ATP5B Is an Essential Factor for Hepatitis B Virus Entry.

Elucidation of the factors responsible for hepatitis B virus (HBV) is extremely important in order to understand the viral life cycle and pathogenesis, and thereby explore potential anti-HBV drugs. The recent determination that sodium taurocholate co-transporting peptide (NTCP) is an essential molecule for the HBV entry into cells led to the development of an HBV infection system in vitro using a human hepatocellular carcinoma (HCC) cell line expressing NTCP; however, the precise mechanism of HBV entry is still largely unknown, and thus it may be necessary to elucidate all the molecules involved. Here, we identified ATP5B as another essential factor for HBV entry. ATP5B was expressed on the cell surface of the HCC cell lines and bound with myristoylated but not with non-myristoylated preS1 2-47, which supported the notion that ATP5B is involved in the HBV entry process. Knockdown of ATP5B in NTCP-expressing HepG2 cells, which allowed HBV infection, reduced HBV infectivity with less cccDNA formation. Taken together, these results strongly suggested that ATP5B is an essential factor for HBV entry into the cells.

Analysis of the Physicochemical Properties, Replication and Pathophysiology of a Massively Glycosylated Hepatitis B Virus HBsAg Escape Mutant.

Mutations in HBsAg, the surface antigen of the hepatitis B virus (HBV), might affect the serum HBV DNA level of HBV-infected patients, since the reverse transcriptase (RT) domain of HBV polymerase overlaps with the HBsAg-coding region. We previously identified a diagnostic escape mutant (W3S) HBV that produces massively glycosylated HBsAg. In this study, we constructed an HBV-producing vector that expresses W3S HBs (pHB-W3S) along with a wild-type HBV-producing plasmid (pHB-WT) in order to analyze the physicochemical properties, replication, and antiviral drug response of the mutant. Transfection of either pHB-WT or W3S into HepG2 cells yielded similar CsCl density profiles and eAg expression, as did transfection of a glycosylation defective mutant, pHB-W3S (N146G), in which a glycosylation site at the 146aa asparagine (N) site of HBs was mutated to glycine (G). Virion secretion, however, seemed to be severely impaired in cases of pHB-W3S and pHB-W3S (N146G), compared with pHB-WT, as determined by qPCR and Southern blot analysis. Furthermore, inhibition of glycosylation using tunicamycinTM on wild-type HBV production also reduced the virion secretion. These results suggested that the HBV core and Dane particle could be formed either by massively glycosylated or glycosylation-defective HBsAg, but reduced and/or almost completely blocked the virion secretion efficiency, indicating that balanced glycosylation of HBsAg is required for efficient release of HBV, and mutations inducing an imbalanced glycosylation of HBs would cause the virion to become stuck in the cells, which might be associated with various pathogeneses due to HBV infection.

Chronic Hepatitis B Treatment Strategies Using Polymerase Inhibitor-Based Combination Therapy.

Viral polymerase is an essential enzyme for the amplification of the viral genome and is one of the major targets of antiviral therapies. However, a serious concern to be solved in hepatitis B virus (HBV) infection is the difficulty of eliminating covalently closed circular (ccc) DNA. More recently, therapeutic strategies targeting various stages of the HBV lifecycle have been attempted. Although cccDNA-targeted therapies are attractive, there are still many problems to be overcome, and the development of novel polymerase inhibitors remains an important issue. Interferons and nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are the only therapeutic options currently available for HBV infection. Many studies have reported that the combination of interferons and NRTI causes the loss of hepatitis B surface antigen (HBsAg), which is suggestive of seroconversion. Although NRTIs do not directly target cccDNA, they can strongly reduce the serum viral DNA load and could suppress the recycling step of cccDNA formation, improve liver fibrosis/cirrhosis, and reduce the risk of hepatocellular carcinoma. Here, we review recent studies on combination therapies using polymerase inhibitors and discuss the future directions of therapeutic strategies for HBV infection.

Functional roles of GRP78 in hepatitis B virus infectivity and antigen secretion.

Viruses utilize cellular proteins to mediate their life cycle. However, the hepatitis B virus (HBV) life cycle is still mysterious and remains to be elucidated. Here, GRP78/BiP/HSPA5, a 78 kDa glucose-regulated protein, was identified as a preS2 interacting protein. Pulldown assay showed the interaction of glucose-regulated protein 78 (GRP78) with both the preS2 domain-containing large S and middle S proteins expressed in a human hepatocellular cell line. The immunofluorescence studies revealed that the preS2 colocalized with GRP78. Interestingly, it was found that preS2 specifically bound to the ATPase domain of GRP78. To understand how GRP78 plays a role in HBV infection, stably GRP78-expressing cells were established, which promoted HBV infectivity and replication. In contrast, knockdown of GRP78 changed the HBV antigen secretion but not the viral DNA amplification. Taken together, these results suggest that GRP78 should interact with preS2 via the ATPase domain and modulate both the HBV infectivity and HBV antigen secretion.