Hepatitis C virus E2-CD81 interaction induces hypermutation of the immunoglobulin gene in B cells

Abstract

Hepatitis C virus (HCV) is one of the leading causes of chronic liver diseases and B-lymphocyte proliferative disorders, including mixed cryoglobulinemia and B-cell lymphoma. It has been suggested that HCV infects human cells through the interaction of its envelope glycoprotein E2 with a tetraspanin molecule CD81, the putative viral receptor. Here, we show that the engagement of B cells by purified E2 induced double-strand DNA breaks specifically in the variable region of immunoglobulin (V(H)) gene locus, leading to hypermutation in the V(H) genes of B cells. Other gene loci were not affected. Preincubation with the anti-CD81 monoclonal antibody blocked this effect. E2-CD81 interaction on B cells triggered the enhanced expression of activation-induced cytidine deaminase (AID) and also stimulated the production of tumor necrosis factor alpha. Knockdown of AID by the specific small interfering RNA blocked the E2-induced double-strand DNA breaks and hypermutation of the V(H) gene. These findings suggest that HCV infection, through E2-CD81 interaction, may modulate host's innate or adaptive immune response by activation of AID and hypermutation of immunoglobulin gene in B cells, leading to HCV-associated B-cell lymphoproliferative diseases.

FIG.1.

CD81 interacts with recombinant HCV E2 protein. (A) Lysates from Sf9 cells expressing HCV E2 or E1 proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. E2 or E1 was detected by immunoblotting with anti-E2 MAb or His probe, respectively. Monomeric and aggregated E2 species are indicated. (B) Binding of E2 to Raji and Hep-CD81 cells in the presence or absence of various antibodies. The percentage of cells binding E2 was measured by FACS. Average values from two replicates are presented. (C) Dose-response curve for E2 binding to Raji cells. Different amounts of E2 from genotypes 1a and 1b were used in the binding experiment as in panel B. (D) Morphological changes of Raji cells at 48 h after treatment with E1, E2, or various antibodies.

FIG. 2.

E2 binding induces DSBs in Raji cells and PBMC, as determined by ligation-mediated PCR (LM-PCR). (A) Cells were treated with E1, E2 (genotypes 1a and 1b), or anti-CD81 antibody, and the cellular DNA was used for LM-PCR for detecting DSBs in general or in V_H or p53 specifically (see Materials and Methods). DNA from HCV-infected or uninfected Raji cells was used as a control. HCV RNA was detected with RT-PCR. Control PCR with β-actin served as an internal control. (B) DSBs in cells pretreated with (+) or without (−) an inhibitory anti-CD81 antibody before binding with E1, E2, or a stimulatory anti-CD81 antibody. (C) Comparison of DSBs in E2 (1a)-treated or HCV-infected Raji cells. DNA samples were serially diluted. (D) DSBs in PBMC. The conditions of treatment were the same as in panel A.

FIG. 3.

Induction of AID and error-prone DNA polymerases in Raji cells and PBMC by E2 binding. (A) RNA samples from Raji cells at different time points after the various treatments were used for semiquantitative RT-PCR amplification of AID, polymerase ζ, polymerase ι, and β-actin. The cDNA of different dilutions (no dilution, 1:5 and 1:25) was used for PCR amplification. H₂O, water control for PCR. (B) AID expression in PBMC. (C) AID and polymerase ι protein expression in Raji cells as detected by immunoblotting. (D) Real-time quantification of polymerase ζ mRNA in Raji cells. The degrees of enhancement after the various treatments are shown.

FIG. 4.

Effects of silencing of AID and polymerases ι and ζ on DSBs. (A to D) The silencing of AID and polymerase ι expression by siRNA transfection in Raji cells as determined by RT-PCR of RNA (A and C) and immunoblotting of proteins (B and D). Samples were collected 2 days after stimulation by E2. (E and F) The expression of polymerase ζ mRNA in Raji cells transfected with the specific antisense oligodeoxynucleotide (AS) as determined by semiquantitative RT-PCR (E) or real-time quantitative RT-PCR (F). (G) E2-induced DSBs in Raji cells after siRNA or antisense DNA transfection as in panels A to F. (H) DSB formation in HepG2 or Hep-CD81 cells treated with E1, E2 or anti-CD81 antibody. (I) Semiquantitative RT-PCR of AID and β-actin transcripts in HepG2 or Hep-CD81 cells after the various treatments.

FIG. 5.

E2-CD81 interaction induces TNF-α production by Raji cells. (A) TNF-α production as determined by ELISA at various time points after E2 or antibody binding. (B) TNF-α production after HCV infection. (C) Detection of HCV RNA in infected cells by RT-PCR. M, mock.

FIG. 6.

A postulated signaling pathway for induction of hypermutation of Ig gene in B cells or hepatocytes by E2 binding. BCR, B-cell receptor complex.