Entry and Egress of Varicella Virus Blocked by Same Anti-gH Monoclonal Antibody

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The trafficking of varicella-zoster virus (VZV) gH was investigated under both infection and transfection conditions. In initial endocytosis assays performed in infected cells, the three glycoproteins gE, gI, and gB served as positive controls for internalization from the plasma membrane. Subsequently, we discovered that gH in VZV-infected cells was also internalized and followed a similar trafficking pattern. This observation was unexpected because all herpesvirus gH homologues have short endodomains not known to contain trafficking motifs. Further investigation demonstrated that VZV gH, when expressed alone with its chaperone gL, was capable of endocytosis in a clathrin-dependent manner, independent of gE, gI, or gB. Upon inspection of the short gH cytoplasmic tail, we discovered a putative tyrosine-based endocytosis motif (YNKI). When the tyrosine was replaced with an alanine, endocytosis of gH was blocked. Utilizing an endocytosis assay dependent on biotin labeling, we further documented that endocytosis of VZV gH was antibody independent. In control experiments, we showed that gE, gI, and gB also internalized in an antibody-independent manner. Alignment analysis of the VZV gH cytoplasmic tail to other herpesvirus gH homologues revealed two important findings: (i) herpes simplex virus type 1 and 2 homologues lacked an endocytosis motif, while all other alphaherpesvirus gH homologues contained a potential motif, and (ii) the VZV gH and simian varicella virus gH cytoplasmic tails were likely longer in length (18 amino acids) than predicted in the original sequence analyses (12 and 16 amino acids, respectively). The longer tails provided the proper context for a functional endocytosis motif. Varicella-zoster virus (VZV) glycoprotein H (gH) is one of seven recognized glycoproteins in VZV (16).The product of open reading frame 37, gH is a 118-kDa type I transmembrane protein with a large ectodomain of 812 residues and a cytoplasmic tail that has been estimated at between 12 and 14 amino acids. VZV gH contains an immunodominant complement-independent neutralization epitope (67). Monoclonal antibodies against gH are able to block entry, egress, and cell-to-cell spread of the virus in cell culture (67,83). These results demonstrate a role for gH in both entry and cell-to-cell spread. In addition, VZV gH, like herpes simplex virus type 1 (HSV-1), requires the formation of a heterodimeric complex with gL for complete maturation and cell surface expression (22,46). Among the human herpesviruses, gH is highly conserved, and many of its properties are common throughout the herpesvirus family. This glycoprotein is essential for penetration and cell-to-cell spread in pseudorabies virus (5, 78), HSV-1 (26), and Epstein-Barr virus (37, 66). The functional importance of the gH-gL complex formation is echoed in other herpesviruses, including HSV-1 (46), pseudorabies virus (53), Epstein-Barr virus (102), human cytomegalovirus (52, 88), human herpesvirus 6 (56), and human herpesvirus 7 (71).VZV gH is considered the major VZV fus...

mentioning

confidence: 75%

A Functional YNKI Motif in the Short Cytoplasmic Tail of Varicella-Zoster Virus Glycoprotein gH Mediates Clathrin-Dependent and Antibody-Independent Endocytosis

Pasieka

Marešová

Grose

2003

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“…VZV gH has fusogenic activities that induce cell-tocell fusion when coexpressed with gL (11,35,44). Further, VZV gH undergoes endocytosis in both VZV-infected and gHϩgL-transfected cells (42).…”

Section: Resultsmentioning

confidence: 99%

Regulation of Varicella-Zoster Virus-Induced Cell-to-Cell Fusion by the Endocytosis-Competent Glycoproteins gH and gE

Pasieka

Marešová

Shiraki

et al. 2004

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The gH glycoprotein of varicella-zoster virus (VZV) is a major fusogen. The realigned short cytoplasmic tail of gH (18 amino acids) harbors a functional endocytosis motif (YNKI) that mediates internalization in both VZV-infected and transfected cells (T. J. Pasieka, L. Maresova, and C. Grose, J. Virol. 77: 4194-4202, 2003). During subsequent confocal microscopy studies of endocytosis-deficient gH mutants, we observed that cells transfected with the gH tail mutants exhibited marked fusion. Therefore, we postulated that VZV gH endocytosis served to regulate cell-to-cell fusion. Subsequent analyses of gH؉gL transfection fusion assays by the Kolmogorov-Smirnov statistical test demonstrated that expression of the endocytosis-deficient gH mutants resulted in a statistically significant enhancement of cell-to-cell fusion (P < 0.0001) compared to wild-type gH. On the other hand, coexpression of VZV gE, another endocytosis-competent VZV glycoprotein, was able to temper the fusogenicity of the gH endocytosis mutants by facilitating internalization of the mutant gH protein from the cell surface. When the latter results were similarly analyzed, there was no longer any enhanced fusion by the endocytosis-deficient gH mutant protein. In summary, these studies support a role for gH endocytosis in regulating the cell surface expression of gH and thereby regulating gH-mediated fusion. The data also confirm and extend prior observations of a gE-gH interaction during viral glycoprotein trafficking in a VZV transfection system. Varicella-zoster virus (VZV) is an extremely cell-associated alphaherpesvirus. Unlike many herpesviruses, VZV is not released into the medium overlying cultured cells (15, 50). Instead, the virus spreads through fusion of one infected cell plasma membrane to that of an adjacent cell. Syncytium formation is induced by the expression of fusogenic viral glycoproteins and results in the formation of large polykaryons that can include hundreds of nuclei (19). We have previously documented cell-to-cell fusion in transfected HeLa cells coexpressing gH and gL (11,30). For the related herpes simplex virus type 1 (HSV-1) and HSV-2, syncytium formation requires expression of the quartet gH, gL, gD, and gB (48). Pseudorabies virus and human herpesvirus type 8 fusion require the coexpression of gH, gL, and gB, but not gD (26,43). Of further interest, other glycoproteins may further modulate virus induced fusion activity (1).All wild-type strains of VZV are highly fusogenic within human skin cells at the base of the typical vesicular lesions of chickenpox, as well as in cultured cells. Studies conducted in this laboratory over several years have demonstrated that the amount of VZV fusion in cell culture is affected by at least three factors: incubation temperature, cell type, and virus strain (8). Fusion was found to be more prominent in epidermal cells and neuroectodermally derived human melanoma cells than in fibroblasts. In addition, when the amount of fusion was compared at 37°C versus 33°C, cells incubated at the lowe...

“…Lytic infection requires membrane fusion, an event governed by a core complex consisting of conserved glycoproteins gB, gH, and gL. Along with membrane fusion, VZV gH and gL are also involved in virus egress and are essential for virus replication (1)(2)(3)(4). In addition to mediating virus entry, VZV glycoproteins can traffic from infected cells to uninfected cells (5).…”

Section: Varicella-zoster Virus (Vzv) Is a Ubiquitous Highly Cell-asmentioning

confidence: 99%

Human Anti-Varicella-Zoster Virus (VZV) Recombinant Monoclonal Antibody Produced after Zostavax Immunization Recognizes the gH/gL Complex and Neutralizes VZV Infection

Birlea

Owens

Eshleman

et al. 2013

Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). V aricella-zoster virus (VZV), the etiologic agent of varicella (chickenpox) and zoster (shingles), is an exclusively human pathogen and a member of the Herpesviridae family of enveloped DNA viruses. Herpesviruses cause both lytic and latent infections. Lytic infection requires membrane fusion, an event governed by a core complex consisting of conserved glycoproteins gB, gH, and gL. Along with membrane fusion, VZV gH and gL are also involved in virus egress and are essential for virus replication (1-4). In addition to mediating virus entry, VZV glycoproteins can traffic from infected cells to uninfected cells (5). VZV glycoproteins induce strong humoral immune responses both in naturally infected individuals and in varicella or zoster vaccine recipients (6-10). While VZV gE is the most immunogenic and predominant glycoprotein in VZV-infected cell membranes, antibodies to VZV gH are the major neutralizing antibodies (11-16). It is likely that neutralizing activity by the gH/gL complex effectively prevents cell-to-cell virus spread (5,(17)(18)(19)(20).Analysis of VZV attachment and membrane fusion requires highly specific neutralizing antibodies. Hybridoma cell lines and phage display libraries produce human anti-VZV gE and gH monoclonal antibodies (MAbs) that neutralize virus infection (21-24). Monoclonal antibodies that recognize the VZV gH/gL protein complex hold promise in therapies involving passive transfer of neutralizing VZV antibodies (15,16,25,26). Here we present a new method for constructing a recombinant human monoclonal anti-VZV antibody and show that this antibody detects a conformational epitope on the gH/gL complex and neutralizes virus. MATERIALS AND METHODS Cells and virus.Human lung fibroblast (HFL) and human embryonic kidney (HEK-EBNA 293) cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with 4 mM L-glutamine (DMEM; Sigma-Aldrich, St. Louis, MO) and 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA). VZV was propagated by cocultivating infected cells with uninfected cells as described previously (27). Infected HFL cultures were harvested at the height of virus-induced cytopathic effect, usually at 3 days postinfection (dpi).Construction of recombinant antibody. Blood was collected 7 days after immunization...