C-B: KSHV Genetics

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The proposed KSHV Genetics Core has four distinct purposes and goals encompassing many aspects of KSHV genetics. The first two major goals are based on new directions and genetic approaches, that were initiated and supported partially under the previous Laboratory Core, in which the Core leader and several of the proposed Core B personnel have been actively engaged over the past two years. These projects involve: (1) To provide and assist all PPG Investigators with successful reagents and proven methodology to generate KSHV (HHV8) gene knock-out, insertion or point mutant viruses in a bacterially manipulated GFP/HygR/BAC system, and (2) To provide them with the means to both propagate these mutant viruses and assay for their biological and biochemical activities, as well as if necessary the ability to complement these mutants, the latter in collaboration with Core C. Current technology using BAC36 developed by Gao et al has clearly progressed to the point where generation of KSHV mutants by bacterial genetic approaches has been demonstrated to be feasible. Furthermore, the ability to both latently infect and lytically reactivate KSHV in human microvascular dermal endothelial cells (DMVEC or TIME cells) as developed within our group, has created the realistic opportunity to be able to work with, propagate and functionally assay such mutants, even when impaired in either latent or lytic cycle functions. There are still many difficulties and technical challenges involved in KSHV genetics, but by pooling the expertise, experience and reagents already developed in individual laboratories into a shared resource actively involved in and dedicated to accomplishing routine KSHV genetic manipulations, we fully anticipate succeeding in this endeavor. The other two much smaller goals include: (3) Maintenance of existing collections of KSHV genomic, cDNA, riboprobe and expression plasmid clones accumulated by members of the PPG in association with previous versions of the Laboratory Core, and (4) Maintenance of our large International collection of all known strains and subtypes of KSHV genomes. Although little new activity is planned or expected in terms of adding to these two collections, this provides a formal mechanism to help maintain them in a viable and accessible form, as well as to be able to provide selected samples to members of the PPG or the KSHV research community as needed. All three individual projects plan to utilize the KSHV Genetics Core to generate KSHV BAC36 mutants that are relevant to their projects, including vMIR1, 2, vFLIP, and vCYC (Project 1); LANA1 functional domain mutants (Project 2); and vCCL1, 2 and vGPCR (Project 3).