Masamichi Muramatsu

Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

Hepatitis B virus (HBV) is one of the major etiological pathogens for liver cirrhosis and hepatocellular carcinoma. Chronic HBV infection is a key factor in these severe liver diseases. During infection, HBV forms a nuclear viral episome in the form of covalently closed circular DNA (cccDNA). Current therapies are not able to efficiently eliminate cccDNA from infected hepatocytes. cccDNA is a master template for viral replication that is formed by the conversion of its precursor, relaxed circular DNA (rcDNA). However, the host factors critical for cccDNA formation remain to be determined. Here, we assessed whether one potential host factor, flap structure-specific endonuclease 1 (FEN1), is involved in cleavage of the flap-like structure in rcDNA. In a cell culture HBV model (Hep38.7-Tet), expression and activity of FEN1 were reduced by siRNA, shRNA, CRISPR/Cas9-mediated genome editing, and a FEN1 inhibitor. These reductions in FEN1 expression and activity did not affect nucleocapsid DNA (NC-DNA) production, but did reduce cccDNA levels in Hep38.7-Tet cells. Exogenous overexpression of wild-type FEN1 rescued the reduced cccDNA production in FEN1-depleted Hep38.7-Tet cells. Anti-FEN1 immunoprecipitation revealed the binding of FEN1 to HBV DNA. An in vitro FEN activity assay demonstrated cleavage of 5′-flap from a synthesized HBV DNA substrate. Furthermore, cccDNA was generated in vitro when purified rcDNA was incubated with recombinant FEN1, DNA polymerase, and DNA ligase. Importantly, FEN1 was required for the in vitro cccDNA formation assay. These results demonstrate that FEN1 is involved in HBV cccDNA formation in cell culture system, and that FEN1, DNA polymerase, and ligase activities are sufficient to convert rcDNA into cccDNA in vitro.

A Single Adaptive Mutation in Sodium Taurocholate Cotransporting Polypeptide Induced by Hepadnaviruses Determines Virus Species-specificity

Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates from fish to human. Hepadnaviruses and their hosts have a long history of acquiring adaptive mutations. However, there are no reports providing direct molecular evidence for such a coevolutionary “arms race” between hepadnaviruses and their hosts. Here, we present evidence suggesting the adaptive evolution of the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, has been influenced by virus infection. Evolutionary analysis of the NTCP-encoding genes from 20 mammals showed that most NTCP residues are highly conserved among species, exhibiting evolution under negative selection (dN/dS < 1); this observation implies that the evolution of NTCP is restricted by maintaining its original protein function. However, 0.7 % of NTCP amino acid (aa) residues exhibit rapid evolution under positive selection (dN/dS > 1). Notably, a substitution at aa 158, a positively selected residue, converting the human NTCP to a monkey-type sequence abrogated the capacity to support HBV infection; conversely, a substitution at this residue converting the monkey Ntcp to the human sequence was sufficient to confer HBV susceptibility. Together, these observations suggested that positive selection at aa 158 was induced by virus infection. Moreover, the aa 158 sequence determined attachment of the HBV envelope protein to host cell, demonstrating the mechanism whereby HBV infection would create positive selection at this residue in NTCP. In summary, we provide the first evidence in agreement with the function of hepadnavirus as a driver for inducing an adaptive mutation in host receptor. Importance Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates, with a long infectious history (hundreds of millions of years). Such a long history generally allows adaptive mutations in hosts to escape from infection, while simultaneously allowing adaptive mutations in viruses to overcome host barriers. However, there is no published molecular evidence for such a coevolutionary “arms race” between hepadnaviruses and hosts. In the present study, we performed coevolutionary phylogenetic analysis between hepadnaviruses and the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, combined with virological experimental assays for investigating the biological significance of NTCP sequence variation. Our data provide the first molecular evidences supporting that HBV-related hepadnaviruses drive adaptive evolution in the NTCP sequence, including a mechanistic explanation of how NTCP mutations determine host viral susceptibility. Our novel insights enhance our understanding of how hepadnaviruses evolved with their hosts, permitting the acquisition of strong species-specificity.

Keratinocyte differentiation induces APOBEC3A, 3B, and mitochondrial DNA hypermutation

Mitochondrial DNA (mtDNA) mutations are found in many types of cancers and suspected to be involved in carcinogenesis, although the mechanism has not been elucidated. In this study, we report that consecutive C-to-T mutations (hypermutations), a unique feature of mutations induced by APOBECs, are found in mtDNA from cervical dysplasia and oropharyngeal cancers. In vitro, we found that APOBEC3A (A3A) and 3B (A3B) expression, as well as mtDNA hypermutation, were induced in a cervical dysplastic cell line W12 when cultured in a differentiating condition. The ectopic expression of A3A or A3B was sufficient to hypermutate mtDNA. Fractionation of W12 cell lysates and immunocytochemical analysis revealed that A3A and A3B could be contained in mitochondrion. These results suggest that mtDNA hypermutation is induced upon keratinocyte differentiation, and shed light on its molecular mechanism, which involves A3s. The possible involvement of mtDNA hypermutations in carcinogenesis is also discussed.

Evaluation of in vitro screening and diagnostic kits for hepatitis C virus infection

BACKGROUNDnTo detect infection by hepatitis C virus (HCV), a reliable kit with high sensitivity and specificity is indispensable. Detection kits for anti-HCV antibodies (anti-HCV) are used for screening, and quantification kits for HCV RNA and core antigen are used for definite diagnosis of HCV infection.nnnOBJECTIVESnWe evaluated the performance of these kits using International Standards and a regional reference panel with HCV negative and positive specimens.nnnSTUDY DESIGNnIn vitro diagnostic kits (10 anti-HCV, two HCV RNA, and three HCV core antigen) were included.nnnRESULTSnNearly all specimens in the regional reference panel were correctly identified by all anti-HCV detection kits (one false-positive was observed in one kit). Both HCV RNA quantification kits also correctly identified and quantified HCV RNA titers, without genotype-specific differences. Among the HCV core antigen kits, International Standard values were inconsistent. The sensitivities of these kits were insufficient to detect HCV in positive specimens in the regional reference panel.nnnCONCLUSIONSnIn vitro diagnostic kits assessing anti-HCV and HCV RNA have sufficient sensitivities and specificities to screen and detect HCV infection. However, HCV core antigen quantification kits have some limitations in their sensitivities and consistencies for diagnosis of HCV infection. Quality control with International Standards and a regional reference panel is important to maintain the performances of diagnostic kits for HCV infection and to verify the clinical reliability of these kits.

IL-1β/ATF3-mediated induction of Ski2 expression enhances hepatitis B virus x mRNA degradation

Hepatitis B virus (HBV) -x protein is a transcriptional regulator required for the HBV life cycle. HBx also induces complications in the host such as hepatocellular carcinoma. We previously showed that HBx mRNA is degraded by the Ski2/RNA exosome complex. In the present study, we report the regulation of this system through the control of Ski2 expression. We identified interleukin (IL) -1β as an inducer of expression from the Ski2 promoter. IL-1β induced the expression of ATF3 transcription factor, which in turn binds to cyclic AMP-responsive element sequence in the Ski2 promoter and is responsible for Ski2 promoter induction by IL-1β. We previously reported that Ski2 expression increases HBx mRNA degradation; consistent with those data, we showed here that HBx mRNA is degraded in response to IL-1β treatment. Interestingly, HBx also significantly induced Ski2 expression. To our knowledge, this is the first report to show activation of the Ski2/RNA exosome complex by both the host and HBV. Understanding the regulation of the Ski2/RNA exosome system is expected to facilitate prevention of HBx-mediated complications through targeting the posttranscriptional degradation of HBx mRNA; and will also help shedding a light on the role of RNA decay systems in inflammation.

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.

Evaluation of antiviral effects of novel NS5A inhibitors in hepatitis C virus cell culture system with full-genome infectious clones

Abstract Nonstructural protein 5A (NS5A) inhibitors of hepatitis C virus (HCV) are known to have potent anti‐viral effects; however, these inhibitors have limited activities on strains with resistant‐associated substitutions or non‐genotype 1 strains. To overcome these shortcomings, novel NS5A inhibitors have been developed and approved for clinical application. The aim of this study was to evaluate the anti‐viral effect of novel NS5A inhibitors (derivatives of odalasvir) on HCV genotype 2 strains in a cell culture system. Chimeric JFH‐1 viruses replaced with NS5A of genotypes 1 and 2 were utilized to assess the genotype‐specific potencies of NS5A inhibitors. We also examined full‐genome infectious clones of JFH‐1, J6cc, and J8cc to confirm the effects of NS5A inhibitors on genotype 2 strains. All chimeric viruses were capable of replication at similar levels in cell culture. We examined the anti‐viral effects of derivatives of the novel NS5A inhibitor and compared with the first‐generation NS5A inhibitor, daclatasvir (DCV). These compounds inhibited replication of chimeric JFH‐1 viruses with NS5A of genotypes 1 and 2 at low concentrations in comparison with DCV. The EC50 values of J6cc and J8cc to these compounds were more than 100‐fold lower than that of DCV. By long‐term culture in the presence of these compounds, we obtained highly resistant variants and identified the responsible substitutions. In conclusion, novel NS5A inhibitors displayed improved potency against HCV genotype 2 strains compared with DCV. However, the activity of these compounds was impaired by emerging resistance‐associated substitutions. HighlightsThe novel NS5A inhibitor displayed improved potency against HCV genotype 2 strains in compared with the first‐generation.The resistance viruses to the novel NS5A inhibitor were emerged after long‐term culture with the compounds.The responsible resistance‐associated substitutions to the novel NS5A inhibitor were identified in the NS5A region.The emergence of resistant viruses impairs the activity of the novel NS5A inhibitor.