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. 2015 Jul 23;12(5):864–878. doi: 10.1016/j.celrep.2015.06.063

Figure 3.

Figure 3

The CD81-Binding Partner SRFBP1 Is Expressed in Human Liver and Required for HCV Infection

(A) SRFBP1 transcript levels in primary human hepatocytes are up to 6-fold higher than in Huh-7.5 cells. Absolute transcript numbers of SRFBP1 in hepatocytes from five donors (D1–D5) and in two independent passages of human hepatoma cells (Huh-7.5) were determined in technical triplicates and displayed as mean + SD.

(B) HCV (JcR2A) infectivity increases in a dose-dependent manner in Huh-7.5 FLuc cells upon overexpression of full-length SRFBP1. Cells were transduced with lentiviruses encoding SRFBP1 or a blasticidin resistance gene (empty vector), 72 hr later infected with HCV, and infectivity measured 48 hpi by luciferase assay. Immunoblot analysis of lysates 72 post-transduction shows dose-dependent SRFBP1 overexpression (green). Actin served as loading control (red). The immunoblot is representative of three biological replicates.

(C) HCV (JcR2A) infectivity is reduced in Huh-7.5 FLuc cells 48 hp silencing of SRFBP1 or CD81. We used a pool of three siRNAs or individual siRNAs targeting the indicated ORF position and measured infectivity at 48 hpi by luciferase assay. Two scrambled siRNAs (1 and 2) served as controls. Immunoblot analysis confirms reduced SRFBP1 protein levels 48 hp RNAi. Mean + SD of three technical replicates are shown. Infectivity data and immunoblot are representative of three biological replicates.

(D) Lentiviral transduction with siRNA-resistant SRFBP1 rescues HCV infection in SRFBP1-silenced Huh-7.5 FLuc cells. Cells were transfected with siRNAs (SRFBP1: siRNA 394), 24 hr later transduced with blasticidin resistance gene encoding lentivirus (siSRFBP1) or siRNA-resistant SRFBP1 encoding lentivirus (siSRFBP1 compl.), and 24 hr later infected with HCV (JcR2A). Infectivity at 48 hpi measured by luciferase assay is shown.

(E) SRFBP1 is dispensable for HCV replication, assembly, and release. Huh-7.5 FLuc cells were transfected with genomic HCV RNA (JcR2A) and the indicated gene silenced 5 hr later (SRFBP1: siRNA 394). At 72 and 96 hp transfection (hpt), supernatants were harvested, cells lysed, and replication efficiency in lysates measured by luciferase assay (upper panel). Viability of HCV-replicating cells upon RNAi was determined using the cellular FLuc reporter at 72 or 96 hpt (middle panel). Supernatants from HCV-transfected and SRFBP1-silenced cells were titrated on naive Huh-7.5 cells to determine virus particle assembly and release rates (bottom panel). Values were normalized to a scrambled siRNA control. Unless stated otherwise, all experiments are displayed as mean + SD of three independent biological replicates each performed in technical triplicates. See also Figure S3.