RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency
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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2017-02-01
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| Series: | Pathogens |
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| Online Access: | http://www.mdpi.com/2076-0817/6/1/9 |
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| author | Alexis L. Santana Darby G. Oldenburg Varvara Kirillov Laraib Malik Qiwen Dong Roman Sinayev Kenneth B. Marcu Douglas W. White Laurie T. Krug |
| author_facet | Alexis L. Santana Darby G. Oldenburg Varvara Kirillov Laraib Malik Qiwen Dong Roman Sinayev Kenneth B. Marcu Douglas W. White Laurie T. Krug |
| author_sort | Alexis L. Santana |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T20:52:30Z |
| format | Article |
| id | doaj.art-17919aa401754c3f9e0f0285890b723f |
| institution | Directory Open Access Journal |
| issn | 2076-0817 |
| language | English |
| last_indexed | 2024-04-11T20:52:30Z |
| publishDate | 2017-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pathogens |
| spelling | doaj.art-17919aa401754c3f9e0f0285890b723f2022-12-22T04:03:48ZengMDPI AGPathogens2076-08172017-02-0161910.3390/pathogens6010009pathogens6010009RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell LatencyAlexis L. Santana0Darby G. Oldenburg1Varvara Kirillov2Laraib Malik3Qiwen Dong4Roman Sinayev5Kenneth B. Marcu6Douglas W. White7Laurie T. Krug8The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, NY 10016, USAGundersen Health System, La Crosse, WI 54601, USADepartment of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USADepartment of Computer Science, Stony Brook University, Stony Brook, NY 11794, USAProgram in Molecular and Cellular Biology, Stony Brook University, Stony Brook, NY 11794, USAApplied Mathematics and Statistics, Stony Brook University, Stony Brook, NY 11794, USADepartment of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USAGundersen Health System, La Crosse, WI 54601, USADepartment of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USARTA, 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.http://www.mdpi.com/2076-0817/6/1/9gammaherpesviruslatencyreactivationNF-kappaB |
| spellingShingle | Alexis L. Santana Darby G. Oldenburg Varvara Kirillov Laraib Malik Qiwen Dong Roman Sinayev Kenneth B. Marcu Douglas W. White Laurie T. Krug RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency Pathogens gammaherpesvirus latency reactivation NF-kappaB |
| title | RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency |
| title_full | RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency |
| title_fullStr | RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency |
| title_full_unstemmed | RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency |
| title_short | RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency |
| title_sort | rta occupancy of the origin of lytic replication during murine gammaherpesvirus 68 reactivation from b cell latency |
| topic | gammaherpesvirus latency reactivation NF-kappaB |
| url | http://www.mdpi.com/2076-0817/6/1/9 |
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