The major basic research focus of this laboratory involves this project, with the following goals: 1) to identify, map and characterize varicella-zoster virus genes and proteins active in latent or productive infections. 2) to define the temporal sequence of gene expression. 3) to determine the interaction of antiviral drugs with viral gene products though a molecular analysis of drug-resistant mutants. To accomplish these ends we have constructed a variety of recombinant libraries of the complete VZV genome. During the past year we have concluded studies of the finer mapping and directionality of viral transcripts. We mapped the viral IE 175 immediate early gene. We have transfected that gene into monkey cells and developed cell lines which stably express it. In the cell lines the expression of the gene appears to be autoregulated. Using assays by which we could measure the activity of viral gene promotors we detected a number of VZV genes that are transactivating and transactivatible. During the past year we have also completed sequence analysis of the thymidine kinase locus of ten VZV strains, including six that are resistant to acyclovir by virtue of mutations in that gene. We identified the nature and location of base mutations which render this gene product nonfunctional. Included in the analyses is the first acyclovir-resistant VZV strain recovered from a human. During the past year we have successfully established a highly sensitive and specific in situ hybridization system using 35S labelled synthetic RNA probes. Such probes VZV RNA expression in the trigeminal ganglia of six of 16 subjects. Continuing efforts will be directed at better mapping and characterizing the RNA expressed during VZV latency.
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