Single amino acid mutation of SR-BI decreases infectivity of hepatitis C virus derived from cell culture in a cell culture model.

ABSTRACT: To investigate the effect of a single amino acid mutation in human class B scavenger receptor I (SR-BI) on the infectivity of cell culture-derived hepatitis C virus (HCVcc) in SR-BI knock-down Huh7-siSR-BI cells.Site-directed mutagenesis was used to construct the SR-BI S112F mutation, and the mutation was confirmed by nucleotide sequencing. SR-BI knock-down Huh7-siSR-BI cells were transfected with SR-BI S112F, SR-BI wild type (WT) and control plasmids, and then infected with HCVpp (HCV pseudoparticles) and hepatitis C virus derived from cell culture (HCVcc). A fluorescence assay was performed to analyze the effect of the S112F mutation on HCV entry; quantitative real-time PCR, immunofluorescence, and Western blot assays were used to analyze the effect of the S112F mutation on HCV infectivity. CHO cells expressing WT and SR-BI S112F were incubated with the HCV E2 protein expressed in HEK 293T cells, and flow cytometry was performed to examine the ability of SR-BI S112F to bind to the HCV E2 protein. Huh7-siSR-BI cells were transfected with SR-BI WT and the S112F mutant, and then DiI-HDL was added and images captured under the microscope to assess the ability of SR-BI S112F to take up HDL.The SR-BI S112F mutation was successfully constructed. The S112F mutation decreased the expression of the SR-BI mRNA and protein. SR-BI S112F decreased HCV entry and HCVcc infectivity in Huh7-siSR-BI cells. The S112F mutation impaired the binding of SR-BI to HCV E2 protein and decreased the HDL uptake of SR-BI.The S112F single amino acid mutation in SR-BI decreased the levels of the SR-BI mRNA and protein, as well as the ability of SR-BI to bind to the HCV E2 protein. Amino acid 112 in SR-BI plays important roles in HCV entry and the infectivity of HCVcc in vitro.

SUBMITTER: Gao R 

PROVIDER: S-EPMC5537182 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature