Translation of hepatitis A virus RNA in vitro: aberrant internal initiations influenced by 5' noncoding region.
Translation of hepatitis A virus RNA in vitro: aberrant internal initiations influenced by 5' noncoding region.
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甲型肝炎病毒 RNA 的体外翻译：受 5 非编码区影响的异常内部起始。
DOI：
10.1016/0042-6822(91)90612-f
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发表时间：
1991
期刊：
影响因子：
3.7
通讯作者：
Ehrenfeld,E
中科院分区：
文献类型：
--
作者：
Jia,XY;Scheper,G;Brown,D;Updike,W;Harmon,S;Richards,O;Summers,D;Ehrenfeld,E
用兔网织红细胞裂解液体外扩增甲型肝炎病毒（HAV）RNA。从全长HAV RNA合成的蛋白质模式是高度复杂的，由范围从<20，000到> 200，000 Da的连续多肽谱组成。通过改变孵育时间、离子或其他反应参数，或通过将HeLa或BS-C-1细胞提取物添加到翻译反应中，模式没有显著改变。在3C编码区中用突变改造的质粒产生的转录物指导与从野生型序列转录的多肽产物相同的总体模式的合成，这表明通过3C的蛋白质加工不产生复杂的蛋白质产物集。翻译仅含有HAV P3编码区的RNA，直接与HAV 5′非编码区相邻，产生一组蛋白产物，这些产物与全长HAV RNA合成的产物的子集精确匹配。用3D、VP 1和2C序列特异性抗血清通过免疫沉淀分析P3 RNA、全长RNA和含突变体3C的RNA的翻译产物;对几种产物进行N-末端序列分析，结果表明兔网织红细胞裂解物中HAV RNA的翻译主要起始于大量的内部AUG密码子，尤其是P3编码区的那些。从P1和P2区域的研究中心启动少量产品。后者的蛋白质进行一些蛋白水解加工，在身份不明的网站，催化的3C蛋白质序列。用脑心肌炎病毒5′端序列替换HAV 5′非编码区增加了正确多蛋白起始位点的起始，减少并改变了内部起始产生的产物。
Hepatitis A virus (HAV) RNAs were translatedin vitroin rabbit reticulocyte lysates. The pattern of proteins synthesized from full-length HAV RNA was highly complex, consisting of a continuous spectrum of polypeptides ranging from <20,000 to >200,000 Da. The pattern was not significantly altered by varying incubation times, ion, or other reaction parameters, or by the addition of HeLa or BS-C-1 cell extracts to the translation reactions. Plasmids engineered with mutations in the 3C coding region produced transcripts which directed the synthesis of the same overall pattern of polypeptide products as those transcribed from wild-type sequences, suggesting that protein processing by 3C did not generate the complex set of protein products. Translation of RNA containing only the P3 coding region of HAV, directly adjacent to the HAV 5′ noncoding region, generated a set of protein products which precisely matched a subset of those synthesized from full-length HAV RNA. The translation products of P3 RNA, full-length RNA, and mutant 3C-containing RNAs were analyzed by immunoprecipitation with antisera specific for 3D, VP1, and 2C sequences; several products were subjected to N-terminal sequence analysis.All together, the results demonstrate that translation of HAV RNA in rabbit reticulocyte lysates initiates predominantly at a large number of internal AUG codons, especially those in the P3 coding region. A minor population of products is initiated from sites in the P1 and P2 regions. The latter proteins undergo some proteolytic processing, at unidentified sites, catalyzed by 3C protein sequences. Replacement of the HAV 5′ noncoding region with encephalomyocarditis virus 5′ end sequences increased initiation at the correct polyprotein start site and both reduced and altered the products generated by internal initiation.
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