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

Abstract

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. Infections with the hepatitis B virus (HBV), an enveloped pararetrovirus, cause about 1 million deaths per year, as current therapies rarely achieve a cure. Understanding the HBV life cycle and concomitant host cell interactions is instrumental to develop new antiviral concepts. Here, we proceeded to dissect the roles of the autophagy machinery in virus propagation. By using RNA interference and overexpression studies in HBV-replicating cell lines, we identified the autophagic Atg5-12/16L1 elongation complex along with Atg10 and Atg3 to be an essential scaffold for HBV nucleocapsid assembly/stability. Deficits in Atg5-12/16L1 and Atg10/Atg3, which normally drive autophagophore membrane expansion, strongly impaired progeny virus yields. HBV gained access to Atg5-12/16L1 via interaction of its core protein with the Atg12 moiety of the complex. In contrast, subsequent autophagosome maturation and closure events were unnecessary for HBV replication, as evidenced by inhibition of Atg8/LC3 conjugation. Interfering with the HBV/Atg12 cross talk may be a tool for virus control.

Hepatitis B subviral envelope particles use the COPII machinery for intracellular transport via selective exploitation of Sec24A and Sec23B

“…Stabilized Sec23B then associates with Sec24, thereby supporting COPII‐mediated autophagosome formation (Jeong et al, ). HBV and even the sole expression of its S protein had been shown to induce autophagy (Doring, Zeyen, Bartusch, & Prange, ; J. Li et al, ; Sir et al, ). Accordingly, it is tempting to speculate that the HBV‐induced autophagy may likewise increase Sec23B levels to benefit SVP egress.…”

“…The mRNA was treated with 5 U of RNase‐free, recombinant DNase I (Hoffmann‐La Roche), and cDNA synthesis was performed by using the qScript cDNA Synthesis Kit (Quanta BioSciences). Quantitative reverse transcription PCR (qRT‐PCR) reactions were performed as described (Doring et al, ). For data analysis, the comparative cycle threshold method ( C T ) was used, and data were reported as the fold change normalized to an endogenous reference gene (β ‐ actin).…”

Manipulation of autophagy by (+) RNA viruses

“…occur in concert at the same sites. Although utilisation of autophagosomes as assembly sites has been recorded for some DNA-viruses e.g., Hepatitis B virus (HBV) [61], experimental data with +RNA RNA viruses are scant. However, atleast for DENV, recent evidence indicates that autophagy might actually assist in assembly of progeny virions, without them serving as replication organelles.…”

Porcine Circovirus Type 2 ORF5 Protein Induces Autophagy to Promote Viral Replication via the PERK-eIF2α-ATF4 and mTOR-ERK1/2-AMPK Signaling Pathways in PK-15 Cells

“…Some viruses can promote their own replication and maturation by converting the Atg5/Atg12/Atg16L complex to LC3, which is the major component of autophagy (Doring et al, 2018).…”