There are two important sets of human adenovirus (Ad) type of 5 gene products whose functions in Ad growth cycle are either not known or only partially understood. These are the two polypeptides encoded by the early gene L1, at 30.0 to 39.0 map units and the two virus encoded small RNA genes, VAI and VAII. In hopes that specific understanding of the precise function of these viral products will shed light on both the process of adenoviral infection, and on normal cellular defenses against viruses, the following experiments are planned. 1. To investigate the L1 gene, and Ad5 mutant with a defective L1 gene will be constructed and propagated. Because it is anticipated that this mutant will be defective, a suitable trans complementing helper system will be developed. The phenotype of the mutant will then be analyzed to determine the L1 gene function(s). 2. To define the precise mechanism action of the VA RNAs, our present studies on VA RNA-mediated translational control in Ad infected human and monkey cells will be continued along several lines: (a) To define the mechanism by which the VAI RNA blocks the activation of eIF-2 alpha kinase in virus infected cells, host proteins (the known dsRNA dependent kinase and/or other host proteins) which bind to VAI RNA will be indentified. Interaction of these host proteins with WT and mutant VAI RNAs will be examined to determine how interaction with the viral small RNAs affects their function. For kinase studies, an antibody to this protein will be raised and the gene for the protein will be cloned. The kinase cDNA will be expressed in lambda gt11. If such proteins retain biologic activity, genetic approaches will be used to construct a structure-function map of the kinase. (b) To determine precisely the sequence elements and structures of VAI molecule that are critical for translation control, Ad5 mutants with single base substitutions in defined regions of the VAI RNA will be generated and analyzed in vivo for translation defect and in vitro for interaction with the host factors. (c) This laboratory recently showed that the SV40 virus can substitute for the VA RNA function in monkey cells if these cells are preinfected with SV40. The viral genome will be dissected genetically to determine which of the SV40 genes is responsible for this unusual complementation between different DNA viruses. The biochemical basis of this complementation will be determined. (d) To find out if the VAII RNA can fully substitute for the VAI RNA, a VAI mutant which contains multiple copies of the VAII will be constructed and analysed.
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