Antibiotic-induced gut bacteria depletion has no effect on HBV replication in HBV immune tolerance mouse model

Commensal microbiota is closely related to Hepatitis B virus(HBV)infection.Gut bacteria maturation accelerates HBV immune clearance in hydrodynamic injection(HDI)HBV mouse model.However,the effect of gut bacteria on HBV replication in recombinant adeno-associated virus(AAV)-HBV mouse model with immune tolerance remains obscure.We aim to investigate its role on HBV replication in AAV-HBV mouse model.C57BL/6 mice were administrated with broad-spectrum antibiotic mixtures(ABX)to deplete gut bacteria and intravenously injected with AAV-HBV to establish persistent HBV replication.Gut microbiota community was analyzed by fecal qPCR assay and 16S ribosomal RNA(rRNA)gene sequencing.HBV replication markers in blood and liver were determined by ELISA,qPCR assay and Western blot at indicated time points.Immune response in AAV-HBV mouse model was activated through HDI of HBV plasmid or poly(I:C)and then detected by quantifying the percentage of IFN-γ^(+)/CD8^(+)T cells in the spleen via flow cytometry as well as the splenic IFN-γmRNA level via qPCR assay.We found that antibiotic exposure remarkably decreased gut bacteria abundance and diversity.Antibiotic treatment failed to alter the levels of serological HBV antigens,intrahepatic HBV RNA transcripts and HBc protein in AAV-HBV mouse model,but contributed to HBsAg increase after breaking of immune tolerance.Overall,our data uncovered that antibiotic-induced gut bacteria depletion has no effect on HBV replication in immune tolerant AAV-HBV mouse model,providing new thoughts for elucidating the correlation between gut bacteria dysbiosis by antibiotic abuse and clinical chronic HBV infection.

Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B

Hepatitis B virus(HBV) infection is one of the major problems that threatens global health. There have been many studies on HBV, but the relationship between HBV and host factors is largely unexplored and more studies are needed to clarify these interactions. Filamin B is an actin-binding protein that acts as a cytoskeleton protein, and it is involved in cell development and several signaling pathways. In this study, we showed that filamin B interacted with HBV core protein,and the interaction promoted HBV replication. The interaction between filamin B and core protein was observed in HEK293T, Huh7 and HepG2 cell lines by co-immunoprecipitation and co-localization immnofluoresence. Overexpression of filamin B increased the levels of HBV total RNAs and pre-genome RNA(pg RNA), and improved the secretion level of hepatitis B surface antigen(HBsAg) and hepatitis B e antigen(HBeAg). In contrast, filamin B knockdown inhibited HBV replication, decreased the level of HBV total RNAs and pgRNA, and reduced the secretion level of HBsAg and HBeAg. In addition, we found that filamin B and core protein may interact with each other via four blocks of argentine residues at the C-terminus of core protein. In conclusion, we identify filamin B as a novel host factor that can interact with core protein to promote HBV replication in hepatocytes. Our study provides new insights into the relationship between HBV and host factors and may provide new strategies for the treatment of HBV infection.

Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression

Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The ex- pression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibi- tion on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

The role of micro RNAs in hepatocyte metabolism and hepatitis B virus replication

Though efficient vaccines against hepatitis B virus(HBV) and antiviral therapies are available,chronic HBV infection is still a global health problem. The process of HBV infection and HBV life cycle are extensively studied in last decades, however, the mechanisms of HBV-induced alterations of host cell metabolisms and host factors involved in modulating of viral replication are not fully understood. Thus, it is an important issue to examine these specific HBV-host interactions for development of novel strategies for antiviral therapies. Recently, microRNAs(miRNAs), a class of post-transcriptional regulatory small RNA, seem to be the relevant fine tuning factors of various cellular activities and pathways, including cell growth, metabolism, and viral replication. In this review, we summarize the up to date knowledge concerning the virus-host interactions and emphasizing on the role of miRNAs in regulation of HBV replication and host cell metabolism.

Hepatitis B virus evades immune recognition via RNA adenosine deaminase ADAR1-mediated viral RNA editing in hepatocytes

HBV is considered as a“stealth”virus that does not invoke interferon(IFN)responses;however,the mechanisms by which HBV bypasses innate immune recognition are poorly understood.In this study,we identified adenosine deaminases acting on RNA 1(ADAR1),which is a key factor in HBV evasion from IFN responses in hepatocytes.Mechanically,ADAR1 interacted with HBV RNAs and deaminated adenosine(A)to generate inosine(I),which disrupted host immune recognition and thus promoted HBV replication.Loss of ADAR1 or its deficient deaminase activity promoted IFN responses and inhibited HBV replication in hepatocytes,and blocking the IFN signaling pathways released the inhibition of HBV replication caused by ADAR1 deficiency.Notably,the HBV X protein(HBx)transcriptionally promoted ADAR1 expression to increase the threshold required to trigger intrinsic immune activation,which in turn enhanced HBV escape from immune recognition,leading to persistent infection.Supplementation with 8-azaadenosine,an ADAR1 inhibitor,efficiently enhanced liver immune activation to promote HBV clearance in vivo and in vitro.Taken together,our results delineate a molecular mechanism by which HBx promotes ADAR1-derived HBV immune escape and suggest a targeted therapeutic intervention for HBV infection.