Varicella-zoster virus (VZV) causes a disseminated illness, varicella, and then becomes latent in sensory ganglia. Reactivation leads to zoster, an exanthem that is restricted to the innervated field of the ganglion in which reactivation occurs. We have developed a model that recapitulates the scenarios of latency and reactivation of VZV in human dorsal root ganglia. In latency, 6 of the 68 genes of VZV are expressed, all of which are either immediate early or early viral genes in the VZV cascade. In latent neuronal infection, the latency-associated proteins encoded by ORFs 4, 21,27, 62, and 63 are restricted to the cytoplasm, although they are regulatory proteins that are predominately nuclear during lytic infection. The model consists of neurons isolated from the guinea pig intestine. When purified neurons are infected with cell-free VZV, latent infection ensues with survival of neurons for weeks, despite the presence of cytoplasmic VZV proteins. Superinfection of latently infected neurons with an adenovirus encoding the ICP0 of herpes simplex virus (HSV) reactivates VZV, as it does for HSV. Reactivation is evidenced by translocation of the VZV latency associated proteins to the nucleus, production of late gene products such as glycoproteins, and neuronal death. This model will be utilized to explore the hypothesis that a block in nuclear transport of specific proteins governs maintenance of latent VZV infection. We will examine whether ORF61p (the VZV ortholog of HSV ICP0) of VZV induces reactivation. We will use array technology to examine changes in gene expression in VZV latency and reactivation. Following mutagenesis, the potential roles of ORF4 (the ortholog of HSV ICP 27) and ORF29p, the major DNA-binding protein in reactivation will be examined. Because our experiments suggest that ORF 29p is secreted, and we have demonstrated its presence in axons during varicella but not in zoster, we will identify the targeting sequence that drives secretion of ORF29p. Finally we will identify the nuclear localization signal of ORF 29p and determine whether nuclear translocation is a prerequisite for reactivation from latency. ? ? ?
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