Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein
Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein
复制标题
DOI：
10.1128/jvi.73.9.7349-7356.1999
复制
发表时间：
1999-09-01
影响因子：
5.4
通讯作者：
Digard, P
中科院分区：
文献类型：
--
作者：
Medcalf, L;Poole, E;Digard, P
流感病毒的负义节段RNA基因组被转录成加帽和多腺苷酸化的mRNA以及全长复制中间体（cRNA）。调节这两种转录形式的机制尚不清楚，尽管有几条证据暗示病毒核蛋白（NP）的作用。特别是，温度变化和生化分析的温度敏感性病毒A/WSN/33 ts 56和A/FPV/罗斯托克/34/吉森tsG 81含有点突变的NP编码区内的复制转录在非允许温度下的特定缺陷。为了鉴定功能缺陷，我们将相关突变引入流感病毒株A/PR/8/34的NP中。这两种突变体在瞬时转染实验中对流感病毒基因表达都是温度敏感的，但在转染细胞中正常定位和积累。同样，突变体保留的能力，自我关联和相互作用的病毒聚合酶复合体是否在允许或非允许的温度下合成。相比之下，当在非允许温度下表达时，突变体NP对RNA结合有缺陷，但当在30 ℃下表达时则没有。这表明NP的RNA结合活性是复制转录所必需的。
The negative-sense segmented RNA genome of influenza virus is transcribed into capped and polyadenylated mRNAs, as well as full-length replicative intermediates (cRNAs). The mechanism that regulates the two forms of transcription remains unclear, although several lines of evidence imply a role for the viral nucleoprotein (NP). In particular, temperature-shift and biochemical analyses of the temperature-sensitive viruses A/WSN/33 ts56 and A/FPV/Rostock/34/Giessen tsG81 containing point mutations within the NP coding region have indicated specific defects in replicative transcription at the nonpermissive temperature. To identify the functional defect, we introduced the relevant mutations into the NP of influenza virus strain A/PR/8/34. Both mutants were temperature sensitive for influenza virus gene expression in transient-transfection experiments but localized and accumulated normally in transfected cells. Similarly, the mutants retained the ability to self-associate and interact with the virus polymerase complex whether synthesized at the permissive or the nonpermissive temperatures. In contrast, the mutant NPs were defective for RNA binding when expressed at the nonpermissive temperature but not when expressed at 30 degrees C. This suggests that the RNA-binding activity of NP is required for replicative transcription.