This proposal aims to continue our studies of the molecular biology of Varicella Zoster Virus (VZV). We plan to pursue the investigation of the structure of VZV DNA with special emphasis on the mode of formation and frequency of occurrence of circular forms and forms with inverted UL segments. We will further characterize VZV DNA sequences from which strain differences arise, particularly with reference to possible roles as transcription signals. We will attempt to map the VZV thymidine kinase (TK) and DNA polymerase activities physically on the viral genome by rescue of drug-resistant mutants and by use of special cell lines. We will also attempt to obtain expression of viral proteins from VZV sequences cloned into mammalian virus expression systems including vaccinia and bovine papilloma viruses. Our initial target will be the 92K VZV glycoprotein. We will purify and characterize the major 125K VZV DNA-binding protein, comparing its DNA-binding and DNA polymerase-stimulating activities with that of the HSV ICP8. The genomic location of the 125K protein coding sequences will be determined by marker rescue of HSV1 ICP8 ts mutants by VZV fragments. A similar strategy will be employed to attempt rescue of HSV ts mutants in other important genes such as the TK, DNA polymerase and major capsid proteins using the CaPO4 transfection scheme. We also plan to characterize further a virion-associated protein kinase activity in regard to salt, detergent and substrate concentrations; the role of this kinase activity in the formation of viral phosphoproteins and in virus infectivity will be assessed. Finally, we will continue our attempts to detect the presence of VZV genetic material in human sensory ganglia. We have arranged for a new source of tissue and should be supplied with both trigeminal and dorsal root ganglia. The methods of detection will include in situ hybridization using frozen thin sections of explanted ganglia, dot blots and Southern blots. Southern blots will also be used to examine the conformation of detected sequences. Techniques to be employed include restriction enzyme analysis, CSC1 density gradient centrifugation, nick translation, dot and Southern blotting, gel electrophoresis, DNA transfection, filter binding, in situ hybridization and DNA sequencing.
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