The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection
The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection
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DOI：
10.1128/jvi.71.2.1079-1088.1997
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发表时间：
1997-02-01
影响因子：
5.4
通讯作者：
Muzyczka, N
中科院分区：
文献类型：
--
作者：
Pereira, DJ;McCarty, DM;Muzyczka, N
腺相关病毒(AAV)使用三个启动子P5、P19和P30来调节病毒基因的表达，P5和P19启动子分别指导病毒调节蛋白Rep78和-68以及Rep52和-40的合成，P5 Rep蛋白结合一个22bp的线性序列-Rep结合元件(RBE)，它位于末端重复(Tr)和P5启动子内。在没有辅助病毒的情况下，所有四种Rep蛋白都被证明减少了病毒P5和p19启动子的转录。我重点研究了这些蛋白和rbe在生产性感染过程中控制转录的作用，即在腺病毒存在的情况下，我们发现在腺病毒存在的情况下，P5RBE抑制P5转录，而TR中的RBE激活P5。然而，TRRBE和P5RBE都反式激活p19和p40启动子，P5RBE-Rep复合体在抑制P5的同时可以反式激活p19和p40，这一事实表明Rep78/68既是抑制因子又是反式激活因子，P5的Rep抑制是P5RBE所特有的，因为其他P5启动子元件不支持这种活性。我们还证明了在腺病毒的存在下，不与RBE结合的p19Rep蛋白可以消除Rep78和Rep68对P5启动子的抑制。这可能是由于Rep52与Rep78或Rep68结合而产生Rep78/68-Rep52蛋白复合体，可通过免疫沉淀在体内检测到。最后，鉴定出两个缺乏RBE结合和反式激活但P5抑制阳性的Rep突变体，这些突变体可能定义了与其他促进抑制的蛋白质接触所涉及的相互作用区域，这些观察表明在AAV生产性感染过程中控制P5和p19mRNA水平的机制。
Adeno-associated virus (AAV) uses three promoters, p5, p19, and p30, to regulate viral gene expression, The p5 and p19 promoters direct the synthesis of the viral regulatory proteins, Rep78 and -68 and Rep52 and -40, respectively, The p5 Rep proteins bind a linear 22-bp sequence, the Rep binding element (RBE), that is within both the terminal repeat (TR) and the p5 promoter, In the absence of helper virus, all four Rep proteins have been shown to reduce transcription from the viral p5 and p19 promoters, In this report, me focus on the roles of these proteins and the RBEs in controlling transcription during a productive infection, that is, in the presence of adenovirus, We find that in the presence of adenovirus, the p5 RBE represses p5 transcription while the RBE in the TR activates p5. However, both the TR RBE and the p5 RBE transactivate the p19 and p40 promoters, The fact that the p5 RBE-Rep complex can transactivate p19 and p40 while repressing p5 suggests that Rep78/68 is both a repressor and a transactivator, Rep repression of p5 is specific for the p5 RBE, as other p5 promoter elements do not support this activity, We also demonstrate that in the presence of adenovirus, the p19 Rep proteins, which do not bind to the RBE, can eliminate repression of the p5 promoter by Rep78 and Rep68. This may occur by the association of Rep52 with Rep78 or Rep68 to produce a Rep78/68-Rep52 protein complex which can be detected in vivo by immunoprecipitation, Finally, two Rep mutants that were deficient in RBE binding and transactivation but positive for p5 repression were identified, These mutants may define interaction domains involved in making contacts with other proteins that facilitate repression, These observations suggest a mechanism for controlling the p5 and p19 mRNA levels during a productive AAV infection.