STRUCTURE AND FUNCTION OF POXVIRUS GENES

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The objective is to identify the mechanisms by which poxviruses control their gene expression. The specific aims are as follows. a) To characterize the cowpox virus gene encoding the 160K protein that is the major component of the virus-induced A-type inclusions. The 160K protein is the product of a late gene, and it is one of the most abundant viral proteins synthesized. The structure of the gene will be determined by nucleotide sequence analysis; and the site at which transcrption is initiated will be identified. Site-directed mutagenesis will be used both to identify cis-acting signals involved in the regulation of transcription, and to investigate the functions of the gene product. b) To characterize the cowpox virus gene that encodes a non-structural 38K protein. This protein is synthesized early in infection, and is a product of one of the most strongly expressed early genes. The site at which trasnscription of this gene is initiated will be determined. Site-directed mutagenesis will then be used to identify the regions controlling the temporal regulation of transcription and regions controlling the rate of trasnscription. The function of the protein will also be examined. The 38K protein can direct the production of haemorrhage in virus-induced lesions. Moreover the predicted amino acid sequence of this protein shares some similarity to those of proteins that are inhibitors of serine proteases. Therefore the ability of the 38K protein to effect haemmorhage by the inhibition of serine proteases will be examined. c) To develop genetic complementation systems for use in analyses of the functions of poxvirus genes. Attempts will be made to obtain cells (helper cells) that will express individual genes of poxviruses. Two approaches will be used to achieve this: one will employ retrovirus expression vectors that contain the poxvirus gene of interest; the second will use plasmids in which the poxvirus gene is under the control of a hormonally-regulated promoter element. The helper cells expressing a particular virus gene will then be used as host cells for the isolation and culture of viruses in which the equivalent gene in the virus genome has been inactivated. These systems will be used in the following way: i) to try to isolate viruses that have had an essential gene function inactivated; ii) to examine the effects of altering the amounts of a viral protein in a cell, relative to the amounts of other viral proteins; and iii) to examine the effects of synthesizing a viral gene product at a period in the viral growth cycle when it is not normally produced.