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Journal of Virology, October 2009, p. 9786-9802, Vol. 83, No. 19
0022-538X/09/$08.00+0     doi:10.1128/JVI.00704-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Disruption of LANA in Rhesus Rhadinovirus Generates a Highly Lytic Recombinant Virus{triangledown} ,{dagger}

Kwun Wah Wen,1,2,3 Dirk P. Dittmer,1,2,3* and Blossom Damania1,2,3*

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,1 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,2 Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275993

Received 5 April 2009/ Accepted 2 July 2009

Rhesus monkey rhadinovirus (RRV) is a gammaherpesvirus that is closely related to human Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). RRV is the closest relative to KSHV that has a fully sequenced genome and serves as an in vitro and an in vivo model system for KSHV. The latency-associated nuclear antigen (LANA) protein of both KSHV and RRV plays key roles in the establishment and maintenance of these herpesviruses. We have constructed a RRV recombinant virus (RRV{Delta}LANA/GFP) in which the RRV LANA open reading frame has been disrupted with a green fluorescent protein (GFP) expression cassette generated by homologous recombination. The integrity of the recombinant virus was confirmed by diagnostic PCR, restriction digestion, Southern blot analysis, and whole-genome sequencing. We compared the single-step and multistep replication kinetics of RRV{Delta}LANA/GFP, RRV-GFP, wild-type (WT) RRV H26-95, and a revertant virus using traditional plaque assays, as well as real-time quantitative PCR-based genome quantification assays. The RRV{Delta}LANA/GFP recombinant virus exhibited significantly higher lytic replicative properties compared to RRV-GFP, WT RRV, or the revertant virus. This was observed upon de novo infection and in the absence of chemical inducers such as phorbol esters. In addition, by using a quantitative real-time PCR-based viral array, we are the first to report differences in global viral gene expression between WT and recombinant viruses. The RRV{Delta}LANA/GFP virus displayed increased lytic gene transcription at all time points postinfection compared to RRV-GFP. Moreover, we also examined several cellular genes that are known to be repressed by KSHV LANA and report that these genes are derepressed during de novo lytic infection with the RRV{Delta}LANA/GFP virus compared to RRV-GFP. Finally, we also demonstrate that the RRV{Delta}LANA/GFP virus fails to establish latency in B cells, as measured by the loss of GFP-positive cells and intracellular viral genomes.

* Corresponding author. Mailing address: Lineberger Comprehensive Cancer Center, CB#7295, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 843-6011. Fax: (919) 966-9673. E-mail for B. Damania: damania{at}med.unc.edu. E-mail for D. P. Dittmer: ddittmer{at}med.unc.edu

{triangledown} Published ahead of print on 8 July 2009.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, October 2009, p. 9786-9802, Vol. 83, No. 19
0022-538X/09/$08.00+0     doi:10.1128/JVI.00704-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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