Gene Expression from the ORF50/K8 Region of Kaposi's Sarcoma-Associated Herpesvirus☆

Abstract The ORF50 gene of Kaposi's sarcoma (KS)-associated herpesvirus, or human herpesvirus 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 reactivation. We demonstrate that ORF50 is expressed in a bicistronic message after induction of the viral lytic cycle. ORF50 migrated as a series of polypeptides: the major ones as 119 and 101 kDa, respectively. Using 3′ rapid amplification of cDNA ends, RT-PCR, and cDNA library screening, we demonstrate that the major ORF50 transcript also encodes K8. The ORF50/K8 transcript was resistant to cyclohexamide, whereas the K8 transcript was only partially resistant to cyclohexamide at early timepoints. Both transcripts showed partial resistance after 12 h of phorbol ester induction. Using a GAL4-ORF50 fusion protein expression vector, we demonstrate that the transactivation domain of ORF50 resides within a 160-amino-acid region of the carboxyl portion of the ORF. Upstream regions of both ORF50 and K8 have basal promoter activity in KSHV-infected cells. K8, which had sequence homology to Bzip proteins, did not activate either promoter. However, both promoters were activated after cotransfection of ORF50 in BCBL-1 cells.