ROLE OF HCMV IN KS ASSOCIATED WITH AIDS

本提案的主要目标是研究HCMV在 KS的发病机制与艾滋病有关。 我们的方法是确定 HCMV在KS的诱导中充当“假定的”辅因子。 使用 转化Towne XbaI-E片段的亚片段探针， 我们建议：1）分析KS组织DNA的存在，状态， HCMV转化DNA和myc相关序列的表达。 的 这些序列的存在将通过Southern印迹来确定 通过北方杂交和表达。 分子 涉及HCMV TowneXbaI-E的杂交研究将使用病毒DNA 与细胞DNA缺乏同源性的探针以及含有myc的探针 转化Towne XbaI-E片段的BamHI-J&M亚片段。 （二） 测定正常二倍体SHE细胞和NIH 3 T3中的转化活性 Towne Xbal-E和myc相关序列的细胞（在上述#1中鉴定）。 将使用KS组织DNA进行转染研究， 以鉴定和分离转化基因，并确定任何 与Towne XbaI-E转化片段同源。 3)检测HCMV 分离改变或扩增的转化片段， myc相关序列。 限制性内切酶分析将用于测定 DNA的结构变化。 NIH 3 T3和SHE中的转染测定 将进行细胞培养以确定转化活性。 4)量化 患者外周血淋巴细胞中的HCMV基因组（拷贝数） 健康的同性恋者、无KS的AIDS患者和AIDS患者 KS）。 由于外周血淋巴细胞是可接近的组织， 已经证明在活动性HCMV感染中是病毒的储存库，我们 建议将这些细胞作为建议的HCMV的有用来源进行分析 KS的辅助因子。 将对外周血淋巴细胞进行初步筛查 通过斑点杂交检测HCMV序列的存在， 病毒特异性DNA探针。 病毒基因组的拷贝数 病毒基因组的定量和物理状态（游离或整合）， 将通过Southern印迹法研究外周血淋巴细胞 杂交方法

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.