Role of oxysterol binding protein in hepatitis C virus infection

Abstract

Hepatitis C virus (HCV) RNA genome replicates within the ribonucleoprotein (RNP) complex in the modified membranous structures extended from endoplasmic reticulum. A proteomic analysis of HCV RNP complexes revealed the association of oxysterol binding protein (OSBP) as one of the components of these complexes. OSBP interacted with the N-terminal domain I of the HCV NS5A protein and colocalized to the Golgi compartment with NS5A. An OSBP-specific short hairpin RNA that partially downregulated OSBP expression resulted in a decrease of the HCV particle release in culture supernatant with little effect on viral RNA replication. The pleckstrin homology (PH) domain located in the N-terminal region of OSBP targeted this protein to the Golgi apparatus. OSBP deletion mutation in the PH (DeltaPH) domain failed to localize to the Golgi apparatus and inhibited the HCV particle release. These studies suggest a possible functional role of OSBP in the HCV maturation process.

FIG. 1.

Effect of silencing OSBP on HCV replication and viral particle release. Huh7.5.1 cells were infected with lentiviral vectors encoding an shRNA expression cassette of scrambled shRNA, shRNA-1, and shRNA-2. Four days after lentiviral infection, cells were infected with HCV (JFH1) at an MOI of 1. (A) Western blot analysis of HCV-infected cells with indicated shRNAs at day 6. IB, immunoblotting carried out using indicated antibodies. (B) Quantitative RT-PCR analysis of OSBP mRNA. (C) Quantitative RT-PCR analysis of GAPDH mRNA. (D) Intracellular HCV RNA levels measured by quantitative RT-PCR analysis. (E) Accumulation of extracellular HCV viral RNA in the culture medium as measured by quantitative RT-PCR analysis. (F) Supernatant infectivity assay. The infectivity of cultured supernatant at day 6 was determined as described in Materials and Methods and previously (58). Means and standard errors of at least triplicate measurements are shown. GE, genomic copies.

FIG. 2.

Schematic representation of various OSBP domains. (A) The domain organizations of OSBP and amino acid regions are indicated on the figure. G/A-rich, glycine- and alanine-rich region; PH, binds to PI4P [PtdIns(4)P]; L-Zipper, leucine zipper domain; FF, FFAT; oxysterol-binding domain, binds to oxysterols and cholesterols. The numbering of amino acids is based on human OSBP (NM_002556). (B) OSBP mutants. The coding for the N-PH mutant extends from aa 1 to 208. The ΔPH mutant lacks aa 35 to 273. Mutant W172A carries a point mutation of a conserved tryptophan residue at 172 within the PH domain. Mutant FF/AA contains two phenylalanine residues in the FFAT motif replaced with alanine residues, required for VAP-A association. PP2A, phosphatase 2A; HePTP, tyrosine phosphatase.

FIG. 3.

Subcellular localization of OSBP and OSBP mutants. Huh7 cells were transfected with vectors encoding the FLAG-tagged wild-type (WT) and indicated mutant OSBP genes. Cells were analyzed by confocal immunofluorescence microscopy with anti-FLAG and anti-TGN46 antibodies (see Materials and Methods). Panels in the left column show FLAG-tagged OSBP (red). Panels in the center column show subcellular localization of TGN46 as a marker of the TGN. Panels in the right column show superimposed images of OSBP and TGN46.

FIG. 4.

Effect of OSBP mutants on HCV replication and secretion. Huh7 cells were infected with HCV at an MOI of 0.5, maintained for 8 days, transfected with OSBP expression vector by electroporation, and analyzed after 48 h by RT-PCR (A and B) and Western blot assays (C). (A) Accumulation of HCV RNA in the culture supernatant. (B) The level of intracellular HCV RNA. (C) Western blot analysis of the HCV-infected cells using anti-NS5A (upper panel) and anti-FLAG for the detection of OSBP and mutants (lower panel). (D) Production of chimeric reporter HCV. Huh7.5.1 cells were transfected with both Luc-Jc1 RNA (luciferase reporter virus) and wild-type and mutant OSBP expression vectors, as indicated. Culture supernatants were collected at 72 h after transfection and used to infect naïve Huh7.5.1 cells. Cellular lysates from these secondary infections were assayed at 42 h for luciferase activity to determine the level of infectious reporter virion titer. Means and standard errors of at least triplicate measurements are shown. Expression vectors used for all the experiments are indicated. GE, genomic copies.

FIG. 5.

Mapping of OSBP binding site(s) within the NS5A protein. (A) Schematic representation of wild-type and deletion mutants of NS5A encoded by pEF-NS5A vectors. Various domains of NS5A are shown. Huh7 cells were cotransfected with OSBP (pFLAG-CMV-OSBP), wild-type NS5A (pEF1-NS5A), or NS5A deletion mutants. Wild-type and mutant NS5A expression vectors contain a Myc/His tag. (B) Expression of wild-type NS5A, NS5A deletion mutant proteins (upper panel), and OSBP (middle panel) was analyzed by Western blot assays. For OSBP-NS5A protein-protein interaction studies, cellular lysates were immunoprecipitated (IP) using anti-FLAG antibody (OSBP), followed by immunoblotting (IB) using anti-Myc antibody (lower panel).

FIG. 6.

25-HC stimulates Golgi compartment localization of OSBP and NS5A. Uninfected and HCV (JFH1)-infected Huh7 cells were treated with or without 10 μM 25-HC and incubated for 12 h prior to immunostaining. The immunofluorescence assay was performed as described in Materials and Methods.