Horikami S M, Moyer S A
Department of Immunology and Medical Microbiology, University of Florida School of Medicine, Gainesville 32610.
J Virol. 1991 Oct;65(10):5342-7. doi: 10.1128/JVI.65.10.5342-5347.1991.
A transcription system with detergent-disrupted purified measles virus was developed. Synthesis of authentic, full-length measles virus N, P, M, and F mRNAs by purified virus occurred as identified by dot-blot hybridization analysis of individual measles virus clones and gel electrophoresis. The relative abundance of the first five viral mRNAs synthesized in vitro decreased significantly with their distance from the 3' end. The addition of the soluble protein fraction from uninfected A549 cells stimulated overall viral RNA synthesis but did not alter the relative abundance of each of the mRNAs. Measles virus synthesized in vitro a leader RNA of approximately 55 nucleotides in length, suggesting that like other negative-strand viruses, transcription initiated only at the 3' end of the genome RNA. Purified measles virus also catalyzed RNA editing during the synthesis of the P mRNA as shown by modified primer extension analysis of the mRNA products and by translation of the modified RNA into the V protein in rabbit reticulocyte lysates. These data suggested that the RNA editing activity was virus encoded.
开发了一种用去污剂裂解纯化麻疹病毒的转录系统。通过对单个麻疹病毒克隆的斑点印迹杂交分析和凝胶电泳鉴定,纯化病毒可合成真实的全长麻疹病毒N、P、M和F mRNA。体外合成的前五种病毒mRNA的相对丰度随着它们与3'端距离的增加而显著降低。添加未感染的A549细胞的可溶性蛋白组分可刺激病毒RNA的整体合成,但不会改变每种mRNA的相对丰度。体外合成的麻疹病毒产生了一条长度约为55个核苷酸的前导RNA,这表明与其他负链病毒一样,转录仅在基因组RNA的3'端起始。如对mRNA产物进行的改良引物延伸分析以及在兔网织红细胞裂解物中将修饰的RNA翻译成V蛋白所示,纯化的麻疹病毒在P mRNA合成过程中也催化RNA编辑。这些数据表明RNA编辑活性是由病毒编码的。