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.
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