Defective genomes of herpes simplex virus (HSV) have been previously shown to consist of multiple head to tail reiterations of limited regions of the standard virus DNA. Furthermore, cells co-infected with defective and helper viruses have been shown to overproduce the viral polypeptides which are encoded within the defective genome repeat units. The studies proposed in this application involve the use of such defective HSV genomes to study viral DNA replication and viral gene expression in the HSV infected cell. Specifically, these studies are based on our previous observations that cotransfections of cells with """"""""foster"""""""" helper virus DNA and """"""""seed"""""""" monomeric repeat units of the HSV defective genomes result in the regeneration of concatemeric defective virus DNA molecules which can be packaged into HSV virions. We propose to make use of such a scheme for the regeneration of defective genomes to study cis replication functions of HSV DNA, including replication origins, sequences recognized for the cleavage of concatemeric replicative intermediates, and sequences specifying the packaging of viral DNA into nucleocapsids. Furthermore, we plan to construct an HSV cloning-amplifying vector which will contain a bacterial replication origin and drug resistance markers enabling its growth and selection in bacteria, restriction enzyme sites suitable for the insertion of added DNA sequences, and the set of cis replication functions which will enable its use as seed repeat units. This HSV vector will be used to clone and amplify specific regions of the standard HSV DNA, including the region spanning coordinates 0.37-0.44, known to contain a cluster of DNA replication functions. By use of the newly regenerated defective HSV genomes and deleted derivatives derived from them, we propose to define the domains of specific viral genes, and to study putative recognition sites which determine the regulated scheme of viral gene expression in the HSV infected cell.
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