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Journal of Virology, April 2001, p. 3175-3184, Vol. 75, No. 7
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.7.3175-3184.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Kaposi's Sarcoma-Associated Herpesvirus K-bZIP Protein Is Phosphorylated by Cyclin-Dependent Kinases

Andrew G. Polson,1 Lan Huang,2 David M. Lukac,1 Justin D. Blethrow,3 David O. Morgan,3 Alma L. Burlingame,2 and Don Ganem1,4,*

Departments of Microbiology and Immunology,1 Pharmaceutical Chemistry and Medicine,2 and Physiology3 and Howard Hughes Medical Institute,4 University of California San Francisco, San Francisco, California 94143

Received 29 September 2000/Accepted 8 January 2001

The K8 locus in Kaposi's sarcoma-associated herpesvirus (KSHV) is syntenic with the Epstein-Barr virus (EBV) BZLF (Z) locus and expresses three alternatively spliced transcripts. The fully spliced transcript encodes K-bZIP, the KSHV homologue of the EBV immediate-early transcriptional transactivator Z. Here we show that despite the presence of alternatively spliced transcripts, the protein from the fully spliced RNA, K-bZIP, is the principal product detectable in KSHV-infected B cells. The protein is detected only in lytically infected cells and is localized to the nucleus. We further characterized K-bZIP by determining its phosphorylation status. Phosphoamino acid analysis revealed phosphorylation on serine and threonine. Analysis of the sites of K-bZIP phosphorylation by tandem mass spectrometry revealed that K-bZIP was phosphorylated on Thr 111 and Ser 167. These phosphorylation sites are contained within cyclin-dependent kinase (CDK) recognition sites with the consensus sequence (S/T)PXR, suggesting that K-bZIP could be phosphorylated by CDKs. We tested this hypothesis using an in vitro kinase reaction performed in whole-cell extracts that resemble in vivo conditions more closely than standard in vitro kinase reactions. We found that the three CDK-cyclin complexes we tested phosphorylated K-bZIP but not the control ORF 73 protein, which contains four (S/T)PXR sites. Ectopic expression of K-bZIP cannot reactivate KSHV from latency, and single and double mutants of K-bZIP in which alanines replaced the phosphorylated serine and/or threonine also failed to induce lytic replication. These studies indicate that K-bZIP is a substrate for CDKs and should inform further functional analyses of the protein.

* Corresponding author. Mailing address: Howard Hughes Medical Institute, Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA 94143-0414. Phone: (415) 476-2826. Fax: (415) 476-0939. E-mail: ganem{at}cgl.ucsf.edu.

Journal of Virology, April 2001, p. 3175-3184, Vol. 75, No. 7
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.7.3175-3184.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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