A SINGLE 13-KILOBASE DIVERGENT LOCUS IN THE KAPOSI SARCOMA-ASSOCIATEDHERPESVIRUS (HUMAN HERPESVIRUS-8) GENOME CONTAINS 9 OPEN READING FRAMES THAT ARE HOMOLOGOUS TO OR RELATED TO CELLULAR PROTEINS

Two small fragments of a novel human gammaherpesvirus genome known as Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (H HV-8) have been shown to be present in virtually all AIDS and non-AIDS KS lesions, as well as in body cavity-based lymphomas (BCBL) and in m ulticentric Castleman's disease, We have extended those studies by ide ntifying and sequencing a third fragment of HHV-8 DNA encoding a viral thymidylate synthetase (TS) gene, Use of this viral TS fragment as a probe led to the identification and mapping of a cluster of overlappin g phage lambda clones from a BCBL tumor DNA genomic library that spann ed 48 kb on the left-hand side of the HHV-8 genome between the equival ents of open reading frame 6 (ORF6) and ORF31 of herpesvirus saimiri ( HVS). DNA sequencing of a 17-kb segment encompassing a gammaherpesviru s divergent locus (DL-B) between ORF11 and ORF17 revealed the presence of nine viral ORFs with predicted gene products related to cellular p roteins. These include the complete TS gene and a dihydrofolate reduct ase (DHFR) gene, four novel cytokine genes (encoding viral interleukin -6, viral MIP-1A, viral MIP-1B, and BCK) that have not previously been found to be encoded by a virus, and a bcl-2 homolog, This region in H HV-8 also contains the T1.1 abundant lytic cycle nuclear RNA gene and encompasses two genes (or exons) encoding proteins with C4HC3 zinc fin ger domains of the PHD/leukemia-associated protein subtype. The latter are related to the spliced immediate early IE1 protein of the gamma-2 class herpesvirus bovine herpesvirus type 4 and a similar motif found in HVS ORF12, Although genes for TS and DHFR enzymes are also encoded by HVS (ORF70 and ORF2), both occur at different genomic loci than in HHV-8, and the HHV-8 DHFR protein is much farther diverged from human DHFR than is the HVS version, implying that they were probably acquir ed as host cell cDNAs by independent evolutionary events. Transcripts from the IE1-A, IE1-B, DHFR, and MIP-1B genes were all detected by Nor thern blot hybridization analysis in a BCBL cell line at 12 h after in duction with butyrate but were not present before induction, indicatin g that these are all primarily lytic cycle genes. We conclude that the DL-B locus of gammaherpesviruses displays considerably more variabili ty that previously appreciated and that expression of many of these ge nes is likely to have important implications for HHV-8 biology and the rapy.