{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,11]],"date-time":"2026-03-11T00:47:09Z","timestamp":1773190029478,"version":"3.50.1"},"reference-count":53,"publisher":"American Society for Microbiology","issue":"1","license":[{"start":{"date-parts":[[2003,1,1]],"date-time":"2003-01-01T00:00:00Z","timestamp":1041379200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Virol"],"published-print":{"date-parts":[[2003,1]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded replication-associated protein (RAP, or K8) has been shown to induce both CCAAT\/enhancer binding protein alpha (C\/EBP\u03b1) and p21\n            <jats:sup>CIP-1<\/jats:sup>\n            expression, resulting in G\n            <jats:sub>0<\/jats:sub>\n            \/G\n            <jats:sub>1<\/jats:sub>\n            cell cycle arrest during the lytic cycle. RAP and C\/EBP\u03b1 are also known to interact strongly both in vitro and in lytically infected cells. We recognized two potential consensus C\/EBP binding sites in the RAP promoter and performed electrophoretic mobility shift assay (EMSA) analysis with in vitro-translated C\/EBP\u03b1; this analysis showed that one of these sites has a very high affinity for C\/EBP\u03b1. Luciferase (LUC) assays performed with a target RAP promoter-LUC reporter gene confirmed that C\/EBP\u03b1 can transcriptionally activate the RAP promoter up to 50-fold. Although RAP had no effect on its own promoter by itself, the addition of RAP and C\/EBP\u03b1 together resulted in a threefold increase in activity over that obtained with C\/EBP\u03b1 alone. Importantly, the introduction of exogenous Flag-tagged C\/EBP\u03b1 triggered RAP expression in BCBL-1 cells latently infected with KSHV, as detected by both reverse transcription-PCR and double-label immunofluorescence assay analyses, suggesting the presence of a self-reinforcing loop with C\/EBP\u03b1 and RAP activating each other. The RAP promoter can also be activated 50- to 120-fold by the KSHV lytic-cycle-triggering protein known as replication and transcription activator (RTA). C\/EBP\u03b1 and RTA together cooperated to elevate RAP promoter activity four- to sixfold more than either alone. Furthermore, the addition of RAP, C\/EBP\u03b1, and RTA in LUC reporter cotransfection assays resulted in 7- to 15-fold more activation than that seen with either C\/EBP\u03b1 or RTA alone. Site-specific mutational analysis of the RAP promoter showed that the strong C\/EBP binding site is crucial for C\/EBP\u03b1-mediated transactivation of the RAP promoter. However, the C\/EBP binding site also overlaps the previously reported 16-bp RTA-responsive element (RRE), and the same mutation also both reduced RTA-mediated transactivation and abolished the cooperativity between C\/EBP\u03b1 and RTA. Furthermore, in vitro-translated RTA, although capable of binding directly to the polyadenylated nuclear RNA (PAN) RRE motif, failed to bind to the RAP RRE and interfered with RRE-bound C\/EBP\u03b1 in EMSA experiments. Partial RTA responsiveness but no cooperativity could be transferred to a heterologous promoter containing added consensus C\/EBP binding sites. A chromatin immunoprecipitation assay showed that all three proteins associated specifically with RAP promoter DNA in vivo and that, when C\/EBP\u03b1 was removed from a tetradecanoyl phorbol acetate-treated JSC-1 primary effusion lymphoma cell lysate, the levels of association of RTA and RAP with the RAP promoter were reduced 3- and 13-fold, respectively. Finally, RTA also proved to physically interact with both C\/EBP\u03b1 and RAP, as assayed both in vitro and by immunoprecipitation. Binding to C\/EBP\u03b1 occurred within the N-terminal DNA binding domain of RTA, and deletion of a 17-amino-acid basic motif of RTA abolished both the C\/EBP\u03b1 and DNA binding activities as well as all RTA transactivation and the cooperativity with C\/EBP\u03b1. Therefore, we suggest that RTA transactivation of the RAP RRE is mediated by an interaction with DNA-bound C\/EBP\u03b1 but that full activity requires more than just the core C\/EBP binding site.\n          <\/jats:p>","DOI":"10.1128\/jvi.77.1.600-623.2003","type":"journal-article","created":{"date-parts":[[2002,12,11]],"date-time":"2002-12-11T19:22:58Z","timestamp":1039634578000},"page":"600-623","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":88,"title":["Role of CCAAT\/Enhancer-Binding Protein Alpha (C\/EBP\u03b1) in Activation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic-Cycle Replication-Associated Protein (RAP) Promoter in Cooperation with the KSHV Replication and Transcription Activator (RTA) and RAP"],"prefix":"10.1128","volume":"77","author":[{"given":"Shizhen Emily","family":"Wang","sequence":"first","affiliation":[{"name":"Viral Oncology Program, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21231-1000"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frederick Y.","family":"Wu","sequence":"additional","affiliation":[{"name":"Molecular Virology Laboratories, Department of Pharmacology and Molecular Sciences"},{"name":"Viral Oncology Program, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21231-1000"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masahiro","family":"Fujimuro","sequence":"additional","affiliation":[{"name":"Viral Oncology Program, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21231-1000"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianchao","family":"Zong","sequence":"additional","affiliation":[{"name":"Viral Oncology Program, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21231-1000"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"S. Diane","family":"Hayward","sequence":"additional","affiliation":[{"name":"Molecular Virology Laboratories, Department of Pharmacology and Molecular Sciences"},{"name":"Viral Oncology Program, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21231-1000"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gary S.","family":"Hayward","sequence":"additional","affiliation":[{"name":"Molecular Virology Laboratories, Department of Pharmacology and Molecular Sciences"},{"name":"Viral Oncology Program, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21231-1000"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M101216200"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1101\/gad.14.15.1920"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.74.21.10187-10193.2000"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1182\/blood.V86.7.2708.2708"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.70.11.8218-8223.1996"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.7997879"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.64.7.3358-3369.1990"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.16.9339"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1986.tb04635.x"},{"key":"e_1_3_2_11_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.88.6.2593"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.75.12.5614-5626.2001"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.82.12.4085"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.46.30057"},{"key":"e_1_3_2_15_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.64.3.1227-1232.1990"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.75.10.4475-4481.2001"},{"key":"e_1_3_2_17_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.74.13.6207-6212.2000"},{"key":"e_1_3_2_18_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M011587200"},{"key":"e_1_3_2_19_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.75.19.9509-9516.2001"},{"key":"e_1_3_2_20_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.3289117"},{"key":"e_1_3_2_21_2","doi-asserted-by":"publisher","DOI":"10.1006\/bbrc.1999.1885"},{"key":"e_1_3_2_22_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.64.6.2560-2568.1990"},{"key":"e_1_3_2_23_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.64.3.1143-1155.1990"},{"key":"e_1_3_2_24_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.90.20.9606"},{"key":"e_1_3_2_25_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.73.3.1909-1917.1999"},{"key":"e_1_3_2_26_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.75.15.6786-6799.2001"},{"key":"e_1_3_2_27_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.73.11.9348-9361.1999"},{"key":"e_1_3_2_28_2","doi-asserted-by":"publisher","DOI":"10.1006\/viro.1998.9486"},{"key":"e_1_3_2_29_2","unstructured":"Miller G. 1990. 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