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 herpesvi rus-8) establishes latent and lytic infections in both lymphoid and endothe lial cells and has been associated with diseases of both cell types. The KS HV open reading frame 50 (ORF50) protein is a transcriptional activator tha t 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 bot h necessary and sufficient for activation. The element consists of a conser ved 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 palind rome 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 i mplies that sequence-specific DNA binding is necessary for ORF50-mediated a ctivation through this element. Expression of truncated versions of the ORF 50 protein reveals that DNA binding is mediated by the amino-terminal 272 a mino acids of the polypeptide.