Analysis of the transcription of immediate early regulatory genes of Varicella-Zoster virus and host cell gene expression in VZV-infected cells in vitro and in the SCIDhu mouse model in vivo

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella and herpes zoster. Infection begins with virus entry through the mucosa of the upper respiratory tract, leading to primary viremia and spread of virus by infected T cells to skin, where the virus causes a rash, followed by infection of sensory ganglia, where the virus establishes latency. This work characterizes the host response to VZV infection in cells that are biologically important during VZV pathogenesis, and examines the contribution of specific viral proteins to VZV pathogenesis. Using microarray technology, the host cell transcriptional response to VZV infection was found to be distinct in VZV-infected primary human fibroblasts and T cells in culture, and human skin xenografts in the SCIDhu mouse model. VZV genes were added to the human cDNA microarrays and viral gene transcription was monitored in primary fibroblasts. Infection with a virus containing a point mutation in the IE63 protein resulted in the decreased transcription of several viral genes. The host cell response to infection with this, or another small plaque phenotype mutant virus, was similar to infection with intact VZV, suggesting that the small plaque phenotype resulted from deficiencies in viral gene expression rather than differing host cell responses to the viruses. Transcription of ORF62 and ORF63, which encode the immediate early regulatory proteins IE62 and IE63, was analyzed by cloning the 1.5kb ORF62/63 intergenic region between two different luciferase genes, which acted as reporters for ORF62 or ORF63 expression. This reporter cassette was inserted into the viral genome so that the transcription of ORF62 and ORF63 could be monitored during VZV infection without affecting the growth of the virus. Mutations of binding sites for cellular transregulatory proteins were found to alter the luciferase activity of either or both the ORF62 and ORF63 reporters in fibroblasts. The microarray results also indicated that viral infection altered the regulation of specific host cell signaling pathways, including the inhibition of the NF-kappaB pathway. This pathway, which mediates an inflammatory response to infection, was inhibited by the VZV-induced stabilization of IkappaBalpha levels and the associated cytoplasmic sequestration of NF-kappaB proteins in infected cells.