Increased expression of the N protein of respiratory syncytial virus stimulates minigenome replication but does not alter the balance between the synthesis of mRNA and antigenome
Increased expression of the N protein of respiratory syncytial virus stimulates minigenome replication but does not alter the balance between the synthesis of mRNA and antigenome
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DOI：
10.1006/viro.1997.8734
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发表时间：
1997-09-15
期刊：
影响因子：
3.7
通讯作者：
Collins, PL
中科院分区：
文献类型：
--
作者：
Fearns, R;Peeples, ME;Collins, PL
非分节负链RNA病毒合成RNA的一个流行模型是，转录和RNA复制是由相同的聚合酶复合体执行的，并且在核衣壳N蛋白介导的两个过程之间存在动态平衡。根据该模型，转录一直持续到可溶N蛋白积累到足以启动新生RNA产物的衣壳化，从而以某种方式将聚合酶转换为读透复制模式。利用重组的转录和RNA复制系统对呼吸道合胞病毒（RSV）模型进行了检测，该系统包括一个微型复制子和从转染质粒中表达的细胞内病毒蛋白。初步实验表明，重组RNA的复制效率很高，因此，平均每个被封装的质粒提供的小基因组分子被扩增10到50倍。在存在或不存在M2 ORF1转录延伸因子的情况下，N蛋白单独或与磷蛋白P一起增加。从质粒中获得的N和P蛋白的最大表达量等于或超过了在rsv感染细胞中获得的表达量。N蛋白水平的增加刺激了RNA复制。这与RNA复制依赖于N蛋白衣壳化的观点是一致的，这是该模型的一个假设。在不同N、P表达条件下，M2 ORF1蛋白对RNA复制均无明显影响，证实并扩展了前人的研究结果。然而，没有证据表明正意义RNA合成从转录（mrna合成）到RNA复制（抗基因组合成）发生了重大转变。正义反基因组和mRNA的合成似乎以固定的比例发生，mRNA是迄今为止更丰富的产物。(C) 1997学术出版社。
A popular model for RNA synthesis by nonsegmented negative-strand RNA viruses is that transcription and RNA replication are executed by the same polymerase complex and that there is a dynamic balance between the two processes that is mediated by the nucleocapsid N protein. According to this model, transcription occurs until sufficient soluble N protein accumulates to initiate encapsidation of the nascent RNA product, which somehow switches the polymerase into a readthrough replicative mode. This model was examined for respiratory syncytial virus (RSV) using a reconstituted transcription and RNA replication system that involves a minireplicon and viral proteins that are expressed intracellularly from transfected plasmids. Preliminary experiments showed that reconstituted RNA replication was highly productive, such that on average each molecule of plasmid-supplied minigenome that became encapsidated was amplified 10- to 50-fold. N protein was increased on its own or in concert with the phosphoprotein P and in the presence or absence of the M2 ORF1 transcription elongation factor. The maximum level of N and P protein expression achieved from plasmids equalled or exceeded that obtained in RSV-infected cells. Increased levels of N protein stimulated RNA replication. This is consistent with the idea that RNA replication is dependent on the availability of N protein for encapsidation, which is one postulate of the model. The M2 ORF1 protein had no detectable effect on RNA replication under the various conditions of expression of N and P, which confirmed and extended previous results. However, there was no evidence of a significant switch in positive-sense RNA synthesis from transcription (synthesis of mRNAs) to RNA replication (synthesis of antigenome). The synthesis of positive-sense antigenome and mRNA appeared to occur at a fixed ratio, with mRNA being by far the more abundant product. (C) 1997 Academic Press.