Figure 5: Co-stimulatory molecule B7.2/CD86 is identified as a co-receptor for lymphotropic HCV infection in B lymphocytes.

(a) Expression cloning of lymphotropic HCV receptor. HEK293T cells were transfected with B-cell cDNA library and selected with functional screening using HCVpp(SB) as the ligand and puromycin or other selection antibiotic. To recover B-cell cDNAs in selected (surviving) colonies, DNA was isolated from these selected HEK293T cells and amplified by PCR with specific primers to identify the inserted cDNA library sequence. (b) The blocking effects of anti-B7.2 and anti-CD81 on HCVpp(SB) infection (*P<0.05, t-test, n=3). Error bars represent s.d. (c) B7.2 silencing inhibited HCVpp-SB entry into Raji cells. The HCVpp entry experiment was performed in triplicate (n=3). Error bars represent s.d. (d) Correlation plot between B7.2 levels and relative infectivity. (e) Time course of the blocking effects of anti-B7.2 and anti-CD81 on HCVpp(SB) infection. Synchronized infections were performed on Raji or Huh7.5.1 cells. Values (percent entry) are relative to the signal seen when the antibody was added at 4 h post temperature shift. Cellular HCV RNA levels were detected in Raji cell cultures treated with isotype-matched or anti-B7.2 or anti-CD81 antibody. Fits of t=0 and later data points represent a one-phase exponential association and sigmoidal dose-response (variable slope) for anti-B7.2 (50 μg ml⁻¹) or anti-CD81 (50 μg ml⁻¹) antibody (mean of n=3; error bars, s.d.). (f) Both B7.2 IgV and cytoplasmic regions are required for efficient HCV entry. The Raji cells (B7.2^low) transduced with lentivirus expressing sh-B7.2 were electroporated with GFP-B7.2 (B7.2), GFP-B7.2/CD8 (B7.2/CD8), GFP-ICAM-1 (ICAM-1) or GFP-ICAM-1/CD8 (ICAM-1/CD8) vectors. HCV entry levels were assessed for luciferase activities at 72 h after HCVpp challenge of transfected cells (n=3). Error bars represent s.d.