Becerra S P, Koczot F, Fabisch P, Rose J A
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J Virol. 1988 Aug;62(8):2745-54. doi: 10.1128/JVI.62.8.2745-2754.1988.
The three adeno-associated virus type 2 (AAV2) structural proteins (A, B, and C) are specified by transcripts generated from the most-rightward promoter (p40). Protein C (60 kilodaltons [kDa]), the most abundantly produced, is entirely contained within B (72 kDa) which, in turn, is contained within A (90 kDa). Although neither of the known structures of p40 transcripts, an unspliced 2.6-kilobase (kb) RNA and a spliced 2.3-kb RNA, possesses an AUG-initiated open reading frame that accounts for the synthesis of proteins A and B, recent evidence indicates that B is initiated by a unique eucaryotic initiation codon (ACG) (S.P. Becerra, J.A. Rose, M. Hardy, B. Baroudy, and C.W. Anderson, Proc. Natl. Acad. Sci. USA 82:7919-7923, 1985). In the present study, we analyzed the in vitro translation of AAV capsid proteins from synthetic transcripts and the in vivo expression of AAV mRNA and capsid proteins in 293 cells transfected with AAV DNA constructs. The results demonstrated that AAV transcripts contain only one functional 5' splice donor site, that synthesis of capsid proteins from the unspliced 2.6-kb transcript is very inefficient, that transcripts without the intervening sequence (IVS) (i.e., the 2.3-kb RNA) do not produce protein A but effectively synthesize proteins B and C, and that protein A is actively synthesized from transcripts which contain the last 34 bases of the IVS. Protein A initiates within this 34-base segment in reading frame 1, apparently with the AUG codon at nucleotide 2203, and then elongates into the B and C open reading frame. Because A is inefficiently synthesized from the 2.6-kb transcript, we conclude that an effective A transcript is generated by alternative splicing and that the alternative 3' acceptor site may lie at nucleotide 2200 within a context of...CAG]GTA. The levels of B and C produced by a synthetic transcript devoid of the IVS suggest that the known 2.3-kb RNA is the main source of these proteins and indicate that this single RNA species expresses both proteins by alternative use of their respective initiation codons.
三种2型腺相关病毒（AAV2）结构蛋白（A、B和C）由最右侧启动子（p40）产生的转录本所编码。产量最高的蛋白C（60千道尔顿[kDa]）完全包含在蛋白B（72 kDa）中，而蛋白B又包含在蛋白A（90 kDa）中。虽然p40转录本的两种已知结构，即未剪接的2.6千碱基（kb）RNA和剪接后的2.3-kb RNA，都没有一个能解释蛋白A和B合成的AUG起始开放阅读框，但最近的证据表明，蛋白B是由一个独特的真核起始密码子（ACG）起始的（S.P.贝塞拉、J.A.罗斯、M.哈迪、B.巴鲁迪和C.W.安德森，《美国国家科学院院刊》82:7919 - 7923，1985）。在本研究中，我们分析了合成转录本中AAV衣壳蛋白的体外翻译以及用AAV DNA构建体转染的293细胞中AAV mRNA和衣壳蛋白的体内表达。结果表明，AAV转录本仅包含一个功能性5'剪接供体位点，从未剪接的2.6-kb转录本合成衣壳蛋白的效率非常低，没有间隔序列（IVS）的转录本（即2.3-kb RNA）不产生蛋白A，但能有效合成蛋白B和C，并且蛋白A是从包含IVS最后34个碱基的转录本中活跃合成的。蛋白A在这个34碱基片段内的阅读框1中起始，显然起始于核苷酸2203处的AUG密码子，然后延伸到B和C的开放阅读框。由于从2.6-kb转录本中合成A的效率较低，我们得出结论，有效的A转录本是通过可变剪接产生的，并且可变3'受体位点可能位于……CAG]GTA背景下的核苷酸2200处。缺乏IVS的合成转录本产生的B和C水平表明，已知的2.3-kb RNA是这些蛋白的主要来源，并表明这种单一RNA种类通过交替使用它们各自的起始密码子来表达这两种蛋白。
