Background:  GB virus C (GBV-C) infection is associated with prolonged survival among HIV-infected people. Little is known about GBV-C attachment and entry, although a recent report suggested that the envelope glycoprotein E2 binds to the tetraspanin CD81 on the surface of CD4⁺ T cells. We developed a direct E2 binding assay to determine cell types that bind E2, and to assess the role of CD81 and E2 cell binding.

Methods:  Murine anti-GBV-C E2 and human anti-hepatitis C V (HCV) E2 monoclonal antibodies were used to detect binding of GBV-C or HCV E2 to MOLT-4 cells (a CD4⁺ T-cell line). Purified GBV-C and HCV E2 proteins were truncated at C-terminus and purified from CHO cells. CD81 was detected on cells using commercial anti-CD81 monoclonal antibodies (JS81). Appropriate species and isotype control antibodies were used as controls. E2 binding was measured by flow cytometry as previously described for HCV.

Results:  Using anti-E2 monoclonal antibodies to detect E2 binding, we demonstrated GBV-C E2 (10 µg/mL) bound to MOLT-4, HeLa, Jurkat, and 3T3 cell lines as detected by 4 of 10 anti-E2 monoclonal antibody tested, but not with anti-HCV monoclonal antibodies or isotype control antibodies. E2 binding was not observed using Daudi or CHO cells. Two GBV-C E2 monoclonal antibodies that did not recognize E2 bound to cells were able to neutralize binding of E2 to cells. HCV E2 binding (10 µg/mL) was demonstrated only when anti-HCV E2 monoclonal antibodies was used, and HCV E2 was not detected by anti-GBV-C E2 monoclonal antibodies. Daudi cells expressed high levels of CD81 on the surface, yet E2 binding was not observed. Also, GBV-C E2 binding was not blocked by soluble human or murine CD81 (20 µg/mL), whereas human CD81 blocked more than 95% of HCV E2 binding.

Conclusions:  Using a direct GBV-C E2 binding assay, we have identified cell lines that do or do not bind E2. Several lines of evidence indicate that CD81 is not required for E2 binding. Two monoclonal antibodies neutralized E2 binding to cells, suggesting that E2 antibody may neutralize GBV-C via interference with virus attachment. Further studies are underway using this system to identify a GBV-C E2 receptor.

Keywords: GB Virus C; hepatitis G Virus; E2 protein