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Pathogens 2017, 6(1), 9; doi:10.3390/pathogens6010009

RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency

1
The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, NY 10016, USA
2
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA
3
Gundersen Health System, La Crosse, WI 54601, USA
4
Department of Computer Science, Stony Brook University, Stony Brook, NY 11794, USA
5
Program in Molecular and Cellular Biology, Stony Brook University, Stony Brook, NY 11794, USA
6
Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY 11794, USA
7
Biomedical Research Foundation Academy of Athens (BRFAA), Athens 115 27, Greece
8
Biochemistry and Cell Biology Dept., Stony Brook University, Stony Brook, NY 11794, USA
9
Department of Pathology, Health Sciences Center, Stony Brook University, Stony Brook, NY 11794, USA
*
Author to whom correspondence should be addressed.
Received: 15 November 2016 / Accepted: 10 February 2017 / Published: 16 February 2017
(This article belongs to the Special Issue Herpesviruses)
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Abstract

RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R) that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA. View Full-Text
Keywords: gammaherpesvirus; latency; reactivation; NF-kappaB gammaherpesvirus; latency; reactivation; NF-kappaB
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Santana, A.L.; Oldenburg, D.G.; Kirillov, V.; Malik, L.; Dong, Q.; Sinayev, R.; Marcu, K.B.; White, D.W.; Krug, L.T. RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency. Pathogens 2017, 6, 9.

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