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

Abstract

AIMTo 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.METHODSSite-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.RESULTSThe 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.CONCLUSIONThe 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.

Scavenger receptor class B type I genetic variants associated with disease severity in chronic hepatitis C virus infection

“…Previous studies have shown that some common SR‐BI polymorphisms are associated with differential HCV treatment outcome and viral load 41,42 . In addition, naturally occurring, but very rare SR‐BI coding mutations (specifically S112F and T175A) have reduced HCV entry function 42–44 . Despite the association between SR‐BI polymorphisms and key facets of HCV replication and biology, their potential impact on disease severity has not been defined.…”

“… 41 , 42 In addition, naturally occurring, but very rare SR‐BI coding mutations (specifically S112F and T175A) have reduced HCV entry function. 42 , 43 , 44 Despite the association between SR‐BI polymorphisms and key facets of HCV replication and biology, their potential impact on disease severity has not been defined. To address this shortfall, we used archived DNA extracts obtained from the extensively characterized Trent HCV cohort 45 to investigate the prevalence of relatively high frequency polymorphisms in individuals with severe versus mild HCV‐induced liver disease.…”