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1
Marnata, Caroline, Aure Saulnier, Dimitri Mompelat, Thomas Krey, Lisette Cohen, Célia Boukadida, Lucile Warter, et al. "Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction." Journal of Virology 89, no. 23 (September 23, 2015): 12131–44. http://dx.doi.org/10.1128/jvi.01161-15.
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ABSTRACTHepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts.IMPORTANCEHepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts.
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Sims, Stuart, Kevin Michaelsen, Sara Burkhard, and Cornel Fraefel. "In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and Pegivirus and Construction of Pseudoparticles." Advances in Virology 2021 (July 30, 2021): 1–9. http://dx.doi.org/10.1155/2021/5569844.
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The 5′ untranslated region (5′ UTR) of rodent hepacivirus (RHV) and pegivirus (RPgV) contains sequence homology to the HCV type III internal ribosome entry sites (IRES). Utilizing a monocistronic expression vector with an RNA polymerase I promoter to drive transcription, we show cell-specific IRES translation and regions within the IRES required for full functionality. Focusing on RHV, we further pseudotyped lentivirus with RHV and showed cell surface expression of the envelope proteins and transduction of murine hepatocytes and we then constructed full-length RHV and RPgV replicons with reporter genes. Using the replicon system, we show that the RHV NS3-4A protease cleaves a mitochondrial antiviral signaling protein reporter. However, liver-derived cells did not readily support the complete viral life cycle.
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Walter, Stephanie, Alexander Bollenbach, Juliane Doerrbecker, Stephanie Pfaender, Richard J. P. Brown, Gabrielle Vieyres, Claire Scott, et al. "Ion Channel Function and Cross-Species Determinants in Viral Assembly of Nonprimate Hepacivirus p7." Journal of Virology 90, no. 10 (March 9, 2016): 5075–89. http://dx.doi.org/10.1128/jvi.00132-16.
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ABSTRACTNonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though the two viruses share a similar genomic organization, conservation of the encoded hepaciviral proteins remains undetermined. The HCV p7 protein is localized within endoplasmic reticulum (ER) membranes and is important for the production of infectious particles. In this study, we analyzed the structural and functional features of NPHV p7 in addition to its role during virus assembly. Three-dimensional homology models for NPHV p7 using various nuclear magnetic resonance spectroscopy (NMR) structures were generated, highlighting the conserved residues important for ion channel function. By applying a liposome permeability assay, we observed that NPHV p7 exhibited liposome permeability features similar to those of HCV p7, indicative of similar ion channel activity. Next, we characterized the viral protein using a p7-basedtrans-complementation approach. A similar subcellular localization pattern at the ER membrane was observed, although production of infectious particles was likely hindered by genetic incompatibilities with HCV proteins. To further characterize these cross-species constraints, chimeric viruses were constructed by substituting different regions of HCV p7 with NPHV p7. The N terminus and transmembrane domains were nonexchangeable and therefore constitute a cross-species barrier in hepaciviral assembly. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectioustrans-complemented viral particles. In conclusion, comparison of NPHV and HCV p7 revealed structural and functional homology of these proteins, including liposome permeability, and broadly acting determinants that modulate hepaciviral virion assembly and contribute to the host-species barrier were identified.IMPORTANCEThe recent discovery of new relatives of hepatitis C virus (HCV) enables for the first time the study of cross-species determinants shaping hepaciviral pathogenesis. Nonprimate hepacivirus (NPHV) was described to infect horses and represents so far the closest homolog of HCV. Both viruses encode the same viral proteins; however, NPHV protein functions remain poorly understood. In this study, we aimed to dissect NPHV p7 on a structural and functional level. By using various NMR structures of HCV p7 as templates, three-dimensional homology models for NPHV p7 were generated, highlighting conserved residues that are important for ion channel function. A p7-basedtrans-complementation approach and the construction of NPHV/HCV p7 chimeric viruses showed that the N terminus and transmembrane domains were nonexchangeable. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious viral particles. These results identify species-specific constraints as well as exchangeable determinants in hepaciviral assembly.
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Reed, Karen E., Alexander E. Gorbalenya, and Charles M. Rice. "The NS5A/NS5 Proteins of Viruses from Three Genera of the Family Flaviviridae Are Phosphorylated by Associated Serine/Threonine Kinases." Journal of Virology 72, no. 7 (July 1, 1998): 6199–206. http://dx.doi.org/10.1128/jvi.72.7.6199-6206.1998.
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ABSTRACT Phosphorylation of the expressed NS5A protein of hepatitis C virus (HCV), a member of the Hepacivirus genus of the familyFlaviviridae, has been demonstrated in mammalian cells and in a cell-free assay by an associated kinase activity. In this report, phosphorylation is also shown for the NS5A and NS5 proteins, respectively, of bovine viral diarrhea virus (BVDV) and yellow fever virus (YF), members of the other two established genera in this family. Phosphorylation of BVDV NS5A and YF NS5 was observed in infected cells, transient expression experiments, and a cell-free assay similar to the one developed for HCV NS5A. Phosphoamino acid analyses indicated that all three proteins were phosphorylated by serine/threonine kinases. Similarities in the properties of BVDV NS5A, YF NS5, and HCV NS5A phosphorylation in vitro further suggested that closely related kinases or the same kinase may phosphorylate these viral proteins. Conservation of this trait among three quite distantly related viruses representing three separate genera suggests that phosphorylation of the NS5A/NS5 proteins or their association with cellular kinases may play an important role in the flavivirus life cycle.
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Kim, Hangeun, Budhaditya Mazumdar, Sandip K. Bose, Keith Meyer, Adrian M. Di Bisceglie, Daniel F. Hoft, and Ranjit Ray. "Hepatitis C Virus-Mediated Inhibition of Cathepsin S Increases Invariant-Chain Expression on Hepatocyte Surface." Journal of Virology 86, no. 18 (July 3, 2012): 9919–28. http://dx.doi.org/10.1128/jvi.00388-12.
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Hepatocytes are the main source of hepatitis C virus (HCV) replication and contain the maximum viral load in an infected person. Chronic HCV infection is characterized by weak cellular immune responses to viral proteins. Cathepsin S is a lysosomal cysteine protease and controls HLA-DR–antigen complex presentation through the degradation of the invariant chain. In this study, we examined the effect of HCV proteins on cathepsin S expression and found it to be markedly decreased in dendritic cells (DCs) exposed to HCV or in hepatocytes expressing HCV proteins. The downregulation of cathepsin S was mediated by HCV core and NS5A proteins involving inhibition of the transcription factors interferon regulatory factor 1 (IRF-1) and upstream stimulatory factor 1 (USF-1) in gamma interferon (IFN-γ)-treated hepatocytes. Inhibition of cathepsin S by HCV proteins increased cell surface expression of the invariant chain. In addition, hepatocytes stably transfected with HCV core or NS5A inhibited HLA-DR expression. Together, these results suggested that HCV has an inhibitory role on cathepsin S-mediated major histocompatibility complex (MHC) class II maturation, which may contribute to weak immunogenicity of viral antigens in chronically infected humans.
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Navas, Maria-Cristina, Françoise Stoll-Keller, and Jovan Pavlovic. "Lack of expression of hepatitis C virus core protein in human monocyte-erived dendritic cells using recombinant semliki forest virus." Acta Biológica Colombiana 24, no. 3 (September 1, 2019): 493–502. http://dx.doi.org/10.15446/abc.v24n3.79368.
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Hepatitis C Virus belongs to the Flaviviridae family. One proposed mechanism of HCV persistence in the ability to infect hematopoietic cells, including Dendritic cells (DCs). HCV infection of DCs could impair their functions that represent one of the mechanisms, thus hampering viral clearance by the host immune system. Among HCV-encoded proteins, the highly conserved Core protein has been suggested to be responsible for the immunomodulatory properties of this Hepacivirus. Recombinant viral vectors expressing the HCV Core protein and allowing its transduction and therefore the expression of the protein into DCs could be useful tools for the analysis of the properties of the Core protein. Vaccinia Virus and retrovirus have been used to transduce human DCs. Likewise, gene transfer into DCs using Semliki Forest Virus has been reported. This study aimed to express the HCV Core protein in human monocyte-derived DCs using an SFV vector, in which the subgenomic RNA encoding the structural proteins was replaced by the HCV Core sequence and then analyze the effects of its expression on DCs functions.
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Anggakusuma, Richard J. P. Brown, Dominic H. Banda, Daniel Todt, Gabrielle Vieyres, Eike Steinmann, and Thomas Pietschmann. "Hepacivirus NS3/4A Proteases Interfere with MAVS Signaling in both Their Cognate Animal Hosts and Humans: Implications for Zoonotic Transmission." Journal of Virology 90, no. 23 (September 21, 2016): 10670–81. http://dx.doi.org/10.1128/jvi.01634-16.
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ABSTRACTMultiple novel members of the genusHepacivirushave recently been discovered in diverse mammalian species. However, to date, their replication mechanisms and zoonotic potential have not been explored in detail. The NS3/4A serine protease of hepatitis C virus (HCV) is critical for cleavage of the viral polyprotein. It also cleaves the cellular innate immune adaptor MAVS, thus decreasing interferon (IFN) production and contributing to HCV persistence in the human host. To investigate the conservation of fundamental aspects of the hepaciviral life cycle, we explored if MAVS cleavage and suppression of innate immune signaling represent a common mechanism employed across different clades of the genusHepacivirusto enhance viral replication. To estimate the zoonotic potential of these nonhuman hepaciviruses, we assessed if their NS3/4A proteases were capable of cleaving human MAVS. NS3/4A proteases of viruses infecting colobus monkeys, rodents, horses, and cows cleaved the MAVS proteins of their cognate hosts and interfered with the ability of MAVS to induce the IFN-β promoter. All NS3/4A proteases from nonhuman viruses readily cleaved human MAVS. Thus, NS3/4A-dependent cleavage of MAVS is a conserved replication strategy across multiple clades within the genusHepacivirus. Human MAVS is susceptible to cleavage by these nonhuman viral proteases, indicating that it does not pose a barrier for zoonotic transmission of these viruses to humans.IMPORTANCEVirus infection is recognized by cellular sensor proteins triggering innate immune signaling and antiviral defenses. While viruses have evolved strategies to thwart these antiviral programs in their cognate host species, these evasion mechanisms are often ineffective in a novel host, thus limiting viral transmission across species. HCV, the best-characterized member of the genusHepaciviruswithin the familyFlaviviridae, uses its NS3/4A protease to disrupt innate immune signaling by cleaving the cellular adaptor protein MAVS. Recently, a large number of HCV-related viruses have been discovered in various animal species, including wild, livestock, and companion animals. We show that the NS3/4A proteases of these hepaciviruses from different animals and representing various clades of the genus cleave their cognate host MAVS proteins in addition to human MAVS. Therefore, cleavage of MAVS is a common strategy of hepaciviruses, and human MAVS is likely unable to limit replication of these nonhuman viruses upon zoonotic exposure.
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Hartlage, Alex S., Piyush Dravid, Christopher M. Walker, and Amit Kapoor. "Adenovirus-vectored T cell vaccine for hepacivirus shows reduced effectiveness against a CD8 T cell escape variant in rats." PLOS Pathogens 17, no. 3 (March 18, 2021): e1009391. http://dx.doi.org/10.1371/journal.ppat.1009391.
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There is an urgent need for a vaccine to prevent chronic infection by hepatitis C virus (HCV) and its many genetic variants. The first human vaccine trial, using recombinant viral vectors that stimulate pan-genotypic T cell responses against HCV non-structural proteins, failed to demonstrate efficacy despite significant preclinical promise. Understanding the factors that govern HCV T cell vaccine success is necessary for design of improved immunization strategies. Using a rat model of chronic rodent hepacivirus (RHV) infection, we assessed the impact of antigenic variation and immune escape upon success of a conceptually analogous RHV T cell vaccine. Naïve Lewis rats were vaccinated with a recombinant human adenovirus expressing RHV non-structural proteins (NS)3-5B and later challenged with a viral variant containing immune escape mutations within major histocompatibility complex (MHC) class I-restricted epitopes (escape virus). Whereas 7 of 11 (64%) rats cleared infection caused by wild-type RHV, only 3 of 12 (25%) were protected against heterologous challenge with escape virus. Uncontrolled replication of escape virus was associated with durable CD8 T cell responses targeting escaped epitopes alone. In contrast, clearance of escape virus correlated with CD4 T cell helper immunity and maintenance of CD8 T cell responses against intact viral epitopes. Interestingly, clearance of wild-type RHV infection after vaccination conferred enhanced protection against secondary challenge with escape virus. These results demonstrate that the efficacy of an RHV T cell vaccine is reduced when challenge virus contains escape mutations within MHC class I-restricted epitopes and that failure to sustain CD8 T cell responses against intact epitopes likely underlies immune failure in this setting. Further investigation of the immune responses that yield protection against diverse RHV challenges in this model may facilitate design of broadly effective HCV vaccines.
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Lazarevic, Ivana, Ana Banko, Danijela Miljanovic, and Maja Cupic. "Biological features of hepatitis B virus strains associated with fulminant hepatitis." Future Virology 15, no. 7 (July 2020): 455–69. http://dx.doi.org/10.2217/fvl-2020-0011.
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Accumulating evidence suggests that hepatitis B virus (HBV) biological features may influence the course and clinical manifestations of infection and possibly the development of fulminant hepatitis (FH). Since HBV is not a cytocidal virus, virus-induced liver damage results from an interplay between the virus replication and the host's defense. Therefore, viral factors contributing to enhanced replication, induction of a stronger immune attack or apoptosis of hepatocytes could be crucial in development of FH. Numerous mutations in basal core promoter, pre-C, C and S regions of the HBV genome contribute to development of FH by different mechanisms, including enhanced viral replication, the loss of a decoy for immune response, unbalanced expression of viral proteins and retention of unprocessed cytotoxic proteins in hepatocytes.
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Naas, Turaya, Masoud Ghorbani, Ikuri Alvarez-Maya, Michael Lapner, Rashmi Kothary, Yves De Repentigny, Susantha Gomes, et al. "Characterization of liver histopathology in a transgenic mouse model expressing genotype 1a hepatitis C virus core and envelope proteins 1 and 2." Journal of General Virology 86, no. 8 (August 1, 2005): 2185–96. http://dx.doi.org/10.1099/vir.0.80969-0.
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Hepatitis C virus (HCV) is a major cause of chronic hepatitis and hepatocellular carcinoma worldwide. The purpose of this study was to determine how the HCV structural proteins affect the dynamic structural and functional properties of hepatocytes and measure the extra-hepatic manifestations induced by these viral proteins. A transgenic mouse model was established by expressing core, E1 and E2 proteins downstream of a CMV promoter. HCV RNA was detected using RT-PCR in transgenic mouse model tissues, such as liver, kidney, spleen and heart. Expression of the transgene was analysed by real-time PCR to quantify viral RNA in different tissues at different ages. Immunofluorescence analysis revealed the expression of core, E1 and E2 proteins predominantly in hepatocytes. Lower levels of protein expression were detected in spleen and kidneys. HCV RNA and viral protein expression increased in the liver with age. Histological analysis of liver cells demonstrated steatosis in transgenic mice older than 3 months, which was more progressed with age. Electron microscopy analysis revealed alterations in nuclei, mitochondria and endoplasmic reticulum. HCV structural proteins induce a severe hepatopathy in the transgenic mouse model. These mice became more prone to liver and lymphoid tumour development and hepatocellular carcinoma. In this model, the extra-hepatic effects of HCV, which included swelling of renal tubular cells, were mild. It is likely that the HCV structural proteins mediate some of the histological alterations in hepatocytes by interfering with lipid transport and liver metabolism.
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Johnson, Craig M., Daniel R. Perez, Roy French, William C. Merrick, and Ruben O. Donis. "The NS5A protein of bovine viral diarrhoea virus interacts with the α subunit of translation elongation factor-1." Journal of General Virology 82, no. 12 (December 1, 2001): 2935–43. http://dx.doi.org/10.1099/0022-1317-82-12-2935.
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A cellular protein that interacts with the NS5A polypeptide of bovine viral diarrhoea virus (BVDV) was identified in a yeast two-hybrid screen. The NS5A interactor was identified as the α subunit of bovine translation elongation factor 1A (eEF1A). Cell-free binding studies were performed with chimeric NS5A fused to glutathione S-transferase (GST–NS5A) expressed in bacteria. GST–NS5A bound specifically to both in vitro-translated and mammalian cell-expressed eEF1A. Moreover, purified eEF1A bound specifically to GST–NS5A attached to a solid phase. Conservation of this interaction was then analysed using a set of NS5A proteins derived from divergent BVDV strains encompassing known biotypes and genotypes. NS5A from all BVDV strains tested so far interacted with eEF1A. The conserved association of eEF1A with virus molecules involved in genome replication and the postulated role of pestivirus and hepacivirus NS5A in replication indicate that this interaction may play a role in the replication of BVDV.
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Alzahrani, Nabeel, Ming-Jhan Wu, Saravanabalaji Shanmugam, and MinKyung Yi. "Delayed by Design: Role of Suboptimal Signal Peptidase Processing of Viral Structural Protein Precursors in Flaviviridae Virus Assembly." Viruses 12, no. 10 (September 26, 2020): 1090. http://dx.doi.org/10.3390/v12101090.
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The Flaviviridae virus family is classified into four different genera, including flavivirus, hepacivirus, pegivirus, and pestivirus, which cause significant morbidity and mortality in humans and other mammals, including ruminants and pigs. These are enveloped, single-stranded RNA viruses sharing a similar genome organization and replication scheme with certain unique features that differentiate them. All viruses in this family express a single polyprotein that encodes structural and nonstructural proteins at the N- and C-terminal regions, respectively. In general, the host signal peptidase cleaves the structural protein junction sites, while virus-encoded proteases process the nonstructural polyprotein region. It is known that signal peptidase processing is a rapid, co-translational event. Interestingly, certain signal peptidase processing site(s) in different Flaviviridae viral structural protein precursors display suboptimal cleavage kinetics. This review focuses on the recent progress regarding the Flaviviridae virus genus-specific mechanisms to downregulate signal peptidase-mediated processing at particular viral polyprotein junction sites and the role of delayed processing at these sites in infectious virus particle assembly.
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Hsin, Fu, Yu-Chen Hsu, Yu-Fei Tsai, Shu-Wha Lin, and Helene Minyi Liu. "The transmembrane serine protease hepsin suppresses type I interferon induction by cleaving STING." Science Signaling 14, no. 687 (June 15, 2021): eabb4752. http://dx.doi.org/10.1126/scisignal.abb4752.
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Many viral proteases mediate the evasion of antiviral innate immunity by cleaving adapter proteins in the interferon (IFN) induction pathway. Host proteases are also involved in innate immunity and inflammation. Here, we report that the transmembrane protease hepsin (also known as TMPRSS1), which is predominantly present in hepatocytes, inhibited the induction of type I IFN during viral infections. Knocking out hepsin in mouse embryonic fibroblasts (MEFs) increased the viral infection–induced expression of Ifnb1, an Ifnb1 promoter reporter, and an IFN-sensitive response element promoter reporter. Ectopic expression of hepsin in cultured human hepatocytes and HEK293T cells suppressed the induction of IFNβ during viral infections by reducing the abundance of STING. These effects depended on the protease activity of hepsin. We identified a putative hepsin target site in STING and showed that mutating this site protected STING from hepsin-mediated cleavage. In addition to hepatocytes, several hepsin-producing prostate cancer cell lines showed reduced STING-mediated type I IFN induction and responses. These results reveal a role for hepsin in suppressing STING-mediated type I IFN induction, which may contribute to the vulnerability of hepatocytes to chronic viral infections.
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Oda, K., T. Fujiwara, and Y. Ikehara. "Brefeldin A arrests the intracellular transport of viral envelope proteins in primary cultured rat hepatocytes and HepG2 cells." Biochemical Journal 265, no. 1 (January 1, 1990): 161–67. http://dx.doi.org/10.1042/bj2650161.
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We have studied the effect of brefeldin A (BFA) on the intracellular transport of the envelope proteins of vesicular stomatitis virus (VSV) and sindbis virus in primary cultured rat hepatocytes. BFA (2.5 micrograms/ml) inhibited not only the secretion of plasma proteins into the medium, but also the assembly of both G protein of VSV and E1 and E2 proteins (envelope proteins) of sindbis virus into respective virions. Concomitantly, both the acquisition of endo-beta-N-acetylglucosaminidase H resistance by the G protein and the proteolytic conversion of PE2 to E2 were found to be inhibited in the BFA-treated cells, suggesting that the intracellular transport of the envelope proteins was arrested in the endoplasmic reticulum. Such inhibitory effects of the drug were variable depending upon the culture conditions of the hepatocytes. In the 1-day-cultured cells, even in the presence of the drug, newly synthesized envelope proteins were assembled into the virions after a 3 h chase period, at the same time as secretion of plasma proteins into the medium resumes. In contrast, in 4-day-cultured hepatocytes, BFA continuously blocked the entry of the envelope proteins into the virions and the release of plasma proteins into the medium for at least 5 h. BFA also completely inhibited the exocytotic pathway in HepG2 cells. These results indicate that the duration time of the effect of BFA is different from one cell to another and may change depending upon the culture conditions of the cells.
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McLauchlan, John. "Hepatitis C virus: viral proteins on the move." Biochemical Society Transactions 37, no. 5 (September 21, 2009): 986–90. http://dx.doi.org/10.1042/bst0370986.
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There is now increasing evidence that LDs (lipid droplets) play a central role in the production of infectious HCV (hepatitis C virus) and participate in virus assembly. Two viral proteins, namely core, which forms the capsid, and NS5A (non-structural 5A protein), a component of complexes engaged in viral RNA synthesis, are detected at LD surfaces in infected cells. Interactions between the two proteins may be critical for anchoring RNA replication sites to droplets for initiating virus assembly. The requirements for targeting of core in particular has received considerable attention since the nature of its interaction with LDs could play a key role in determining the efficiency of virion production. As well as attaching to droplets, core is able to alter their intracellular distribution and direct them towards the microtubule organizing centre. Inhibitors that disrupt microtubules block this redistribution by core and there is a concomitant decrease in virus production. Therefore altered dynamics of LDs may contribute to HCV assembly and release. The purpose of targeting LDs by HCV may be linked to their contribution to the formation of VLDLs (very-low-density lipoproteins) in hepatocytes since virus circulating in infected patients is associated with lipoprotein. Thus HCV may utilize the role played by LDs in the formation of lipoprotein particles as part of its life cycle and access this pathway by direct interaction of viral components with these intracellular storage organelles.
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Wang, Jinguo, and Tomasz I. Michalak. "Inhibition by Woodchuck Hepatitis Virus of Class I Major Histocompatibility Complex Presentation on Hepatocytes Is Mediated by Virus Envelope Pre-S2 Protein and Can Be Reversed by Treatment with Gamma Interferon." Journal of Virology 80, no. 17 (September 1, 2006): 8541–53. http://dx.doi.org/10.1128/jvi.00830-06.
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ABSTRACT Presentation of class I major histocompatibility complex (MHC) is severely down-regulated on hepatocytes in chronic hepatitis caused by woodchuck hepatitis virus (WHV). To determine which of the viral proteins mediates class I MHC antigen suppression, cultured normal woodchuck hepatocytes were transfected with the complete WHV genome, sequences encoding individual virus proteins, or whole virus genomes in which transcription of selected proteins was disabled by site-specific mutagenesis. It was found that hepatocyte presentation of class I MHC antigen was significantly inhibited following transfection with complete WHV genome or with viral subgenomic fragments encoding envelope pre-S2 protein or pre-S1 protein, which naturally encompasses pre-S2 amino acid sequence. In contrast, hepatocytes transfected with WHV X gene alone demonstrated a profound enhancement in the class I antigen display, whereas those expressing virus major S protein or nucleocapsid (core) protein were not different from control hepatocytes. Analysis of the mutated WHV sequences confirmed that the envelope pre-S2 protein was responsible for inhibition of the class I MHC antigen display. Interestingly, treatment with recombinant woodchuck gamma interferon (rwIFN-γ) restored the inhibited presentation of the class I antigen. Moreover, the class I antigen suppression was not associated with down-regulation of hepatocyte genes for class I MHC heavy chain, β2-microglobulin, transporters associated with antigen processing, and proteasome subunits. These findings indicate that the defective presentation of class I MHC antigen on hepatocytes transcribing WHV is a consequence of posttranscriptional suppression exerted by virus pre-S2 protein and that this hindrance can be fully reversed by IFN-γ.
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König, Matthias, Heinz-Jürgen Thiel, and Gregor Meyers. "Detection of Viral Proteins after Infection of Cultured Hepatocytes with Rabbit Hemorrhagic Disease Virus." Journal of Virology 72, no. 5 (May 1, 1998): 4492–97. http://dx.doi.org/10.1128/jvi.72.5.4492-4497.1998.
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ABSTRACT The calicivirus rabbit hemorrhagic disease virus (RHDV), which replicates predominantly in the livers of infected rabbits, cannot be propagated in tissue culture. To enable the performance of in vitro studies, rabbit hepatocytes were isolated by liver perfusion and gradient centrifugation. After inoculation with purified RHDV, more than 50% of the cells proved to be infected. Protein analyses led to the detection of 13 RHDV-specific polypeptides within the infected cells. These proteins were assigned to defined regions of the viral genome, resulting in a refined model of RHDV genome organization.
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Inoue, Jun, Kosuke Sato, Masashi Ninomiya, and Atsushi Masamune. "Envelope Proteins of Hepatitis B Virus: Molecular Biology and Involvement in Carcinogenesis." Viruses 13, no. 6 (June 11, 2021): 1124. http://dx.doi.org/10.3390/v13061124.
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The envelope of hepatitis B virus (HBV), which is required for the entry to hepatocytes, consists of a lipid bilayer derived from hepatocyte and HBV envelope proteins, large/middle/small hepatitis B surface antigen (L/M/SHBs). The mechanisms and host factors for the envelope formation in the hepatocytes are being revealed. HBV-infected hepatocytes release a large amount of subviral particles (SVPs) containing L/M/SHBs that facilitate escape from the immune system. Recently, novel drugs inhibiting the functions of the viral envelope and those inhibiting the release of SVPs have been reported. LHBs that accumulate in ER is considered to promote carcinogenesis and, especially, deletion mutants in the preS1/S2 domain have been reported to be associated with the development of hepatocellular carcinoma (HCC). In this review, we summarize recent reports on the findings regarding the biological characteristics of HBV envelope proteins, their involvement in HCC development and new agents targeting the envelope.
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Zhang, Dake, Shicheng Guo, and Steven J. Schrodi. "Mechanisms of DNA Methylation in Virus-Host Interaction in Hepatitis B Infection: Pathogenesis and Oncogenetic Properties." International Journal of Molecular Sciences 22, no. 18 (September 12, 2021): 9858. http://dx.doi.org/10.3390/ijms22189858.
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Hepatitis B virus (HBV), the well-studied oncovirus that contributes to the majority of hepatocellular carcinomas (HCC) worldwide, can cause a severe inflammatory microenvironment leading to genetic and epigenetic changes in hepatocyte clones. HBV replication contributes to the regulation of DNA methyltransferase gene expression, particularly by X protein (HBx), and subsequent methylation changes may lead to abnormal transcription activation of adjacent genes and genomic instability. Undoubtedly, the altered expression of these genes has been known to cause diverse aspects of infected hepatocytes, including apoptosis, proliferation, reactive oxygen species (ROS) accumulation, and immune responses. Additionally, pollutant-induced DNA methylation changes and aberrant methylation of imprinted genes in hepatocytes also complicate the process of tumorigenesis. Meanwhile, hepatocytes also contribute to epigenetic modification of the viral genome to affect HBV replication or viral protein production. Meanwhile, methylation levels of HBV integrants and surrounding host regions also play crucial roles in their ability to produce viral proteins in affected hepatocytes. Both host and viral changes can provide novel insights into tumorigenesis, individualized responses to therapeutic intervention, disease progress, and early diagnosis. As such, DNA methylation-mediated epigenetic silencing of cancer-related genes and viral replication is a compelling therapeutic goal to reduce morbidity and mortality from liver cancer caused by chronic HBV infection. In this review, we summarize the most recent research on aberrant DNA methylation associated with HBV infection, which is involved in HCC development, and provide an outlook on the future direction of the research.
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Balasubramanian, Anuradha, Ramesh K. Ganju, and Jerome E. Groopman. "HCV and HIV Envelope Proteins Co-Operatively Induce Fas-Mediated Apoptosis Via a Novel Stat1 Signaling Pathway." Blood 104, no. 11 (November 16, 2004): 604. http://dx.doi.org/10.1182/blood.v104.11.604.604.
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Abstract Hepatitis C virus (HCV) co-infects approximately 40% of patients with human immunodeficiency virus (HIV). HCV/HIV co-infected patients often have progressive liver disease that can lead to cirrhosis and death. We observed that hepatocytes exposed to HCV and HIV envelope proteins undergo apoptosis via an ‘innocent bystander’ mechanism due to cell surface binding of viral proteins independent of direct viral infection. HCV envelope protein E2 (1.5 nM) and HIV envelope protein gp120 (0.8 nM) derived from M-tropic and T-tropic viruses induce significant apoptosis in both hepatocytic cell lines and primary hepatocytes, while either of these viral proteins alone does not. Now, we have elucidated the signaling mechanisms that mediate this effect. HCV-E2 and HIV-gp120 were found to significantly upregulate Fas ligand (FasL). We then examined the Stat family of proteins known to participate in FasL and apoptotic pathways. We observed an increased DNA binding activity of Stat1 upon HCV-E2 and HIV-gp120 stimulation. Furthermore, overexpression of wild type Stat1αincreased apoptosis and FasL expression in HepG2 cells, whereas a C-terminal domain deleted mutant, Stat1β, decreased HCV-E2 and HIV-gp120 mediated apoptosis and FasL upregulation. Overexpression of Stat1αand Stat1β in primary hepatocytes confirmed that Stat1αenhanced apoptosis upon HCV-E2 and HIV-gp120 treatment. We observed a tyrosine dependent activation of Stat1 and a subsequent serine phosphorylation of Stat1. TYK2, lyn kinase, RAFTK and MAP kinases were activated upstream of Stat1. In addition, Stat1 associated with the death domain-containing adapter protein TRADD. TRADD is known to induce inflammatory signaling through the NFκB pathway. Here, the association of Stat1 with TRADD would reduce the availability of TRADD to induce NFκB. Thus, Stat1 sequestration of downstream apoptotic signaling molecules would block the host inflammatory response. Further characterization of Fas-mediated apoptosis revealed that caspase 3 and caspase 7 were activated following HCV-E2 and HIV-gp120 stimulation. However, we were not able to detect significant activity of either caspase 8 or caspase 9. We also found a loss in mitochondrial membrane potential upon HCV-E2 and HIV-gp120 stimulation, which leads to the release of cytochrome C and AIF into the cytosol. Taken together, these studies indicate that the viral proteins of HCV and HIV co-operate in causing the apoptosis of hepatocytes, independent of direct infection, by induction of novel Stat1 downstream signaling pathways at the expense of a normal host inflammatory response.
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Tellinghuisen, Timothy L., Matthew S. Paulson, and Charles M. Rice. "The NS5A Protein of Bovine Viral Diarrhea Virus Contains an Essential Zinc-Binding Site Similar to That of the Hepatitis C Virus NS5A Protein." Journal of Virology 80, no. 15 (August 1, 2006): 7450–58. http://dx.doi.org/10.1128/jvi.00358-06.
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ABSTRACT The recent demonstration that the NS5A protein of hepatitis C virus (HCV) contains an unconventional zinc-binding site with the format Cx17CxCx20C and the presence of a similar sequence element in the NS5A proteins of members of the Pestivirus genus has led to the hypothesis that the NS5A protein of the pestivirus bovine viral diarrhea virus (BVDV) is a zinc-binding protein. A method for the expression and partial purification of BVDV NS5A was developed, and the partially purified protein was analyzed for zinc content by atomic absorption spectroscopy. BVDV NS5A was found to coordinate a single zinc atom per protein molecule. Mutation of any of the four cysteines of the predicted zinc-binding motif eliminated zinc coordination. Furthermore, analysis of mutations at these cysteine residues in the context of a BVDV replicon system indicated that these residues were absolutely essential for RNA replication. The recently determined crystal structure of the N-terminal zinc-binding domain of the HCV NS5A protein, combined with secondary structure predictions of the region surrounding the mapped BVDV zinc-binding region, indicates that the BVDV zinc-binding motif fits the general template Cx22CxCx24C and likely comprises a three-stranded antiparallel β-sheet fold. These data highlight the similarities between the Hepacivirus and Pestivirus NS5A proteins and suggest that both proteins perform a not-yet-defined function in RNA replication that requires coordination of a single zinc atom.
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Hartlage, Alex S., and Amit Kapoor. "303. A Surrogate Rodent Model for Studying Hepatitis C Virus-specific CD8 T-cell Impairment and Vaccine Prevention." Open Forum Infectious Diseases 6, Supplement_2 (October 2019): S163. http://dx.doi.org/10.1093/ofid/ofz360.376.
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Abstract Background Virus-specific CD8 T cells are essential for control of acute hepatitis C virus (HCV) infections, yet spontaneously fail in most patients leading to lifelong chronicity and increased risk for severe liver diseases. Efforts to study HCV-specific CD8 T-cell impairment have been hampered by a lack of small animal models. Recently, we established a rat model of chronic HCV-like infection using a hepacivirus homolog identified in Rattus norvegicus. The nature of virus-specific CD8 T-cell immunity in this model has yet to be determined. Methods Using two MHC class I tetramers against epitopes located in the E1 and NS5B proteins, we tracked the induction and phenotype of virus-specific CD8 T cells during chronic infection. Responses to infection were similarly analyzed in immune rats that had been vaccinated against the NS3-5B proteins, a strategy that is effective in this experimental setting. Results Virus-specific CD8 T cells expanded vigorously in liver shortly after infection but did not develop into functional effectors based upon failure to produce cytokines (IFNγ, TNFα, IL-2, IL-4, IL-10, IL-17A) following peptide stimulation. Notably, subversion of responses was not due to viral escape from T-cell recognition, but rather an intrinsic defect in the antiviral response. Indeed, these populations expressed the inhibitory receptor programed cell death-1 and other markers consistent with an arrested effector-like state precluded from long-term memory formation (CD127-CD27+CD28+CD62L-GranzymeB+). In contrast, adenoviral immunization of naïve rats protected virus-specific T cells from functional impairment after infection and supported memory response development, including against the E1 epitope not encoded by vaccine. Conclusion Together, our findings reveal a spontaneous failure of virus-specific CD8 T cells following rat hepacivirus challenge that is highly reminiscent of human HCV infections. Furthermore, these results highlight the utility and significance of this model for understanding mechanisms of HCV persistence and protective immunity necessary for the development of effective vaccines and immune interventions. Disclosures All authors: No reported disclosures.
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Chandra, Sunandini, Raju Kalaivani, Manoj Kumar, Narayanaswamy Srinivasan, and Debi P. Sarkar. "Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes." Molecular Biology of the Cell 28, no. 26 (December 15, 2017): 3801–14. http://dx.doi.org/10.1091/mbc.e17-06-0400.
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Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion–mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206—an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.
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Meuleman, Philip, Louis Libbrecht, Stefan Wieland, Rita De Vos, Nagy Habib, Anna Kramvis, Tania Roskams, and Geert Leroux-Roels. "Immune Suppression Uncovers Endogenous Cytopathic Effects of the Hepatitis B Virus." Journal of Virology 80, no. 6 (March 15, 2006): 2797–807. http://dx.doi.org/10.1128/jvi.80.6.2797-2807.2006.
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ABSTRACT It is generally accepted that the host's immune response rather than the virus itself is causing the hepatocellular damage seen in acute and chronic hepatitis B virus (HBV) infections. However, in situations of severe immune suppression, chronic HBV patients may develop a considerable degree of liver disease. To examine whether HBV has direct cytopathic effects in severely immune compromised hosts, we have infected severe combined immune deficient mice (uPA-SCID), harboring human liver cells, with HBV. Serologic analysis of the plasma of HBV-infected animals revealed the presence of extremely high amounts of viral genomes and proteins. Histological analysis of the livers of uPA-SCID chimeras infected with HBV for more than 2 months showed that the majority of human hepatocytes had a ground-glass appearance, stained intensely for viral proteins, and showed signs of considerable damage and cell death. This histopathologic pattern closely resembles the picture observed in the livers of immunosuppressed HBV patients. These lesions were not observed in animals infected with HBV for less than 1 month. Ultrastructural analysis of long-term-infected hepatocytes showed a highly increased presence of cylindrical HBsAg structures, core particles, and Dane particles compared to short-term-infected hepatocytes. These long-term-infected hepatocytes also contained elevated amounts of HBV cccDNA. In conclusion, HBV causes dramatic intracellular changes and hepatocellular damage in the human hepatocytes that reside in a severely immune deficient mouse. These lesions show much resemblance to the ones encountered in immunosuppressed chronic HBV patients. Our observations indicate that HBV may be directly cytopathic in conditions of severe immune suppression.
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Codran, Audrey, Cathy Royer, Daniel Jaeck, Michèle Bastien-Valle, Thomas F. Baumert, Marie Paule Kieny, Carlos Augusto Pereira, and Jean-Pierre Martin. "Entry of hepatitis C virus pseudotypes into primary human hepatocytes by clathrin-dependent endocytosis." Journal of General Virology 87, no. 9 (September 1, 2006): 2583–93. http://dx.doi.org/10.1099/vir.0.81710-0.
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Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Studies of the early steps of HCV infection have been hampered by the lack of convenient in vitro or in vivo models. Although several cell-surface molecules that mediate the binding of HCV envelope proteins to target cells have been identified, mechanisms of viral entry into human hepatocytes are still poorly understood. Vesicular stomatitis virus/HCV pseudotyped viruses expressing the HCV envelope glycoproteins on the viral envelope were generated and it was found that their entry into human hepatocytes required co-expression of E1 and E2 on the pseudotype surface. Neutralization of pseudotype infection by anti-HCV antibodies suggested that cellular entry was mediated by HCV envelope glycoproteins and by previously characterized cell-surface molecules, including CD81. An entry assay based on the release of a fluorochrome from labelled HCV pseudotypes provided evidence for a pH-dependent fusion of the pseudotype envelope with a cellular compartment. By using a panel of endocytosis inhibitors, it is postulated that penetration of HCV into primary cultures of hepatocytes takes place by clathrin-mediated endocytosis.
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Lavillette, Dimitri, Eve-Isabelle Pécheur, Peggy Donot, Judith Fresquet, Jennifer Molle, Romuald Corbau, Marlène Dreux, François Penin, and François-Loïc Cosset. "Characterization of Fusion Determinants Points to the Involvement of Three Discrete Regions of Both E1 and E2 Glycoproteins in the Membrane Fusion Process of Hepatitis C Virus." Journal of Virology 81, no. 16 (May 30, 2007): 8752–65. http://dx.doi.org/10.1128/jvi.02642-06.
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ABSTRACT Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH.
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Wu, Yi-Hsuan, Yi Yang, Ching-Hung Chen, Chia-Jen Hsiao, Tian-Neng Li, Kuan-Ju Liao, Koichi Watashi, Bor-Sen Chen, and Lily Hui-Ching Wang. "Aerobic glycolysis supports hepatitis B virus protein synthesis through interaction between viral surface antigen and pyruvate kinase isoform M2." PLOS Pathogens 17, no. 3 (March 15, 2021): e1008866. http://dx.doi.org/10.1371/journal.ppat.1008866.
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As an intracellular pathogen, the reproduction of the hepatitis B virus (HBV) depends on the occupancy of host metabolism machinery. Here we test a hypothesis if HBV may govern intracellular biosynthesis to achieve a productive reproduction. To test this hypothesis, we set up an affinity purification screen for host factors that interact with large viral surface antigens (LHBS). This identified pyruvate kinase isoform M2 (PKM2), a key regulator of glucose metabolism, as a binding partner of viral surface antigens. We showed that the expression of viral LHBS affected oligomerization of PKM2 in hepatocytes, thereby increasing glucose consumption and lactate production, a phenomenon known as aerobic glycolysis. Reduction of PKM2 activity was also validated in several different models, including HBV-infected HepG2-NTCP-C4 cells, adenovirus mediated HBV gene transduction and transfection with a plasmid containing complete HBV genome on HuH-7 cells. We found the recovery of PKM2 activity in hepatocytes by chemical activators, TEPP-46 or DASA-58, reduced expressions of viral surface and core antigens. In addition, reduction of glycolysis by culturing in low-glucose condition or treatment with 2-deoxyglucose also decreased expressions of viral surface antigen, without affecting general host proteins. Finally, TEPP-46 largely suppressed proliferation of LHBS-positive cells on 3-dimensional agarose plates, but showed no effect on the traditional 2-dimensional cell culture. Taken together, these results indicate that HBV-induced metabolic switch may support its own translation in hepatocytes. In addition, aerobic glycolysis is likely essential for LHBS-mediated oncogenesis. Accordingly, restriction of glucose metabolism may be considered as a novel strategy to restrain viral protein synthesis and subsequent oncogenesis during chronic HBV infection.
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Seitz, Stefan, Jelena Habjanič, Anne K. Schütz, and Ralf Bartenschlager. "The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle." Annual Review of Virology 7, no. 1 (September 29, 2020): 263–88. http://dx.doi.org/10.1146/annurev-virology-092818-015508.
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New hepatitis B virions released from infected hepatocytes are the result of an intricate maturation process that starts with the formation of the nucleocapsid providing a confined space where the viral DNA genome is synthesized via reverse transcription. Virion assembly is finalized by the enclosure of the icosahedral nucleocapsid within a heterogeneous envelope. The latter contains integral membrane proteins of three sizes, collectively known as hepatitis B surface antigen, and adopts multiple conformations in the course of the viral life cycle. The nucleocapsid conformation depends on the reverse transcription status of the genome, which in turn controls nucleocapsid interaction with the envelope proteins for virus exit. In addition, after secretion the virions undergo a distinct maturation step during which a topological switch of the large envelope protein confers infectivity. Here we review molecular determinants for envelopment and models that postulate molecular signals encoded in the capsid scaffold conducive or adverse to the recruitment of envelope proteins.
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Hui, Alvin, Eda Altan, Nathan Slovis, Caitlin Fletcher, Xutao Deng, and Eric Delwart. "Circovirus in Blood of a Febrile Horse with Hepatitis." Viruses 13, no. 5 (May 20, 2021): 944. http://dx.doi.org/10.3390/v13050944.
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Circoviruses infect vertebrates where they can result in a wide range of disease signs or in asymptomatic infections. Using viral metagenomics we analyzed a pool of five sera from four healthy and one sick horse. Sequences from parvovirus-H, equus anellovirus, and distantly related to mammalian circoviruses were recognized. PCR identified the circovirus reads as originating from a pregnant mare with fever and hepatitis. That horse’s serum was also positive by real time PCR for equine parvovirus H and negative for the flavivirus equine hepacivirus. The complete circular genome of equine circovirus 1 strain Charaf (EqCV1-Charaf) was completed using PCR and Sanger sequencing. EqCV1 replicase showed 73–74% identity to those of their closest relatives, pig circoviruses 1/2, and elk circovirus. The closest capsid proteins were from the same ungulate circoviruses with 62–63% identity. The overall nucleotide identity of 72% to its closest relative indicates that EqCV1 is a new species in the Circovirus genus, the first reported in genus Equus. Whether EqCV1 alone or in co-infections can result in disease and its prevalence in different equine populations will require further studies now facilitated using EqCV1′s genome sequence.
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Hossain, Md Golzar, Sharmin Akter, Eriko Ohsaki, and Keiji Ueda. "Impact of the Interaction of Hepatitis B Virus with Mitochondria and Associated Proteins." Viruses 12, no. 2 (February 4, 2020): 175. http://dx.doi.org/10.3390/v12020175.
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Around 350 million people are living with hepatitis B virus (HBV), which can lead to death due to liver cirrhosis and hepatocellular carcinoma (HCC). Various antiviral drugs/nucleot(s)ide analogues are currently used to reduce or arrest the replication of this virus. However, many studies have reported that nucleot(s)ide analogue-resistant HBV is circulating. Cellular signaling pathways could be one of the targets against the viral replication. Several studies reported that viral proteins interacted with mitochondrial proteins and localized in the mitochondria, the powerhouse of the cell. And a recent study showed that mitochondrial turnover induced by thyroid hormones protected hepatocytes from hepatocarcinogenesis mediated by HBV. Strong downregulation of numerous cellular signaling pathways has also been reported to be accompanied by profound mitochondrial alteration, as confirmed by transcriptome profiling of HBV-specific CD8 T cells from chronic and acute HBV patients. In this review, we summarize the ongoing research into mitochondrial proteins and/or signaling involved with HBV proteins, which will continue to provide insight into the relationship between mitochondria and HBV and ultimately lead to advances in viral pathobiology and mitochondria-targeted antiviral therapy.
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Chabrolles, Hélène, Héloïse Auclair, Serena Vegna, Thomas Lahlali, Caroline Pons, Maud Michelet, Yohann Couté, et al. "Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production." PLOS Pathogens 16, no. 11 (November 12, 2020): e1008593. http://dx.doi.org/10.1371/journal.ppat.1008593.
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Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.
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Banerjee, Arup, Keith Meyer, Budhaditya Mazumdar, Ratna B. Ray, and Ranjit Ray. "Hepatitis C Virus Differentially Modulates Activation of Forkhead Transcription Factors and Insulin-Induced Metabolic Gene Expression." Journal of Virology 84, no. 12 (March 31, 2010): 5936–46. http://dx.doi.org/10.1128/jvi.02344-09.
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ABSTRACT Chronic hepatitis C virus (HCV) infection is often associated with insulin resistance and hepatic steatosis. Insulin regulates gene expression of key enzymes in glucose and lipid metabolism by modulating the activity of specific Forkhead box transcriptional regulators (FoxO1 and FoxA2) via the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway in the liver. In this study, we observed that HCV infection of human hepatocytes impaired insulin-induced FoxO1 translocation from the nucleus to the cytoplasm and significantly reduced accumulation of FoxA2 in the nucleus. Phosphorylation of FoxO1 at Ser256, a downstream target for Akt, was inhibited in hepatocytes infected with HCV or expressing the core protein or full-length (FL) genome of HCV. Further, an interaction between FoxO1 and 14-3-3 protein, important for FoxO1 translocation, was inhibited in HCV core-expressing cells. Hepatocytes infected with HCV, expressing the core protein alone or polyprotein displayed an increased level of glucose-6-phosphatase (G6P) mRNA. On the other hand, microsomal triglycerol transfer protein (MTP) activity and apolipoprotein B (ApoB) secretion were significantly reduced in hepatocytes expressing HCV proteins. Together, these observations suggest that HCV infection or ectopic expression of the core protein either alone or together with other viral proteins from an FL gene construct differentially modulates FoxO1 and FoxA2 activation and affects insulin-induced metabolic gene regulation in human hepatocytes.
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Yu, Xianming, and Janet E. Mertz. "Critical Roles of Nuclear Receptor Response Elements in Replication of Hepatitis B Virus." Journal of Virology 75, no. 23 (December 1, 2001): 11354–64. http://dx.doi.org/10.1128/jvi.75.23.11354-11364.2001.
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ABSTRACT Functional analysis of the roles of the nuclear receptor response elements (NRREs) in the transcription and replication of hepatitis B virus (HBV) in the context of its whole genome has been hampered by the extensive overlapping of the NRREs with the regions encoding viral proteins. We introduced point mutations that inactivate the NRREs individually without altering the open reading frames of viral proteins. These mutations in the context of a plasmid containing 1.2 copies of the HBV genome were transiently transfected into the human hepatoma cell line Huh7. Inactivation of the NRRE in either the preC promoter (NRREpreC) or enhancer I (NRREenhI) led to moderate reductions in synthesis of viral RNAs. Concurrent inactivation of both NRREs led to 7- to 8-fold reductions in synthesis of the preC, pregenomic, and preS RNAs and a 15-fold reduction in synthesis of the S RNA. The accumulation of viral DNA in the cytoplasmic nucleocapsids and virion particles in the culture medium was also reduced seven- to eightfold. These results suggest that these NRREs are critical for the efficient propagation of HBV in hepatocytes. In cotransfection experiments we also found that overexpression of PPARα-RXRα in the presence of their respective ligands led to a fourfold increase in pregenomic RNA synthesis and a four- to fivefold increase in viral DNA synthesis, while it had little or no effect on synthesis of the other viral RNAs. Similar effects were observed with overexpression of PPARγ-RXRα in the presence of their respective ligands. This activation was dependent on NRREpreC, because the increase in synthesis of viral RNA and DNA was not observed when this site was mutated. Likewise, no activation of synthesis of pregenomic RNA and viral DNA by PPARα-RXRα was observed in a naturally occurring NRREpreC − mutant of HBV. Our results suggest that interactions between nuclear receptors and NRREs present in the HBV genome may play critical roles in regulating its transcription and replication during HBV infection of hepatocytes.
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Douam, Florian, Louis-Marie Bobay, Guillemette Maurin, Judith Fresquet, Noémie Calland, Carine Maisse, Tony Durand, François-Loïc Cosset, Cyrille Féray, and Dimitri Lavillette. "Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients." Journal of Virology 90, no. 2 (November 4, 2015): 992–1008. http://dx.doi.org/10.1128/jvi.02516-15.
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ABSTRACTHepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCV JFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders.IMPORTANCEHepatitis C virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytesin vitroandin vivo, highlighting the existence of lymphotropism determinants within HCV viral proteins. We isolated HCV envelope glycoproteins from patient B lymphocytes that conferred to a nonlymphotropic HCV the ability to enter B cells, thus providing a platform for characterization of HCV entry into lymphocytes. This unusual tropism was accompanied by a loss of entry function into hepatocytes, suggesting that HCV lymphotropic variants likely constitute a distinct but parallel source for viral persistence and immune escape within chronically infected patients. Moreover, the level of genetic divergence of B-cell-derived envelopes correlated with their degree of lymphotropism, underlining a long-term specialization of some viral populations for B-lymphocytes. Consequently, the clearance of both hepatotropic and nonhepatotropic HCV populations may be important for effective treatment of chronically infected patients.
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Lundin, Marika, Magnus Monné, Anders Widell, Gunnar von Heijne, and Mats A. A. Persson. "Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B." Journal of Virology 77, no. 9 (May 1, 2003): 5428–38. http://dx.doi.org/10.1128/jvi.77.9.5428-5438.2003.
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ABSTRACT Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in Flaviviridae.
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Lu, Mengji, Beate Lohrengel, Gero Hilken, Thekla Kemper, and Michael Roggendorf. "Woodchuck Gamma Interferon Upregulates Major Histocompatibility Complex Class I Transcription but Is Unable To Deplete Woodchuck Hepatitis Virus Replication Intermediates and RNAs in Persistently Infected Woodchuck Primary Hepatocytes." Journal of Virology 76, no. 1 (January 1, 2002): 58–67. http://dx.doi.org/10.1128/jvi.76.1.58-67.2002.
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ABSTRACT Gamma interferon (IFN-γ) is an important mediator with multiple functions in the host defense against viral infection. IFN-γ, in concert with tumor necrosis factor alpha (TNF-α), leads to a remarkable reduction of intrahepatic replication intermediates and specific mRNAs of hepatitis B virus (HBV) by a noncytolytic mechanism in the transgenic mouse model. Thus, it is rational to evaluate the potential value of IFN-γ for the treatment of chronic HBV infection. In the present study, we expressed recombinant woodchuck IFN-γ (wIFN-γ) in Escherichia coli and mammalian cells. wIFN-γ protected woodchuck cells against infection of murine encephalomyocarditis virus in a species-specific manner. It upregulated the mRNA level of the woodchuck major histocompatibility complex class I (MHC-I) heavy chain in permanent woodchuck WH12/6 cells and regulated differentially the gene expression. However, the level of the replication intermediates and specific RNAs of woodchuck hepatitis virus (WHV) in persistently WHV-infected primary woodchuck hepatocytes did not change despite a treatment with 1,000 U of wIFN-γ per ml or with a combination of wIFN-γ and woodchuck TNF-α. Rather, hepatocytes derived from chronic carriers had an elevated level of the MHC-I heavy-chain mRNAs, most probably due to the exposure to inflammatory cytokines in vivo. Treatment with high doses of wIFN-γ led to an abnormal cell morphology and loss of hepatocytes. Thus, wIFN-γ regulates the gene expression in woodchuck hepatocytes but could not deplete WHV replication intermediates and mRNAs in persistently infected hepatocytes. The cellular response to wIFN-γ may be changed in hepatocytes from chronically WHV-infected woodchucks. It should be clarified in the future whether the continuous exposure of hepatocytes to inflammatory cytokines or the presence of viral proteins leads to changes of the cellular response to wIFN-γ.
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Barrera, Azeneth, Bernadette Guerra, Helen Lee, and Robert E. Lanford. "Analysis of Host Range Phenotypes of Primate Hepadnaviruses by In Vitro Infections of Hepatitis D Virus Pseudotypes." Journal of Virology 78, no. 10 (May 15, 2004): 5233–43. http://dx.doi.org/10.1128/jvi.78.10.5233-5243.2004.
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ABSTRACT Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.
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Dandri, Maura, Antonio Bertoletti, and Marc Lütgehetmann. "Innate immunity in hepatitis B and D virus infection: consequences for viral persistence, inflammation, and T cell recognition." Seminars in Immunopathology 43, no. 4 (May 21, 2021): 535–48. http://dx.doi.org/10.1007/s00281-021-00864-x.
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AbstractChronic infections with human hepatitis viruses continue to be a major health burden worldwide. Despite the availability of an effective prophylactic vaccine against the hepatitis B virus (HBV) and of antiviral agents efficiently suppressing HBV replication, more than 250 million people are currently chronically infected with this hepatotropic DNA virus, and resolution of chronic hepatitis B (CHB) is rarely achieved. Moreover, coinfection with the hepatitis D virus (HDV), a human RNA satellite virus requiring the envelope proteins of HBV for productive viral spreading, substantially aggravates the disease course of CHB. The molecular mechanisms by which these viruses interact with each other and with the intrinsic innate responses of the hepatocytes are not fully understood. While HBV appears to avoid innate immune recognition, HDV elicits a strong enhancement of innate responses. Notwithstanding, such induction does not hamper HDV replication but contributes to liver inflammation and pathogenesis. Intriguingly, HDV appears to influence the ability of T cells to recognize infected hepatocytes by boosting antigen presentation. This review focuses on current knowledge regarding how these viruses can shape and counteract the intrinsic innate responses of the hepatocytes, thus affecting the immune system and pathogenesis. Understanding the distinct strategies of persistence that HBV and HDV have evolved is central for advancing the development of curative therapies.
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Gearhart, Tricia L., and Michael J. Bouchard. "The Hepatitis B Virus X Protein Modulates Hepatocyte Proliferation Pathways To Stimulate Viral Replication." Journal of Virology 84, no. 6 (January 6, 2010): 2675–86. http://dx.doi.org/10.1128/jvi.02196-09.
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ABSTRACT Worldwide, there are over 350 million people who are chronically infected with the human hepatitis B virus (HBV); chronic HBV infections are associated with the development of hepatocellular carcinoma (HCC). The results of various studies suggest that the HBV X protein (HBx) has a role in the development of HBV-associated HCC. HBx can regulate numerous cellular signal transduction pathways, including those that modulate cell proliferation. Many previous studies that analyzed the impact of HBx on cell proliferation pathways were conducted using established or immortalized cell lines, and when HBx was expressed in the absence of HBV replication, and the precise effect of HBx on these pathways has often differed depending on experimental conditions. We have studied the effect of HBx on cell proliferation in cultured primary rat hepatocytes, a biologically relevant system. We demonstrate that HBx, both by itself and in the context of HBV replication, affected the levels and activities of various cell cycle-regulatory proteins to induce normally quiescent hepatocytes to enter the G1 phase of the cell cycle but not to proceed to S phase. We linked HBx regulation of cell proliferation to cytosolic calcium signaling and HBx stimulation of HBV replication. Cumulatively, our studies suggest that HBx induces normally quiescent hepatocytes to enter the G1 phase of the cell cycle and that this calcium-dependent HBx activity is required for HBV replication. These studies identify an essential function of HBx during HBV replication and a mechanism that may connect HBV infections to the development of HCC.
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Chen, Zhi, Yang-Xia Li, Hai-Jing Fu, Yan-Li Ren, Ling Zou, Shi-Zhen Shen, Ping Chen, Ting Sun, and Chun-Hong Huang. "Hepatitis B Virus Core Antigen Stimulates IL-6 Expression via p38, ERK and NF-κB Pathways in Hepatocytes." Cellular Physiology and Biochemistry 41, no. 1 (2017): 91–100. http://dx.doi.org/10.1159/000455954.
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Background: Hepatitis B virus (HBV) causes both acute and chronic liver injury. Viral proteins are involved in the pathological progress. Hepatitis B core antigen (HBcAg), a component of viral nucleocapsid, is not only essential for HBV lifecycle, but also exhibits strong immunogenicity. The cytoplasmic location of HBcAg in liver biopsy is associated with liver injury and inflammation, but the exact mechanisms remain to be elaborated. Methods: Huh7, SMMC-7721 and L-02 cells were transfected with pEGFP-N1-HBcAg to establish an intracellular HBcAg expression model. The mRNA and protein levels of Interleukin (IL)-6 were detected by qPCR and ELISA respectively. The signaling pathway-related proteins were investigated by western blot and immunofluorescence assay. Results: HBcAg increased the expression and secretion of IL-6 through activating extracellular signal-related kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-κB). These activations can be blocked by specific inhibitors of the three pathways. Conclusions: HBcAg actives p38, ERK1/2 and NF-κB to enhance the production of IL-6 in hepatocytes. This provides a molecular mechanism to explain the association of cytoplasmic HBcAg with severe liver injury and inflammation.
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Machida, Keigo, Kevin T. H. Cheng, Vicky M. H. Sung, Ki Jeong Lee, Alexandra M. Levine, and Michael M. C. Lai. "Hepatitis C Virus Infection Activates the Immunologic (Type II) Isoform of Nitric Oxide Synthase and Thereby Enhances DNA Damage and Mutations of Cellular Genes." Journal of Virology 78, no. 16 (August 15, 2004): 8835–43. http://dx.doi.org/10.1128/jvi.78.16.8835-8843.2004.
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ABSTRACT Hepatitis C virus (HCV) infection causes hepatitis, hepatocellular carcinoma, and B-cell lymphomas in a significant number of patients. Previously we have shown that HCV infection causes double-stranded DNA breaks and enhances the mutation frequency of cellular genes, including proto-oncogenes and immunoglobulin genes. To determine the mechanisms, we studied in vitro HCV infection of cell culture. Here we report that HCV infection activated the immunologic (type II) isoform of nitric oxide (NO) synthase (NOS), i.e., inducible NOS (iNOS), thereby inducing NO, which in turn induced DNA breaks and enhanced the mutation frequencies of cellular genes. Treatment of HCV-infected cells with NOS inhibitors or small interfering RNA specific for iNOS abolished most of these effects. Expression of the core protein or nonstructural protein 3 (NS3), but not the other viral proteins, in B cells or hepatocytes induced iNOS and DNA breaks, which could be blocked by NOS inhibitors. The core protein also enhanced the mutation frequency of cellular genes in hepatocytes derived from HCV core transgenic mice compared with that in control mice. The iNOS promoter was activated more than fivefold in HCV-infected cells, as revealed by a luciferase reporter assay driven by the iNOS promoter. Similarly, the core and NS3 proteins also induced the same effects. Therefore, we conclude that HCV infection can stimulate the production of NO through activation of the gene for iNOS by the viral core and NS3 proteins. NO causes DNA breaks and enhances DNA mutation. This sequence of events provides a mechanism for HCV pathogenesis and oncogenesis.
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Chouteau, P., J. Le Seyec, I. Cannie, M. Nassal, C. Guguen-Guillouzo, and P. Gripon. "A Short N-Proximal Region in the Large Envelope Protein Harbors a Determinant That Contributes to the Species Specificity of Human Hepatitis B Virus." Journal of Virology 75, no. 23 (December 1, 2001): 11565–72. http://dx.doi.org/10.1128/jvi.75.23.11565-11572.2001.
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ABSTRACT Infection by hepatitis B virus (HBV) is mainly restricted to humans. This species specificity is likely determined at the early phase of the viral life cycle. Since the envelope proteins are the first viral factors to interact with the cell, they represent attractive candidates for controlling the HBV host range. To investigate this assumption, we took advantage of the recent discovery of a second virus belonging to the primateOrthohepadnavirus genus, the woolly monkey HBV (WMHBV). A recombinant plasmid was constructed for the expression of all WMHBV envelope proteins. In additional constructs, N-terminal sequences of the WMHBV large envelope protein were substituted for their homologous HBV counterparts. All wild-type and chimeric WMHBV surface proteins were properly synthesized by transfected human hepatoma cells, and they were competent to replace the original HBV proteins for the production of complete viral particles. The resulting pseudotyped virions were evaluated for their infectious capacity on human hepatocytes in primary culture. Virions pseudotyped with wild-type WMHBV envelope proteins showed a significant loss of infectivity. By contrast, infectivity was completely restored when the first 30 residues of the large protein originated from HBV. Analysis of smaller substitutions within this domain limited the most important region to a stretch of only nine amino acids. Reciprocally, replacement of this motif by WMHBV residues in the context of the HBV L protein significantly reduced infectivity of HBV. Hence this short region of the L protein contributes to the host range of HBV.
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Ebert, Gregor, Simon Preston, Cody Allison, James Cooney, Jesse G. Toe, Michael D. Stutz, Samar Ojaimi, et al. "Cellular inhibitor of apoptosis proteins prevent clearance of hepatitis B virus." Proceedings of the National Academy of Sciences 112, no. 18 (April 20, 2015): 5797–802. http://dx.doi.org/10.1073/pnas.1502390112.
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Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.
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Mancinelli, Romina, Luigi Rosa, Antimo Cutone, Maria Stefania Lepanto, Antonio Franchitto, Paolo Onori, Eugenio Gaudio, and Piera Valenti. "Viral Hepatitis and Iron Dysregulation: Molecular Pathways and the Role of Lactoferrin." Molecules 25, no. 8 (April 24, 2020): 1997. http://dx.doi.org/10.3390/molecules25081997.
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The liver is a frontline immune site specifically designed to check and detect potential pathogens from the bloodstream to maintain a general state of immune hyporesponsiveness. One of the main functions of the liver is the regulation of iron homeostasis. The liver detects changes in systemic iron requirements and can regulate its concentration. Pathological states lead to the dysregulation of iron homeostasis which, in turn, can promote infectious and inflammatory processes. In this context, hepatic viruses deviate hepatocytes’ iron metabolism in order to better replicate. Indeed, some viruses are able to alter the expression of iron-related proteins or exploit host receptors to enter inside host cells. Lactoferrin (Lf), a multifunctional iron-binding glycoprotein belonging to the innate immunity, is endowed with potent antiviral activity, mainly related to its ability to block viral entry into host cells by interacting with viral and/or cell surface receptors. Moreover, Lf can act as an iron scavenger by both direct iron-chelation or the modulation of the main iron-related proteins. In this review, the complex interplay between viral hepatitis, iron homeostasis, and inflammation as well as the role of Lf are outlined.
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Sung, Vicky M. H., and Michael M. C. Lai. "Murine Retroviral Pseudotype Virus Containing Hepatitis B Virus Large and Small Surface Antigens Confers Specific Tropism for Primary Human Hepatocytes: a Potential Liver-Specific Targeting System." Journal of Virology 76, no. 2 (January 15, 2002): 912–17. http://dx.doi.org/10.1128/jvi.76.2.912-917.2002.
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ABSTRACT We have developed a system for producing murine leukemia virus (MLV) pseudotyped with human hepatitis B virus (HBV) large (L) and small (S) surface antigens (HBsAg) for targeting primary human hepatocytes. Using the MLV(HBV) pseudotype virus containing a β-galactosidase reporter gene, we demonstrated that this pseudotype virus exhibits strict tropism for primary human hepatocytes, similar to the natural target cell specificity of HBV. It does not infect any of the established tissue culture cell lines, including human hepatoma cell lines (HepG2 and Huh-7), or rat primary hepatocytes. The infectivity of MLV(HBV) for human hepatocytes was inhibited by anti-HBs antibody. The L form of HBsAg was both necessary and sufficient for virus infectivity, but the presence of both L and S forms enhanced the surface expression of HBsAg and thus increased virus production. The middle form of HBsAg was not necessary. This pseudotype virus bypasses the requirement for the liver-specific transcription factors for HBV replication, enabling direct study of HBV tissue tropism conferred by the viral envelope proteins. This virus also offers a potential liver-specific targeting system for gene therapy.
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Péneau, Camille, Jessica Zucman-Rossi, and Jean-Charles Nault. "Genomics of Viral Hepatitis-Associated Liver Tumors." Journal of Clinical Medicine 10, no. 9 (April 22, 2021): 1827. http://dx.doi.org/10.3390/jcm10091827.
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Virus-related liver carcinogenesis is one of the main contributors of cancer-related death worldwide mainly due to the impact of chronic hepatitis B and C infections. Three mechanisms have been proposed to explain the oncogenic properties of hepatitis B virus (HBV) infection: induction of chronic inflammation and cirrhosis, expression of HBV oncogenic proteins, and insertional mutagenesis into the genome of infected hepatocytes. Hepatitis B insertional mutagenesis modifies the function of cancer driver genes and could promote chromosomal instability. In contrast, hepatitis C virus promotes hepatocellular carcinoma (HCC) occurrence mainly through cirrhosis development whereas the direct oncogenic role of the virus in human remains debated. Finally, adeno associated virus type 2 (AAV2), a defective DNA virus, has been associated with occurrence of HCC harboring insertional mutagenesis of the virus. Since these tumors developed in a non-cirrhotic context and in the absence of a known etiological factor, AAV2 appears to be the direct cause of tumor development in these patients via a mechanism of insertional mutagenesis altering similar oncogenes and tumor suppressor genes targeted by HBV. A better understanding of virus-related oncogenesis will be helpful to develop new preventive strategies and therapies directed against specific alterations observed in virus-related HCC.
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Sprinzl, Martin Franz, Heike Oberwinkler, Heinz Schaller, and Ulrike Protzer. "Transfer of Hepatitis B Virus Genome by Adenovirus Vectors into Cultured Cells and Mice: Crossing the Species Barrier." Journal of Virology 75, no. 11 (June 1, 2001): 5108–18. http://dx.doi.org/10.1128/jvi.75.11.5108-5118.2001.
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ABSTRACT For the study of hepatitis B virus infection, no permissive cell line or small animal is available. Stably transfected cell lines and transgenic mice which contain hepadnavirus genomes produce virus, but—unlike in natural infection—from an integrated viral transcription template. To transfer hepadnavirus genomes across the species barrier, we developed adenovirus vectors in which 1.3-fold-overlength human and duck hepatitis B virus genomes were inserted. The adenovirus-mediated genome transfer efficiently initiated hepadnavirus replication from an extrachromosomal template in established cell lines, in primary hepatocytes from various species, and in the livers of mice. Following the transfer, hepatitis B virus proteins, genomic RNA, and all replicative DNA intermediates were detected. Detection of covalently closed circular DNA in hepatoma cell lines and in primary hepatocytes indicated that an intracellular replication cycle independent from the transferred linear viral genome was established. High-titer hepatitis B virions were released into the culture medium of hepatoma cells and the various primary hepatocytes. In addition, infectious virions were secreted into the sera of mice. In conclusion, adenovirus-mediated genome transfer initiated efficient hepatitis B virus replication in cultured liver cells and in the experimental animals from an extrachromosomal template. This will allow development of small-animal systems of hepatitis B virus infection and will facilitate study of pathogenicity of wild-type and mutant viruses as well as of virus-host interaction and new therapeutic approaches.
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Chigbu, DeGaulle, Ronak Loonawat, Mohit Sehgal, Dip Patel, and Pooja Jain. "Hepatitis C Virus Infection: Host–Virus Interaction and Mechanisms of Viral Persistence." Cells 8, no. 4 (April 25, 2019): 376. http://dx.doi.org/10.3390/cells8040376.
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Hepatitis C (HCV) is a major cause of liver disease, in which a third of individuals with chronic HCV infections may develop liver cirrhosis. In a chronic HCV infection, host immune factors along with the actions of HCV proteins that promote viral persistence and dysregulation of the immune system have an impact on immunopathogenesis of HCV-induced hepatitis. The genome of HCV encodes a single polyprotein, which is translated and processed into structural and nonstructural proteins. These HCV proteins are the target of the innate and adaptive immune system of the host. Retinoic acid-inducible gene-I (RIG-I)-like receptors and Toll-like receptors are the main pattern recognition receptors that recognize HCV pathogen-associated molecular patterns. This interaction results in a downstream cascade that generates antiviral cytokines including interferons. The cytolysis of HCV-infected hepatocytes is mediated by perforin and granzyme B secreted by cytotoxic T lymphocyte (CTL) and natural killer (NK) cells, whereas noncytolytic HCV clearance is mediated by interferon gamma (IFN-γ) secreted by CTL and NK cells. A host–HCV interaction determines whether the acute phase of an HCV infection will undergo complete resolution or progress to the development of viral persistence with a consequential progression to chronic HCV infection. Furthermore, these host–HCV interactions could pose a challenge to developing an HCV vaccine. This review will focus on the role of the innate and adaptive immunity in HCV infection, the failure of the immune response to clear an HCV infection, and the factors that promote viral persistence.
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Bonvicini, Francesca, Claudia Filippone, Elisabetta Manaresi, Marialuisa Zerbini, Monica Musiani, and Giorgio Gallinella. "HepG2 hepatocellular carcinoma cells are a non-permissive system for B19 virus infection." Journal of General Virology 89, no. 12 (December 1, 2008): 3034–38. http://dx.doi.org/10.1099/vir.0.2008/004341-0.
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Parvovirus B19 has been associated with liver dysfunction and has been considered a potential aetiological agent of fulminant hepatitis and hepatitis-associated aplastic anaemia. The possible effects of B19 virus infection on the liver have been investigated using HepG2 hepatocellular carcinoma cells as a model system, but the reported results are inconsistent. To investigate this relationship further, this study followed the course of B19 virus infection of HepG2 cells in terms of viral DNA, RNA and protein production by quantitative PCR, RT-PCR and immunofluorescence assays. The data showed that B19 virus is able to bind and possibly enter HepG2 cells, but that viral genome replication or transcription is not supported and that viral proteins are not produced. As far as HepG2 cells can be considered a representative model system, any possible pathogenic role of B19 virus on the liver cannot be ascribed to infection or to a direct cytopathic effect on hepatocytes.
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Kang, Yubin, Litao Xie, Diane Thi Tran, Colleen S. Stein, Melissa Hickey, Beverly L. Davidson, and Paul B. McCray. "Persistent expression of factor VIII in vivo following nonprimate lentiviral gene transfer." Blood 106, no. 5 (September 1, 2005): 1552–58. http://dx.doi.org/10.1182/blood-2004-11-4358.
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Abstract Hemophilia A is a clinically important coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)–based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudo-typed FIV efficiently and showed excellent hepatocyte tropism. The GP64-pseudotyped vector was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human α1-antitrypsin promoter) further enhanced transgene expression in hepatocytes. We generated a GP64-pseudotyped FIV vector encoding the B domain–deleted human FVIII coding region driven by the liver-specific promoter, with 2 beneficial point mutations in the A1 domain. Intravenous vector administration conferred sustained FVIII expression in hemophilia A mice for several months without the generation of anti–human FVIII antibodies and resulted in partial phenotypic correction. These findings demonstrate the utility of GP64-pseudotyped FIV lentiviral vectors for targeting hepatocytes to correct disorders associated with deficiencies of secreted proteins.