Gene expression from the ORF50/K8 region of Kaposi's sarcoma-associated herpesvirus

The ORF50 gene of Kaposi's sarcoma (KS)-associated herpesvirus, or human he rpesvirus 8 (KSHV), activates viral replication and is weakly homologous to the herpesvirus family of R transactivators; therefore, the transcription and :translation events from this region of KSHV are key events in viral re activation. We demonstrate that ORF50 is expressed in a bicistronic message after induction of the viral lytic cycle. ORF50 migrated as a series of po lypeptides; the major ones as 119 and 101 kDa, respectively. Using 3' rapid amplification of cDNA ends, RT-PCR, and CDNA library screening, we demonst rate that the major ORF50 transcript also encodes K8. The ORF50/K8 transcri pt was resistant to cyclohexamide, whereas the K8 transcript was only parti ally resistant to cyclohexamide at early timepoints. Both transcripts showe d partial resistance after 12 h of phorbol ester induction. Using a GAL4-OR F50 fusion protein expression vector, we demonstrate that the transactivati on domain of ORF50 resides within a 160-amino-acid region of the carboxyl p ortion of the ORF. Upstream regions of both ORF50 and K8 have basal promote r activity in KSHV-infected cells. K8, which had sequences homology to Bzip proteins, did not activate either promoter. However, both promoters were a ctivated after cotransfection of ORF50 in BCBL-1 cells. (C) 1999 Academic P ress.