Analysis of a neutralizing antibody for human herpesvirus 6B reveals a role for glycoprotein Q1 in viral entry

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.     

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

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‐6A gQ1 and gQ2 are critical for human CD46 usage

Based on genetic and antigenic differences and on their cell tropism, human herpes virus‐6 (HHV‐6) has been classified into two variants, HHV‐6A and HHV‐6B. Recently, these variants were re‐classified as two different species. The HHV‐6A glycoprotein complex, gH/gL/gQ1/gQ2 binds to its cellular receptor, CD46; however, the corresponding complex in HHV‐6B rarely binds to CD46. To determine which viral molecules in the glycoprotein complex determine HHV‐6A‐CD46 binding, each molecule of the HHV‐6A complex (i.e., gH, gL, gQ1, or gQ2) was replaced with the corresponding HHV‐6B molecule, and the ability of the replaced protein to be incorporated into the complex and the ability of the complex to bind CD46 were examined. It was found that when all four glycoproteins were expressed, they were able to form a tetrameric complex. However, a complex formed by HHV‐6A gH/gL/gQ1/gQ2 complexes replaced with HHV‐6B gQ1 or gQ2 scarcely bind CD46, whereas HHV‐6A complexes in which gH or gL was replaced with the HHV‐6B molecules did bind it. These results indicate that HHV‐6A gQ1 and gQ2 play an important role in CD46 binding.     

Complementation of the function of glycoprotein H of human herpesvirus 6 variant A by glycoprotein H of variant B in the virus life cycle

Human herpesvirus 6 (HHV-6) is a T-cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6 variant A (HHV-6A) and HHV-6B, based on genetic, antigenic, and cell tropisms, although the homology of their entire genomic sequences is nearly 90%. The HHV-6A glycoprotein complex gH/gL/gQ1/gQ2 is a viral ligand that binds to the cellular receptor human CD46. Because gH has 94.3% amino acid identity between the variants, here we examined whether gH from one variant could complement its loss in the other. Recently, we successfully reconstituted HHV-6A from its cloned genome in a bacterial artificial chromosome (BAC) (rHHV-6ABAC). Using this system, we constructed HHV-6ABAC DNA containing the HHV-6B gH (BgH) gene instead of the HHV-6A gH (AgH) gene in Escherichia coli. Recombinant HHV-6ABAC expressing BgH (rHHV-6ABAC-BgH) was successfully reconstituted. In addition, a monoclonal antibody that blocks HHV-6B but not HHV-6A infection neutralized rHHV-6ABAC-BgH but not rHHV-6ABAC. These results indicate that HHV-6B gH can complement the function of HHV-6A gH in the viral infectious cycle.     

Human herpesvirus 6 glycoprotein complex formation is required for folding and trafficking of the gH/gL/gQ1/gQ2 complex and its cellular receptor binding

Human herpesvirus 6 (HHV-6) is a T-cell-tropic betaherpesvirus. A glycoprotein (g) complex that is unique to HHV-6, gH/gL/gQ1/gQ2, is a viral ligand for its cellular receptor, human CD46. However, whether complex formation or one component of the complex is required for CD46 binding and how the complex is transported in cells are open questions. Furthermore, in HHV-6-infected cells the gQ1 protein modified with N-linked glycans is expressed in two forms with different molecular masses: an 80-kDa form (gQ1-80K) and a 74-kDa form (gQ1-74K). Only gQ1-80K, but not gQ1-74K, forms the complex with gQ2, gH, and gL, and this four-component complex is incorporated into mature virions. Here, we characterized the molecular context leading to the maturation of gQ1 by expressing combinations of the individual gH/gL/gQ1/gQ2 components in 293T cells. Surprisingly, only when all four molecules were expressed was a substantial amount of gQ1-80K detected, indicating that all three of the other molecules (gQ2, gH, and gL) were necessary and sufficient for gQ1 maturation. We also found that only the tetrameric complex, and not its subsets, binds to CD46. Finally, a gQ2-null virus constructed in the BAC (bacterial artificial chromosome) system could not be reconstituted, indicating that gQ2 is essential for virus growth. These results show that gH, gL, gQ1, and gQ2 are all essential for the trafficking and proper folding of the gH/gL/gQ1/gQ2 complex and, thus, for HHV-6 infection.     

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

Human 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.     

Discovery of a second form of tripartite complex containing gH-gL of human herpesvirus 6 and observations on CD46

The human herpesvirus 6 (HHV-6) glycoprotein H (gH)-glycoprotein L (gL) complex associates with glycoprotein Q (gQ) (Y. Mori, P. Akkapaiboon, X. Yang, and K. Yamanishi, J. Virol. 77:2452-2458, 2003), and the gH-gL-gQ complex interacts with human CD46 (Y. Mori, X. Yang, P. Akkapaiboon, T. Okuno, and K. Yamanishi, J. Virol. 77:4992-4999, 2003). Here, we show that the HHV-6 U47 gene, which is a positional homolog of the human cytomegalovirus glycoprotein O (gO) gene, encodes a third component of the HHV-6 gH-gL-containing envelope complex. A monoclonal antibody (MAb) against the amino terminus of HHV-6 gO reacted in immunoblots with protein species migrating at 120 to 130 kDa and 74 to 80 kDa in lysates of HHV-6-infected cells and with a 74- to 80-kDa protein species in purified virions. The 80-kDa form of gO was coimmunoprecipitated with an anti-gH MAb, but an anti-gQ MAb, which coimmunoprecipitated gH, did not coprecipitate gO. Furthermore, the gH-gL-gO complex did not bind to human CD46, indicating that the complex was not a ligand for CD46. These findings suggested that the viral envelope contains at least two kinds of tripartite complexes, gH-gL-gQ and gH-gL-gO, and that the gH-gL-gO complex may play a role different from that of gH-gL-gQ during viral infection. This is the first report of two kinds of gH-gL complexes on the viral envelope in a member of the herpesvirus family.     

脂筏在人类疱疹病毒6型装配中的作用

为了探讨脂筏在人类疱疹病毒6型(HHV-6)装配中的作用,用HHV-6 GS株感染HSB2细胞,用非离子去污剂Triton X-100提取脂筏成分,利用Western blot分析HHV-6包膜糖蛋白与脂筏的相关性.并用免疫荧光双标记的方法,从分子共定位的角度研究HHV-6糖蛋白B(gB)与GPI(glycosyl-phosphatidyl inosital)锚固蛋白CD59分子以及神经节苷脂GMI(monosialotetrahexosyl ganglioside)分子之间的表达与分布关系.结果发现HHV-6包膜糖蛋白B、H、L、Q1和Q2(gB、gH、gL、gQ1和gQ2)分布在脂筏部位.激光共聚焦显微镜可观察到CD59分子及GM1均与HHV-6包膜糖蛋白B有着相同的分布,即脂筏提供HHV-6装配的平台.关于脂筏在人类疱疹病毒6型装配中的作用,这是第一次报道.     

Human herpesvirus 6 variant A glycoprotein H-glycoprotein L-glycoprotein Q complex associates with human CD46

Human CD46 is a cellular receptor for human herpesvirus 6 (HHV-6). Virus entry into host cells requires a glycoprotein H (gH)-glycoprotein L (gL) complex. We show that the CD46 ectodomain blocked HHV-6 infection and bound a complex of gH-gL and the 80-kDa U100 gene product, designated glycoprotein Q, indicating that the complex is a viral ligand for CD46.     

Human herpesvirus 6 variant A but not variant B induces fusion from without in a variety of human cells through a human herpesvirus 6 entry receptor,CD46

Human herpesvirus 6 (HHV-6) is a lymphotropic betaherpesvirus that productively infects T cells and monocytes. HHV-6 isolates can be differentiated into two groups, variants A and B (HHV-6A and HHV-6B). Here, we show a functional difference between HHV-6A and -6B in that HHV-6A induced syncytium formation of diverse human cells but HHV-6B did not. The syncytium formation induced by HHV-6A was observed 2 h after infection; moreover, it was found in the presence of cycloheximide, indicating that HHV-6A induced fusion from without (FFWO) in the target cells. Furthermore, the fusion event was dependent on the expression of the HHV-6 entry receptor, CD46, on the target cell membrane. In addition, we determined that short consensus repeat 2 (SCR2), -3, and -4 of the CD46 ectodomain were essential for the formation of the virus-induced syncytia. Monoclonal antibodies against glycoproteins B and H of HHV-6A inhibited the fusion event, indicating that the syncytium formation induced by HHV-6A required glycoproteins H and B. These findings suggest that FFWO, which HHV-6A induced in a variety of cell lines, may play an important role in the pathogenesis of HHV-6A, not only in lymphocytes but also in various tissues, because CD46 is expressed ubiquitously in human tissues.     

GP96 Interacts with HHV-6 during Viral Entry and Directs It for Cellular Degradation

CD46 and CD134 mediate attachment of Human Herpesvirus 6A (HHV-6A) and HHV-6B to host cell, respectively. But many cell types interfere with viral infection through rapid degradation of viral DNA. Hence, not all cells expressing these receptors are permissive to HHV-6 DNA replication and production of infective virions suggesting the involvement of additional factors that influence HHV-6 propagation. Here, we used a proteomics approach to identify other host cell proteins necessary for HHV-6 binding and entry. We found host cell chaperone protein GP96 to interact with HHV-6A and HHV-6B and to interfere with virus propagation within the host cell. In human peripheral blood mononuclear cells (PBMCs), GP96 is transported to the cell surface upon infection with HHV-6 and interacts with HHV-6A and -6B through its C-terminal end. Suppression of GP96 expression decreased initial viral binding but increased viral DNA replication. Transient expression of human GP96 allowed HHV-6 entry into CHO-K1 cells even in the absence of CD46. Thus, our results suggest an important role for GP96 during HHV-6 infection, which possibly supports the cellular degradation of the virus.     

CD46 is a cellular receptor for human herpesvirus 6

Human herpesvirus 6 (HHV-6) is the etiologic agent of exanthema subitum, causes opportunistic infections in immunocompromised patients, and has been implicated in multiple sclerosis and in the progression of AIDS. Here, we show that the two major HHV-6 subgroups (A and B) use human CD46 as a cellular receptor. Downregulation of surface CD46 was documented during the course of HHV-6 infection. Both acute infection and cell fusion mediated by HHV-6 were specifically inhibited by a monoclonal antibody to CD46; fusion was also blocked by soluble CD46. Nonhuman cells that were resistant to HHV-6 fusion and entry became susceptible upon expression of recombinant human CD46. The use of a ubiquitous immunoregulatory receptor opens novel perspectives for understanding the tropism and pathogenicity of HHV-6.     

The human herpesvirus 6 U100 gene product is the third component of the gH-gL glycoprotein complex on the viral envelope

The human herpesvirus 6 (HHV-6) variant A U100 gene encodes the third component of the glycoprotein H (gH)-glycoprotein L (gL)-containing complex. Glycosidase digestion analysis showed that the U100 gene products are glycoproteins consisting of an 80-kDa protein with complex N-linked oligosaccharides and a 74-kDa protein with immature, high-mannose N-linked oligosaccharides. Based on these characteristics, we designated the U100 gene products glycoprotein Q (gQ). Only the 80-kDa form of gQ was coimmunoprecipitated with an anti-gH antibody, suggesting that the 80-kDa protein associates with the gH-gL complex in HHV-6-infected cells. Furthermore, the complex was detected in purified virions, suggesting that it may play an important role in viral entry.     

A chimeric protein of simian immunodeficiency virus envelope glycoprotein gp140 and Escherichia coli aspartate transcarbamoylase

The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R(2)) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C(6)R(6) ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.     

Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46

Human herpesvirus 6 (HHV-6) employs the complement regulator CD46 (membrane cofactor protein) as a receptor for fusion and entry into target cells. Like other known herpesviruses, HHV-6 encodes multiple glycoproteins, several of which have been implicated in the entry process. In this report, we present evidence that glycoprotein H (gH) is the viral component responsible for binding to CD46. Antibodies to CD46 co-immunoprecipitated an approximately 110-kDa protein band specifically associated with HHV-6-infected cells. This protein was identified as gH by selective depletion with an anti-gH monoclonal antibody, as well as by immunoblot analysis with a rabbit hyperimmune serum directed against a gH synthetic peptide. In reciprocal experiments, a monoclonal antibody against HHV-6 gH was found to co-immunoprecipitate CD46. Studies using monoclonal antibodies directed against specific CD46 domains, as well as engineered constructs lacking defined CD46 regions, demonstrated a close correspondence between the CD46 domains involved in the interaction with gH and those previously shown to be critical for HHV-6 fusion (i.e. short consensus repeats 2 and 3).     

Human Herpesvirus 6 and Measles Virus Employ Distinct CD46 Domains for Receptor Function

We employed a quantitative cell fusion assay to identify structural domains of CD46 required for its function as a receptor for human herpesvirus 6 (HHV-6). We examined the activities of recombinant variants of CD46, including different isoforms as well as engineered truncations and molecular chimeras with decay-accelerating factor, a related protein in the family of regulators of complement activation (RCA). We observed strong receptor activity for all four CD46 isoforms, which differ in the membrane-proximal extracellular and cytoplasmic domains, indicating that the critical determinants for HHV-6 receptor activity reside outside the C-terminal portion of CD46. Analysis of the short consensus repeat (SCR) regions that comprise most of the extracellular portion of CD46 indicated a strong dependence on SCRs 2 and 3 and no requirement for SCRs 1 or 4. Fusion-inhibition studies with SCR-specific monoclonal antibodies supported the essential role of SCRs 2 and 3 in HHV-6 receptor activity. These findings contrast markedly with fusion mediated by measles virus glycoproteins for which we observed a strict dependence on SCRs 1 and 2, consistent with previous reports. These results expand the emerging notion that CD46 and other members of the RCA family are co-opted in distinct manners by different infectious pathogens.     

Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus.

A monoclonal antibody (MCI20.6) which inhibited measles virus (MV) binding to host cells was previously used to characterize a 57- to 67-kDa cell surface glycoprotein as a potential MV receptor. In the present work, this glycoprotein (gp57/67) was immunopurified, and N-terminal amino acid sequencing identified it as human membrane cofactor protein (CD46), a member of the regulators of complement activation gene cluster. Transfection of nonpermissive murine cells with a recombinant expression vector containing CD46 cDNA conferred three major properties expected of cells permissive to MV infection. First, expression of CD46 enabled MV to bind to murine cells. Second, the CD46-expressing murine cells were able to undergo cell-cell fusion when both MV hemagglutinin and MV fusion glycoproteins were expressed after infection with a vaccinia virus recombinant encoding both MV glycoproteins. Third, M12.CD46 murine B cells were able to support MV replication, as shown by production of infectious virus and by cell biosynthesis of viral hemagglutinin after metabolic labeling of infected cells with [35S]methionine. These results show that the human CD46 molecule serves as an MV receptor allowing virus-cell binding, fusion, and viral replication and open new perspectives in the study of MV pathogenesis.     

Induction of cell-cell fusion from without by human herpesvirus 6B

Human herpesvirus (HHV) 6A induce fusion from without (FFWO), whereas HHV-6B is believed to be ineffective in this process. Here, we demonstrate that HHV-6B induces rapid fusion in both epithelial cells and lymphocytes. The fusion was identified 1 h postinfection, could be inhibited by antibodies to HHV-6B gH and to the cellular receptor CD46, and was dependent on virus titer but independent of de novo protein synthesis and UV inactivation of the virus. Comparisons indicate that HHV-6A is only 10-fold more effective in inducing FFWO than HHV-6B. These data demonstrate that HHV-6B can induce FFWO in epithelial cells and lymphocytes.