Analysis of a Neutralizing Antibody for Human Herpesvirus 6B Reveals a Role for Glycoprotein Q1 in Viral Entry

Abstract

Human herpesvirus 6 (HHV-6) is a T cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6A and HHV-6B, based on differences in their genetic, antigenic, and growth characteristics and cell tropisms. The function of HHV-6B should be analyzed more in its life cycle, as more than 90% of people have the antibodies for HHV-6B but not HHV-6A. It has been shown that the cellular receptor for HHV-6A is human CD46 and that the viral ligand for CD46 is the envelope glycoprotein complex gH/gL/gQ1/gQ2; however, the receptor-ligand pair used by HHV-6B is still unknown. In this study, to identify the glycoprotein(s) important for HHV-6B entry, we generated monoclonal antibodies (MAbs) that inhibit infection by HHV-6B. Most of these MAbs were found to recognize gQ1, indicating that HHV-6B gQ1 is critical for virus entry. Interestingly, the recognition of gQ1 by the neutralizing MAb was enhanced by coexpression with gQ2. Moreover, gQ1 deletion or point mutants that are not recognized by the MAb could nonetheless associate with gQ2, indicating that although the MAb recognized the conformational epitope of gQ1 exposed by the gQ2 interaction, this epitope was not related to the gQ2 binding domain. Our study shows that HHV-6B gQ1 is likely a ligand for the HHV-6B receptor, and the recognition site for this MAb will be a promising target for antiviral agents.Human herpesvirus 6 (HHV-6) was first isolated from patients with lymphocytic disorders in1986 (36) and was subsequently shown to be the causative agent of exanthem subitum (ES) (48). Currently, HHV-6 can be classified into two variants, HHV-6A and HHV-6B, based on differences in genetic, antigenic, and growth characteristics and cell tropisms (1,5,7,8). HHV-6B causes infant ES, and more than 90% of people have antibodies (Abs) against HHV-6B (31, 38), while the pathogenesis of HHV-6A is still unknown. Recently, it was shown that a reactivation of HHV-6B causes encephalitis in immunocompromised hosts (13,45,46) and possibly enhances the severity of drug-induced sensitivity syndrome (14).Human CD46, a regulator of the complement activation receptor expressed on all nuclear cells, is a receptor for HHV-6 (37), and its viral ligand is the envelope glycoprotein complex gH/gL/gQ1/gQ2 (3, 28). Although this complex can bind CD46 (28), those of some clinical isolates, including laboratory strains of HHV-6B, do not bind it (24, 26). The gQ gene is unique because it is conserved only among HHV-6A, HHV-6B, and 15,19). Recently, we successfully reconstituted a virus from the HHV-6 genome (43) and found that HHV-6 gQ1 is essential for virus growth and probably for entry.As monoclonal antibodies (MAbs) against gH and gB inhibit virus-induced cell fusion and infection, gH and gB are thought to be fusogenic candidates (39). In addition, as it is common to herpesviruses generally, gH homologues expressed on viral envelopes form a complex with gL homologues (18,20,21).In addition to gH/gL/gQ1/gQ2, another gH/gL complex, gH/ gL/gO, is present in the viral envelopes of both ...

Identification of the Human Herpesvirus 6A gQ1 Domain Essential for Its Functional Conformation

“…In a virus neutralization assay, a 4,000-fold dilution of purified Mab AgQ 1-1 (0.89 mg/ml) completely blocked HHV-6A strain GS infection at 4.0 ϫ 10Recently, we also produced a neutralizing Mab against HHV-6B gQ1 (BgQ1) and determined the critical domain on BgQ1 for that Mab's recognition (27). Here, we similarly sought to identify the recognition site(s) for Mab AgQ 1-1.…”

“…The monoclonal antibodies (Mabs) designated AgQ 1-1 and gH F5 were obtained by immunizing mouse footpads with UV-inactivated HHV-6A (strain GS) virions (27). Lymphocytes from the popliteal lymph nodes were subsequently retrieved and fused with the nonproducing myeloma cell line SP2.…”

“…The hybridoma cells secreting Mabs for HHV-6A were selected by immunofluorescence antibody assay (IFA). Next, the selected Mabs were examined with the neutralizing assay described elsewhere (27). The Mabs designated AgQ1-119, AgL4, gH1-1, and AgQ2B were described previously (24).…”

“…The virus neutralization assay was performed as described previously (27). Virus titers were determined by measuring the 50% tissue culture infective dose (TCID 50 ) (31).…”

“…Since the average amino acid identity between HHV-6A U1102 and HHV-6B HST was about 94% (26), gQ1 and gQ2 appear to be key genes for determining various biological differences between HHV-6A and -B. Although the function of HHV-6B gQ1 was unknown, we previously obtained an HHV-6B-specific neutralizing antibody that recognizes a conformational epitope for gQ1, indicating that HHV-6B gQ1, like the gQ1 of HHV-6A, plays a key role in virus infection, especially in cell entry (27). Therefore, to elucidate the viral entry mechanism, it was important to analyze the structure and function of gQ1 in more detail.…”