Wang Shizhen Emily, Wu Frederick Y, Chen Honglin, Shamay Meir, Zheng Qizhi, Hayward Gary S
Viral Oncology Program of the Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231-1000, USA.
J Virol. 2004 Apr;78(8):4248-67. doi: 10.1128/jvi.78.8.4248-4267.2004.
Kaposi's sarcoma-associated herpesvirus (KSHV) maintains a latent infection in primary effusion lymphoma (PEL) cells, but treatment with tetradecanoyl phorbol acetate (TPA) can trigger the full lytic-cycle replication in some of these cells. During lytic-cycle replication, the KSHV-encoded replication and transcription activator (RTA or ORF50), the mRNA transport and accumulation protein (MTA), and the replication-associated protein (RAP) all play crucial roles in expression of downstream viral genes as well as in mediation of viral DNA replication. The cellular CCAAT/enhancer-binding protein alpha (C/EBP alpha) is induced in TPA-treated PEL cells and contributes to transactivation of the promoters for all of these genes through both direct binding and cooperative interactions with RTA and RAP targeted to upstream C/EBP sites. However, little is known about how RTA expression is triggered initially at the earliest stages after TPA induction when the C/EBP alpha levels are still limited. Treatment with TPA proved to significantly induce both AP1 DNA-binding activity and levels of activated phosphorylated cJUN in PEL cells and ectopic expression of cJUN-plus-cFOS-induced RTA protein expression in PEL cells. Cotransfected cJUN plus cFOS or TPA treatment transactivated the KSHV RTA, RAP, and MTA promoters in an AP1-binding site-dependent manner in all three promoters. Chromatin immunoprecipitation assays confirmed that cJUN associates with these KSHV target promoters in PEL cells as early as 4 h after TPA treatment. Furthermore, the KSHV RTA and RAP proteins both interact with cJUN or both cJUN and cFOS in vitro or by coimmunoprecipitation from induced PEL cells and enhance cJUN-plus-cFOS-mediated transactivation of these viral promoters. Both increased phosphorylated cJUN and AP1 DNA-binding activity was detected as early as 1 h after TPA treatment in PEL cells, suggesting that AP1 activity may be crucial for very early activation of the RAP, MTA, and RTA promoters during the KSHV lytic cycle. Finally, expression of RTA alone increased cJUN protein levels severalfold in DG75 cells but did not induce cJUN phosphorylation. Therefore, we suggest that the initiating effects of TPA via the AP1 pathway in PEL cells need to be amplified by RTA for full lytic-cycle induction.
卡波西肉瘤相关疱疹病毒（KSHV）在原发性渗出性淋巴瘤（PEL）细胞中维持潜伏感染，但用十四酰佛波醇乙酸酯（TPA）处理可在其中一些细胞中触发完整的裂解周期复制。在裂解周期复制过程中，KSHV编码的复制和转录激活因子（RTA或ORF50）、mRNA转运和积累蛋白（MTA）以及复制相关蛋白（RAP）在下游病毒基因的表达以及病毒DNA复制的介导中都起着关键作用。细胞CCAAT/增强子结合蛋白α（C/EBPα）在TPA处理的PEL细胞中被诱导，并通过与靶向上游C/EBP位点的RTA和RAP直接结合及协同相互作用，促进所有这些基因启动子的反式激活。然而，在TPA诱导后最早阶段，当C/EBPα水平仍然有限时，RTA表达最初是如何被触发的，目前知之甚少。事实证明，TPA处理可显著诱导PEL细胞中的AP1 DNA结合活性以及活化的磷酸化cJUN水平，并且cJUN加cFOS的异位表达可诱导PEL细胞中RTA蛋白表达。共转染的cJUN加cFOS或TPA处理以AP1结合位点依赖的方式反式激活所有三个启动子中的KSHV RTA、RAP和MTA启动子。染色质免疫沉淀分析证实，TPA处理后4小时，cJUN就与PEL细胞中的这些KSHV靶启动子结合。此外，KSHV RTA和RAP蛋白在体外或通过从诱导的PEL细胞中共免疫沉淀，都与cJUN或cJUN和cFOS相互作用，并增强cJUN加cFOS介导的这些病毒启动子的反式激活。在PEL细胞中，TPA处理后1小时就检测到磷酸化cJUN和AP1 DNA结合活性增加，这表明AP1活性可能对KSHV裂解周期中RAP、MTA和RTA启动子的极早期激活至关重要。最后，单独的RTA表达使DG75细胞中的cJUN蛋白水平增加了几倍，但未诱导cJUN磷酸化。因此，我们认为TPA通过AP1途径在PEL细胞中的起始作用需要被RTA放大，以实现完整的裂解周期诱导。
