Functional Characterization of the HveA Homolog Specified by African Green Monkey Kidney Cells with a Herpes Simplex Virus Expressing the Green Fluorescence Protein

Foster, Timothy P.; Chouljenko, Vladimir N.; Kousoulas, Konstantin G.

doi:10.1006/viro.1999.9743

Cited by 24 publications

(23 citation statements)

References 22 publications

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“…The ability of HveA-Y23F to mediate HSV entry indicates that interactions with the hy-droxyl group of HveA-Y23 are not critical for HveA function. This result is supported by the fact that the primate HveA homolog functions as an HSV entry mediator and has phenylalanine in place of tyrosine at position 23 (17). From this result, we predict that gD-M11 is important for gD binding to HveA because gD-M11 interacts with the HveA-Y23 phenyl ring (Fig.…”

Section: Discussionmentioning

confidence: 67%

Structure-Based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM)

Connolly

Landsburg

Carfı́

et al. 2002

J Virol

107

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Binding of herpes simplex virus (HSV) envelope glycoprotein D (gD) to a cell surface receptor is an essential step of virus entry. We recently determined the crystal structure of gD bound to one receptor, HveA. HveA is a member of the tumor necrosis factor receptor family and contains four characteristic cysteine-rich domains (CRDs). The first two CRDs of HveA are necessary and sufficient for gD binding. The structure of the gD-HveA complex reveals that 17 amino acids in HveA CRD1 and 4 amino acids in HveA CRD2 directly contact gD. To determine the contribution of these 21 HveA residues to virus entry, we constructed forms of HveA mutated in each of these contact residues. We determined the ability of the mutant proteins to bind gD, facilitate virus entry, and form HveA oligomers. Our results point to a binding hot spot centered around HveA-Y23, a residue that protrudes into a crevice on the surface of gD. Both the hydroxyl group and phenyl group of HveA-Y23 contribute to HSV entry. Our results also suggest that an intermolecular ␤-sheet formed between gD and HveA residues 35 to 37 contributes to binding and that a C37-C19 disulfide bond in CRD1 is a critical component of HveA structure necessary for gD binding. The results argue that CRD2 is required for gD binding mainly to provide structural support for a gD binding site in CRD1. Only one mutant, HveA-R75A, exhibited enhanced gD binding. While some mutations influenced complex formation, the majority did not affect HSV entry, suggesting that most contact residues contribute to HveA receptor function collectively rather than individually. This structure-based dissection of the gD-HveA binding site highlights the contribution of key residues within HveA to gD binding and HSV entry and defines a target region for the design of small-molecule inhibitors.In humans, herpes simplex virus (HSV) typically causes mucosal lesions and then spreads to the peripheral nervous system, where it establishes latent infections in sensory neurons. HSV encodes at least 11 membrane glycoproteins, and four of these (gD, gH, gL, and gB) are essential for entry of virions into mammalian cells. Expression of these four glycoproteins alone can facilitate cell fusion (4,33,37,41,44).The current model for virus entry posits the following series of events. Initially gC and/or gB binds cell surface heparan sulfate proteoglycans (41). Although binding of gC is a highaffinity interaction (K D ϭ 10 Ϫ8 M) (38), it is not essential for virus entry (41,42). This gC binding step is followed by binding of gD to one of several cell surface receptors. This essential step leads to pH-independent membrane fusion of the viral envelope with the cell plasma membrane, a process facilitated by gD, gH, gL, and gB (41). Some experiments suggest that the gH/gL heterodimer and/or gB is the viral fusogen, but how their fusogenic activity is triggered by the gD-receptor interaction is unknown (18,22).Several cellular receptors for HSV have been identified through expression cloning and homology searches, includin...

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Section: Discussionmentioning

confidence: 67%

Structure-Based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM)

Connolly

Landsburg

Carfı́

et al. 2002

J Virol

107

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“…The isolates were then screened for the loss of ability to use each of the known HSV entry receptors. Although Vero cells express monkey versions of nectin-1 and HVEM and both are functional HSV-1 entry receptors (10,11), all of the mutants obtained retained full entry activity with nectin-1 but not HVEM. This finding suggests that HVEM is not a functional entry receptor in Vero cells or that HSV-1 mutants capable of using HVEM for entry, but not nectin-1, may be rarer than the converse.…”

Section: Discussionmentioning

confidence: 98%

“…The virus isolates screened were first selected for their ability to form plaques on Vero (African green monkey kidney) cells, which are known to express primate forms of HVEM and nectin-1 (10,11). Sequencing of the gD genes identified mutations that significantly reduced or eliminated functional interactions with both HVEM and 3-O-S HS, whereas entry activity with nectin-1 was left intact.…”

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confidence: 99%

Random mutagenesis of the gene encoding a viral ligand for multiple cell entry receptors to obtain viral mutants altered for receptor usage

Yoon

Spear²

2004

Proc. Natl. Acad. Sci. U.S.A.

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Herpes simplex virus type 1 (HSV-1) can enter cells expressing any one of multiple entry receptors, including the herpesvirus entry mediator (HVEM), nectin-1, and sites in heparan sulfate generated by specific 3-O-sulfotransferases. The viral ligand for these receptors is glycoprotein D (gD). To define structural requirements for functional interactions of gD with its receptors and to obtain viral mutants altered for receptor usage, we generated a library of HSV-1 mutants with random mutations in the gD gene. Viral isolates selected on a monkey cell line (Vero) were screened for the loss of ability to infect cells expressing each of the HSV-1 receptors. The 10 HSV-1 mutants obtained had 12 mutations in gD, affecting 11 amino acids. All mutations reduced or abrogated viral entry through HVEM and 3-O-sulfated heparan sulfate, indicating that similar features of gD are critical for functional interactions with both these receptors. None of the mutations reduced viral entry through nectin-1, whereas a subset of the mutations conferred ability to use nectin-2 as an entry receptor. These and other results show that features of gD, including conformation of the N terminus, critical for functional interactions with HVEM͞3-O-sulfated heparan sulfate, differ from those critical for interactions with nectin-1. Herpes simplex virus (HSV) can bind to cells by interaction with heparan sulfate but entry requires interaction of envelope glycoprotein D (gD) with any one of three classes of cell-surface receptors (1, 2). These receptors include the herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor receptor family; nectin-1 and nectin-2, cell adhesion molecules in the Ig superfamily; and specific sites in heparan sulfate generated by certain isoforms of 3-O-sulfotransferase (the product is referred to as 3-O-sulfated heparan sulfate, 3-O-S HS). Binding of gD to one of these receptors triggers fusion of the viral envelope with a cell membrane, an event that requires also the action of envelope glycoprotein B, glycoprotein H, and glycoprotein L (1). We and others have engaged in targeted mutagenesis studies to define the residues in gD that are critical for physical and functional interactions with the several HSV entry receptors (3-6). These studies have been guided in part by serendipity and in part by an x-ray structure of a truncated form of HSV type 1 (HSV-1) gD in complex with a truncated form of HVEM (7).An approach for random mutagenesis of gD and screening of viral mutants for altered receptor preferences, unbiased except for the requirement that the mutants retain ability to use at least one receptor, was needed to obtain the range of mutants desired and to define the structural determinants of gD critical for interactions with each receptor. We adapted existing technology to develop a system for precise gene replacement at the gD locus in the HSV-1 genome and to generate a library of viral mutants altered in the gD gene, a library that could be screened for the desired phenotypes. This system relied...

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“…Samples were electrophoretically separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with anti-protC monoclonal antibody HPC-4 at a 1:50 dilution (ATCC CRL HB-9892). Subsequently, blots were incubated for 1 h with a peroxidase-conjugated secondary antibody at a 1:50,000 dilution and then visualized on X-ray film by chemiluminescence (Pierce Chemicals, Rockford, Ill.) (11,14). All antibody dilutions and buffer washes were performed in TBS supplemented with 0.135 M CaCl 2 and 0.11 M MgCl 2 (TBS-Ca/Mg).…”

Section: Cells and Virusesmentioning

confidence: 99%

Plasma Membrane Topology of Syncytial Domains of Herpes Simplex Virus Type 1 Glycoprotein K (gK): the UL20 Protein Enables Cell Surface Localization of gK but Not gK-Mediated Cell-to-Cell Fusion

Foster

Álvarez

Kousoulas

2003

J Virol

Self Cite

117

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Most spontaneously occurring mutations that cause extensive herpes simplex virus type 1 (HSV-1)-induced cell fusion are single amino acid changes within glycoprotein K (gK). Despite the strong genetic association of gK with virus-induced cell fusion, its direct involvement in cellular membrane fusion has been controversial, largely due to previously unsuccessful efforts to detect gK expression on virion and cellular surfaces. Recently, we showed that gK is expressed on HSV-1 virions and functioned in virus entry (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). To determine whether gK is expressed on cellular surfaces, as well as its membrane topology, we generated the recombinant viruses gKV5DI, gKV5DII, gKV5DIII, and gKV5DIVcontaining insertions of the V5 antigenic epitope within each of four domains of gK predicted to localize either in the cytoplasmic side or in the extracytoplasmic side of cellular membranes. Immunohistochemical and confocal microscopy analyses of infected cells showed that both wild-type and syncytial forms of gK were expressed on cell surfaces. Analysis of the topology of the V5-tagged gK revealed that gK domains I and IV were located extracellularly, whereas domains II and III were localized intracellularly. Transiently expressed gK failed to localize in cellular plasma membranes. In contrast, infection of gKtransfected cells with the gK-null virus ⌬gK enabled expression of gK on cell surfaces, as well as gK-mediated membrane fusion. Transient-coexpression experiments revealed that the UL20 protein enabled cell surface expression of gK, but not gK-mediated cell-to-cell fusion, indicating that additional viral proteins are required for expression of the gK syncytial phenotype.Viral glycoproteins are key determinants of membrane fusion events throughout the life cycle of herpesviruses. Herpes simplex viruses (HSVs) specify at least 11 glycoproteins:

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Functional Characterization of the HveA Homolog Specified by African Green Monkey Kidney Cells with a Herpes Simplex Virus Expressing the Green Fluorescence Protein

Cited by 24 publications

References 22 publications

Structure-Based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM)

Structure-Based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM)

Random mutagenesis of the gene encoding a viral ligand for multiple cell entry receptors to obtain viral mutants altered for receptor usage

Plasma Membrane Topology of Syncytial Domains of Herpes Simplex Virus Type 1 Glycoprotein K (gK): the UL20 Protein Enables Cell Surface Localization of gK but Not gK-Mediated Cell-to-Cell Fusion

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