PMC

A Pool of SRFBP1 Localizes to CD81 on Endosomes and Is Recruited to CD81 upon HCV Glycoprotein Exposure
(A) SRFBP1 localizes to the trans-GOLGI, endosomes, and actin. Huh-7.5 cells were stained for SRFBP1; the trans-GOLGI marker p230; and the endosomal markers EEA1, LBPA, and LAMP1 as described in A. F and G actin were stained with Alexa-conjugated phalloidin and DNase I, respectively.
(B) Pearson’s correlation coefficient for SRFBP1 and indicated cellular proteins calculated by intensity correlation analysis. Each symbol represents an individual frame; horizontal lines indicate the mean ± SEM.
(C) SRFBP1 localizes to CD81 on endosomes. Huh-7.5 cells were transfected with expression plasmids for EGFP-Rab4, -Rab5, and -Rab7 and stained for SRFBP1 and CD81. Colocalization of SRFBP1, CD81, and Rab proteins across a section (yellow line) is depicted in the upper panels. Arrowheads indicate colocalization.
(D) SRFBP1 and CD81 colocalize at early endosomes. Quantification of SRFBP1, CD81, and Rab triple-positive puncta is shown. Box and whisker plot showing median, minimum, and maximum values from six independent frames.
(E) Bioinformatics prediction of two weak amphipathic helices for SRFBP1 (black bars) with the second helix (aas 108–128) showing a small hydrophobic face of five amino acids (FLLVI). The hydrophobic face is highlighted in light gray in the primary sequence and in yellow in the helix model. Two cysteine residues (aa 254 and aa 300), which could serve as palmitoylation sites, are indicated by arrowheads.
(F) Membrane flotation assay suggests membrane association of SRFBP1. Huh-7.5 cells were transduced with mycDDK-tagged SRFBP1, 48 hr later lysed in hypotonic buffer, and analyzed by Nycodenz gradient ultracentrifugation followed by immunoblot analysis against SRFBP1, GAPDH, and CLDN1. TX-100-treated lysates served as solubilization control. L, precleared lysate; M, marker; P, pellet after lysate preclearing. One out of three independent experiments is shown.
(G) Exposure to soluble HCV glycoprotein (eE2) increases SRFBP1-CD81 colocalization in Huh-7.5 cells. Cells were incubated with eE2, with eE2 and an E2 blocking antibody (α-E2), or with PBS (mock) for 15 min; fixed; and stained for SRFBP1 and CD81 as described in A. Arrowheads indicate colocalization.
(H) Pearson’s correlation coefficient for SRFBP1 and CD81 calculated by intensity correlation analysis. Each symbol represents an individual frame; horizontal lines indicate the mean ± SEM; p value is indicated.
Representative images; inserts show magnification; scale bars 10 μm (A and C) and 20 μm (G). See also .

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 .

SRFBP1 Is a Pan-genotypic and HCV-Specific Host Entry Factor
(A) SRFBP1 is dispensable for VSV infection. SRFBP1-silenced Huh-7.5 cells were infected with VSV^∗M_Q (MOI 0.1) and analyzed for GFP expression by flow cytometry 20 hpi. Histogram is representative of biological triplicates (left panel). Quantification of VSV^∗M_Q infectivity 20 hpi in SRFBP1-silenced cells is determined as percentage of GFP-positive cells (middle panel) or by normalization of the mean fluorescence intensity (MFI) of VSV-infected SRFBP1- or CD81-silenced cells to MFI of scrambled siRNA-transfected cells (right panel).
(B) SRFBP1 is dispensable for coronavirus infection. SRFBP1-silenced cells were infected with HCoV229E-luc (MOI 0.1) and RLuc activity in cell lysates measured 24 hpi. Infectivity relative to a scrambled siRNA control is shown.
(C) Immunoblot analysis of SRFBP1 and CD81 48 hp siRNA transfection. Huh-7.5 cells were transfected with siRNA or transduced with the indicated pWPI expression construct as in (A)–(H), 48 hr later lysed, and analyzed by immunoblot. Actin served as loading control. ^∗, residual SRFBP1 signal.
(D) Bicistronic translational reporter assay with HCV IRES-driven RLuc and cap-dependent FLuc (see also A and S6B). SRFBP1 silencing and overexpression was performed as in (C), and 48 hr later, cells were transfected with translational reporter RNA. Eight hours after reporter transfection, luciferase activity in lysates was monitored.
(E) Early replication reporter assay using a subgenomic HCV genome expressing FLuc. SRFBP1 silencing and overexpression was performed as in (C), and 48 hr later, JFH-SGR-FLuc RNA was transfected into cells; cells lysed after 8 hr; and luciferase activity monitored. A polymerase mutant JFH-SGR-FLuc replicon (ΔGDD) was used to assess translation of HCV genomes independent of de novo replication. See also C and S6D for additional controls.
(F) SRFBP1 is required in a plasma membrane fusion assay of HCV infection. Huh-7.5 cells silenced for SRFBP1 were pretreated with concanamycin A (5 nM; 1 hr) to block vacuolar type H⁺-ATPases, incubated with HCV (JcR2A) for 2 hr at 4°C in the presence of concanamycin A, shifted to 37°C, and washed with a pH 5 or pH 7 buffer for 5 min. After incubation with concanamycin A for 4 hr, medium was changed and endosomal acidification independent infectivity measured at 48 hpi. See also E and S6F for additional controls.
(G) Lentiviral pseudotypes infect Huh-7.5 cells independently of SRFBP1. Cells in which SRFBP1 had been silenced (48 hr) were infected with HIV-1 pseudotypes encoding FLuc and displaying glycoproteins from HCV genotype 1 (H77), HCV genotype 2 (J6), VSV, or no glycoprotein. At 72 hpi, cells were lysed and FLuc activity measured. Infectivity was calculated by subtraction of background read for glycoprotein-free particles and relative to VSVG particles.
(H) Silencing of SRFBP1 reduces infectivity of chimeric HCV viruses with glycoproteins from all seven genotypes. Huh-7.5 FLuc cells were subjected to siRNA-mediated silencing followed by infection with intergenotypic HCV chimeras (MOI 0.1) expressing RLuc. Forty-eight hours post-infection, infectivity was determined by RLuc activity measurement. Cells treated with CD81 targeting or scrambled siRNAs served as controls. SRFBP1-targeting siRNA 394 was used in all experiments. Data from three to five biological replicates are displayed as mean + SD. See also .

HCV Life Cycle and Possible Role for SRFBP1 during HCV Entry
Our data point toward a role for SRFBP1 in the last step of entry; i.e., nucleocapsid uncoating.