Fodor E, Palese P, Brownlee G G, García-Sastre A
Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, USA.
J Virol. 1998 Aug;72(8):6283-90. doi: 10.1128/JVI.72.8.6283-6290.1998.
We have engineered influenza A/WSN/33 viruses which have viral RNA (vRNA) segments with altered base pairs in the conserved double-stranded region of their vRNA promoters. The mutations were introduced into the segment coding for the neuraminidase (NA) by using a reverse genetics system. Two of the rescued viruses which share a C-G-->A-U double mutation at positions 11 and 12' at the 3' and 5' ends of the NA-specific vRNA, respectively, showed approximately a 10-fold reduction of NA levels. The mutations did not dramatically affect the NA-specific vRNA levels found in virions or the NA-specific vRNA and cRNA levels in infected cells. In contrast, there was a significant decrease in the steady-state levels of NA-specific mRNAs in infected cells. Transcription studies in vitro with ribonucleoprotein complexes isolated from the two transfectant viruses indicated that transcription initiation of the NA-specific segment was not affected. However, the majority of NA-specific transcripts lacked poly(A) tails, suggesting that mutations in the double-stranded region of the influenza virus vRNA promoter can attenuate polyadenylation of mRNA molecules. This is the first time that a promoter mutation in an engineered influenza virus has shown a differential effect on influenza virus RNA transcription and replication.
我们构建了甲型流感病毒A/WSN/33，其病毒RNA（vRNA）片段在vRNA启动子保守双链区域的碱基对发生了改变。通过反向遗传学系统将突变引入编码神经氨酸酶（NA）的片段中。两种拯救出的病毒，分别在NA特异性vRNA的3'和5'末端的第11位和12'位共享一个C-G到A-U的双重突变，其NA水平降低了约10倍。这些突变并未显著影响病毒粒子中NA特异性vRNA的水平，也未影响感染细胞中NA特异性vRNA和cRNA的水平。相比之下，感染细胞中NA特异性mRNA的稳态水平显著下降。对从两种转染病毒中分离出的核糖核蛋白复合物进行的体外转录研究表明，NA特异性片段的转录起始不受影响。然而，大多数NA特异性转录本缺乏聚腺苷酸尾巴，这表明流感病毒vRNA启动子双链区域的突变可减弱mRNA分子的聚腺苷酸化。这是首次在工程化流感病毒中发现启动子突变对流感病毒RNA转录和复制产生差异效应。
