FIG 3.

VAP function in HCV infection is not solely through OSBP. (A) HCV RO morphology in VAPA/VAPB knockout cells. Huh7.5.1/T7 cells stably expressing T7 RNA polymerase were transduced with the indicated sgRNA with Cas9 or shRNA and then transfected with constructs expressing the NS3-5B/GFP polyprotein encoding NS5A-GFP under the control of the T7 promoter. Forty-eight hours later, cells were immunostained for GFP (green) with DAPI (blue) nuclear counterstaining. The lengths of the NS5A-positive membranous structures in each group were quantitated with ImageJ, and the average length is indicated in pixels as means±SD. Bar, 10 μm. KO, knockout. KD, kinase dead. (B) Coimmunoprecipitation of VAPA with HCV NS5A protein. 293T cells stably expressing a negative-control shRNA or an shRNA targeting OSBP (shOSBP) were cotransfected to express HCV NS5A and HA-tagged VAPA. Forty-eight hours later, cells were lysed and immunoprecipitated with HA antibody, followed by immunoblotting with the indicated antibodies. (C) Huh7.5.1 cells stably transduced with wild-type or mutant OSBP were infected with the JFH-1 strain of HCV. Three days later, cell homogenates (L) were separated into a crude membrane pellet and a water-soluble supernatant (W) by centrifugation. The pellet was treated with cold 1% NP-40 and spun again; the detergent-soluble (S) fraction was removed, and the detergent-resistant membrane (R) pellet was resuspended with RIPA buffer containing 0.1% SDS. Samples were analyzed by SDS-PAGE, followed by immunoblotting for the indicated proteins.