KS ASSOCIATED DNA VIRUS

已分离出两个DNA序列，命名为KS 330 Bam和KS 627 Bam 通过代表性差异分析（RDA）从KS病变中提取。南部 印迹杂交和PCR分析表明，这些序列是 特别是与KS以及罕见艾滋病的子集有关， 相关体腔淋巴瘤。这些序列也存在于 经典KS病变和来自HIV阴性男同性恋者的KS病变提示 KS临床亚型的统一病因学。这些序列 这是第一个发现KS的可靠分子标记。该627 bp片段具有 与γ疱疹病毒被膜基因核苷酸同源。一个2166 bp的区域 包括330 bp的片段进行测序，发现有两个开放的 与γ疱疹病毒衣壳ORF同源的阅读框（ORF）。这些KS 相关序列似乎是一种可传播的 DNA病毒，基因组约270 kb，与 γ疱疹病毒亚科Epstein-Barr病毒（EBV）和松鼠猴疱疹病毒 （HSVSA）。EBV阳性细胞系BCBL 1，来源于一种 已发现阳性体腔淋巴瘤是双重感染的 平均每个细胞有50个拷贝， 用于描述病毒特征的工具。研究表明，新的 发现人类疱疹病毒是KS和一些艾滋病淋巴瘤的病因。 虽然这种药剂的正式分类尚未确定， 确定，它在这里被指定为卡波西肉瘤相关 疱疹样病毒（KSHV）。 该提案的战略，为表征这种病毒，并确定其 致癌潜力将与其他组使用的方法相似 在疱疹病毒saimiri和EB病毒的研究。 第一，续 双向基因组步移将用于获得序列信息 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 临床上重要的，如疱疹病毒主要衣壳蛋白和 胸苷激酶ORF同源物，将被靶向。第二，就地 将进行KS病变的杂交研究以鉴定 KS病变中感染的细胞类型以及 各种病毒抗原第三，将进行传播研究， 在EBV非允许细胞系中繁殖病毒。这些研究将 导致体外转化实验以鉴定特定病毒 致癌基因结合起来，这些研究将开始 这种新的和新出现的人类病原体的表征。这些 研究将直接为未来的治疗和 针对艾滋病相关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.