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

Abstract

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...

The Neutralizing Linear Epitope of Human Herpesvirus 6A Glycoprotein B Does Not Affect Virus Infectivity

“…It has been reported that for herpes simplex virus (HSV) to induce cell fusion, coexpression of gB, gD, and gH-gL is required in HSV-permissive cells (27); likewise, cell fusion induced by pseudorabies virus (PrV) requires gB and gH-gL (28), Epstein-Barr virus (EBV) requires gB, gH, gL, and gp42 (29), human cytomegalovirus (HCMV) requires gB and gH-gL (30), and HHV-8 requires gB and gH-gL (10). For HHV-6A, it has been reported that coexpression of gB, gH, gL, gQ1, and gQ2 mediates cell fusion in 293T cells (31). In this study, to examine whether gB epitope mutations affect the cell fusion mediated by HHV-6A glycoproteins, we generated an HHV-6A fusion assay system.…”

Cranial nerve III and/or VI function abnormalities associated with active herpesvirus infection: Clinical experience and literature review

“…There are also other immune modulation mechanisms enabling suppression of immune responses in infection: activation of CD4+ and CD8+ T cell proliferation [34][35][36]; activation of expression of the cytokines that inhibit IL-2 synthesis by activated T cells, and subsequent deactivation of T cells; suppression of the growth and differentiation of marrow progenitors; and reduction of HLA class I expression on dendritic cells [34,37]. It has been demonstrated that, without viral replication, HHV7, HHV6A and some strains of HHV6B can induce cell-cell fusion between cells expressing human CD46 [38,39].…”

Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion

“…For OvHV-2 it is not known whether gB and gH/gL are sufficient or whether additional proteins are needed to induce membrane fusion. Previous studies have successfully demonstrated the role of several herpesvirus glycoproteins in membrane fusion using a virus-free in vitro cell-cell fusion assay (10,11,(22)(23)(24)(25). Therefore, a virus free cell-cell fusion assay was used to examine which OvHV-2 glycoproteins mediate membrane fusion.…”