Hepatitis C virus infection increases autophagosome stability by suppressing lysosomal fusion through an Arl8b-dependent mechanism.

ABSTRACT

Autophagy is a conserved cellular process involving intracellular membrane trafficking and degradation. Pathogens, including hepatitis C virus (HCV), often exploit this process to promote their own survival. The aim of this study was to determine the mechanism by which HCV increases steady-state autophagosome numbers while simultaneously inhibiting flux through the autophagic pathway. Using the lysosomal inhibitor bafilomycin A1, we showed that HCV-induced alterations in autophagy result from a blockage of autophagosome degradation rather than an increase in autophagosome generation. In HCV-infected cells, lysosome function was normal, but a tandem RFP-GFP-LC3 failed to reach the lysosome even under conditions that activate autophagy. Autophagosomes and lysosomes isolated from HCV-infected cells were able to fuse with each other normally in vitro, suggesting that the cellular fusion defect resulted from trafficking rather than an inability of vesicles to fuse. Arl8b is an Arf-like GTPase that specifically localizes to lysosomes and plays a role in autophagic flux through its effect on lysosomal positioning. At basal levels, Arl8b was primarily found in a perinuclear localization and co-localized with LC3-positive autophagosomes. HCV infection increased the level of Arl8b 3-fold and redistributed Arl8b to a more diffuse, peripheral pattern that failed to co-localize with LC3. Knockdown of Arl8b in HCV-infected cells restored autophagosome-lysosome fusion and autophagic flux to levels seen in control cells. Thus, HCV suppresses autophagic flux and increases the steady-state levels of autophagosomes by increasing the expression of Arl8b, which repositions lysosomes and prevents their fusion with autophagosomes.