HCV infection activates the proteasome via PA28γ acetylation and heptamerization to facilitate the degradation of RNF2, a catalytic component of polycomb repressive complex 1.

We previously reported that hepatitis C virus (HCV) infection or HCV core protein expression induces HOX gene expression by impairing histone H2A monoubiquitination via a proteasome-dependent reduction in the level of RNF2, a key catalytic component of polycomb repressive complex 1 (H. Kasai, K. Mochizuki, T. Tanaka, A. Yamashita, et al., J Virol 95:e01784-20, 2021, https://doi.org/10.1128/jvi.01784-20). In this study, we aimed to investigate the mechanism by which HCV infection accelerates RNF2 degradation. Yeast two-hybrid screening and an immunoprecipitation assay revealed that RNF2 is a PA28γ-binding protein. The proteasome activator PA28γ destabilized the RNF2 protein in a proteasome-dependent manner, since RNF2 degradation was impaired by PA28γ knockout or MG132 treatment. HCV infection or core protein expression reduced the levels of RNF2 and histone H2A K119 monoubiquitination and induced the expression of HOX genes in the presence of PA28γ, while PA28γ knockout reversed these changes. Treatment with a lysine acetyltransferase inhibitor inhibited the acetylation of PA28γ at K195 and the degradation of the RNF2 protein, while treatment with a lysine deacetylase inhibitor accelerated these events in a PA28γ-dependent manner. RNF2 protein degradation was increased by expression of the acetylation mimetic PA28γ mutant but not by expression of the acetylation-defective mutant or the proteasome activation-defective mutant. Furthermore, HCV infection or core protein expression facilitated the interaction between PA28γ and the lysine acetyltransferase CBP/p300 and then accelerated PA28γ acetylation and heptazmerization to promote RNF2 degradation. These data suggest that HCV infection accelerates the acetylation-dependent heptamerization of PA28γ to increase the proteasomal targeting of RNF2.IMPORTANCEHCV is a causative agent of HCV-related liver diseases, including hepatic steatosis, cirrhosis, and hepatocellular carcinoma. PA28γ, which, in heptameric form, activates the 20S core proteasome for the degradation of PA28γ-binding proteins, is responsible for HCV-related liver diseases. HCV core protein expression or HCV infection accelerates RNF2 degradation, leading to the induction of HOX gene expression via a decrease in the level of H2Aub on HOX gene promoters. However, the mechanism of RNF2 degradation in HCV-infected cells has not been clarified. The data presented in this study suggest that PA28γ acetylation and heptamerization are promoted by HCV infection or by core protein expression to activate the proteasome for the degradation of RNF2 and are responsible for HCV propagation. This study provides novel insights valuable for the development of therapies targeting both HCV propagation and HCV-related diseases.

SARS-CoV-2 papain-like protease inhibits ISGylation of the viral nucleocapsid protein to evade host anti-viral immunity.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.

Identification of neutralizing epitopes in the preS2 domain of the hepatitis B virus.

Hepatitis B virus (HBV) infection is a major public health problem. The sodium taurocholate cotransporting polypeptide (NTCP) has been identified as an essential HBV receptor. Human hepatocytes are infected with HBV via binding between the preS1 region of the HBV large envelope protein and the NTCP. However, the role of preS2 in HBV entry is not well understood. In this study, we induced anti-preS2 serum in mice by DNA immunization, and showed that the resulting antiserum neutralized HBV infectivity. Competition assays using overlapping peptides suggested that the neutralizing epitope is located in the N-terminal region of preS2. In addition, monoclonal antibodies targeting the N-terminal region of preS2 neutralized HBV infectivity, indicating that these domains are critical epitopes for viral neutralization. These findings provide new insights into the HBV entry machinery while suggesting a novel modality for the prevention and treatment of HBV infection.

Neutralization of hepatitis B virus with vaccine-escape mutations by hepatitis B vaccine with large-HBs antigen.

Although the current hepatitis B (HB) vaccine comprising small-HBs antigen (Ag) is potent and safe, attenuated prophylaxis against hepatitis B virus (HBV) with vaccine-escape mutations (VEMs) has been reported. We investigate an HB vaccine consisting of large-HBsAg that overcomes the shortcomings of the current HB vaccine. Yeast-derived large-HBsAg is immunized into rhesus macaques, and the neutralizing activities of the induced antibodies are compared with those of the current HB vaccine. Although the antibodies induced by the current HB vaccine cannot prevent HBV infection with VEMs, the large-HBsAg vaccine-induced antibodies neutralize those infections. The HBV genotypes that exhibited attenuated neutralization via these vaccines are different. Here, we show that the HB vaccine consisting of large-HBsAg is useful to compensate for the shortcomings of the current HB vaccine. The combined use of these HB vaccines may induce antibodies that can neutralize HBV strains with VEMs or multiple HBV genotypes.

Hydrophobic Alpha-Helical Short Peptides in Overlapping Reading Frames of the Coronavirus Genome.

In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

SARS-CoV-2 ORF6 disrupts nucleocytoplasmic trafficking to advance viral replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF6 is an antagonist of interferon (IFN)-mediated antiviral signaling, achieved through the prevention of STAT1 nuclear localization. However, the exact mechanism through which ORF6 prevents STAT1 nuclear trafficking remains unclear. Herein, we demonstrate that ORF6 directly binds to STAT1 with or without IFN stimulation, resulting in the nuclear exclusion of STAT1. ORF6 also recognizes importin α subtypes with different modes, in particular, high affinity to importin α1 but a low affinity to importin α5. Although ORF6 potentially disrupts the importin α/importin ß1-mediated nuclear transport, thereby suppressing the nuclear translocation of the other classical nuclear localization signal-containing cargo proteins, the inhibitory effect of ORF6 is modest when compared with that of STAT1. The results indicate that the drastic nuclear exclusion of STAT1 is attributed to the specific binding with ORF6, which is a distinct strategy for the importin α1-mediated pathway. Combined with the results from a newly-produced replicon system and a hamster model, we conclude that SARS-CoV-2 ORF6 acts as a virulence factor via regulation of nucleocytoplasmic trafficking to accelerate viral replication, resulting in disease progression.

Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening.

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-ß but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-ß. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.

Establishment of monoclonal antibodies broadly neutralize infection of hepatitis B virus.

Antibodies against hepatitis B virus S protein can protect against hepatitis B virus (HBV) infection. Therefore, hepatitis B immunoglobulin (HBIG), which contains HBsAb, is used clinically as a therapy for HBV infection. In this study, a series of monoclonal antibodies that recognize multiple HBV genotypes was obtained. All the antibodies recognized conformational epitopes of S protein, but not linear epitopes. Several antibodies neutralized HBV infection and exhibited strong affinities and neutralizing activities. Antigenic epitope analysis demonstrated that they recognized residue Ile152 of S protein, which is localized outside the "a" determinant. Ile152 is highly conserved, and a mutation in this residue resulted in reduced expression of large hepatitis B surface proteins (L protein), suggesting that the amino acid at this position is involved in the expression of L protein. In addition, the antibodies neutralized the infection of hepatitis D virus possessing a Gly145 mutation to Arg in S protein, which is a well-known escape mutation against HBIG treatment. Using mouse monoclonal antibodies, a humanized antibody possessing affinities and neutralizing activities similar to those of the original mouse antibody was successfully established. The antibodies generated in this study may have the potential for use in alternative antibody therapies for HBV infection.

Inhibitory effect of a novel thiazolidinedione derivative on hepatitis B virus entry.

The development of novel antivirals to treat hepatitis B virus (HBV) infection is still needed because currently available drugs do not completely eradicate chronic HBV in some patients. Recently, troglitazone and ciglitazone, classified among the compounds including the thiazolidinedione (TZD) moiety, were found to inhibit HBV infection, but these compounds are not clinically available. In this study, we synthesized 11 TZD derivatives, compounds 1-11, and examined the effect of each compound on HBV infection in HepG2 cells expressing NTCP (HepG2/NTCP cells). Among the derivatives, (Z)-5-((4'-(naphthalen-1-yl)-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (compound 6) showed the highest antiviral activity, with an IC50 value of 0.3 µM and a selectivity index (SI) of 85, but compound 6 did not affect HCV infection. Treatment with compound 6 inhibited HBV infection in primary human hepatocytes (PHHs) but did not inhibit viral replication in HepG2.2.15 cells or HBV DNA-transfected Huh7 cells. Moreover, treatment with compound 6 significantly impaired hepatitis delta virus (HDV) infection and inhibited a step in HBV particle internalization but did not inhibit attachment of the preS1 lipopeptide or viral particles to the cell surface. These findings suggest that compound 6 interferes with HBV infection via inhibition of the internalization process.

Amino Acid Polymorphism in Hepatitis B Virus Associated With Functional Cure.

BACKGROUND & AIMS: To provide an adequate treatment strategy for chronic hepatitis B, it is essential to know which patients are expected to have a good prognosis and which patients do not require therapeutic intervention. Previously, we identified the substitution of isoleucine to leucine at amino acid 97 (I97L) in the hepatitis B core region as a key predictor among patients with stable hepatitis. In this study, we attempted to identify the point at which I97L affects the hepatitis B virus (HBV) life cycle and to elucidate the underlying mechanisms governing the stabilization of hepatitis. METHODS: To confirm the clinical features of I97L, we used a cohort of hepatitis B e antigen-negative patients with chronic hepatitis B infected with HBV-I97 wild-type (wt) or HBV-I97L. The effects of I97L on viral characteristics were evaluated by in vitro HBV production and infection systems with the HBV reporter virus and cell culture-generated HBV. RESULTS: The ratios of reduction in hepatitis B surface antigen and HBV DNA were higher in patients with HBV-I97L than in those with HBV-I97wt. HBV-I97L exhibited lower infectivity than HBV-I97wt in both infection systems with reporter HBV and cell culture-generated HBV. HBV-I97L virions exhibiting low infectivity primarily contained a single-stranded HBV genome. The lower efficiency of cccDNA synthesis was demonstrated after infection of HBV-I97L or transfection of the molecular clone of HBV-I97L. CONCLUSIONS: The I97L substitution reduces the level of cccDNA through the generation of immature virions with single-stranded genomes. This I97L-associated low efficiency of cccDNA synthesis may be involved in the stabilization of hepatitis.

DsRNA Sequencing for RNA Virus Surveillance Using Human Clinical Samples.

Although viruses infect various organs and are associated with diseases, there may be many unidentified pathogenic viruses. The recent development of next-generation sequencing technologies has facilitated the establishment of an environmental viral metagenomic approach targeting the intracellular viral genome. However, an efficient method for the detection of a viral genome derived from an RNA virus in animal or human samples has not been established. Here, we established a method for the efficient detection of RNA viruses in human clinical samples. We then tested the efficiency of the method compared to other conventional methods by using tissue samples collected from 57 recipients of living donor liver transplantations performed between June 2017 and February 2019 at Kyushu University Hospital. The viral read ratio in human clinical samples was higher by the new method than by the other conventional methods. In addition, the new method correctly identified viral RNA from liver tissues infected with hepatitis C virus. This new technique will be an effective tool for intracellular RNA virus surveillance in human clinical samples and may be useful for the detection of new RNA viruses associated with diseases.

Establishment of a Cell Culture Model Permissive for Infection by Hepatitis B and C Viruses.

Compared with each monoinfection, coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is well known to increase the risks of developing liver cirrhosis and hepatocellular carcinoma. However, the mechanism by which HBV/HCV coinfection is established in hepatocytes is not well understood. Common cell culture models for coinfection are required to examine viral propagation. In this study, we aimed to establish a cell line permissive for both HBV and HCV infection. We first prepared a HepG2 cell line expressing sodium taurocholate cotransporting polypeptide, an HBV receptor, and then selected a cell line highly permissive for HBV infection, G2/NT18-B. After transduction with a lentivirus-encoding microRNA-122, the cell line harboring the highest level of replicon RNA was selected and then treated with anti-HCV compounds to eliminate the replicon RNA. The resulting cured cell line was transduced with a plasmid-encoding CD81. The cell line permissive for HCV infection was cloned and then designated the G2BC-C2 cell line, which exhibited permissiveness for HBV and HCV propagation. JAK inhibitor I potentiated the HCV superinfection of HBV-infected cells, and fluorescence-activated cell-sorting analysis indicated that HBV/HCV double-positive cells accounted for approximately 30% of the coinfected cells. Among several host genes tested, cyclooxygenase-2 showed synergistic induction by coinfection compared with each monoinfection. Conclusion: These data indicate that our in vitro HBV/HCV coinfection system provides an easy-to-use platform for the study of host and viral responses against coinfection and the development of antiviral agents targeting HBV and HCV.

N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus.

BACKGROUND AND AIMS: An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. APPROACH AND RESULTS: We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. CONCLUSIONS: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.

Induction of HOX Genes by Hepatitis C Virus Infection via Impairment of Histone H2A Monoubiquitination.

Hepatitis C virus (HCV) infection causes liver pathologies, including hepatocellular carcinoma (HCC). Homeobox (HOX) gene products regulate embryonic development and are associated with tumorigenesis, although the regulation of HOX genes by HCV infection has not been clarified in detail. We examined the effect of HCV infection on HOX gene expression. In this study, HCV infection induced more than half of the HOX genes and reduced the level of histone H2A monoubiquitination on lysine 119 (K119) (H2Aub), which represses HOX gene promoter activity. HCV infection also promoted proteasome-dependent degradation of RNF2, which is an E3 ligase mediating H2A monoubiquitination as a component of polycomb repressive complex 1. Since full-genomic replicon cells but not subgenomic replicon cells exhibited reduced RNF2 and H2Aub levels and induction of HOX genes, we focused on the core protein. Expression of the core protein reduced the amounts of RNF2 and H2Aub and induced HOX genes. Treatment with LY-411575, which can reduce HCV core protein expression via signal peptide peptidase (SPP) inhibition without affecting other viral proteins, dose-dependently restored the amounts of RNF2 and H2Aub in HCV-infected cells and impaired the induction of HOX genes and production of viral particles but not viral replication. The chromatin immunoprecipitation assay results also indicated infection- and proteasome-dependent reductions in H2Aub located in HOX gene promoters. These results suggest that HCV infection or core protein induces HOX genes by impairing histone H2A monoubiquitination via a reduction in the RNF2 level.IMPORTANCE Recently sustained virologic response can be achieved by direct-acting antiviral (DAA) therapy in most hepatitis C patients. Unfortunately, DAA therapy does not completely eliminate a risk of hepatocellular carcinoma (HCC). Several epigenetic factors, including histone modifications, are well known to contribute to hepatitis C virus (HCV)-associated HCC. However, the regulation of histone modifications by HCV infection has not been clarified in detail. In this study, our data suggest that HCV infection or HCV core protein expression impairs monoubiquitination of histone H2A K119 in the homeobox (HOX) gene promoter via destabilization of RNF2 and then induces HOX genes. Several lines of evidence suggest that the expression of several HOX genes is dysregulated in certain types of tumors. These findings reveal a novel mechanism of HCV-related histone modification and may provide information about new targets for diagnosis and prevention of HCC occurrence.

Identification of Two Critical Neutralizing Epitopes in the Receptor Binding Domain of Hepatitis B Virus preS1.

Hepatitis B virus (HBV) infection is a major public health problem. Human hepatocytes are infected with HBV via binding between the preS1 region in the large envelope protein of HBV and sodium taurocholate cotransporting polypeptide. Although several monoclonal antibodies (MAbs) that recognize the receptor binding domain in preS1 and neutralize HBV infection have been isolated, details of neutralizing epitopes are not understood. In this study, we generated 13 MAbs targeting the preS1 receptor binding domain from preS1-specific memory B cells derived from DNA immunized mice. The MAbs were classified into three groups according to the epitope regions, designated epitopes I-III. A virus neutralization assay revealed that MAbs recognizing epitopes I and III neutralized HBV infection, suggesting that these domains are critical epitopes for viral neutralization. In addition, a neutralization assay against multiple genotypes of HBV revealed that epitope I is a semi-pangenotypic neutralizing epitope, whereas epitope III is a genotype-specific epitope. We also showed that neutralizing MAbs against preS1 could neutralize HBV bearing vaccine-induced escape mutation. These findings provide insight into novel immunoprophylaxis for the prevention and treatment of HBV infection.IMPORTANCE The HBV preS1 2-47 aa region (preS1/2-47) is essential for virus binding with sodium taurocholate cotransporting polypeptide. Several MAbs targeting preS1/2-47 have been reported to neutralize HBV infection; however, which region in preS1/2-47 contains the critical neutralizing epitope for HBV infection is unclear. Here, we generated several MAbs targeting preS1/2-47 and found that MAbs recognizing the N- or C-terminus of preS1/2-47 remarkably neutralized HBV infection. We further confirmed the neutralizing activity of anti-preS1 MAbs against HBV with vaccine escape mutation. These data clarified the relationship between the antibody epitope and the virus neutralizing activity and also suggested the potential ability of a vaccine antigen containing the preS1 region to overcome the weakness of current HB vaccines comprising the small S protein.

Anti-viral effects of interferon-λ3 on hepatitis B virus infection in cell culture.

AIM: Interferon (IFN)-λ3 is known to have antiviral effects against various pathogens. Recently, it has been reported that the production of IFN-λ3 in colon cells after the administration of nucleotide analogs is expected to reduce hepatitis B surface antigen in chronic hepatitis B patients. Here, we aimed to prove the antiviral effects of IFN-λ3 on hepatitis B virus (HBV) by using an in vitro HBV production and infection system. METHODS: We used HepG2.2.15-derived HBV as an inoculum and the replication-competent molecular clone of HBV as a replication model. RESULTS: By administering IFN-λ3 to HepG2 cells transfected with the HBV molecular clone, the production of hepatitis B surface antigen and hepatitis B core-related antigen was reduced dose-dependently. IFN-λ3 treatment also reduced the number of HBV-positive cells and the synthesis of covalently closed circular DNA after infection of HepG2.2.15-derived HBV to sodium taurocholate cotransporting polypeptide-transduced HepG2 cells. The inhibitory effect on HBV infection by IFN-λ3 was confirmed by using a recombinant a HBV reporter virus system. To elucidate the underlying mechanisms of the anti-HBV effect of IFN-λ3, we assessed the transcription of HBV RNA and the production of core-associated HBV DNA in HBV molecular clone-transfected HepG2 cells, and found that both parameters were reduced by IFN-λ3. CONCLUSIONS: We observed that the administration of IFN-λ3 inhibits HBV infection and the production of HBV proteins at the HBV RNA transcription level. This finding provides novel insight into the treatment of chronic hepatitis B patients with the administration or induction of IFN-λ3.

USP15 Participates in Hepatitis C Virus Propagation through Regulation of Viral RNA Translation and Lipid Droplet Formation.

Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs.IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.

Roles of the 5' Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication.

The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5'-terminal domain (I); three additional domains (I', II, and III), which are homologous to domains I, II, and III, respectively, of HCV; and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5' UTR was lower than that of the HCV 5' UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5' UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domains I and I' of EHcV (the 5'-proximal EHcV-specific region) improved RNA stability and provided the HCV SGR with microRNA 122 (miR-122)-independent replication capability, while EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domains I and I' improves RNA stability and viral replication regardless of miR-122 expression. The 5'-proximal EHcV-specific region may represent an inherent mechanism to facilitate viral replication in nonhepatic tissues.IMPORTANCE EHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs in horses, donkeys, and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5' untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5'-proximal EHcV-specific region enhances viral replication and RNA stability in a miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5' UTR contributes to the differences in tissue tropism observed between these hepaciviruses.

Seroepidemiology of non-primate hepacivirus (NPHV) in Japanese native horses.

Non-primate hepacivirus (NPHV) is recently identified as a closely related homologue of hepatitis C virus. The previous studies showed a high prevalence of NPHV infection among Japanese domestic horses originated from abroad. The historical distribution of NPHV among horses in Japan, therefore, is still unknown. In this study, seroepidemiological study of NPHV was conducted using 335 sera from five breeds of Japanese native horses. These horses are maintained as the pedigree and are reared apart from other horse breeds. The detection of antibodies against NPHV were conducted by western blot analysis using the recombinant protein of the NPHV core protein. The antibodies against NPHV were detected in all five breeds, 83 out of 335 (23.4%) horses. These results suggested that NPHV was circulating among Japanese native horses.

Cinnamic acid derivatives inhibit hepatitis C virus replication via the induction of oxidative stress.

Several cinnamic acid derivatives have been reported to exhibit antiviral activity. In this study, we prepared 17 synthetic cinnamic acid derivatives and screened them to identify an effective antiviral compound against hepatitis C virus (HCV). Compound 6, one of two hit compounds, suppressed the viral replications of genotypes 1b, 2a, 3a, and 4a with EC50 values of 1.5-8.1 µM and SI values of 16.2-94.2. The effect of compound 6 on the phosphorylation of Tyr705 in signal transducer and activator of transcription 3 (STAT3) was investigated because a cinnamic acid derivative AG490 was reported to suppress HCV replication and the activity of Janus kinase (JAK) 2. Compound 6 potently suppressed HCV replication, but it did not inhibit the JAK1/2-dependent phosphorylation of STAT3 Tyr705 at the same concentration. Furthermore, a pan-JAK inhibitor tofacitinib potently impaired phosphorylation of STAT3 Tyr 705, but it did not inhibit HCV replication in the replicon cells and HCV-infected cells at the same concentration, supporting the notion that the phosphorylated state of STAT3 Tyr705 is not necessarily correlated with HCV replication. The production of reactive oxygen species (ROS) was induced by treatment with compound 6, whereas N-acetyl-cysteine restored HCV replication and impaired ROS production in the replicon cells treated with compound 6. These data suggest that compound 6 inhibits HCV replication via the induction of oxidative stress.