Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication.
Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication.
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DOI：
10.1371/journal.ppat.1000462
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发表时间：
2009-05
期刊：
影响因子：
6.7
通讯作者：
Ortín J
中科院分区：
文献类型：
--
作者：
Jorba N;Coloma R;Ortín J
甲型流感病毒基因组由8个负极性的单链RNA片段组成。每一个都包括在一个核糖核蛋白颗粒(VRNP)中，该颗粒含有聚合酶复合体和一些核蛋白(NP)单体。病毒RNA的复制是通过形成互补的正极性RNP(CRNP)来进行的，CRNP是产生许多后代vRNP的中间产物。转录的启动是通过帽子抢夺机制进行的，根据该机制，聚合酶窃取细胞内有上限的寡核苷酸，并将其作为引物复制vRNP模板。转录终止过早地发生在聚腺苷酸化信号处，聚合酶重复复制该信号以产生3‘-末端的PolyA。在这里，我们研究了病毒RNA复制和转录的机制。我们使用高效的体内重组RNP转录/复制系统和定义明确的RNA复制或转录缺陷的聚合酶突变体来研究模板RNP中存在的聚合酶复合体和新合成的聚合酶复合体在复制和转录过程中的作用。反式互补实验结果表明，可溶性聚合酶复合体可以反式合成子代RNA，并整合到子代vRNPs中，但只能在顺式中检测到转录。这些结果与病毒RNA复制的新模型是一致的，在该模型中，模板RNP将被可溶的聚合酶复合体反式复制，而与复制酶不同的聚合酶复合体将指导子代vRNA的包裹。相反，vRNP的转录将发生在顺式系统中，驻留的聚合酶复合体将负责mRNA的合成和多聚腺苷酸化。甲型流感病毒每年都会流行，偶尔也会大流行呼吸道疾病。目前流行的禽流感病毒可能引起新的大流行，因此人们对了解病毒如何复制其基因组越来越感兴趣。它由八个RNA分子组成，每个分子都与一个聚合酶复合体结合，并以核糖核蛋白复合体(RNPs)的形式被多个核蛋白拷贝所包裹。这些结构负责病毒RNA的复制和转录，但这些过程的详细机制尚不完全清楚。在这里，我们报告了使用Experent体外和体内转录和复制重组系统以及转录缺陷或复制缺陷的聚合点突变体进行遗传互补实验的结果。这些结果与病毒复制的新模型相一致，在该模型中，不同于亲代RNP中的聚合酶负责反式RNA复制，而后代RNP与不同于执行复制的聚合酶相关联。相反，转录是由驻留在RNP中的聚合酶在顺式中进行的。
The influenza A viruses genome comprises eight single-stranded RNA segments of negative polarity. Each one is included in a ribonucleoprotein particle (vRNP) containing the polymerase complex and a number of nucleoprotein (NP) monomers. Viral RNA replication proceeds by formation of a complementary RNP of positive polarity (cRNP) that serves as intermediate to generate many progeny vRNPs. Transcription initiation takes place by a cap-snatching mechanism whereby the polymerase steals a cellular capped oligonucleotide and uses it as primer to copy the vRNP template. Transcription termination occurs prematurely at the polyadenylation signal, which the polymerase copies repeatedly to generate a 3′-terminal polyA. Here we studied the mechanisms of the viral RNA replication and transcription. We used efficient systems for recombinant RNP transcription/replication in vivo and well-defined polymerase mutants deficient in either RNA replication or transcription to address the roles of the polymerase complex present in the template RNP and newly synthesised polymerase complexes during replication and transcription. The results of trans-complementation experiments showed that soluble polymerase complexes can synthesise progeny RNA in trans and become incorporated into progeny vRNPs, but only transcription in cis could be detected. These results are compatible with a new model for virus RNA replication, whereby a template RNP would be replicated in trans by a soluble polymerase complex and a polymerase complex distinct from the replicative enzyme would direct the encapsidation of progeny vRNA. In contrast, transcription of the vRNP would occur in cis and the resident polymerase complex would be responsible for mRNA synthesis and polyadenylation. The influenza A viruses produce annual epidemics and occasional pandemics of respiratory disease. There is great concern about a potential new pandemic being caused by presently circulating avian influenza viruses, and hence increasing interest in understanding how the virus replicates its genome. This comprises eight molecules of RNA, each one bound to a polymerase complex and encapsidated by multiple copies of the nucleoprotein, in the form of ribonucleoprotein complexes (RNPs). These structures are responsible for virus RNA replication and transcription but the detailed mechanisms of these processes are not fully understood. We report here the results of genetic complementation experiments using proficient in vitro and in vivo recombinant systems for transcription and replication, and polymerase point mutants that are either transcription-defective or replication-defective. These results are compatible with a new model for virus replication whereby a polymerase distinct from that present in the parental RNP is responsible for RNA replication in trans and the progeny RNP is associated to a polymerase distinct from that performing replication. In contrast, transcription is carried out in cis by the polymerase resident in the RNP.
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