DNA binding by Kaposi's sarcoma-associated herpesvirus lytic switch protein is necessary for transcriptional activation of two viral delayed early promoters.
Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus-8) establishes latent and lytic infections in both lymphoid and endothelial cells and has been associated with diseases of both cell types. The KSHV open reading frame 50 (ORF50) protein is a transcriptional activator that plays a central role in the reactivation of lytic viral replication from latency. Here we identify and characterize a DNA binding site for the ORF50 protein that is shared by the promoters of two delayed early genes (ORF57 and K-bZIP). Transfer of this element to heterologous promoters confers on them high-level responsiveness to ORF50, indicating that the element is both necessary and sufficient for activation. The element consists of a conserved 12-bp palindromic sequence and less conserved sequences immediately 3' to it. Mutational analysis reveals that sequences within the palindrome are critical for binding and activation by ORF50, but the presence of a palindrome itself is not absolutely required. The 3' flanking sequences also play a critical role in DNA binding and transactivation. The strong concordance of DNA binding in vitro with transcriptional activation in vivo strongly implies that sequence-specific DNA binding is necessary for ORF50-mediated activation through this element. Expression of truncated versions of the ORF50 protein reveals that DNA binding is mediated by the amino-terminal 272 amino acids of the polypeptide.
Lukac
D M
DM
Howard Hughes Medical Institute and Department of Microbiology, University of California Medical Center, San Francisco, California 94143-0414, USA.
Garibyan
L
L
Kirshner
J R
JR
Palmeri
D
D
Ganem
D
D
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D050976:Basic-Leucine Zipper Transcription Factors; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D019288:Herpesvirus 8, Human / Q000378:metabolism*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D016385:TATA Box; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000235:genetics*
D050976:Basic-Leucine Zipper Transcription Factors; D002352:Carrier Proteins; D017874:Immediate-Early Proteins; C118265:K8 protein, Human herpesvirus 8; C415247:ORF57 protein, Herpesvirus saimiri; D012097:Repressor Proteins; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; D014764:Viral Proteins; D004247:DNA