Cassinotti P, Weitz M, Tratschin J D
Department of Virology, University of Berne, Switzerland.
Virology. 1988 Nov;167(1):176-84.
We have mapped a minor spliced transcript with mRNA properties which is derived from the capsid gene promoter (P40) of the adeno-associated virus (AAV) genome. By S1 nuclease mapping as well as primer extension analysis this mRNA was found to occur by splicing at the same donor site (nucleotide, NT 1907) but at an alternative acceptor site when compared to the major, 2.3-kb spliced P40 transcript which encodes two of the three AAV capsid proteins, namely VP2 and VP3. This minor acceptor site is located at NT 2200, i.e., 27 NT upstream of the acceptor site used to generate the VP2/VP3 mRNA. We have also sequenced the AAV genome in this region and have found one important error in the sequence published by A. Srivastava, E. Lusby, and K.I. Berns (1983, J. Virol., 45, 555-564): residue at NT 2429 has to be deleted from the reported sequence. This correction of the sequence reveals that the open reading frame (ORF) which encodes all three capsid proteins extends from NT 2203 to NT 4321. Furthermore, this entire ORF is contained in the minor but not in the major spliced P40 transcript. We provide evidence that the largest AAV structural protein VP1 is translated from this hitherto undetected spliced transcript by initiation at its first AUG (NT 2203) and termination at NT 4321. The calculated molecular weight of this VP1 polypeptide is 78,247 Da.
我们绘制了一种具有mRNA特性的次要剪接转录本图谱，该转录本源自腺相关病毒（AAV）基因组的衣壳基因启动子（P40）。通过S1核酸酶图谱分析以及引物延伸分析发现，与编码三种AAV衣壳蛋白中的两种（即VP2和VP3）的主要2.3 kb剪接P40转录本相比，这种mRNA通过在相同的供体位点（核苷酸，NT 1907）剪接，但在一个替代的受体位点发生剪接。这个次要受体位点位于NT 2200，即用于产生VP2 / VP3 mRNA的受体位点上游27个核苷酸处。我们还对该区域的AAV基因组进行了测序，发现在A. Srivastava、E. Lusby和K.I. Berns（1983年，《病毒学杂志》，45卷，555 - 564页）发表的序列中有一个重要错误：报道序列中NT 2429处的残基必须删除。序列的这种校正表明，编码所有三种衣壳蛋白的开放阅读框（ORF）从NT 2203延伸至NT 4321。此外，整个ORF包含在次要但不包含在主要剪接的P40转录本中。我们提供的证据表明，最大的AAV结构蛋白VP1是从这种迄今未检测到的剪接转录本翻译而来的，起始于其第一个AUG（NT 2203），终止于NT 4321。这种VP1多肽的计算分子量为78,247 Da。
