title: RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency.
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.
authors.affiliation: The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, NY 10016, USA. Alexis.santana@nyumc.org.|Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA. Alexis.santana@nyumc.org.
authors.forename: Alexis L
authors.initials: AL
authors.lastname: Santana
authors.affiliation: Gundersen Health System, La Crosse, WI 54601, USA. darby.oldenburg@gmail.com.
authors.forename: Darby G
authors.initials: DG
authors.lastname: Oldenburg
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA. varvara.kirillov@stonybrook.edu.
authors.forename: Varvara
authors.initials: V
authors.lastname: Kirillov
authors.affiliation: Department of Computer Science, Stony Brook University, Stony Brook, NY 11794, USA. laraib.malik@stonybrook.edu.
authors.forename: Laraib
authors.initials: L
authors.lastname: Malik
authors.affiliation: Program in Molecular and Cellular Biology, Stony Brook University, Stony Brook, NY 11794, USA. qiwen.dong@stonybrook.edu.
authors.forename: Qiwen
authors.initials: Q
authors.lastname: Dong
authors.affiliation: Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY 11794, USA. roman@rsinayev.com.
authors.forename: Roman
authors.initials: R
authors.lastname: Sinayev
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA. kenneth.marcu@stonybrook.edu.|Biomedical Research Foundation Academy of Athens (BRFAA), Athens 115 27, Greece. kenneth.marcu@stonybrook.edu.|Biochemistry and Cell Biology Dept., Stony Brook University, Stony Brook, NY 11794, USA. kenneth.marcu@stonybrook.edu.|Department of Pathology, Health Sciences Center, Stony Brook University, Stony Brook, NY 11794, USA. kenneth.marcu@stonybrook.edu.
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Marcu
authors.affiliation: Gundersen Health System, La Crosse, WI 54601, USA. DWWhite@gundersenhealth.org.
authors.forename: Douglas W
authors.initials: DW
authors.lastname: White
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA. laurie.krug@stonybrook.edu.
authors.forename: Laurie T
authors.initials: LT
authors.lastname: Krug
keywords: NF-kappaB; gammaherpesvirus; latency; reactivation