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Journal of Virology, December 2007, p. 13519-13532, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.00832-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Overexpression of the Kaposi's Sarcoma-Associated Herpesvirus Transactivator K-Rta Can Complement a K-bZIP Deletion BACmid and Yields an Enhanced Growth Phenotype{triangledown}

Taeko Kato-Noah,1 Yiyang Xu,2 Cyprian C. Rossetto,1 Kelly Colletti,1 Iva Papousková,1 and Gregory S. Pari1*

Department of Microbiology and the Cell and Molecular Biology Program, University of Nevada School of Medicine, Reno, Nevada 89557,1 G. W. Hooper Foundation, Box 0552, University of California, San Francisco, San Francisco, California 94143-05522

Received 18 April 2007/ Accepted 24 September 2007

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (HHV8) ORF50 encodes a transactivator, K-Rta, which functions as the switch from latent to lytic virus replication. K-bZIP interacts with K-Rta and can repress its transactivation activity for some viral promoters. Both K-Rta and K-bZIP are required for origin-dependent DNA replication. To determine the role of K-bZIP in the context of the viral genome, we generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a deletion in the K-bZIP open reading frame. This BACmid, BAC36{Delta}K8, displayed an enhanced growth phenotype with respect to virus production and accumulation of virus-encoded mRNAs measured by real-time PCR when K-Rta was used to induce the virus lytic cycle. Conversely, induction of the virus lytic cycle using tetradecanoyl phorbol acetate/n-butyrate resulted in no virus production and an aberrant gene expression pattern from BAC36{Delta}K8-containing cells compared to wild-type (wt) BAC. This null virus phenotype was efficiently complemented by the expression of K-bZIP in trans, restoring virus production to wt BAC levels. Immunofluorescence staining revealed that subcellular localization of K-Rta was unchanged; however, a disruption of LANA subcellular localization was observed in cells harboring BAC36{Delta}K8, suggesting that K-bZIP influences LANA localization. Coimmunoprecipitation experiments confirmed that K-bZIP interacts with LANA in BCBL-1 cells and in cotransfection assays. Lastly, the chromatin immunoprecipitation assay revealed that, in an environment where K-Rta is overexpressed and in the absence of K-bZIP, K-Rta binds to CAAT enhancer binding protein {alpha} sites within oriLyt, suggesting that it is K-Rta that supplies an essential replication function and that K-bZIP may serve to augment or facilitate the interaction of K-Rta with oriLyt.

* 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 3 October 2007.

Journal of Virology, December 2007, p. 13519-13532, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.00832-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Rossetto, C., Yamboliev, I., Pari, G. S. (2009). Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 K-bZIP Modulates Latency-Associated Nuclear Protein-Mediated Suppression of Lytic Origin-Dependent DNA Synthesis. J. Virol. 83: 8492-8501 [Abstract] [Full Text]  
  • Lefort, S., Flamand, L. (2009). Kaposi's Sarcoma-Associated Herpesvirus K-bZIP Protein Is Necessary for Lytic Viral Gene Expression, DNA Replication, and Virion Production in Primary Effusion Lymphoma Cell Lines. J. Virol. 83: 5869-5880 [Abstract] [Full Text]  


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