Trempe J P, Carter B J
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
J Virol. 1988 Sep;62(9):3356-63. doi: 10.1128/JVI.62.9.3356-3363.1988.
Fine-structure mapping of the capsid-specific mRNAs from adeno-associated virus (AAV) revealed an alternate splicing pattern in these RNAs. S1 nuclease and primer extension analyses showed that splicing of these mRNAs occurs at acceptor sites at nucleotide 2228 (major splice) or 2201 (minor splice). Both splice acceptors were ligated to the same 55-nucleotide leader in mature mRNAs. Both species were present in equal amounts in mRNA derived from AAV plasmid-transfected cells. However, when adenovirus infection accompanied the DNA transfection, the major splice predominated over the minor splice. Using cDNA clones of both the major and minor spliced mRNAs, we demonstrated that the largest AAV capsid protein, VP1, was derived from the minor spliced mRNA. The other capsid proteins, VP2 and VP3, came predominantly from the major spliced mRNA. These results, which describe the previously undetected minor splice, provide a mechanism for the production of all three AAV virion proteins.
腺相关病毒(AAV)衣壳特异性mRNA的精细结构图谱显示这些RNA存在可变剪接模式。S1核酸酶和引物延伸分析表明,这些mRNA的剪接发生在核苷酸2228(主要剪接)或2201(次要剪接)的受体位点。在成熟mRNA中,两种剪接受体都与相同的55个核苷酸的前导序列相连。在源自AAV质粒转染细胞的mRNA中,这两种类型的mRNA含量相等。然而,当腺病毒感染伴随DNA转染时,主要剪接产物比次要剪接产物占优势。利用主要和次要剪接mRNA的cDNA克隆,我们证明最大的AAV衣壳蛋白VP1源自次要剪接的mRNA。其他衣壳蛋白VP2和VP3主要来自主要剪接的mRNA。这些描述了先前未检测到的次要剪接的结果,为三种AAV病毒粒子蛋白的产生提供了一种机制。