Collis P S, O'Donnell B J, Barton D J, Rogers J A, Flanegan J B
Department of Immunology and Medical Microbiology, College of Medicine, University of Florida, Gainesville 32610-0266.
J Virol. 1992 Nov;66(11):6480-8. doi: 10.1128/JVI.66.11.6480-6488.1992.
Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis.
全长和亚基因组脊髓灰质炎病毒RNA在体外转录并转染到HeLa细胞中，以研究病毒RNA在体内的复制情况。通过共转染程序和Northern（RNA）印迹分析，分析了具有缺失突变的RNA在有无辅助RNA的情况下的复制能力。这种方法的一个优点是，无需首先分离条件致死突变体，就可以对病毒RNA复制和遗传互补进行表征。衣壳编码区（P1）存在大的框内缺失的亚基因组RNA比全长病毒RNA转录本复制效率更高。在共转染实验中，全长和亚基因组RNA的复制水平均略有降低，并且似乎相互干扰彼此的复制。相比之下，P1中具有类似大小的框外缺失的亚基因组RNA在转染细胞中单独或在存在辅助RNA的情况下均不复制。对于包含跨越P1、P2和P3编码区的大框内缺失的RNA转录本，也观察到了类似的结果。P1-2A编码序列中存在框内缺失的突变RNA能够自我复制，但水平显著降低。与在反式中提供2A的辅助RNA共转染后，该RNA的复制得到了完全互补。然而，P1-2A-2B框内缺失完全阻断了RNA复制，并且无法得到互补。对照实验表明，当RNA转录本在体外翻译时，所有预期的病毒蛋白均被合成并加工。因此，我们的结果表明，2A是一种反式作用蛋白，而2B以及可能的其他病毒蛋白在脊髓灰质炎病毒RNA在体内的复制过程中是顺式作用的。我们的数据支持一种脊髓灰质炎病毒RNA复制模型，该模型将正链RNA分子的翻译与负链RNA合成的复制复合体的形成直接联系起来。
