After completing my clinical training in infectious diseases, my last 7 months have been spent pursuing basic herpesviral research under the mentorship of Drs. Stuart P. Adler and Michael McVoy, two experienced herpesviral investigators. This experience has instilled me with a strong desire to pursue an academic career in basic viral pathogenesis research with a clinical focus. The research facilities here have proven more than sufficient for me to complete preliminary work on the project proposed- a detailed analysis of the processes of herpesvirus genome cleavage, packaging and circularization.
Specific aims of the project are to: 1) Define essential cis DNA elements at cleavage sites and determine the processes they mediate: cleavage, packaging, or circularization. Recombinant gpCMVs containing mutations at one to two cleavage sites will be constructed. Cleavage, packaging, or circularization functions will then be determined for the mutagenized site. 2) Purify, identify, and express the trans acting proteins that bind to the cis elements. Gel shift assays will be performed on both infected and uninfected cell lysates to identify DNA binding proteins using oligonucleotides containing sequences determined to be essential in section 1. DNA binding domains will be fine mapped by mutagenesis of oligo sequences. The ability of synthetic oligonucleotides that compete for DNA binding sites of proteins to inhibit viral replication in tissue culture will be tested. Binding proteins will be purified on DNA affinity columns and partially sequenced to infer the DNA sequences of the open reading frames encoding the protein of interest. These sequences will be used to construct probes that will be used to screen a gpCMV cDNA library. Proteins encoded by these cDNAs will be expressed in recombinant baculoviruses. 3) Determine the biochemical activities of these protein components. Activities may include site specific endonuclease activity (cleavage) ligation of DNA ends (circularization), and DNA translocation (packaging). These functions will be tested in 2 ways: (a) Biochemical assays for cleavage an circularization using artificial plasmid substrates and baculovirus expressed recombinant proteins. (B) construction of recombinant viruses with knockout mutations and analysis of DNA replicative intermediates formed in the absence of the protein of interest. It is hoped that these studies will extend our knowledge of these critical functions of all herpes viruses and assist in the development of antiviral agents. Coupled with intensive graduate level basic science course work, journal club, conference attendance, and frequent interaction with mentors and other investigator, I feel this comprehensive proposal will allow me to become a capable independent investigator.
