FIG 8.

Lys²³ and Lys¹²¹ of VPS4A are ubiquitylation acceptor sites for HCV-mediated VPS4A polyubiquitylation. (A) Amino acid sequence alignment of VPS4A (GenBank accession number NM_013245.3) and VPS4B (GenBank accession number NM_004869.4). Common Lys residues are highlighted in blue, specific Lys residues for VPS4A are highlighted in pink, and specific Lys residues for VPS4B are highlighted in yellow. Numbers indicate the position of amino acid residues. (B and C) Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. At 3 h postinfection, cells were transfected with expression plasmids for each FLAG-tagged VPS4A single-point mutant (B) or double-point mutant (C) together with HA-tagged Ub plasmid. At 4 days transfection, cells were harvested, and cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (1st panel) or anti-FLAG PAb (2nd panel). Input samples were immunoblotted with anti-HA PAb (3rd panel), anti-FLAG PAb (4th panel), anti-NS3 MAb (5th panel), and anti-GAPDH MAb (6th panel), respectively. The level of GAPDH served as a loading control. HC, immunoglobulin heavy chain.