Rab33B and its autophagic Atg5/12/16L1 effector assist in hepatitis B virus naked capsid formation and release

Abstract

SummaryHepatitis B virus morphogenesis is accompanied by the production and release of non-enveloped capsids/nucleocapsids. Capsid particles are formed inside the cell cytosol by multimerization of core protein subunits and ultimately exported in an uncommon coatless state. Here, we investigated potential roles of Rab GTPases in capsid formation and trafficking by using RNA interference and overexpression studies. Naked capsid release does not require functions of the endosomeassociated Rab5, Rab7 and Rab27 proteins, but depends on functional Rab33B, a GTPase participating in autophagosome formation via interaction with the Atg5-Atg12/Atg16L1 complex. During capsid formation, Rab33B acts in conjunction with its effector, as silencing of Atg5, Atg12 and Atg16L1 also impaired capsid egress. Analysis of capsid maturation steps revealed that Rab33B and Atg5/12/16L1 are required for proper particle assembly and/or stability. In support, the capsid protein was found to interact with Atg5 and colocalize with Atg5/12/16L1, implicating that autophagy pathway functions are involved in capsid biogenesis. However, a complete and functional autophagy pathway is dispensable for capsid release, as judged by knockdown analysis of Atg8/LC3 family members and pharmaceutical ablation of canonical autophagy. Experiments aimed at analysing the capsid release-stimulating activity of the Alix protein provide further evidence for a link between capsid formation and autophagy.

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

“…Thereby, however, we obtained no hints for a direct physical interaction between core and Rab33B. Rather, a link may be established by the autophagic Atg5/12/16 complex, a known effector of active Rab33B, that we previously identified as a core-interacting partner [18]. In support, the depletion of Atg5/12/16 likewise prevents core membrane association [31].…”

“…Due to the Rab33B RNAi-induced downregulation of core in HBV-replicating cells, we failed to unequivocally analyze the distribution of core under these conditions (data not shown). For less understood reasons, the Rab33B RNAi-induced core protein reduction is less pronounced upon sole expression of core [18] (see also the discussion). We therefore studied membrane association of core synthesized in the absence of the other HBV proteins, especially of the pre-core protein.…”

“…The pathway of release of naked capsids, devoid of a membrane coat, is independent of ESCRT functions but depends on Alix [16], a multifunctional protein with key roles in membrane biology. In addition, this pathway is assisted by the cellular Rab33B GTPase [18]. …”

“…Previously, we showed that HBV naked capsid formation and egress require Rab33B in conjunction with its Atg5/12/16L1 effector, while a complete and functional autophagy pathway is dispensable [18]. Although the precise mechanistic insights are still lacking, Rab33B and Atg5/12/16L1 together with autophagic membrane platforms may provide a scaffold for HBV naked capsid maturation and assembly.…”

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

“…However, discrimination of HBeAg from the cccDNA-independently produced core protein is problematic owing to their largely identical amino acid sequences; furthermore, not only HBeAg but also non-enveloped capsids are found in the culture supernatant [79]; this holds also for DHBV (Dörnbrack, K.; Costa, C.; Nassal, M.; unpublished data). To overcome this problem, others [80] and we (Dörnbrack, K.; Costa, C.; Verrier, E.; Nassal, M; unpublished data) have engineered coding sequences for the small hemagglutinine (HA) tag into the precore regions of HBV and/or DHBV such that only HBeAg becomes HA-tagged and the negative impact on replication via the precore-overlapping ε sequence remains limited.…”