Masamune, Atsushi

Currently, the hepatocellular trafficking pathways that are used by the hepatitis B virus (HBV) during viral infection and shedding are poorly defined. It is known that the HBV uses late endosomal and multivesicular body (MVB) compartments for assembly and release. The intraluminal vesicles (ILVs) generated within MVBs have also been implicated in the late synthesis stages of a variety of pathogenic viruses. We recently observed that the HBV within infected hepatocytes appears to associate with the tetraspanin protein CD63, known to be a prominent and essential component of ILVs. Immunofluorescence microscopy of HBV‐expressing cells showed that CD63 colocalized with HBV proteins (large hepatitis B surface antigens [LHBs] and hepatitis B core) and labeled an exceptionally large number of secreted extracellular vesicles of uniform size. Small interfering RNA (siRNA)–mediated depletion of CD63 induced a substantial accumulation of intracellular LHBs protein but did not alter the levels of either intracellular or extracellular HBV DNA, nor pregenomic RNA. Consistent with these findings, we found that markedly less LHBs protein was associated with the released HBV particles from CD63 siRNA‐treated cells. Importantly, the HBV viral particles that were shed from CD63‐depleted cells were substantially less infective than those collected from control cells with normal CD63 levels. Conclusion: These findings implicate the tetraspanin protein CD63 as a marker and an important component in the formation and release of infectious HBV particles.

HBV infection is a worldwide health problem, but the mechanisms of how HBV utilizes cellular machinery for its life cycle are poorly understood. In particular, it has been unclear how the viral components and virions are transported among the organelles. The HBV budding site has been reported to be the ER or MVB, but it has not been clearly determined. In this study, siRNA-based screening of Rab proteins using HBV-expressing cells showed that Rab5B, one of the Rab5 isoforms, has important roles in late steps of the HBV life cycle. Although Rab5 is known to work on early endosomes, this study showed that Rab5B plays a role in the transport of LHBs between the ER and MVB. Furthermore, it affects the transcription of LHBs. This is the first report on the mechanisms of HBV envelope protein transport among the organelles, and the results provide important insights into the therapeutic control of HBV infection.