A versatile method to profile hepatitis B virus DNA integration.

BACKGROUND: HBV DNA integration into the host genome is frequently found in HBV-associated HCC tissues and is associated with hepatocarcinogenesis. Multiple detection methods, including hybrid capture-sequencing, have identified integration sites and provided clinical implications; however, each has advantages and disadvantages concerning sensitivity, cost, and throughput. Therefore, methods that can comprehensively and cost-effectively detect integration sites with high sensitivity are required. Here, we investigated the efficiency of RAISING (Rapid Amplification of Integration Site without Interference by Genomic DNA contamination) as a simple and inexpensive method to detect viral integration by amplifying HBV-integrated fragments using virus-specific primers covering the entire HBV genome. METHODS AND RESULTS: Illumina sequencing of RAISING products from HCC-derived cell lines (PLC/PRF/5 and Hep3B cells) identified HBV-human junction sequences as well as their frequencies. The HBV-human junction profiles identified using RAISING were consistent with those determined using hybrid capture-sequencing, and the representative junctions could be validated by junction-specific nested PCR. The comparison of these detection methods revealed that RAISING-sequencing outperforms hybrid capture-sequencing in concentrating junction sequences. RAISING-sequencing was also demonstrated to determine the sites of de novo integration in HBV-infected HepG2-NTCP cells, primary human hepatocytes, liver-humanized mice, and clinical specimens. Furthermore, we made use of xenograft mice subcutaneously engrafted with PLC/PRF/5 or Hep3B cells, and HBV-human junctions determined by RAISING-sequencing were detectable in the plasma cell-free DNA using droplet digital PCR. CONCLUSIONS: RAISING successfully profiles HBV-human junction sequences with smaller amounts of sequencing data and at a lower cost than hybrid capture-sequencing. This method is expected to aid basic HBV integration and clinical diagnosis research.

Selective inhibition of hepatitis B virus internalization by oxysterol derivatives.

Given that the current approved anti-hepatitis B virus (HBV) drugs suppress virus replication and improve hepatitis but cannot eliminate HBV from infected patients, new anti-HBV agents with different mode of action are urgently needed. In this study, we identified a semi-synthetic oxysterol, Oxy185, that can prevent HBV infection in a HepG2-based cell line and primary human hepatocytes. Mechanistically, Oxy185 inhibited the internalization of HBV into cells without affecting virus attachment or replication. We also found that Oxy185 interacted with an HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP), and inhibited the oligomerization of NTCP to reduce the efficiency of HBV internalization. Consistent with this mechanism, Oxy185 also inhibited the hepatitis D virus infection, which relies on NTCP-dependent internalization, but not hepatitis A virus infection, and displayed pan-genotypic anti-HBV activity. Following oral administration in mice, Oxy185 showed sustained accumulation in the livers of the mice, along with a favorable liver-to-plasma ratio. Thus, Oxy185 is expected to serve as a useful tool compound in proof-of-principle studies for HBV entry inhibitors with this novel mode of action.

Hepatocellular organellar abnormalities following elimination of hepatitis C virus.

BACKGROUND AND AIMS: The future development of hepatocellular carcinoma (HCC) in patients after sustained virologic response (SVR) is an important issue. The purposes of this study were to investigate pathological alterations in organelle of the liver of SVR patients and to characterize organelle abnormalities that may be related to carcinogenesis after SVR. METHODS: The ultrastructure of liver biopsy specimens from patients with chronic hepatitis C (CHC) and SVR were compared to cell and mouse models and assessed semi-quantitatively using transmission electron microscopy. RESULTS: Hepatocytes in patients with CHC showed abnormalities in the nucleus, mitochondria, endoplasmic reticulum, lipid droplet, and pericellular fibrosis, comparable to those seen in hepatitis C virus (HCV)-infected mice and cells. DAA treatment significantly reduced organelle abnormalities such as the nucleus, mitochondria, and lipid droplet in the hepatocytes of patients and mice after SVR, and cured cells, but it did not change dilated/degranulated endoplasmic reticulum and pericellular fibrosis in patients and mice after SVR. Further, samples from patients with a post-SVR period of >1 year had significantly larger numbers of abnormalities in the mitochondria and endoplasmic reticulum than those of <1 year. A possible cause of organelle abnormalities in patients after SVR could be oxidative stress of the endoplasmic reticulum and mitochondria associated with abnormalities of the vascular system due to fibrosis. Interestingly, abnormal endoplasmic reticulum was associated with patients with HCC for >1 year after SVR. CONCLUSIONS: These results indicate that patients with SVR exhibit a persistent disease state and require long-term follow-up to detect early signs of carcinogenesis.

HBV with precore and basal core promoter mutations exhibits a high replication phenotype and causes ER stress-mediated cell death in humanized liver chimeric mice.

BACKGROUND AND AIMS: Mutations within the precore (PC) and basal core promoter (BCP) regions of the HBV genome are associated with fulminant hepatitis and HBV reactivation. These mutations may enhance viral replication, but little is known about whether they directly induce damage to the liver. We investigated mechanisms of direct cytopathic effects induced by the infection with PC/BCP mutants in the absence of immune response in vitro and in vivo . APPROACH AND RESULTS: Mice with humanized livers and hepatocytes derived from humanized mice were infected with either wild-type or mutant-type PC/BCP HBV, and the HBV replication and human hepatocyte damage were evaluated. HBV proliferated vigorously in mice with PC/BCP-mutant infection, and the severe loss of human hepatocytes with a slight human ALT elevation subsequently occurred only in PC/BCP mutant mice. In PC/BCP mutant infection, the accumulation of HBsAg in humanized livers colocalized with the endoplasmic reticulum, leading to apoptosis through unfolded protein response in HBV-infected hepatocytes. RNA-sequencing revealed the molecular characteristics of the phenotype of PC/BCP mutant infection in a humanized mouse model. Reduced ALT elevation and higher HBV DNA levels in this model are consistent with characteristics of HBV reactivation, indicating that the hepatocyte damage in this model might mimic HBV reactivation followed by hepatocyte damage under immunosuppressive conditions. CONCLUSION: PC and BCP mutations were associated with enhanced viral replication and cell death induced by ER stress using HBV infection models. These mutations might be associated with liver damage in patients with fulminant hepatitis or HBV reactivation.

Independent evolution of multi-dominant viral genome species observed in a hepatitis C virus carrier.

The viral genome quasispecies composition of hepatitis C virus (HCV) could have important implications to viral pathogenesis and resistance to anti-viral treatment. The purpose of the present study was to profile the HCV RNA quasispecies. We developed a strategy to determine the full-length HCV genome sequences co-existing within a single patient serum by using next-generation sequencing technologies. The isolated viral clones were divided into the groups that can be distinguished by core amino acid 70 substitution. Subsequently, we determined HCV full-length genome sequences of three independent dominant species co-existing in the sequential serum with a 7-year interval. From phylogenetic analysis, these dominant species evolved independently. Our study demonstrated that multiple dominant species co-existed in patient sera and evolved independently.

Patient-derived monoclonal antibody neutralizes HCV infection in vitro and vivo without generating escape mutants.

In recent years, new direct-acting antivirals for hepatitis C virus (HCV) have been approved, but hepatitis C continues to pose a threat to human health. It is important to develop neutralizing anti-HCV antibodies to prevent medical and accidental infection, such as might occur via liver transplantation of chronic HCV patients and needle-stick accidents in the clinic. In this study, we sought to obtain anti-HCV antibodies using phage display screening. Phages displaying human hepatocellular carcinoma patient-derived antibodies were screened by 4 rounds of biopanning with genotype-1b and -2a HCV envelope E2 protein adsorbed to magnetic beads. The three antibodies obtained from this screen had reactivity against E2 proteins derived from both genotype-1b and -2a strains. However, in epitope analysis, these antibodies did not recognize linear peptides from an overlapping E2 epitope peptide library, and did not bind to denatured E2 protein. In addition, these antibodies showed cross-genotypic neutralizing activity against genotype-1a, -1b, -2a, and -3a cell culture-generated infectious HCV particles (HCVcc). Moreover, emergence of viral escape mutants was not observed after repeated rounds of passaging of HCV-infected cells in the presence of one such antibody, e2d066. Furthermore, injection of the e2d066 antibody into human hepatocyte-transplanted immunodeficient mice inhibited infection by J6/JFH-1 HCVcc. In conclusion, we identified conformational epitope-recognizing, cross-genotypic neutralizing antibodies using phage display screening. Notably, e2d066 antibody did not select for escape mutant emergence in vitro and demonstrated neutralizing activity in vivo. Our results suggested that these antibodies may serve as prophylactic and therapeutic agents.

Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus.

In microbiological research, it is important to understand the time course of each step in a pathogen's lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3-4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5' UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.

Fungal Secondary Metabolite Exophillic Acid Selectively Inhibits the Entry of Hepatitis B and D Viruses.

Current anti-hepatitis B virus (HBV) drugs are suppressive but not curative for HBV infection, so there is considerable demand for the development of new anti-HBV agents. In this study, we found that fungus-derived exophillic acid inhibits HBV infection with a 50% maximal inhibitory concentration (IC50) of 1.1 µM and a 50% cytotoxic concentration (CC50) of >30 µM in primary human hepatocytes. Exophillic acid inhibited preS1-mediated viral attachment to cells but did not affect intracellular HBV replication. Exophillic acid appears to target the host cells to reduce their susceptibility to viral attachment rather than acting on the viral particles. We found that exophillic acid interacted with the HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP). Exophillic acid impaired the uptake of bile acid, the original function of NTCP. Consistent with our hypothesis that it affects NTCP, exophillic acid inhibited infection with HBV and hepatitis D virus (HDV), but not that of hepatitis C virus. Moreover, exophillic acid showed a pan-genotypic anti-HBV effect. We thus identified the anti-HBV/HDV activity of exophillic acid and revealed its mode of action. Exophillic acid is expected to be a potential new lead compound for the development of antiviral agents.

Induction of neutralizing antibodies against hepatitis C virus by a subviral particle-based DNA vaccine.

Direct-acting antivirals (DAAs) have been introduced for the treatment of hepatitis C virus (HCV); however, there is still no available vaccine for preventing HCV infection. We previously reported on a Japanese encephalitis virus (JEV) subviral particle (SVP)-based vaccine with insertion of the HCV E2 neutralization epitope at three positions (SVP-E2/Tri). In this study, we utilized this SVP platform for DNA immunization. In addition, we explored further sites permitting the insertion of HCV epitopes without impairing viral assembly and secretion to elicit higher titers of neutralizing antibodies, and we identified three new positions for foreign epitope insertion. Successful secretion of SVPs with the insertion of HCV epitopes at five positions (SVP-E2/Pent) was seen from transfected cells. Compared to SVP-E2/Tri, sera from mice immunized with the plasmid expressing SVP-E2/Pent showed more neutralization activity against HCV, and less neutralization activity against JEV, suggesting that the additional insertion of HCV epitopes contributed to the induction of antibodies against the inserted peptide, whereas the neutralizing epitopes against JEV were disrupted. This study provides a potentially effective novel DNA vaccine platform.

IFN-γ‒Induced APOBEC3B Contributes to Merkel Cell Polyomavirus Genome Mutagenesis in Merkel Cell Carcinoma.

Merkel cell polyomavirus (MCPyV) is the causative agent of an aggressive skin tumor, Merkel cell carcinoma. The viral genome is integrated into the tumor genome and harbors nonsense mutations in the helicase domain of large T antigen. However, the molecular mechanisms by which the viral genome gains the tumor-specific mutations remain to be elucidated. Focusing on host cytosine deaminases APOBEC3s, we find that A3A, A3B, or A3G introduces A3-specific mutations into episomal MCPyV genomes in MCPyV-replicating 293-derivative cells. Sequence analysis of MCPyV genomes retrieved from the NCBI database revealed a decrease of TpC dinucleotide, a preferred target for A3A and A3B, in the 3'-region of the large T antigen‒coding sequence. The viral DNA isolated from tumors contained mutated cytosines, with a remarkable bias toward TpC dinucleotide. Analysis of publicly available microarray data showed that expression of IFN-γ and cytotoxic T lymphocyte markers was positively correlated with the A3A, A3B, and A3G levels in MCPyV-positive but not in MCPyV-negative tumors. Finally, IFN-γ treatment induced A3B and A3G expression in the MCPyV-positive Merkel cell carcinoma cell line MS-1. These results suggest that the IFN-γ-A3B axis plays pivotal roles in evolutionally shaping MCPyV genomic sequences and in generating tumor-specific large T antigen mutations during development of Merkel cell carcinoma.

Development of an intervention system for linkage-to-care and follow-up for hepatitis B and C virus carriers.

BACKGROUND: Poor connections in the cascade of viral hepatitis care have been discussed around the world. In 2011 in Japan, 500,000 to 1.25 million hepatitis B and C virus carriers needed to consult with hepatologists, so linkage-to-care (LTC) needs to be promoted. Therefore, in this study, to improve LTC and care-seeking behaviors, we attempted to establish a community-based intervention system and evaluate its effectiveness by analyzing behavior modifications. METHODS: In a model city, Okazaki (population: 387,887 as of 2019), LTC was encouraged among HBV and HCV carriers by annually mailed brochures, and their care-seeking behaviors were followed up through questionnaires for 8 years (2012-2019). Their behavior modifications and demographic characteristics were analyzed anonymously in cooperation with community health workers, hepatologists, and researchers. RESULTS: Through regional HBsAg and anti-HCV screening, 333 HBV and 208 HCV carriers were identified. Before the intervention, only 34.7% (25/72) of HBV- and 34.3% (24/70) of HCV-positive individuals had consulted with hepatologists. However, in 2019, after the intervention, these proportions increased to 79.8% (91/114) and 91.2% (52/57), respectively. Access to outpatient care and treatment uptake also continuously improved. However, individuals over 70 years of age were significantly less likely to engage in care-seeking behaviors (p < 0.05), and significantly fewer HCV-positive females received treatment (p = 0.03). CONCLUSIONS: A paper-based reiterative intervention encouraging LTC and follow-up successfully improved the care-seeking behaviors of hepatitis virus-positive individuals and enabled their behavior modifications to be monitored. Further trials are required to advance the system by age- and gender-specific interventions.

NTCP Oligomerization Occurs Downstream of the NTCP-EGFR Interaction during Hepatitis B Virus Internalization.

Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and noncompetitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, coimmunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affects the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms. IMPORTANCE Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.

Prolonged Gut Dysbiosis and Fecal Excretion of Hepatitis A Virus in Patients Infected with Human Immunodeficiency Virus.

Hepatitis A virus (HAV) causes transient acute infection, and little is known of viral shedding via the duodenum and into the intestinal environment, including the gut microbiome, from the period of infection until after the recovery of symptoms. Therefore, in this study, we aimed to comprehensively observe the amount of virus excreted into the intestinal tract, the changes in the intestinal microbiome, and the level of inflammation during the healing process. We used blood and stool specimens from patients with human immunodeficiency virus who were infected with HAV during the HAV outbreak in Japan in 2018. Moreover, we observed changes in fecal HAV RNA and quantified the plasma cytokine level and gut microbiome by 16S rRNA analysis from clinical onset to at least 6 months after healing. HAV was detected from clinical onset up to a period of more than 150 days. Immediately after infection, many pro-inflammatory cytokines were elicited, and some cytokines showed different behaviors. The intestinal microbiome changed significantly after infection (dysbiosis), and the dysbiosis continued for a long time after healing. These observations suggest that the immunocompromised state is associated with prolonged viral shedding into the intestinal tract and delayed recovery of the intestinal environment.

Cellular OCIAD2 protein is a proviral factor for hepatitis C virus replication.

Hepatitis C virus (HCV) nonstructural protein NS4B is necessary for HCV replication. Our previous research found that NS4B-associated cellular proteins PREB and Surfeit 4 are involved in HCV replication. However, the molecular mechanism of HCV replication is not fully understood. Here we identified cellular ovarian cancer immunoreactive antigen domain containing 2 (OCIAD2) protein as a novel NS4B-associated HCV host cofactor by screening with small interfering RNA. Knockdown of OCIAD2 reduced significantly the HCV replication in a dose-dependent and genotype-independent manner. Further research showed that OCIAD2 was recruited into the HCV RNA replication complex by the interaction with NS4B. Interestingly, HCV replication induced OCIAD2 expression. In turn, overexpression of wild OCIAD2 also promoted virus replication whereas that of OCIAD2 mutant lacking the ability to bind NS4B exerted no effect on HCV replication. We also examined whether OCIAD2 interacted with other proteins participating in the HCV RNA replication complex including viral proteins NS5A, NS5B, and cellular proteins PREB, Surfeit 4. The results showed that OCIAD2 interacted with PREB and NS5A, but not NS5B or Surfeit 4. Our findings provide new insights into the function of OCIAD2 and HCV replication mechanism.

Identification of natural compounds extracted from crude drugs as novel inhibitors of hepatitis C virus.

Natural product-derived crude drugs are expected to yield an abundance of new drugs to treat infectious diseases. Hepatitis C virus (HCV) is an oncogenic virus that significantly impacts public health. In this study, we sought to identify anti-HCV compounds in extracts of natural products. A total of 110 natural compounds extracted from several herbal medicine plants were examined for antiviral activity against HCV. Using a Huh7-mCherry-NLS-IPS reporter system for HCV infection, we first performed a rapid screening for anti-HCV compounds extracted from crude drugs. The compounds threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-butoxypropan-1-ol (#106) and medioresinol (#110), which were extracted from Crataegus cuneate, exhibited anti-HCV activity and significantly inhibited HCV production in a dose-dependent manner. Analyses using HCV pseudoparticle and subgenomic replicon systems indicated that compounds #106 and #110 specifically inhibit HCV RNA replication but not viral entry or translation. Interestingly, compound #106 also inhibited the replication and production of hepatitis A virus. Our findings suggest that C. cuneate is a new source for novel anti-hepatitis virus drug development.

Oncogenic transcriptomic profile is sustained in the liver after the eradication of the hepatitis C virus.

Hepatocellular carcinoma (HCC) developing after hepatitis C virus (HCV) eradication is a serious clinical concern. However, molecular basis for the hepatocarcinogenesis after sustained virologic response (SVR) remains unclear. In this study, we aimed to unveil the transcriptomic profile of post-SVR liver tissues and explore the molecules associated with post-SVR carcinogenesis. We analysed 90 RNA sequencing datasets, consisting of non-cancerous liver tissues including 20 post-SVR, 40 HCV-positive and 7 normal livers, along with Huh7 cell line specimens before and after HCV infection and eradication. Comparative analysis demonstrated that cell cycle- and mitochondrial function-associated pathways were altered only in HCV-positive non-cancerous liver tissues, whereas some cancer-related pathways were up-regulated in the non-cancerous liver tissues of both post-SVR and HCV-positive cases. The persistent up-regulation of carcinogenesis-associated gene clusters after viral clearance was reconfirmed through in vitro experiments, of which, CYR61, associated with liver fibrosis and carcinogenesis in several cancer types, was the top enriched gene and co-expressed with cell proliferation-associated gene modules. To evaluate whether this molecule could be a predictor of hepatocarcinogenesis after cure of HCV infection, we also examined 127 sera from independent HCV-positive cohorts treated with direct-acting antivirals (DAAs), including 60 post-SVR-HCC patients, and found that the elevated serum Cyr61 was significantly associated with early carcinogenesis after receiving DAA therapy. In conclusion, some oncogenic transcriptomic profiles are sustained in liver tissues after HCV eradication, which might be a molecular basis for the liver cancer development even after viral clearance. Among them, up-regulated CYR61 could be a possible biomarker for post-SVR-HCC.

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.

MCPIP1 reduces HBV-RNA by targeting its epsilon structure.

Hepatitis B virus (HBV) is the major causative factor of chronic viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. We previously demonstrated that a proinflammatory cytokine IL-1ß reduced the level of HBV RNA. However, the mechanism underlying IL-1ß-mediated viral RNA reduction remains incompletely understood. In this study, we report that immune regulator Monocyte chemotactic protein-1-induced protein 1 (MCPIP1) can reduce HBV RNA in hepatocytes. MCPIP1 expression level was higher in the liver tissue of HBV-infected patients and mice. Overexpression of MCPIP1 decreased HBV RNA, whereas ablating MCPIP1 in vitro enhanced HBV production. The domains responsible for RNase activity or oligomerization, were required for MCPIP1-mediated viral RNA reduction. The epsilon structure of HBV RNA was important for its antiviral activity and cleaved by MCPIP1 in the cell-free system. Lastly, knocking out MCPIP1 attenuated the anti-HBV effect of IL-1ß, suggesting that MCPIP1 is required for IL-1ß-mediated HBV RNA reduction. Overall, these results suggest that MCPIP1 may be involved in the antiviral effect downstream of IL-1ß.

Sphingomyelin Is Essential for the Structure and Function of the Double-Membrane Vesicles in Hepatitis C Virus RNA Replication Factories.

Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses.IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.

EBV-LMP1 induces APOBEC3s and mitochondrial DNA hypermutation in nasopharyngeal cancer.

An Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a principal oncogene that plays a pivotal role in EBV-associated malignant tumors including nasopharyngeal cancer (NPC). Recent genomic landscape studies revealed that NPC also contained many genomic mutations, suggesting the role of LMP1 as a driver gene for the induction of these genomic mutations. Nonetheless, its exact mechanism has not been investigated. In this study, we report that LMP1 alters the expression profile of APOBEC3s(A3s), host deaminases that introduce consecutive C-to-U mutations (hypermutation). In vitro, LMP1 induces APOBEC3B (A3B) and 3F(A3F), in a nasopharyngeal cell line, AdAH. Overexpression of LMP1, A3B, or A3F induces mtDNA hypermutation, which is also detectable from NPC specimens. Expression of LMP1 and A3B in NPC was correlated with neck metastasis. These results provide evidence as to which LMP1 induces A3s and mtDNA hypermutation, and how LMP1 facilitates metastasis is also discussed.