Induction of varicella-zoster virus-neutralizing antibodies in mice by co-infection with recombinant vaccinia viruses expressing the gH or gL gene

Varicella-zoster virus (VZV) is an extremely cell-associated alphaherpesvirus; VZV infection is spread almost exclusively via cell membrane fusion. The envelope glycoprotein H (gH) is highly conserved among the herpesviruses. A virus-encoded chaperone, glycoprotein L (gL), associates with gH, and the gH:gL complex is required for gH maturation and membrane expression. We recently demonstrated that in the VZV system, the gH:gL complex facilitated cell membrane fusion and extensive polykaryon formation in transfected cells (K. M. Duus, C. Hatfield, and C. Grose, Virology 210:429-440, 1995). To further define the functions of the unusual VZV gL chaperone protein, we have performed a series of mutagenesis experiments with both gH and gL and analyzed the mutants by laser scanning confocal microscopy in a transfection-based fusion assay. We established the fact that immature gH exited the endoplasmic reticulum (ER) when coexpressed with either gE or gI and appeared on the cell surface in a patch pattern. A similar effect was observed on the cell surface with gH with a cytoplasmic tail mutagenized to closely resemble the vaccinia virus hemagglutinin cytoplasmic tail. Site-directed mutagenesis of the five gL cysteine residues demonstrated that four of five cysteines participated in the gL chaperone function required for proper maturation of gH. On the other hand, the same gL mutants facilitated transport of immature gH to the cell surface, where patching occurred. Studies of gL processing demonstrated that maturation did not require transport beyond the medial-Golgi; furthermore, gL was not detected in the outer cell membrane, nor was it secreted into the medium. Colocalization studies with 3,3-dihexyloxa-cabocyanine iodide and N-(e-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-erythro-sphingosine confirmed that gL was found primarily in the ER and cis/medial-Golgi when expressed alone. When all of these data were considered, they suggested a posttranslational gH:gL regulation model whereby the gL chaperone modulated gH expression via retrograde flow from the Golgi to the ER. In this schema, mature gL returns to the ER, where it escorts immature gH from the ER to the Golgi; thereafter, mature gH is transported from the trans-Golgi to the outer cell membrane, where it acts as a major fusogen.

Section: Discussionmentioning

confidence: 99%

Multiple regulatory effects of varicella-zoster virus (VZV) gL on trafficking patterns and fusogenic properties of VZV gH

Duus

Grose

1996

“…The Escherichia coli xanthine-guanidine phosphoribosyl transferase (gpt) gene under the control of the VV I 3 promoter served as a selection marker for recombinant viruses (5). The construction of the single recombinant viruses expressing glycoproteins gB (VV-gB), gE (VV-gE), gH (VV-gH), and gL (VV-gL), as well as double recombinant viruses VV-gHϩgL and VV-gEϩgB, was described in detail previously (26,29,35). Likewise, VZV cloning strategies in the VV T7-pTM1 expression system were previously published (59).…”

Section: Methodsmentioning

confidence: 99%

Varicella-Zoster Virus gB and gE Coexpression, but Not gB or gE Alone, Leads to Abundant Fusion and Syncytium Formation Equivalent to Those from gH and gL Coexpression

Marešová

Pasieka

Grose

2001

Varicella-zoster virus (VZV) is distinguished from herpes simplex virus type 1 (HSV-1) by the fact that cell-to-cell fusion and syncytium formation require only gH and gL within a transient-expression system. In the HSV system, four glycoproteins, namely, gH, gL, gB, and gD, are required to induce a similar fusogenic event. VZV lacks a gD homologous protein. In this report, the role of VZV gB as a fusogen was investigated and compared to the gH-gL complex. First of all, the VZV gH-gL experiment was repeated under a different set of conditions; namely, gH and gL were cloned into the same vaccinia virus (VV) genome. Surprisingly, the new expression system demonstrated that a recombinant VV-gH؉gL construct was even more fusogenic than seen in the prior experiment with two individual expression plasmids containing gH and gL (K. M. Duus and C. Grose, J. Virol. 70:8961-8971, 1996). Recombinant VV expressing VZV gB by itself, however, effected the formation of only small syncytia. When VZV gE and gB genes were cloned into one recombinant VV genome and another fusion assay was performed, extensive syncytium formation was observed. The degree of fusion with VZV gE-gB coexpression was comparable to that observed with VZV gH-gL: in both cases, >80% of the cells in a monolayer were fused. Thus, these studies established that VZV gE-gB coexpression greatly enhanced the fusogenic properties of gB. Control experiments documented that the fusion assay required a balance between the fusogenic potential of the VZV glycoproteins and the fusion-inhibitory effect of the VV infection itself.Varicella-zoster virus (VZV) is a highly fusogenic virus, but the degree of fusion is dependent on the cell substrate in which the virus is propagated (20). In human fibroblast cells, fusion formation is limited to a small number of nuclei per syncytium. In contrast, in human melanoma cells, all VZV strains examined to date exhibit fusion formation in which the entire monolayer is eventually involved. Polykaryon formation also occurs during primary VZV infection in human epidermal cells. Therefore, fusion formation appears to be related to cells of ectodermal origin (20). The question of which glycoproteins are involved in VZV-induced fusion was addressed in two earlier reports in which transfection studies were carried out with the VZV gH and gL genes (13,14). Transfection with gH alone caused little or no syncytium formation. In contrast, cotransfection with gH and gL genes led to multiple foci of fusion within the monolayer, where syncytia from 6 to 25 nuclei were easily detected. This set of experiments also documented the utility of confocal microscopy as an instrument to detect syncytium formation and glycoprotein expression within a polykaryon.Of interest, the VZV results differed markedly from the herpes simplex virus type 1 (HSV-1) data, which showed that cotransfection with four glycoprotein genes, namely those of gH, gL, gB, and gD, was required for syncytium formation (42,55). In the latter case, each syncytium often included 10 to 20 ...

“…Individuals who have had primary infection with VZV or herpes simplex virus (HSV), the most closely related human alphaherpesvirus, have humoral and cellular immunity against gH (1,56). Immunization of mice with a recombinant vaccinia virus expressing VZV gH and its chaperone, glycoprotein L (gL), induced specific antibodies capable of neutralizing VZV in vitro (28,37). Immunization of mice with purified HSV gH/gL protein resulted in the production of neutralizing antibodies and protected mice from HSV challenge (5,44), and administration of an anti-HSV gH monoclonal antibody (MAb) protected mice from HSV challenge (16).…”

mentioning

confidence: 99%

Anti-Glycoprotein H Antibody Impairs the Pathogenicity of Varicella-Zoster Virus in Skin Xenografts in the SCID Mouse Model

Vleck¹,

Oliver²,

Reichelt³

et al. 2010

Varicella-zoster virus (VZV) infection is usually mild in healthy individualsVaricella-zoster virus (VZV) causes chicken pox (varicella) upon primary infection. Lifelong latency is established in neurons of the sensory ganglia, and reactivation leads to shingles (herpes zoster) (1). Disease is usually inconsequential in immunocompetent people but can be severe in immunocompromised patients. The current prophylaxis for these high-risk individuals exposed to VZV is high-titer immunoglobulin to VZV administered within 96 h of exposure. This prophylaxis does not always prevent disease, but the severity of symptoms and mortality rates are usually reduced (32).Glycoprotein H (gH) is a type 1 transmembrane protein that is required for virus-cell and cell-cell spread in all herpesviruses studied (12,15,24,26). gH is an important target of the host immune system. Individuals who have had primary infection with VZV or herpes simplex virus (HSV), the most closely related human alphaherpesvirus, have humoral and cellular immunity against gH (1, 56). Immunization of mice with a recombinant vaccinia virus expressing VZV gH and its chaperone, glycoprotein L (gL), induced specific antibodies capable of neutralizing VZV in vitro (28,37). Immunization of mice with purified HSV gH/gL protein resulted in the production of neutralizing antibodies and protected mice from HSV challenge (5, 44), and administration of an anti-HSV gH monoclonal antibody (MAb) protected mice from HSV challenge (16). Antibodies to HSV and Epstein-Barr virus gH effectively neutralize during virus penetration but not during adsorption in vitro, indicating an essential role for gH in the fusion of viral and cellular membranes but not in initial attachment of the virus to the cell (18, 33).Anti-gH MAb 206, an immunoglobulin G1 (IgG1) antibody which recognizes a conformation-dependent epitope on the mature glycosylated form of gH, neutralizes VZV infection in vitro in the absence of complement (35). MAb 206 inhibits cell-cell fusion in vitro, based on reductions in the number of infected cells and the number of infected nuclei within syncytia, and appears to inhibit the ability of virus particles to pass from the surface of an infected epithelial cell to a neighboring cell via cell extensions (8,35,43). When infected cells were treated with MAb 206 for 48 h postinfection (hpi), virus egress and syncytium formation were not apparent, but they were evident within 48 h after removal of the antibody, suggesting that the effect of the antibody was reversible and that there was a requirement for new gH synthesis and trafficking to produce cell-cell fusion. Conversely, nonneutralizing antibodies to glycoproteins E (gE) and I (gI), as well as an antibody to immediate-early protein 62 (IE62), had no effect on VZV spread (46).Like that of other herpesviruses, VZV entry into cells is presumed to require fusion of the virion envelope with the cell membrane or endocytosis followed by fusion. One of the hallmarks of VZV infection is cell fusion and formation of syncytia (8)....