Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing

Abstract

Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expression. By analyzing HBV infection cycle steps, we identified a hitherto unknown membrane targeting module in the highly basic C-terminal domain of the NC-forming core protein. Rab33B inactivation reduced core membrane association, suggesting that membrane platforms participate in HBV assembly reactions. Biochemical and immunofluorescence analyses provided further hints that the viral core, rather than the envelope, is the main target for Rab33B intervention. Rab33B-deficiency reduced core protein levels without affecting viral transcription and hampered core/NC sorting to envelope-positive, intracellular compartments. Together, these results indicate that Rab33B is an important player in intracellular HBV trafficking events, guiding core transport to NC assembly sites and/or NC transport to budding sites.

Small Interfering RNA Screening for the Small GTPase Rab Proteins Identifies Rab5B as a Major Regulator of Hepatitis B Virus Production

“…If another screening method detecting only HBV particles with envelope using anti-HBs antibody were used, the result might be different. A previous study showed that Rab33B controls HBV assembly by regulating formation and/or transport of nucleocapsids (29). The study showed that knockdown of Rab33B reduced the amount of Dane particles, whereas the screening of the present study indicated that Rab33B knockdown did not affect the amount of HBV DNA but increased HBsAg in the culture supernatant.…”

Hepatitis B Virus Subverts the Autophagy Elongation Complex Atg5-12/16L1 and Does Not Require Atg8/LC3 Lipidation for Viral Maturation

“…We thus hypothesize that HBV may co-opt the Atg5-12/16L1 conjugate in a phagophore-tethered state. As supportive evidence, we recently showed that the Rab33B GTPase, an effector of the Atg5-12/16L1 conjugate, is a positive regulator of HBV assembly, guiding NC assembly/stability (26,50). Like all Rab proteins, Rab33B exerts its function in a membrane-tethered form and had been implicated in the regulation of autophagy via fusion of Rab33B-decorated, Golgi apparatus-derived vesicles with the Atg5-12/16L1 complex in order to supply lipids for the expanding phagophore (50).…”

“…Our results pose the question of whether HBV may use the Atg5-12/16L1 conjugate in a noncanonical manner (i.e., as a distinct function mediated by individual Atg proteins) or a canonical (i.e., autophagy-like) manner, with the latter involving autophagic membranes. Previously, we showed that the HBV core is able to associate with intracellular membranes (26), albeit the functional relevance is yet unknown. In the case of many other viruses, like retroviruses, membrane targeting of viral components helps to concentrate and drive the assembly reaction (47).…”

“…The faulty trafficking of core in Atg12-KD cells prompted us to study the core-membrane association. Previously, we identified a membrane-targeting site within the basic C terminus of core that may confer its targeting to budding sites (26). For membrane flotation analyses, HuH-7 cells were treated with control siRNA duplexes, the Atg5-specific siRNA, or a mixture of Atg5-, Atg12-, and Atg16L1-specific siRNAs prior to transfection with an expression plasmid carrying only core (pCore).…”

“…Originally, we interpreted the necessity of Rab33B and its effector as a prosurvival mechanism specific for HBV naked capsids (25). By expanding these analyses, we recently showed that Rab33B is likewise crucial for the production of HBV particles by guiding core transport to NC assembly sites and/or NC transport to budding sites (26). Inspired by these observations, we here investigated the roles of autophagy pathway steps in HBV morphogenesis, with a special emphasis on the Atg5-12/16L1 elongation complex.…”