Human herpesvirus 8 molecular mimicry of ephrin ligands facilitates cell entry and triggers EphA2 signaling

Human herpesvirus 8 (HHV-8) is an oncogenic virus that enters cells by fusion of the viral and endosomal cellular membranes in a process mediated by viral surface glycoproteins. One of the cellular receptors hijacked by HHV-8 to gain access to cells is the EphA2 tyrosine kinase receptor, and the mechanistic basis of EphA2-mediated viral entry remains unclear. Using X-ray structure analysis, targeted mutagenesis, and binding studies, we here show that the HHV-8 envelope glycoprotein complex H and L (gH/gL) binds with subnanomolar affinity to EphA2 via molecular mimicry of the receptor’s cellular ligands, ephrins (Eph family receptor interacting proteins), revealing a pivotal role for the conserved gH residue E52 and the amino-terminal peptide of gL. Using FSI-FRET and cell contraction assays, we further demonstrate that the gH/gL complex also functionally mimics ephrin ligand by inducing EphA2 receptor association via its dimerization interface, thus triggering receptor signaling for cytoskeleton remodeling. These results now provide novel insight into the entry mechanism of HHV-8, opening avenues for the search of therapeutic agents that could interfere with HHV-8–related diseases.

Herpesviruses are known to hijack cellular receptors to enter cells, but this study shows that human herpesvirus 8 takes this to another level by using its envelope glycoprotein complex gH/gL to mimic the EphA2 receptor’s natural ligands, ephrins.     

人类疱疹病毒7型糖蛋白gB、gH、gL、gO介导细胞融合

人类疱疹病毒 7 型(HHV-7)的感染依赖于包膜糖蛋白在病毒生命周期的多个阶段发挥功能. 这些蛋白质可以介导病毒吸附,病毒包膜和宿主细胞膜融合以及病毒在细胞间的接触传播. 将表达 HHV-7 糖蛋白的 293T 细胞与 HHV-7 易感的SupT1 细胞共培养,检测虫荧光素酶报告基因的表达,以鉴定介导膜融合的 HHV-7 糖蛋白. 研究发现,HHV-7 糖蛋白 gB、gH、gL、gO 能介导 293T 细胞与 SupT1 细胞的融合,且融合可被抗 CD4 单抗所抑制. 结果表明,糖蛋白 gB、gH、gL、gO对于 HHV-7 引发的膜融合是必需的,其中某个蛋白质或所形成的蛋白质复合物可能是 CD4 的配体.     

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.     

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.     

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.     

Antibody evasion by the N terminus of murid herpesvirus-4 glycoprotein B

Herpesviruses characteristically transmit infection from immune hosts. Although their success in escaping neutralization by pre-formed antibody is indisputable, the underlying molecular mechanisms remain largely unknown. Glycoprotein B (gB) is the most conserved component of the herpesvirus entry machinery and its N terminus (gB-NT) is a common neutralization target. We used murid herpesvirus-4 to determine how gB-NT contributes to the virus-antibody interaction. Deleting gB-NT had no obvious impact on virus replication, but paradoxically increased virion neutralization by immune sera. This reflected greater antibody access to neutralization epitopes on gH/gL, with which gB was associated. gB-NT itself was variably protected against antibody by O-linked glycans; on virions from epithelial cells it was protected almost completely. gB-NT therefore provides a protective and largely protected cover for a vulnerable part of gH/gL. The conservation of predicted glycosylation sites in other mammalian herpesvirus gB-NTs suggests that this evasion mechanism is widespread. Interestingly, the gB-NT glycans that blocked antibody binding could be targeted for neutralization instead by a lectin, suggesting a means of therapeutic counterattack.     

Soluble Human Cytomegalovirus gH/gL/pUL128–131 Pentameric Complex,but Not gH/gL,Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes

Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128–131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128–131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128–131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)₂ homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0–6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4–8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128–131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process.     

Identification and characterization of a novel structural glycoprotein in pseudorabies virus, gL.

Herpesvirus envelope glycoproteins play important roles in the interaction between virions and target cells. In the alphaherpesvirus pseudorabies virus (PrV), seven glycoproteins that all constitute homologs of glycoproteins found in herpes simplex virus type 1 (HSV-1) have been characterized, including a homolog of HSV-1 glycoprotein H (gH). Since HSV-1 gH is found associated with another essential glycoprotein, gL, we analyzed whether PrV also encodes a gL homolog. DNA sequence analysis of a corresponding part of the UL region adjacent to the internal inverted repeat in PrV strains Kaplan and Becker revealed the presence of two open reading frames (ORF). Deduced proteins exhibited homology to uracil-DNA glycosylase encoded by HSV-1 ORF UL2 (54% identity) and gL encoded by HSV-1 ORF UL1 (24% identity), respectively. To identify the PrV UL1 protein, rabbit antisera were prepared against two synthetic oligopeptides that were predicted by computer analysis to encompass antigenic epitopes. Sera against both peptides reacted in Western blots of purified virions with a 20-kDa protein. The specificity of the reaction was demonstrated by peptide competition. Since the PrV UL1 sequence did not reveal the presence of a consensus N-linked glycosylation site, concanavalin A affinity chromatography and enzymatic deglycosylation of virion glycoproteins were used to ascertain that the PrV UL1 product is O glycosylated. Therefore, we designated this protein PrV gL. Analysis of mutant PrV virions lacking gH showed that concomitantly with the absence of gH, gL was also missing in purified virions. In summary, we identified and characterized a novel structural PrV glycoprotein, gL, which represents the eighth PrV glycoprotein described. In addition, we show that virion location of PrV gL is dependent on the presence of PrV gH.     

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.     

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.     

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.     

Characterization of a novel third member of the human cytomegalovirus glycoprotein H-glycoprotein L complex.

A prerequisite for understanding the molecular function of the human cytomegalovirus (HCMV) gH (UL75)-gL (UL115) complex is a detailed knowledge of the structure of this complex in its functional form, as it is present in mature virions. The gH protein is known to be a component of a 240-kDa envelope complex designated as gCIII (D. R. Gretch, B. Kari, L. Rasmussen, R. C. Gehrz, and M. F. Stinski, J. Virol. 62:875-881, 1988). However, the exact composition of the gCIII complex remains unknown. In this report, we attempted reconstitution of the gCIII complex by coexpression of gH and gL in the baculovirus expression system. Formation of recombinant gH-gL complexes of approximately 115 kDa was demonstrated; however, no higher-molecular-mass (approximately 240-kDa) recombinant gH-gL complexes were detected, suggesting that the presence of gH and gL alone is not sufficient for reconstitution of the gCIII complex. To identify other mammalian and/or HCMV factors which may be necessary for gCIII formation, immunoprecipitates of gH and gL from HCMV-infected fibroblasts and purified HCMV virions were examined. This analysis did reveal a number of coprecipitating proteins which associate either transiently or integrally with gH and gL. One coprecipitating protein of 145 kDa was shown to be an integral component of gCIII, along with gH and gL. Characterization of the 145-kDa protein demonstrates that it is structurally and antigenically unrelated to gH and gL and that it appears to be virally encoded. Together, these data indicate that the 145-kDa protein is a third novel component of the mature HCMV gH-gL complex.     

Human herpesvirus 8 glycoprotein B (gB), gH, and gL can mediate cell fusion

Herpesvirus entry into cells and herpesvirus-induced cell fusion are related processes in that virus penetration proceeds by fusion of the viral envelope and cell membrane. To characterize the human herpesvirus 8 (HHV-8) glycoproteins that can mediate cell fusion, a luciferase reporter gene activation assay was used. Chinese hamster ovary (CHO) cells expressing the HHV-8 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with human cells transfected with T7 RNA polymerase. Because HHV-8 glycoprotein B (gB) expressed in CHO cells localizes to the perinuclear region, a truncated form of gB (designated gB(MUT)) that lacks putative endocytosis signals was constructed by deletion of the distal 58 amino acids of the cytoplasmic tail. HHV-8 gB(MUT) was expressed efficiently on the surface of CHO cells. HHV-8 gB, gH, and gL could mediate the fusion of CHO cells with two different human cell types, embryonic kidney cells and B lymphocytes. Substituting gB(MUT) for gB significantly enhanced the fusion of CHO cells with human embryonic kidney cells but not B lymphocytes. Thus, two human cell types known to be susceptible to HHV-8 entry were also suitable targets for cell fusion induced by HHV-8 gB, gH, and gL. For human embryonic kidney cells and B cells at least, optimal fusion was noted with the expression of all three HHV-8 glycoproteins.     

Maturation of Human Herpesvirus 6A Glycoprotein O Requires Coexpression of Glycoprotein H and Glycoprotein L

Glycoprotein O (gO) is conserved among betaherpesviruses, but little is known about the maturation process of gO in human herpesvirus 6 (HHV-6). We found that HHV-6 gO maturation was accompanied by cleavage of its carboxyl terminus and required coexpression of gH and gL, which promoted the export of gO out of the endoplasmic reticulum (ER). Finally, we also found that gO was not required for HHV-6A growth in T cells.     

Identification of the human herpesvirus 6 glycoprotein H and putative large tegument protein genes.

Determination of the nucleotide sequences of two molecular clones of human herpesvirus 6 (HHV-6) (strain GS) and comparison with those of human cytomegalovirus (HCMV) has allowed the identification of the genes for the glycoprotein H (gH) and the putative large tegument protein of HHV-6. Two molecular clones of fragments of HHV-6, the BamHI-G fragment (7,981 bp) of the clone termed pZVB43 and a HindIII fragment (8,717 bp) of the clone termed pZVH14, represent approximately 10% of the HHV-6 genome (16,689). An open reading frame within the BamHI-G fragment was designated the gH gene of HHV-6 because of the extensive sequence similarity of its predicted product (79,549 Da) to the HCMV gH gene product. The predicted product (239,589 Da) of an open reading frame within clone pZVH14 showed homology to the predicted product of the proposed gene of HCMV representing the large tegument protein. Computer analyses indicated a closer relationship of the predicted peptides of these HHV-6 genes to those of HCMV than to those of the other human herpesviruses Epstein-Barr virus, herpes simplex virus type 1, and varicella-zoster virus. The gH gene was more conserved among the human herpesvirus group, while significant sequence similarity of the tegument gene could be found only with that of HCMV. The data reported here with one conserved gene (gH) and a more divergent gene (tegument) support previous reports that HHV-6 and HCMV are more closely related to each other than to the other well-characterized human herpesviruses.     

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.     

Extreme dependence of gH and gL expression on ORF57 and association with highly unusual codon usage in rhesus monkey rhadinovirus

Standard vectors for high-level expression elicited undetectable levels of the gH and gL glycoproteins of rhesus monkey rhadinovirus (RRV) following transient-transfection assays under a variety of conditions. These same vectors and conditions yielded high levels of RRV gB expression. Unlike other genes of RRV, both the gH and gL genes were noted to have a highly aberrant, suboptimal codon usage. High levels of RRV gH and gL expression were achieved by two alternative means: codon optimization or coexpression of RRV ORF57. The failure of gH and gL to be expressed in the absence of ORF57 and in the absence of codon optimization could not be explained by the failure of RNA to egress from the nucleus. Rather, the defect in gH and gL expression appeared to be cytoplasmic in nature. It is not clear at the present time whether the aberrant codon usage for gH and gL of RRV is an intentional regulatory strategy used by the virus or whether it is driven by some external force, such as intrinsic immunity. In any event, our results indicate that the need of ORF57 for gH and gL expression can be circumvented by codon optimization, that RRV ORF57 acts principally to allow translation of gH and gL RNA in the cytoplasm, and that this activity of ORF57 is related in some way to the aberrant codon usage of the gH and gL RNAs.     

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).     

The Epstein-Barr virus (EBV) BZLF2 gene product associates with the gH and gL homologs of EBV and carries an epitope critical to infection of B cells but not of epithelial cells.

Glycoprotein gp85, the product of the BXLF2 open reading frame (ORF), is the gH homolog of Epstein-Barr virus (EBV) and has been implicated in penetration of virus into B cells. Like its counterparts in other herpesviruses, it associates with a gL homolog, gp25, which is the product of the BKRF2 ORF. Unlike the gH homologs of other herpesviruses, however, gp85 also complexes with two additional glycoproteins of 42 and 38 kDa. Glycoproteins gp42 and gp38 were determined to be alternatively processed forms of the BZLF2 gene product. Coexpression of EBV gH and gL facilitated transport of gH to the cell surface and resulted in formation of a stable complex of gH and gL. It also restored expression of an epitope recognized by monoclonal antibody E1D1, which immunoprecipitates the native gH complex but not recombinant gH expressed in isolation. Coexpression of gH, gL, and the BZLF2 ORF restored expression of an epitope recognized by a second monoclonal antibody, F-2-1, which immunoprecipitates the native gH-gL-gp42/38 complex but not the complex of recombinant gH and gL alone. The epitope recognized by antibody F-2-1 was mapped to the BZLF2 gene product itself. Antibody F-2-1 inhibited the ability of EBV to infect B lymphocytes but had no effect on the ability of the virus to infect the epithelial cell line SVK-CR2. In contrast, antibody E1D1 had no effect on infection of the B-cell line but inhibited infection of the epithelial cell line. These results indicate that penetration of the two cell types by EBV involves differential use of the gH-gL-gp42/38 complex and suggest the hypothesis that the BZLF2 gene product has evolved as a unique adaptation to infection of B lymphocytes by EBV.