An authentic 3′ noncoding region is necessary for efficient poliovirus replication
An authentic 3′ noncoding region is necessary for efficient poliovirus replication
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DOI：
10.1128/jvi.79.18.11962-11973.2005
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发表时间：
2005-09-01
影响因子：
5.4
通讯作者：
Semler, BL
中科院分区：
文献类型：
--
作者：
Brown, DM;Cornell, CT;Semler, BL
小核糖核酸病毒RNA复制涉及负链中间体的特异性合成，随后在大量细胞mRNA存在下积累正链病毒RNA。在此之前，为了鉴定启动负链RNA合成所需的顺式作用元件，我们从人鼻病毒和脊髓灰质炎病毒基因组RNA中删除了整个3'非编码区。这些缺失突变的转录本显示了严重的延迟RNA积累后转染的HeLa细胞。有趣的是，在随后的HeLa细胞感染中，缺失突变脊髓灰质炎病毒仅显示出RNA合成的中度缺陷。这些数据表明，转染后细胞病变效应产生的延迟可能是由于RNA复制缺陷被最初几轮RNA合成期间产生的补偿突变积累所克服。在这项研究中，我们对缺失突变病毒的整个基因组进行了测序，发现与亲本克隆相比只有两个核苷酸变化。对这些序列变异体的转染分析表明，序列的改变并没有为3'非编码区缺失突变复制缺陷提供补偿功能。缺失突变表型的进一步检查显示，RNA转染后的严重复制缺陷部分是由于体外衍生的缺失突变转录本中存在的非病毒末端序列。我们的数据表明，携带完整的3'非编码区缺失突变的脊髓灰质炎病毒RNA具有感染性（而不仅仅是准感染性）。
Picornavirus RNA replication involves the specific synthesis of negative-strand intermediates followed by an accumulation of positive-strand viral RNA in the presence of a multitude of cellular mRNAs. Previously, in an effort to identify cis-acting elements required for initiation of negative-strand RNA synthesis, we deleted the entire 3' noncoding regions from human rhinovirus and poliovirus genomic RNAs. These deletion mutation transcripts displayed a severe delay in RNA accumulation following transfection of HeLa cells. Interestingly, in subsequent infection of HeLa cells, the deletion-mutant poliovirus displayed only a moderate deficiency in RNA synthesis. These data suggested that the delay in the production of cytopathic effects after transfection may have been due to an RNA replication defect overcome by the accumulation of a compensatory mutation(s) generated during initial rounds of RNA synthesis. In this study, we have sequenced the entire genome of the deletion-mutant virus and found only two nucleotide changes from the parental clone. Transfection analysis of these sequence variants revealed that the sequence changes did not provide compensatory functions for the 3' noncoding region deletion mutation replication defect. Further examination of the deletion mutant phenotype revealed that the severe replication defect following RNA transfection is due, in part, to nonviral terminal sequences present in the in vitro-derived deletion mutation transcripts. Our data suggest that poliovirus RNA harboring a complete 3' noncoding region deletion mutation is infectious (not merely quasi-infectious).