RESUMEN
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.
Asunto(s)
Herpes Zóster/virología , Herpesvirus Humano 3/fisiología , Proteínas del Envoltorio Viral/fisiología , Antígenos Virales/fisiología , Regulación Viral de la Expresión Génica , Humanos , Replicación ViralRESUMEN
Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains. Single-chain antibody fragments (scAb) derived from MAb hu206 were produced in Escherichia coli. These scAb retained the binding properties of the whole antibody. However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206. Shortening the peptide linker joining the V(H) to the V(kappa) domain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy. The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.