional diversification of the plant CHS superfamily of genes.
Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, sev... more Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, several Arabidopsis genes, including polyketide synthase A (PKSA), which encodes an anther-specific chalcone synthase-like enzyme (ASCL), have been shown to be involved in sporopollenin biosynthesis. The genome of the moss Physcomitrella patens contains putative orthologs of the Arabidopsis sporopollenin biosynthesis genes.
Background: Physcomitrella PpORS is an ancient member of the plant type III polyketide synthase (... more Background: Physcomitrella PpORS is an ancient member of the plant type III polyketide synthase (PKS) family. Results: PpORS, produced in nonprotonemal moss cells, synthesizes pentaketide 2Ј-oxoalkylresorcinols using a unique substrate binding site. Conclusion: PpORS is a novel very long chain 2Ј-oxoalkylresorcinol synthase. Significance: This is the first step toward understanding the co-evolution of the type III PKS family and land plants.
Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections remain major health problems world... more Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections remain major health problems worldwide, with 400−500 million chronically infected people worldwide. Chronic infection results in liver cirrhosis and hepatocellular carcinoma, the second leading cause of cancer death. Current treatments for HBV limit viral replication without efficiently curing infection. HCV treatment has markedly progressed with the licensing of direct-acting antivirals (DAAs) for HCV cure, yet limited access for the majority of patients is a major challenge. Preventative and curative treatment strategies, aimed at novel targets, are needed for both viruses. Viral entry represents one such target, although detailed knowledge of the entry mechanisms is a prerequisite. For HBV, the recent discovery of the NTCP cell entry factor enabled the establishment of an HBV cell culture model and showed that cyclosporin A and Myrcludex B are NTCP-targeting entry inhibitors. Advances in the understanding of HCV entry revealed it to be a complex process involving many factors, offering several antiviral targets. These include viral envelope proteins E1 and E2, virion-associated lipoprotein ApoE, and cellular factors CD81, SRBI, EGFR, claudin-1, occludin, and the cholesterol transporter NPC1L1. Small molecules targeting SR-BI, EGFR, and NPC1L1 have entered clinical trials, whereas other viral-and host-targeted small molecules, peptides, and antibodies show promise in preclinical models. This review summarizes the current understanding of HBV and HCV entry and describes novel antiviral targets and compounds in different stages of clinical development. Overall, proof-of-concept studies indicate that entry inhibitors are a promising class of antivirals to prevent and treat HBV and HCV infections.
Since their discovery, tremendous progress has been made in our understanding of the roles of cla... more Since their discovery, tremendous progress has been made in our understanding of the roles of claudins in tight junction physiology. In addition, interactions between claudins and other cellular proteins have highlighted their novel roles in cell physiology. Moreover, the importance of claudins is becoming apparent in the pathophysiology of several diseases, including viral infections. Notable is the discovery of CLDN1 as an essential host factor for hepatitis C virus (HCV) entry, which led to detailed characterization of CLDN1 and its association with tetraspanin CD81 for the initiation of HCV infection. CLDN1 has also been shown to facilitate dengue virus entry. Furthermore, owing to the roles of claudins in forming anatomical barriers, several viruses have been shown to alter claudin expression at the tight junction. This review summarizes the role of claudins in viral infection, with particular emphasis on HCV.
Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a con... more Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a ...
Despite advances in hepatitis C treatment, substantial clinical hurdles remain to achieve univers... more Despite advances in hepatitis C treatment, substantial clinical hurdles remain to achieve universal cure and global control of infection. Saeed et al. identified SEC14L2 as a host factor permitting replication of clinical HCV isolates in cell culture, providing a novel system to model infection of patient-derived viruses.
Hepatitis C virus (HCV) remains a major global health burden, with more than 130 million individu... more Hepatitis C virus (HCV) remains a major global health burden, with more than 130 million individuals chronically infected and at risk for the development of hepatocellular carcinoma (HCC). The recent clinical licensing of direct-acting antivirals enables viral cure. However, limited access to therapy and treatment failure in patient subgroups warrants a continuing effort to develop complementary antiviral strategies. Furthermore, once fibrosis is established, curing HCV infection does not eliminate the risk for HCC. High-throughput approaches and screens have enabled the investigation of virus-host interactions on a genome-wide scale. Gain- and loss-of-function screens have identified essential host-dependency factors in the HCV viral life cycle, such as host cell entry factors or regulatory factors for viral replication and assembly. Network analyses of systems-scale data sets provided a comprehensive view of the cellular state following HCV infection, thus improving our understand...
Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a con... more Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a ...
Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcino... more Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus (HBV) and hepatitis D virus (HDV) use the sodium taurocholate co-transporting polypeptide (NTCP, a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high-throughput infectious cell culture model enabling functional genomics of HDV entry and infection. Using a targeted RNAi entry screen we identified glypican 5 (GPC5) as a common host cell entry factor for HBV and HDV. These findings advance our understanding of virus cell entry and open new avenues for curative therapies. Since glypicans have been shown to play a role in the control of cell division and growth regulation, virus-GPC5 interactions may also play a role in the pathogenesis of virus-induced liver disease and cancer. This article is protected by copyright. All rights reserved.
Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcino... more Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus (HBV) and hepatitis D virus (HDV) use the sodium taurocholate co-transporting polypeptide (NTCP, a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high-throughput infectious cell culture model enabling functional genomics of HDV entry and infection. Using a targeted RNAi entry screen we identified glypican 5 (GPC5) as a common host cell entry factor for HBV and HDV. These findings advance our understanding of virus cell entry and open new avenues for curative therapies. Since glypicans have been shown to play a role in the control of cell division and growth regulation, virus-GPC5 interactions may also play a role in the pathogenesis of virus-induced liver disease and cancer. This article is protected by copyright. All rights reserved.
Hepatitis C virus (HCV) causes persistent infection in the majority of infected individuals. The ... more Hepatitis C virus (HCV) causes persistent infection in the majority of infected individuals. The mechanisms of persistence and clearance are only partially understood. Antibodies against host cell entry receptors have been shown to inhibit HCV infection in cell culture and animal models. In this study we aimed to investigate whether anti-receptor antibodies are induced during infection in humans in vivo and whether their presence is associated with outcome of infection. We established an ELISA using a recombinant CD81- claudin-1 (CLDN1) fusion protein to detect and quantify antibodies directed against extracellular epitopes of the HCV CD81-CLDN1 co-receptor complex. The presence of anti-receptor antibodies was studied in serum of patients from a well-defined cohort of a single-source HCV outbreak of pregnant women and several control groups including uninfected pregnant women, patients with chronic hepatitis B and D virus (HBV/HDV) infection and healthy individuals. The virus-neutralizing activity of antibodies was determined using recombinant cell culture-derived HCV (HCVcc). Our results demonstrate that HCV infected patients have statistically significantly higher anti-CD81/CLDN1 antibody titers during the early phase of infection than controls, the titers being significantly higher in resolvers compared to persisters. Functional studies using immunoadsorption and HCV cell culture models demonstrate that HCV-neutralizing anti-receptor antibodies are induced in early phase of HCV infection but not in control groups. The virus-neutralizing properties of these antibodies suggest a role for control of viral infection in conjunction with anti-viral responses. Characterization of these anti-receptor antibodies opens new avenues to prevent and treat HCV infection. This article is protected by copyright. All rights reserved.
Hepatitis C virus (HCV) is a positive strand RNA virus that primarily infects human hepatocytes. ... more Hepatitis C virus (HCV) is a positive strand RNA virus that primarily infects human hepatocytes. HCV infections constitute a global health problem with 80 million people currently chronically infected. Recent studies reported that cholesterol 25-hydroxylase (CH25H) is expressed as an interferon-stimulated gene (ISG) and mediates antiviral activities against different enveloped viruses through the production of 25-hydroxycholesterol (25HC). However, the intrinsic regulation of human CH25H (hCH25H) expression within the liver as well as its mechanistic effects on HCV infectivity remains elusive. In this study, we characterized the expression of hCH25H using liver biopsies and primary human hepatocytes (PHH). In addition, the antiviral properties of this protein and its enzymatic product, 25HC, were further characterized against HCV in tissue culture. hCH25H mRNA levels were significantly upregulated both in HCV-positive liver biopsies and HCV-infected PHH. The expression of hCH25H in PHH was primarily and transiently induced by type-I interferon (IFN). Transient expression of hCH25H in human hepatoma cells restricted HCV infection in a genotype-independent manner. This inhibition required the enzymatic activity of CH25H. We observed an inhibition of viral membrane fusion during the entry process by 25HC, which was not due to a virucidal effect. Yet the primary effect by 25HC on HCV was at the level of RNA replication which was observed by using subgenomic replicons of two different genotypes. Further analysis using electron microscopy revealed that 25HC inhibited formation of the membranous web (MW), the HCV replication factory, independent of RNA replication. In conclusion, HCV infection causes the upregulation of interferon-inducible CH25H in vivo. Its product 25HC restricts HCV primarily at the level of RNA replication by preventing the formation of viral replication factory. This article is protected by copyright. All rights reserved.
Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current... more Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current antiviral therapy fails to clear infection in a substantial proportion of cases. Drug development is focused on nonstructural proteins required for RNA replication. Individuals undergoing orthotopic liver transplantation face rapid, universal reinfection of the graft. Therefore, antiviral strategies targeting the early stages of infection are urgently needed for the prevention of HCV infection. In this study, we identified the polyphenol, epigallocatechin-3-gallate (EGCG), as an inhibitor of HCV entry. Green tea catechins, such as EGCG and its derivatives, epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), have been previously found to exert antiviral and antioncogenic properties. EGCG had no effect on HCV RNA replication, assembly, or release of progeny virions. However, it potently inhibited Cell-culture-derived HCV (HCVcc) entry into hepatoma cell lines as well as primary human hepatocytes. The effect was independent of the HCV genotype, and both infection of cells by extracellular virions and cell-to-cell spread were blocked. Pretreatment of cells with EGCG before HCV inoculation did not reduce HCV infection, whereas the application of EGCG during inoculation strongly inhibited HCV infectivity. Moreover, treatment with EGCG directly during inoculation strongly inhibited HCV infectivity. Expression levels of all known HCV (co-)receptors were unaltered by EGCG. Finally, we showed that EGCG inhibits viral attachment to the cell, thus disrupting the initial step of HCV cell entry. Conclusion: The green tea molecule, EGCG, potently inhibits HCV entry and could be part of an antiviral strategy aimed at the prevention of HCV reinfection after liver transplantation. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (HA 4393/2-1; to T.V.H.) and (Ci 171/2-1; to S.C.) and from the CIHR and BWF (to L.M.S.). T.
Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of second... more Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of secondary metabolites in bacteria, fungi and plants. Some of these metabolites have played important roles during the early evolution of land plants by providing protection from various environmental assaults including UV irradiation. The genome of the moss, Physcomitrella patens, contains at least 17 putative CHS superfamily genes. Three of these genes (PpCHS2b, PpCHS3 and PpCHS5) exist in multiple copies and all have corresponding ESTs. PpCHS11 and probably also PpCHS9 encode non-CHS enzymes, while PpCHS10 appears to be an ortholog of plant genes encoding anther-specific CHS-like enzymes. It was inferred from the genomic locations of genes comprising it that the moss CHS superfamily expanded through tandem and segmental duplication events. Inferred exon-intron architectures and results from phylogenetic analysis of representative CHS superfamily genes of P. patens and other plants showed tha...
Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with va... more Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide a-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain-and v-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated a-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.
Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of second... more Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of secondary metabolites in bacteria, fungi and plants. Some of these metabolites have played important roles during the early evolution of land plants by providing protection from various environmental assaults including UV irradiation. The genome of the moss, Physcomitrella patens, contains at least 17 putative CHS superfamily genes. Three of these genes (PpCHS2b, PpCHS3 and PpCHS5) exist in multiple copies and all have corresponding ESTs. PpCHS11 and probably also PpCHS9 encode non-CHS enzymes, while PpCHS10 appears to be an ortholog of plant genes encoding anther-specific CHS-like enzymes. It was inferred from the genomic locations of genes comprising it that the moss CHS superfamily expanded through tandem and segmental duplication events. Inferred exon-intron architectures and results from phylogenetic analysis of representative CHS superfamily genes of P. patens and other plants showed that intron gain and loss occurred several times during evolution of this gene superfamily. A high proportion of P. patens CHS genes (7 of 14 genes for which the full sequence is known and probably 3 additional genes) are intronless, prompting speculation that CHS gene duplication via retrotransposition has occurred at least twice in the moss lineage. Analyses of sequence similarities, catalytic motifs and EST data indicated that a surprisingly large number (as many as 13) of the moss CHS superfamily genes probably encode active CHS. EST distribution data and different light responsiveness observed with selected genes provide evidence for their differential regulation. Observed diversity within the moss CHS superfamily and amenability to gene manipulation make Physcomitrella a highly suitable model system for studying expansion and functional diversification of the plant CHS superfamily of genes.
Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, sev... more Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, several Arabidopsis genes, including polyketide synthase A (PKSA), which encodes an anther-specific chalcone synthase-like enzyme (ASCL), have been shown to be involved in sporopollenin biosynthesis. The genome of the moss Physcomitrella patens contains putative orthologs of the Arabidopsis sporopollenin biosynthesis genes.
Background: Physcomitrella PpORS is an ancient member of the plant type III polyketide synthase (... more Background: Physcomitrella PpORS is an ancient member of the plant type III polyketide synthase (PKS) family. Results: PpORS, produced in nonprotonemal moss cells, synthesizes pentaketide 2Ј-oxoalkylresorcinols using a unique substrate binding site. Conclusion: PpORS is a novel very long chain 2Ј-oxoalkylresorcinol synthase. Significance: This is the first step toward understanding the co-evolution of the type III PKS family and land plants.
Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections remain major health problems world... more Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections remain major health problems worldwide, with 400−500 million chronically infected people worldwide. Chronic infection results in liver cirrhosis and hepatocellular carcinoma, the second leading cause of cancer death. Current treatments for HBV limit viral replication without efficiently curing infection. HCV treatment has markedly progressed with the licensing of direct-acting antivirals (DAAs) for HCV cure, yet limited access for the majority of patients is a major challenge. Preventative and curative treatment strategies, aimed at novel targets, are needed for both viruses. Viral entry represents one such target, although detailed knowledge of the entry mechanisms is a prerequisite. For HBV, the recent discovery of the NTCP cell entry factor enabled the establishment of an HBV cell culture model and showed that cyclosporin A and Myrcludex B are NTCP-targeting entry inhibitors. Advances in the understanding of HCV entry revealed it to be a complex process involving many factors, offering several antiviral targets. These include viral envelope proteins E1 and E2, virion-associated lipoprotein ApoE, and cellular factors CD81, SRBI, EGFR, claudin-1, occludin, and the cholesterol transporter NPC1L1. Small molecules targeting SR-BI, EGFR, and NPC1L1 have entered clinical trials, whereas other viral-and host-targeted small molecules, peptides, and antibodies show promise in preclinical models. This review summarizes the current understanding of HBV and HCV entry and describes novel antiviral targets and compounds in different stages of clinical development. Overall, proof-of-concept studies indicate that entry inhibitors are a promising class of antivirals to prevent and treat HBV and HCV infections.
Since their discovery, tremendous progress has been made in our understanding of the roles of cla... more Since their discovery, tremendous progress has been made in our understanding of the roles of claudins in tight junction physiology. In addition, interactions between claudins and other cellular proteins have highlighted their novel roles in cell physiology. Moreover, the importance of claudins is becoming apparent in the pathophysiology of several diseases, including viral infections. Notable is the discovery of CLDN1 as an essential host factor for hepatitis C virus (HCV) entry, which led to detailed characterization of CLDN1 and its association with tetraspanin CD81 for the initiation of HCV infection. CLDN1 has also been shown to facilitate dengue virus entry. Furthermore, owing to the roles of claudins in forming anatomical barriers, several viruses have been shown to alter claudin expression at the tight junction. This review summarizes the role of claudins in viral infection, with particular emphasis on HCV.
Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a con... more Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a ...
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