Zheng H, Palese P, García-Sastre A
Department of Microbiology, Mount Sinai School of Medicine, New York, 10029, USA.
Virology. 1996 Mar 1;217(1):242-51. doi: 10.1006/viro.1996.0111.
The genome of influenza A viruses is composed of eight negative-strand RNA segments which contain short noncoding regions at their 3' and 5' ends. The signals required for replication, transcription, and packaging of the viral RNAs are thought to be located in these regions. The highly conserved noncoding nucleotides, which form "panhandle" or "fork" structures by partial complementarity, are important for the transcriptional activity of the viral RNA polymerase. In contrast, the nonconserved noncoding nucleotides located close to the open reading frame of the viral RNAs had not been implicated in RNA transcription. Using a reverse-genetics system, we have now rescued influenza A/WSN/33 viruses whose NA-specific RNA segments have deletions in these nonconserved noncoding regions. Deletion either of the nucleotide residues between the poly(U) stretch and the stop codon at the 5' end or of the nucleotides between position 15 and the start codon at the 3' end did not affect the amount of NA-RNA species found in virions or infected cells. However, a combination of deletions at both the 3' and the 5' ends decreased by 60 times the levels of NA-specific viral RNA found in infected cells at late periods of infection and in virions. This double deletion was also responsible for a fourfold reduction of the steady-state levels of the NA-specific mRNA in infected cells. Viruses whose NA-specific open reading frames were flanked by the noncoding regions of the PB1- or the NS-RNA segments of infuenza A/WSN/33 virus also showed a reduction in the NA-specific viral RNA in virions and in infected cells. The present results demonstrate that the nonconserved nucleotides at the 3' and 5' ends of the NA-RNA segment of influenza A virus play an important role in the replication of this segment.
甲型流感病毒的基因组由八个负链RNA片段组成，这些片段在其3'和5'末端含有短的非编码区。病毒RNA复制、转录和包装所需的信号被认为位于这些区域。高度保守的非编码核苷酸通过部分互补形成“锅柄”或“叉”结构，对病毒RNA聚合酶的转录活性很重要。相比之下，位于病毒RNA开放阅读框附近的非保守非编码核苷酸与RNA转录无关。利用反向遗传学系统，我们现已拯救出甲型/WSN/33流感病毒，其NA特异性RNA片段在这些非保守非编码区存在缺失。删除5'端多聚（U）序列和终止密码子之间的核苷酸残基，或3'端第15位和起始密码子之间的核苷酸，均不影响病毒粒子或感染细胞中NA-RNA种类的数量。然而，3'端和5'端同时缺失会使感染后期感染细胞和病毒粒子中NA特异性病毒RNA的水平降低60倍。这种双重缺失还导致感染细胞中NA特异性mRNA的稳态水平降低四倍。其NA特异性开放阅读框两侧为甲型/WSN/33病毒PB1或NS-RNA片段非编码区的病毒，在病毒粒子和感染细胞中的NA特异性病毒RNA也减少。目前的结果表明，甲型流感病毒NA-RNA片段3'和5'末端的非保守核苷酸在该片段的复制中起重要作用。
