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Kaposi's sarcoma-associated herpesvirus gH/gL: glycoprotein export and interaction with cellular receptors.

ABSTRACT: The attachment, entry, and fusion of Kaposi's sarcoma-associated herpesvirus (KSHV) with target cells are mediated by complex machinery containing, among others, viral glycoprotein H (gH) and its alleged chaperone, gL. We observed that KSHV gH, in contrast to its homologues in several other herpesviruses, is transported to the cytoplasm membrane independently from gL, but not vice versa. Mutational analysis revealed that the N terminus of gH is sufficient for gL interaction. However, the entire extracellular part of gH is required for efficient gL secretion. The soluble ectodomain of gH was sufficient to interact with the surfaces of potential target cells in a heparin-dependent manner, and binding was further enhanced by coexpression of gL. Surface plasmon resonance revealed a remarkably high affinity of gH for glycosaminoglycans. Heparan sulfate (HS) proteoglycans of the syndecan family act as cellular receptors for the gH/gL complex. They promoted KSHV infection, and expression of gH/gL on target cells inhibited subsequent KSHV infection. Whereas gH alone was able to bind to HS, we observed that only the gH/gL complex adhered to heparan sulfate-negative cells at lamellipodium-like structures.

SUBMITTER: Hahn A

PROVIDER: S-EPMC2612313 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Kaposi's sarcoma-associated herpesvirus gH/gL: glycoprotein export and interaction with cellular receptors.

Hahn Alexander A Birkmann Alexander A Wies Effi E Dorer Dominik D Mahr Kerstin K Stürzl Michael M Titgemeyer Fritz F Neipel Frank F

Journal of virology 20081022 1

The attachment, entry, and fusion of Kaposi's sarcoma-associated herpesvirus (KSHV) with target cells are mediated by complex machinery containing, among others, viral glycoprotein H (gH) and its alleged chaperone, gL. We observed that KSHV gH, in contrast to its homologues in several other herpesviruses, is transported to the cytoplasm membrane independently from gL, but not vice versa. Mutational analysis revealed that the N terminus of gH is sufficient for gL interaction. However, the entire ...[more]

PMID: 18945775

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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus associated with Kaposi's sarcoma and two B-cell malignancies. The rhesus monkey rhadinovirus (RRV) is a virus of nonhuman primates that is closely related to KSHV. Eph family receptor tyrosine kinases (Ephs) are cellular receptors for the gH/gL glycoprotein complexes of both KSHV and RRV. Through sequence analysis and mutational screens, we identified conserved residues in the N-terminal domain of KSHV and RRV glycoprotein H that are critical for Eph-binding in vitro. Homology-based structural predictions of the KSHV and RRV gH/gL complexes based on the Epstein-Barr-Virus gH/gL crystal structure located these amino acids in a beta-hairpin on gH, which is likely stabilized by gL and is optimally positioned for protein-protein interactions. Guided by these predictions, we generated recombinant RRV and KSHV strains mutated in the conserved motif as well as an RRV gL null mutant. Inhibition experiments using these mutants confirmed that disruption of the identified Eph-interaction motif or of gL expression resulted in complete detargeting from Ephs. However, all mutants were infectious on all cell types tested, exhibiting normal attachment but a reduction in infectivity of up to one log order of magnitude. While Eph-binding-negative RRV mutants were replication-competent on fibroblasts, their infectivity was comparatively more reduced on endothelial cells with a substantial subpopulation of endothelial cells remaining resistant to infection. Together, this provides evidence for a cell type-specific use of Ephs by RRV. Furthermore, our results demonstrate that gL is dispensable for infection by RRV. Its deletion caused a reduction in infectivity similar to that observed after mutation of Eph-binding residues in gH. Our findings would be compatible with an ability of KSHV and RRV to use other, less efficient entry mediators in lieu of Ephs, although these host factors may not be uniformly expressed by all cells.

Project description:Cell-free and cell-to-cell spread of herpesviruses involves a core fusion apparatus comprised of the fusion protein glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL/gO and gH/gL/pUL128-131 facilitate spread in different cell types. The gO and pUL128-131 components bind distinct receptors, but how the gH/gL portions of the complexes functionally compare is not understood. We previously characterized a panel of gL mutants by transient expression and showed that many were impaired for gH/gL-gB-dependent cell-cell fusion but were still able to form gH/gL/pUL128-131 and induce receptor interference. Here, the gL mutants were engineered into the HCMV BAC clones TB40/e-BAC4 (TB), TR, and Merlin (ME), which differ in their utilization of the two complexes for entry and spread. Several of the gL mutations disproportionately impacted gH/gL/gO-dependent entry and spread over gH/gL/pUL128-131 processes. The effects of some mutants could be explained by impaired gH/gL/gO assembly, but other mutants impacted gH/gL/gO function. Soluble gH/gL/gO containing the L201 mutant failed to block HCMV infection despite unimpaired binding to PDGFRα, indicating the existence of other important gH/gL/gO receptors. Another mutant (L139) enhanced the gH/gL/gO-dependent cell-free spread of TR, suggesting a "hyperactive" gH/gL/gO. Recently published crystallography and cryo-electron microscopy studies suggest structural conservation of the gH/gL underlying gH/gL/gO and gH/gL/pUL128-131. However, our data suggest important differences in the gH/gL of the two complexes and support a model in which gH/gL/gO can provide an activation signal for gB. IMPORTANCE The endemic betaherpesvirus HCMV circulates in human populations as a complex mixture of genetically distinct variants, establishes lifelong persistent infections, and causes significant disease in neonates and immunocompromised adults. This study capitalizes on our recent characterizations of three genetically distinct HCMV BAC clones to discern the functions of the envelope glycoprotein complexes gH/gL/gO and gH/gL/pUL128-13, which are promising vaccine targets that share the herpesvirus core fusion apparatus component, gH/gL. Mutations in the shared gL subunit disproportionally affected gH/gL/gO, demonstrating mechanistic differences between the two complexes, and may provide a basis for more refined evaluations of neutralizing antibodies.

Project description:The core, conserved function of the herpesvirus gH/gL is to promote gB-mediated membrane fusion during entry, although the mechanism is poorly understood. The human cytomegalovirus (HCMV) gH/gL can exist as either the gH/gL/gO trimer or the gH/gL/UL128/UL130/UL131 (gH/gL/UL128-131) pentamer. One model suggests that gH/gL/gO provides the core fusion role during entry into all cells within the broad tropism of HCMV, whereas gH/gL/UL128-131 acts at an earlier stage, by a distinct receptor-binding mechanism to enhance infection of select cell types, such as epithelial cells, endothelial cells, and monocytes/macrophages. To further study the distinct functions of these complexes, mutants with individual charged cluster-to-alanine (CCTA) mutations of gH and gL were combined to generate a library of 80 mutant gH/gL heterodimers. The majority of the mutant gH/gL complexes were unable to facilitate gB-mediated membrane fusion in transient-expression cell-cell fusion experiments. In contrast, these mutants supported the formation of gH/gL/UL128-131 complexes that could block HCMV infection in receptor interference experiments. These results suggest that receptor interactions with gH/gL/UL128-131 involve surfaces contained on the UL128-131 proteins but not on gH/gL. gH/gL/UL128-131 receptor interference could be blocked with anti-gH antibodies, suggesting that interference is a cell surface phenomenon and that anti-gH antibodies can block gH/gL/UL128-131 in a manner that is distinct from that for gH/gL/gO.Interest in the gH/gL complexes of HCMV (especially gH/gL/UL128-131) as vaccine targets has far outpaced our understanding of the mechanism by which they facilitate entry and contribute to broad cellular tropism. For Epstein-Barr virus (EBV), gH/gL and gH/gL/gp42 are both capable of promoting gB fusion for entry into epithelial cells and B cells, respectively. In contrast, HCMV gH/gL/gO appears to be the sole fusion cofactor that promotes gB fusion activity, whereas gH/gL/UL128-131 expands cell tropism through a distinct yet unknown mechanism. This study suggests that the surfaces of HCMV gH/gL are critical for promoting gB fusion but are dispensable for gH/gL/UL128-131 receptor interaction. This underscores the importance of gH/gL/gO in HCMV entry into all cell types and reaffirms the complex as a candidate target for vaccine development. The two functionally distinct forms of gH/gL present in HCMV make for a useful model with which to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion.

Project description:Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is a gammaherpesvirus associated with several human malignancies. DNA methylation at CpG dinucleotides is an epigenetic mark dysregulated in many cancer types and in KSHV-infected cells. Several previous studies have analyzed in detail the CpG methylation of the KSHV episomal genomes, but little is known about the impact of KSHV on the human genome. Our knowledge of cellular CpG methylation in the context of KSHV infection is currently limited to four hypermethylated human gene promoters. Therefore, we undertook a comprehensive CpG methylation analysis of the human methylome in KSHV-infected cells and KSHV-associated primary effusion lymphoma (PEL). We performed Infinium HumanMethylation450K and MethylationEpic BeadChip arrays and identified panels of hyper- and hypomethylated cellular promoters in KSHV-infected cells. We combined our genome-wide methylation analysis with high-throughput RNA sequencing (RNA-seq) to add functional outcomes to the virally induced methylation changes. We were able to correlate many downregulated genes with promoter hypermethylation and upregulated genes with hypomethylation. In addition, we show that treating the cells with a demethylating agent leads to reexpression of these downregulated genes, indicating that, indeed, DNA methylation plays a role in the repression of these human genes. Comparison between de novo infection and PEL suggests that the virus induces initial hypermethylation followed by a slow increase in genome-wide hypomethylation. This study extends our understanding of the relationship between epigenetic changes induced by KSHV infection and tumorigenesis.IMPORTANCE In cancer cells, certain promoters become aberrantly methylated, contributing to the phenotype of the tumor. KSHV infection seems to modify cellular CpG methylation, but only a few methylated promoters have been identified in KSHV-infected cells. Here, we investigated the CpG methylation of the human genome in KSHV-associated primary effusion lymphoma (PEL) and KSHV-infected cells. We have identified many hyper- and hypomethylated gene promoters and correlated their methylation with cellular gene expression. These differentially methylated cellular promoters can distinguish KSHV-positive cells from uninfected cells and may serve as the foundation for the use of these differentially methylated regions as potential biomarkers for KSHV-associated malignancies. Drugs that reverse these cancerous methylation patterns have the potential to inhibit tumor growth. Here, we show that treating PEL cells with a demethylating drug (5-aza-2'-deoxycytidine) led to inhibition of cell growth, raising the possibility of testing this drug for the treatment of PEL.

Dataset Information

Kaposi's sarcoma-associated herpesvirus gH/gL: glycoprotein export and interaction with cellular receptors.

Publications

Kaposi's sarcoma-associated herpesvirus gH/gL: glycoprotein export and interaction with cellular receptors.

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