Mutations in the N-terminal region of influenza virus PB2 protein affect virus RNA replication but not transcription
Mutations in the N-terminal region of influenza virus PB2 protein affect virus RNA replication but not transcription
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DOI：
10.1128/jvi.77.9.5098-5108.2003
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发表时间：
2003-05-01
影响因子：
5.4
通讯作者：
Ortín, J
中科院分区：
文献类型：
--
作者：
Gastaminza, P;Perales, B;Ortín, J
流感病毒PB2突变体是通过改变蛋白N-末端与eIF4E蛋白的氨基酸对齐的保守位置而制备的，参与帽子识别。这些突变基因被用来在体内重组病毒核糖核蛋白(RNPs)，其生物学活性通过(I)体内病毒RNA、cRNA和mRNA积累的检测，(Ii)体外帽子依赖的转录，(Iii)纯化的重组RNPs的帽子抢夺来确定。结果表明，PB2的W49A、F130A和R142a突变降低或取消了突变的RNPs在体内合成RNA的能力，但没有实质上改变它们在体外转录或进行帽子抢夺的能力。一些突变(F130Y、R142a和R142K)被挽救到传染性病毒中。F130Y变异病毒的复制速度快于野生型病毒，而R142a和R142K变异病毒在感染周期中表现出延迟的cRNA和病毒RNA的积累，但初级转录动力学正常，这取决于放线菌胺感染细胞中病毒mRNA的积累。这些结果表明，PB2的N-末端区域在病毒RNA复制中起作用。
PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.