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J Virol. 2012 Sep;86(18):9683-95. doi: 10.1128/JVI.00881-12. Epub 2012 Jun 27.

Quantitative analysis of the bidirectional viral G-protein-coupled receptor and lytic latency-associated nuclear antigen promoter of Kaposi's sarcoma-associated herpesvirus.

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1
Curriculum in Genetics and Molecular Biology, Comprehensive Cancer Center, Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes sustained latent persistence in susceptible cells. This is dependent on the latency-associated nuclear antigen (LANA). Understanding how LANA transcription is regulated thus aids our fundamental understanding of KSHV biology. Two hundred ninety-four base pairs are sufficient to regulate LANA transcription in response to the viral RTA protein and RBPjκ. The same region controls K14/viral G-protein-coupled receptor (vGPCR) transcription in the opposite direction. We used a quantitative analysis in conjunction with specific nucleotide substitutions and defined gain-of-function and loss-of-function RTA mutants to dissect this region. We used a bidirectional reporter driving red and green luciferase to study the LANApi and K14p promoters simultaneously. This established that LANApi/K14p functions as a canonical bidirectional promoter. Both were TATA dependent. K14p was favored by ∼50-fold in this context. Eliminating the distal LANApi TATA box increased maximal output and lowered the induction threshold (T) of K14p even further. Two RBPjκ binding sites were independently required; however, at high concentrations of RTA, direct interactions with an RTA-responsive element (RRE) could complement the loss of one RBPjκ binding site. Intracellular Notch (ICN) was no longer able to activate RBPjκ in the viral context. This suggests a model whereby KSHV alters ICN-RBPjκ gene regulation. When the architecture of this pair of head-to-head RBPjκ binding sites is changed, the sites now respond exclusively to the viral transactivator RTA and no longer to the host mediator ICN.

PMID:
22740392
PMCID:
PMC3446609
DOI:
10.1128/JVI.00881-12
[Indexed for MEDLINE]
Free PMC Article
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