Herpes simplex viruses are endemic in the population and are responsible for a variety of clinical diseases some of which are life threatening, especially in immunocompromised individuals or in newborns. The herpes simplex virus type-1 (HSV-1) genome contains three origins of replication and encodes seven viral genes that are essential for viral genome replication in vivo. However actual mode of viral DNA replication and the role of recombination in this process are not well understood. Even less well understood are the processes of viral genome maturation and encapsidation. The objective of this proposal is to provide a better understanding of the events of genome processing and packaging through the genetic and biochemical analysis of seven viral genes that have been implicated in these processes. Preliminary evidence indicates that UL12, alkaline nuclease, plays an important role in genome maturation and encapsidation. Null mutants in UL12 synthesize viral DNA and initiate cleavage and packaging events; however, the DNA containing capsids which accumulate in the nucleus do not mature into the cytoplasm. The other six viral genes under study (UL6, UL15, UL25, UL28, UL32 and UL33) have been implicated in genome cleavage and packaging at a different stage. Mutants in each of these genes have a common phenotype: they are able to carry out viral DNA synthesis but are unable to cleave or package viral genomes when grown under nonpermissive conditions.
The first aim i s to generate the necessary reagents to carry out the subsequent aims; these include constructs for the expression of each protein in a variety of expression systems and the generation of and characterization of specific antisera.
The second aim i s to isolate or further characterize null mutants in each gene. Most of the existing mutants in these genes for both HSV and pseudorabies (PRV) are temperature sensitive (ts); however, because of potential difficulties with ts mutants such as leak and interference with the function of wild type alleles, it is important to isolate effective null mutants in each gene.
The third aim i s to establish the location of the putative processing/packaging genes within virions and within infected cells. Specifically we plan to use indirect immunofluorescence and confocal microscopy to characterize the intracellular localization both with respect to each other and with respect to replication and capsid protein complexes. In the fourth aim putative protein-protein interactions between the seven proteins will be analyzed by biochemical and genetic methods. Long term aims include assaying each putative processing/packaging protein for predicted activities such as ATPase, terminase, and DNA binding and carrying out a detailed structure-function analysis of each gene. It is anticipated that an analysis of the processing/packaging proteins will not only provide an understanding of the mechanisms of their action but may also lead to the development of novel strategies for antiviral therapy.
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