Wang X S, Srivastava A
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
J Virol. 1998 Jun;72(6):4811-8. doi: 10.1128/JVI.72.6.4811-4818.1998.
The Rep proteins encoded by the adeno-associated virus type 2 (AAV) play a crucial role in the rescue, replication, and integration of the viral genome. In the absence of a helper virus, little expression of the AAV Rep proteins occurs, and the AAV genome fails to undergo DNA replication. Since previous studies have established that expression of the Rep78 and Rep68 proteins from the viral p5 promoter is controlled by the Rep-binding site (RBS) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RBS and the YBS in the p5 promoter. These plasmids were transfected in HeLa or 293 cells and analyzed for the potential to undergo AAV DNA rescue and replication. Our studies revealed that (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in the RBS resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 but not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gene products was insufficient to mediate rescue and/or replication of the AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions that were biologically active in secondary infection of HeLa cells in the presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantly affected stable integration only in 293 cells, presumably because rescue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or KB cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RBS interaction plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficient to confer autonomous replication competence to AAV in 293 cells.
2型腺相关病毒（AAV）编码的Rep蛋白在病毒基因组的拯救、复制和整合过程中发挥着关键作用。在没有辅助病毒的情况下，AAV Rep蛋白几乎不表达，AAV基因组也无法进行DNA复制。由于先前的研究已证实，病毒p5启动子驱动的Rep78和Rep68蛋白的表达受Rep结合位点（RBS）和YY1因子结合位点（YBS）的调控，我们构建了多个重组AAV质粒，这些质粒的p5启动子中的RBS和YBS存在突变和/或缺失。将这些质粒转染到HeLa或293细胞中，并分析其进行AAV DNA拯救和复制的潜力。我们的研究表明：（i）野生型AAV基因组在293细胞中发生了低水平的拯救和自主复制，但在HeLa细胞中未发生；（ii）RBS中的突变导致p5启动子的表达增强，从而使AAV基因组在293细胞中更有效地进行拯救和/或复制，但在HeLa细胞中则不然；（iii）在没有腺病毒共感染的情况下，仅含YBS突变的质粒几乎不发生拯救和/或复制；（iv）腺病毒E1A基因产物的表达不足以介导HeLa细胞中AAV基因组的拯救和/或复制；（v）293细胞中自主复制的AAV基因组成功包装进成熟的子代病毒粒子中，这些病毒粒子在腺病毒存在的情况下对HeLa细胞进行二次感染时具有生物活性；（vi）稳定转染含有新霉素抗性基因的重组AAV质粒仅在293细胞中显著影响稳定整合，推测是因为这些质粒中的AAV基因组在293细胞中发生了拯救和自主复制，而在HeLa或KB细胞中未发生。这些数据表明，在没有腺病毒的情况下，AAV Rep蛋白与RBS的相互作用在下调p5启动子驱动的病毒基因表达中起主导作用，并且这种相互作用的扰动足以赋予AAV在293细胞中的自主复制能力。
