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Journal of Virology, September 2009, p. 8492-8501, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00922-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 K-bZIP Modulates Latency-Associated Nuclear Protein-Mediated Suppression of Lytic Origin-Dependent DNA Synthesis{triangledown}

Cyprian Rossetto, Irena Yamboliev, and Gregory S. Pari*

Department of Microbiology and Immunology and the Cell and Molecular Biology Graduate Program, University of Nevada—Reno, Reno, Nevada

Received 8 May 2009/ Accepted 16 June 2009

The original cotransfection replication assay identified eight human herpesvirus 8 (HHV8)-encoded proteins required for origin-dependent lytic DNA replication. Previously, we demonstrated that under conditions where K-Rta is overexpressed, a K-bZIP knockout bacmid displayed an aberrant subcellular localization pattern for the latency-associated nuclear protein (LANA). Additionally, these same studies demonstrated that K-bZIP interacts with LANA in the absence of K-Rta and that K-bZIP does not directly participate in, but may facilitate, the initiation of lytic DNA synthesis. We developed a modification of the transient cotransfection replication assay wherein both lytic (oriLyt) and latent (terminal repeat) DNA replication are evaluated simultaneously. We now show that LANA represses origin-dependent lytic DNA replication in a dose dependent manner when added to the cotransfection replication assay. This repression was overcome by increasing amounts of a K-bZIP expression plasmid in the cotransfection mixture or by dominant-negative inhibition of the interaction of LANA with K-bZIP by the overexpression of the K-bZIP-LANA binding domain. Chromatin immunoprecipitation assays show that LANA interacts with oriLyt in the absence of K-bZIP expression, suggesting that suppression of lytic replication by LANA is mediated by direct binding. The interaction of K-bZIP with oriLyt was dependent upon the expression of LANA; however, LANA interacted with oriLyt independently of K-bZIP expression. These data suggest that the interaction of LANA with K-bZIP modulates lytic and latent replication and that K-bZIP facilitates lytic DNA replication and modulates the switch from the latent phase of the virus.

* Corresponding author. Mailing address: Department of Microbiology, University of Nevada—Reno, Howard Bldg. 210, Reno, NV 89557. Phone: (775) 784-4824. Fax: (775) 327-2332. E-mail: gpari{at}medicine.nevada.edu

{triangledown} Published ahead of print on 24 June 2009.

Journal of Virology, September 2009, p. 8492-8501, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00922-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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