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 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.
Seaman
W T
WT
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA.
Ye
D
D
Wang
R X
RX
Hale
E E
EE
Weisse
M
M
Quinlivan
E B
EB
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D005814:Genes, Viral / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000737:chemistry / Q000235:genetics; D019288:Herpesvirus 8, Human / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014775:Virus Activation / Q000187:drug effects
D050976:Basic-Leucine Zipper Transcription Factors; D002352:Carrier Proteins; C118265:K8 protein, Human herpesvirus 8; D010455:Peptides; D012333:RNA, Messenger; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014764:Viral Proteins; D003513:Cycloheximide; D011392:Proline; D013755:Tetradecanoylphorbol Acetate