Mendelson E, Trempe J P, Carter B J
J Virol. 1986 Dec;60(3):823-32. doi: 10.1128/JVI.60.3.823-832.1986.
Prior genetic analysis provided evidence for trans-acting regulatory proteins (Rep) coded by the left-hand open reading frame (orf-1) of adeno-associated virus (AAV). We have used immunoblotting analysis to identify four protein products of orf-1. Antibodies elicited against an oligopeptide encoded by orf-1 were reacted with extracts of cells that were infected with AAV or transfected with AAV recombinant vectors in the presence or absence of helper adenovirus. The antibody recognized four polypeptides with apparent molecular weights of 78,000, 68,000, 52,000, and 40,000. The 78,000-dalton (78K) (Rep78) and 68K (Rep68) proteins appear to be encoded by the unspliced 4.2-kilobase (kb) and spliced 3.9-kb mRNAs, respectively, transcribed from the p5 promoter. The 52K (Rep52) and 40K (Rep40) proteins appear to be the products of the unspliced 3.6-kb and the spliced 3.3-kb mRNAs, respectively, transcribed from the p19 promoter. Rigorous identification of Rep68 as an AAV-coded protein is compromised by a cross-reacting cellular protein of similar size. All four proteins were expressed in the human cell lines 293, HeLa, HT29, and A549 infected with AAV together with adenovirus. Rep78 and Rep52 were detected at lower levels in cells infected with AAV at high multiplicity in the absence of adenovirus. Human 293 cells transfected with a recombinant AAV vector (pAV2) also expressed Rep proteins in the presence or absence of adenovirus. Mutations introduced into the Rep region of pAV2 further identified the Rep proteins. The amount of each Rep protein varied between nuclear and cytoplasmic extracts, but all four proteins accumulated during the lytic cycle of the viral infection. Other studies have indicated that the Rep proteins have independent trans-acting functions in viral DNA replication and negative and positive regulation of gene expression. Correlation of each trans-acting function with individual Rep proteins will be facilitated with the antibodies described herein.
先前的基因分析为腺相关病毒（AAV）左手开放阅读框（orf-1）编码的反式作用调节蛋白（Rep）提供了证据。我们利用免疫印迹分析鉴定了orf-1的四种蛋白质产物。针对orf-1编码的寡肽产生的抗体与感染了AAV或在有或无辅助腺病毒存在的情况下用AAV重组载体转染的细胞提取物反应。该抗体识别出四种表观分子量分别为78,000、68,000、52,000和40,000的多肽。78,000道尔顿（78K）（Rep78）和68K（Rep68）蛋白似乎分别由从p5启动子转录的未剪接的4.2千碱基（kb）和剪接的3.9-kb mRNA编码。52K（Rep52）和40K（Rep40）蛋白似乎分别是从p19启动子转录的未剪接的3.6-kb和剪接的3.3-kb mRNA的产物。由于存在一种大小相似的交叉反应细胞蛋白，Rep68作为AAV编码蛋白的严格鉴定受到影响。所有这四种蛋白在感染了AAV和腺病毒的人细胞系293、HeLa、HT29和A549中均有表达。在没有腺病毒的情况下，高倍数感染AAV的细胞中Rep78和Rep52的检测水平较低。用重组AAV载体（pAV2）转染的人293细胞在有或无腺病毒的情况下也表达Rep蛋白。引入pAV2的Rep区域的突变进一步鉴定了Rep蛋白。每种Rep蛋白在核提取物和细胞质提取物中的量有所不同，但所有这四种蛋白在病毒感染的裂解周期中都会积累。其他研究表明，Rep蛋白在病毒DNA复制以及基因表达的负调控和正调控中具有独立的反式作用功能。本文所述的抗体将有助于每种反式作用功能与单个Rep蛋白之间的相关性研究。
