Fig 1.
SR-B1 is dispensable for HCV entry into Huh7 cells.
(A) Expressions of CD81, SR-B1, CLDN1, and OCLN in parental, CD81 KO, SR-B1 KO, CLDN1 KO and OCLN KO Huh7 cells were determined by immunoblotting analysis. (B) Cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA levels at 24 h post-infection were determined by qRT-PCR. (C) Infectious titers in the supernatants at 72 h post-infection were determined by focus-forming assay. In all cases, asterisks indicate significant differences (*P<0.05; **P<0.01) versus the results for parental Huh7 cells. “n.d.” indicates HCV titer below 10 FFU/ml.
Fig 2.
Deficiencies of SR-B1 and LDLR impair HCV entry.
(A) Expressions of SR-B1, LDLR and CD81 in parental and SR-KO Huh7 cells transfected with siRNAs targeting LDLR or CD81 were determined by immunoblotting analysis at 48 h post-transfection. (B) Parental and SR-KO Huh7 cells were infected with HCVcc at an MOI of 1 at 48 h post-transfection with siRNAs targeting LDLR or CD81, and intracellular HCV RNA levels were determined at 24 h post-infection by qRT-PCR.
Fig 3.
SR-B1 and LDLR have a redundant role in HCV entry.
(A) Expressions of SR-B1 and LDLR in parental, SR-KO, LD-KO and SR/LD-DKO Huh7 cells were determined by immunoblotting analysis (upper panel). Cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA levels were determined at 24 h post-infection by qRT-PCR (lower panel). (B) Parental, SR-KO, LD-KO and SR/LD-DKO Huh7 cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA levels were determined at 24, 48 and 72 h post-infection by qRT-PCR. (C, D) Fluorescence localizations in parental, SR-KO, LD-KO and SR/LD-KO Huh7 cells were observed with a confocal microscope, upon infection with HCVcc at an MOI of 1 at 12, 24, 36, 48 and 60 h post-infection. The frequency is shown as the ratio of infected cells to total cells. (E) SR-B1 and LDLR were exogenously expressed in parental, SR-KO, LD-KO and SR/LD-DKO Huh7 cells by infection with lentiviral vectors. Expressions of SR-B1 and LDLR in these cells were determined by immunoblotting analysis (left panel). Cells were infected with HCVcc at an MOI of 1 and intracellular HCV RNA levels were determined at 24 h post-infection by qRT-PCR (right panel). (F) SR-B1 and LDLR were exogenously expressed in SR/LD-DKO Huh7 cells by infection with different amount of lentiviral vectors. Expressions of SR-B1 and LDLR in these cells were determined by immunoblotting analysis (upper panel). Cells were infected with HCVcc at an MOI of 1 and intracellular HCV RNA levels were determined at 24 h post-infection by qRT-PCR (lower panel). Asterisks indicate significant differences (*P<0.05; **P<0.01) versus the results for control cells.
Fig 4.
VLDLR has a similar role with SR-B1 and LDLR in HCV entry.
(A) Relative mRNA expression of VLDLR in various tissues was determined using the NextBio Body Atlas application. (B) mRNA expression of SR-B1, LDLR, VLDLR and GAPDH in Huh7 cells and primary human hepatocyte (PHH) were determined by qRT-PCR. Relative expression levels of mRNA were calculated based on the expression level of GAPDH. (C) VLDLR-HA was exogenously expressed in parental, SR-KO, LD-KO and SR/LD-DKO Huh7 cells by infection with lentiviral vectors. Expressions of VLDLR in these cells were determined by immunoblotting analysis (upper panel). Parental, CD81 KO and SR/LD-DKO Huh7 cells expressing SR-B1, LDLR or VLDLR were infected with HCVcc at an MOI of 1 and intracellular HCV RNA levels were determined at 24 h post-infection (lower panel). (D) VLDLR-HA was exogenously expressed in CD81 KO, CLDN1 KO and OCLN KO Huh7 cells by infection with lentiviral vectors. Expressions of VLDLR, CD81, CLDN1 and OCLN in these cells were determined by immunoblotting analysis (upper panel). Cells were infected with HCVcc at an MOI of 1 and intracellular HCV RNA levels were determined at 24 h post-infection by qRT-PCR (lower panel). (E) SR-B1, LDLR and VLDLR were exogenously expressed in SR/LD-DKO Huh7 cells by infection with lentiviral vectors. Cells were infected with Con1-JFH1 or Jc1 at an MOI of 1, and intracellular HCV RNA levels were determined at 24 h post-infection by qRT-PCR. (F) Sera (100μl) from chimeric mice infected with HCV were inoculated into SR/LD-DKO Huh7 cells expressing either SR-B1, LDLR or VLDLR in 24 well plate. Intracellular HCV RNA levels were determined at 72 h post-infection. In all cases, asterisks indicate significant differences (*P<0.05; **P<0.01) versus the results for control cells.
Fig 5.
SR-B1, LDLR and VLDLR participate in the binding step of HCV entry.
(A) Parental, CD81 KO, CLDN KO, OCLN KO and SR/LD-DKO Huh7 cells were inoculated with HCVcc at an MOI of 1 at 4°C for 1 h and washed three times with PBS, and HCV RNA levels were determined after the binding. (B) SR/LD-DKO Huh7 cells expressing SR-B1, LDLR or VLDLR were infected with HCVcc at an MOI of 1 at 4°C for 1 h and washed three times with PBS, and HCV RNA levels were determined after the binding. In all cases, asterisks indicate significant differences (*P<0.05; **P<0.01) versus the results for control cells.
Fig 6.
Lipid binding and lipid uptake of lipoprotein receptors participate in HCV entry.
Schematics were shown for the SR-B1, LDLR and VLDLR mutants (upper panels of A and B, and left panel of C). (A) S112F- and T175A-SR-B1 were generated to examine the significance of lipid binding ability. (B) Seven or eight repeats in the ligand binding domains of LDLR (left) and VLDLR (right) were deleted (ΔLBD). (C) An asparagine residue in the repeat 5 and in the repeats 2 to 7 of LDLR was substituted with tyrosine (mut5 and mut2-7). The wild-type and mutants of SR-B1, LDLR and VLDLR were expressed in SR/LD-DKO Huh7 cells by lentiviral vectors. Expressions of these receptors were detected by immunoblotting (middle panels in A and B, right upper panel in C). These cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA levels at 24 h post-infection were determined by qRT-PCR (lower panels in A and B, right lower panel in C). (D) HCVpp were inoculated into parental, SR/LD-DKO expressing either SR-B1, LDLR or VLDLR and CD81-KO Huh7 cells, and luciferase activities were determined at 48 h post-infection by using a luciferase assay system. In all cases, asterisks indicate significant differences (*P<0.05; **P<0.01) versus the results for cells expressing wild-type receptors.
Fig 7.
Density-dependent entry of LVPs via lipoprotein receptors.
HCV particles in the culture supernatants of Huh7.5.1 cells were harvested at 72 h post-infection with HCVcc and fractionated by using density gradient centrifugation. Infectious titers of each fraction were determined by focus-forming assay and the buoyant density was plotted for each fraction (upper panels). SR/LD-DKO Huh7 cells expressing either SR-B1, LDLR or VLDLR were infected with each fraction and intracellular HCV RNA levels at 24 h post-infection were determined by qRT-PCR (lower panel). Asterisks indicate significant differences (*P<0.05; **P<0.01) versus the results for SR/LD-DKO Huh7 cells expressing SR-B1.