KS ASSOCIATED DNA VIRUS

已分离到两个DNA序列，分别命名为KS330Bam和KS627Bam 通过代表性差异分析(RDA)从KS病变中提取。南方 杂交和聚合酶链式反应分析表明，这些序列是 特别与KS以及罕见艾滋病的一个子集有关- 与体腔相关的淋巴瘤。这些序列也可以在 经典KS皮损和HIV阴性男同性恋者的KS皮损提示 KS临床亚型的统一病因学。这些序列是 发现了第一个可靠的KS分子标记。627个碱基片段具有 与伽马疱疹病毒被膜基因的核苷酸同源性。A区2166个碱基对 包括330个碱基的片段被测序，并发现有两个开放的 与伽马疱疹病毒衣壳ORF同源的阅读框架(ORF)。这些KS 相关序列似乎是可传播的 具有大约270 kb基因组的DNA病毒，与 EB病毒和塞米里疱疹病毒 (HSVSA)。EBV阳性细胞系BCBL1，来源于 以体腔为基础的阳性淋巴瘤被发现双重感染 每个细胞平均有50个拷贝，这是一个重要的 描述病毒特征的工具。研究表明，一种新的 已发现的人类疱疹病毒是KS和一些艾滋病淋巴瘤的病因。 尽管这个特工的正式分类还没有被 确诊为卡波西氏肉瘤相关 为方便和清楚起见，请使用疱疹样病毒(KSHV)。 这项提议的战略是描述这种病毒的特征并确定其 致癌潜力将类似于其他群体使用的方法 在疱疹病毒和EB病毒的研究中。第一，继续 将使用双向基因组步行来获得序列信息 关于该病毒的基因组结构及其与 其他疱疹病毒。特定的基因，可能是生物学上和 临床上很重要，如疱疹病毒主要衣壳蛋白和 胸苷激酶ORF同源物，将成为靶点。第二，就地 将进行KS病变的杂交研究，以确定 KS皮损的感染细胞类型及亚细胞定位 各种病毒抗原。第三，将进行传播研究，以 在EBV不允许的细胞系中传播病毒。这些研究将 导致体外转化实验以鉴定特定病毒 致癌基因。总而言之，这些研究将开始 这种新出现的人类病原体的特征。这些 研究将直接为未来的治疗和 艾滋病相关KS的疫苗干预。

Two DNA sequences, designated KS330Bam and KS627Bam, have been isolated from a KS lesion by representational difference analysis (RDA). Southern blot hybridizations and PCR analyses indicate that these sequences are specifically associated with KS as well as a subset of rare AIDS- associated body cavity-based lymphomas. These sequences are also found in classical KS lesions and KS lesions from HIV-negative gay men suggesting an unified etiology for the clinical subtypes of KS. These sequences are the first reliable molecular markers found for KS. The 627 bp fragment has nucleotide homology to a gamma herpesvirus tegument gene. A 2166 bp region including the 330 bp fragment was sequenced and found to have two open reading frames (ORF) homologous to gamma herpesvirus capsid ORFs. These KS associated sequences appear to be part of the genome of a transmissible DNA virus with an approximately 270 kb genome, homologous to the Gammaherpesvirinae Epstein-Barr virus (EBV) and herpesvirus saimiri (HSVSA). An EBV positive cell line, BCBL1, derived from one of the positive body cavity-based lymphomas has been found to be dually infected with the agent at an average of 50 copies per cell and is an important tool for characterizing the virus. The studies suggest that a newly discovered human herpesvirus is causal for KS and some AIDS lymphomas. Although a formal classification of this agent has not yet been determined, it is designated here as the Kaposi's sarcoma-associated herpes-like virus (KSHV) for purposes of convenience and clarity. This proposal's strategy for characterizing this virus and determining its oncogenic potential will be similar to the approach used by other groups in the study of herpesvirus saimiri and EBV. First, continued bidirectional genomic walking will be used to obtain sequence information on the virus' genomic organization and its phylogenetic relationship to other herpesviruses. Specific genes which may be biologically and clinically important, such as the herpesviral major capsid protein and thymidine kinase ORF homologs, will be targeted. Secondly, in-situ hybridization studies of KS lesions will be performed to identify the infected cell type in KS lesions as well as subcellular localization of various viral antigens. Third, transmission studies will be undertaken to propagate the virus in EBV nonpermissive cell lines. These studies will lead to in vitro transformation experiments to identify specific viral oncogenes. In combination, these sets of studies will begin the characterization of this new and novel emerging human pathogen. These studies will directly lay the groundwork for future therapeutic and vaccine interventions against AIDS-associated KS.