ROLE OF HCMV IN KS ASSOCIATED WITH AIDS

这项提案的主要目标是调查人巨细胞病毒在人类免疫缺陷病毒感染中的作用。 与艾滋病相关的KS的发病机制。我们的方法是确定 在KS的诱导过程中，HCMV是一种“推定的”辅因子。vbl.使用 鉴定转化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、状态和 人巨细胞病毒转化DNA和myc相关序列的表达。这个 这些序列的存在将通过Southern杂交来确定 杂交和Northern印迹杂交表达。分子 涉及HCMV Towne XbaI-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。2) 正常二倍体SHE细胞和NIH3T3转化活性的测定 Towne Xbal-E和myc相关序列的细胞(在上文#1中确定)。 将按顺序使用KS组织DNA进行转染性研究 对转化基因(S)进行鉴定和分离，并测定 与Towne XbaI-E转化片段的同源性。3)检查巨细胞病毒 对改变或扩增的转化片段和 MYC相关序列。将使用限制性内切酶分析来检测 DNA的结构变化。NIH3T3和SHE细胞的转染法 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巨细胞病毒基因组(拷贝数) 群体(健康同性恋者、无KS的艾滋病患者和艾滋病患者 与KS)。由于外周血淋巴细胞是一种可获得的组织， 已被证明是活跃的人巨细胞病毒感染的病毒储存库，我们 建议分析这些细胞，作为拟议的巨细胞病毒的有用来源 KS的辅因子。将对外周血淋巴细胞进行初步筛查 用斑点杂交法检测人巨细胞病毒序列 病毒特异性DNA探针。病毒基因组的拷贝数将是 病毒基因组的量化和物理状态(自由或整合) 外周血淋巴细胞将用Southern印迹法检测 杂交。

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.