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

Previous studies indicated that hepatitis B virus (HBV) stimulates autophagy to favor its production. To understand how HBV co-opts autophagy as a proviral machinery, we studied the roles of key autophagy proteins in HBV-replicating liver cell cultures. RNA interference-mediated silencing of Atg5, Atg12, and Atg16L1, which promote autophagophore expansion and LC3 membrane conjugation, interfered with viral core/nucleocapsid (NC) formation/stability and strongly diminished virus yields. Concomitantly, the core/NC membrane association and their sorting to envelope-positive compartments were perturbed. A close inspection of the HBV/autophagy cross talk revealed that the virus depended on Atg12 covalently conjugated to Atg5. In support of this finding, HBV required the E2-like enzymes Atg10 and Atg3, which catalyze or facilitate Atg5-12 conjugation, respectively. Atg10 and Atg3 knockdowns decreased HBV production, while Atg3 overexpression increased virus yields. Mapping analyses demonstrated that the HBV core protein encountered the Atg5-12/16L1 complex via interaction with the intrinsically disordered region of the Atg12 moiety that is dispensable for autophagy function. The role of Atg12 in HBV replication was confirmed by its incorporation into virions. Although the Atg5-12/16L1 complex and Atg3 are essential for LC3 lipidation and, thus, for autophagosome maturation and closure, HBV propagation did not require LC3. Silencing of LC3B, the most abundant LC3 isoform, did not inhibit but rather augmented virus production. Similar augmenting effects were obtained upon overexpression of a dominant negative mutant of Atg4B that blocked the lipid conjugation of the LC3 isoforms and their GABARAP paralogues. Together, our data indicate that HBV subverts early, nondegradative autophagy components as assembly scaffolds, thereby concurrently avoiding autophagosomal destruction.