Monoclonal antibodies to glycoprotein B differentiate human herpesvirus 6 into two clusters, variants A and B

cHuman herpesvirus 6 is a T lymphotropic herpesvirus, long classified into variants A and B (HHV-6A and HHV-6B) based on differences in sequence and pathogenicity. Recently, however, HHV-6A and HHV-6B were reclassified as different species. Here, we isolated a neutralizing monoclonal antibody (Mab) named AgQ 1-1 that was specific for HHV-6A glycoprotein Q1 (AgQ1), and we showed that amino acid residues 494 to 497 of AgQ1 were critical for its recognition by this Mab. This region was also essential for AgQ1's complex formation with gH, gL, and gQ2, which might be important for viral binding to the cellular receptor, CD46. In addition, amino acid residues 494 to 497 are essential for viral replication. Interestingly, this sequence corresponds to the domain on HHV-6B gQ1 that is critical for recognition by an HHV-6B-specific neutralizing Mab. Within this domain, only Q at position 496 of HHV-6A is distinct from the HHV-6B sequence; however, the mutant AgQ1(Q496E) was still clearly recognized by the Mab AgQ 1-1. Surprisingly, replacement of the adjacent amino acid, in mutant AgQ1(C495A), resulted in poor recognition by Mab AgQ 1-1, and AgQ1(C495A) could not form the gH/gL/gQ1/gQ2 complex. Furthermore, the binding ability of mutant AgQ1(L494A) with CD46 decreased, although it could form the gH/gL/gQ1/gQ2 complex and it showed clear reactivity to Mab AgQ 1-1. These data indicated that amino acid residues 494 to 497 of AgQ1 were critical for the recognition by Mab AgQ 1-1 and essential for AgQ1's functional conformation.

mentioning

confidence: 99%

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

Maeki

Hayashi

Kawabata

et al. 2013

“…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.…”

mentioning

confidence: 99%

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

Kawabata

Oyaizu

Maeki

et al. 2011

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 ...

“…uman herpesvirus 6 (HHV-6), betaherpesvirus subfamily (1), includes two species, A (HHV-6A) and B (HHV-6B) (2)(3)(4). HHV-6B mainly infects immune cells, such as CD4 ϩ T-lymphocytes, monocytes, and dendritic cells, and also causes exanthema subitum during primary infection in children (5).…”

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confidence: 99%

Herpesvirus 6 Glycoproteins B (gB), gH, gL, and gQ Are Necessary and Sufficient for Cell-to-Cell Fusion

Tanaka

Sasajima

Matsumoto

et al. 2013

The human herpesvirus 6 (HHV-6) envelope glycoprotein gH/gL/gQ1/gQ2 complex associates with host cell CD46 as its cellular receptor. Although gB has been suggested to be involved in HHV-6 infection, its function in membrane fusion has remained unclear. Here, we have developed an HHV-6A (strain GS)and HHV-6B (strain Z29) virus-free cell-to-cell fusion assay and demonstrate that gB and the gH/gL/gQ1/gQ2 complex are the minimum components required for membrane fusion by HHV-6. Human herpesvirus 6 (HHV-6), betaherpesvirus subfamily (1), includes two species, A (HHV-6A) and B (HHV-6B) (2-4). HHV-6B mainly infects immune cells, such as CD4 ϩ T-lymphocytes, monocytes, and dendritic cells, and also causes exanthema subitum during primary infection in children (5). HHV-6B can reactivate from latency in immunocompromised patients and cause pneumonitis, hepatitis, and encephalitis (6, 7). However, the molecular basis of HHV-6A pathogenicity is unclear.The association of several viral glycoproteins with their respective cellular receptors induces virus envelope-cell membrane fusion during viral entry. It has been reported that HHV-6 gH/gL forms a complex with gQ1 and gQ2 and that this complex binds to CD46, which has been reported to function as a cellular receptor for HHV-6 (8-11). gB and a gH/gL complex are conserved in all herpesviruses and thought to play a pivotal role in membrane fusion and herpesvirus infection (12-17). Studies of gBs and gHs of other herpesviruses have elucidated the molecular mechanisms of virus envelope-cell membrane fusion (18-21). Although some antibodies against HHV-6 gB have been reported to block HHV-6B infection (22,23), the function of HHV-6 gB during viral infection remains unclear.To identify the requirement of HHV-6 glycoproteins for virusinduced membrane fusion during the virus infection, each of the glycoproteins was amplified and expressed from HHV-6B (Z29). Briefly, the genomic sequences of gH, gL, gO, gQ1, and gQ2 were amplified from total DNA of HHV-6B-infected Molt3 cells (Riken BRC, Tsukuba, Japan) and cloned into pCAGGS-MCS expression vector (24). For detection purposes, the FLAG epitope was inserted in frame at the N termini of gO and gQ2 genes. The fulllength gB gene containing a promoter and poly(A) tail sequences was amplified by recombinant PCR using plasmids containing partial gB sequences (nucleotides [nt] ϩ1 to ϩ1718 and ϩ1713 to ϩ2493). The purified PCR product was used for transient transfection of 293T cells. Expression of transfected genes was analyzed by flow cytometry. gB and the gH/gL complex were detected on the cell surface using anti-gB monoclonal antibody (MAb) and gHA2 antibody, respectively (Fig. 1A) (25). Cells transfected with plasmid encoding gQ1 or N-terminal FLAG-tagged gQ2 ex- HHV-6B-infected (black lines) or mock-infected (gray-shaded areas) Molt-3 cells were stained with anti-gB, anti-gH, or anti-gQ1 MAb followed by staining with anti-mouse IgG antibody and either CD46-Ig or control Ig (VZV gB-Ig) followed by staining with anti-human IgG Fc portion a...