The main goal of this proposal is to investigate the role of HCMV in the pathogenesis of KS associated with AIDS. Our approach is to determine if HCMV serves as a """"""""putative"""""""" cofactor in the induction of KS. Using subfragment probes of the transforming Towne XbaI-E fragment identified by us we propose to: 1) analyze KS tissue DNA for the presence, state, and expression of HCMV transforming DNA and myc-related sequences. The presence of these sequences will be determined by Southern blot hybridization and the expression by Northern blot hybridization. Molecular hybridization studies involving HCMV Towne XbaI-E will employ viral DNA probes lacking homology to cell DNA as well as probes of the myc-containing BamHI-J&M subfragments of the transforming Towne XbaI-E fragment. 2) Determine transforming activity in normal diploid SHE cells and NIH 3T3 cells of Towne Xbal-E and myc-related sequences (identified in #1 above). Transfection studies will be carried out employing KS tissue DNA in order to identify and isolate the transforming gene(s) and to determine any homology to the Towne XbaI-E transformed fragment. 3) Examine HCMV strainsisolated for altered or amplified transforming fragments and myc-related sequences. Restriction enzyme analysis will be used to assay structural alterations in DNA. Transfection assays in NIH 3T3 and SHE cells will be carried out to determine transforming activity. 4) Quantify HCMV genomes (copy number) in peripheral blood lymphocytes from patients groups (healthy homosexuals, AIDS patients without KS, and AIDS patients with KS). Since peripheral blood lymphocytes are an accessible tissue and have been shown to be a reservoir of virus in active HCMV infection, we propose to analyze these cells as useful source of the proposed HCMV cofactor for KS. Peripheral blood lymphocytes will be initially screened for the presence of HCMV sequences by spot hybridization using viral-specific DNA probes. The number of copies of viral genomes will be quatitated and the physical state (free or integrated) of viral genomes in peripheral blood lymphocytes will be investigated by Southern blot hybridization.
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