Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase
Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase
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DOI：
10.1093/nar/30.2.429
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发表时间：
2002-01-15
影响因子：
14.9
通讯作者：
Tiley, L
中科院分区：
文献类型：
--
作者：
Lee, MTM;Bishop, K;Tiley, L
流感病毒基因组RNA的5'端的前11个核苷酸被证明是流感病毒聚合酶特异性结合所必需和足够的。一种新的体外转录测定法，其中聚合酶通过生物素化的5 '-vRNA寡核苷酸与顺磁珠结合，用于研究不同形式的聚合酶的活性。由共表达的PB 1/PB 2/PA蛋白组成的复合物和由PB 1/PA组成的亚复合物结合到5 '-vRNA寡核苷酸，而单独表达的PB 1不结合。富集的5 '-vRNA/PB 1/PB 2/PA复合物对ApG和珠蛋白mRNA在模型3'-vRNA模板上的引发转录具有高度活性。在没有添加引物的情况下，RNA合成产生具有5 '末端三磷酸或二磷酸基团的产物，表明也发生了真正的未引发的转录起始。没有检测到PB 1/PA复合物的转录酶活性。这些结果表明PA在增强PB 1的5'末端结合活性中的作用，PB 2在能够进行帽依赖性和非依赖性合成的聚合酶复合物的组装中的作用，并且NP对于复制转录的启动并不需要。
The first 11 nt at the 5' end of influenza virus genomic RNA were shown to be both necessary and sufficient for specific binding by the influenza virus polymerase. A novel in vitro transcription assay, in which the polymerase was bound to paramagnetic beads via a biotinylated 5'-vRNA oligonucleotide, was used to study the activities of different forms of the polymerase. Complexes composed of co-expressed PB1/PB2/PA proteins and a sub-complex composed of PB1/PA bound to the 5'-vRNA oligonucleotide, whereas PB1 expressed alone did not. The enriched 5'-vRNA/PB1/PB2/PA complex was highly active for ApG and globin mRNA primed transcription on a model 3'-vRNA template. RNA synthesis in the absence of added primers produced products with 5'-terminal tri- or diphosphate groups, indicating that genuine unprimed initiation of transcription also occurred. No transcriptase activity was detected for the PB1/PA complex. These results demonstrate a role for PA in the enhancement of 5' end binding activity of PB1, a role for PB2 in the assembly of a polymerase complex able to perform both cap-dependent and -independent synthesis and that NP is not required for the initiation of replicative transcription.