Mutational Analysis of Conserved Amino Acids in the Influenza A Virus Nucleoprotein
Mutational Analysis of Conserved Amino Acids in the Influenza A Virus Nucleoprotein
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DOI：
10.1128/jvi.02642-08
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发表时间：
2009-05-01
影响因子：
5.4
通讯作者：
Kawaoka, Yoshihiro
中科院分区：
文献类型：
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作者：
Li, Zejun;Watanabe, Tokiko;Kawaoka, Yoshihiro
核蛋白（NP）在病毒生命周期中具有多种功能，具有在甲型、B和C型流感病毒中高度保守的区域。为了更好地理解高度保守的NP氨基酸在病毒复制中的作用，我们进行了全面的突变分析。使用反向遗传学，我们试图产生74个病毒具有NP的保守氨基酸突变。其中，48个突变体病毒被成功拯救; 26个突变体是不可行的，这表明在病毒复制中的相应的NP氨基酸的关键作用。为了鉴定这些NP突变损害的病毒生命周期中的步骤，我们检查了病毒基因组复制/转录、NP定位和病毒RNA片段掺入子代病毒体中。我们确定了15个氨基酸的取代，在NP抑制病毒基因组复制和/或转录，导致具有这些取代的病毒显着的生长缺陷。我们还发现了几个NP突变，影响多个病毒RNA（vRNA）片段的有效掺入后代病毒体，即使一个单一的vRNA片段被有效地纳入。因此，NP中相应的保守氨基酸对于八个vRNA区段的集合的组装和/或掺入可能是关键的。
The nucleoprotein (NP), which has multiple functions during the virus life cycle, possesses regions that are highly conserved among influenza A, B, and C viruses. To better understand the roles of highly conserved NP amino acids in viral replication, we conducted a comprehensive mutational analysis. Using reverse genetics, we attempted to generate 74 viruses possessing mutations at conserved amino acids of NP. Of these, 48 mutant viruses were successfully rescued; 26 mutants were not viable, suggesting a critical role of the respective NP amino acids in viral replication. To identify the step(s) in the viral life cycle that is impaired by these NP mutations, we examined viral-genome replication/transcription, NP localization, and incorporation of viral-RNA segments into progeny virions. We identified 15 amino acid substitutions in NP that inhibited viral-genome replication and/or transcription, resulting in significant growth defects of viruses possessing these substitutions. We also found several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficiently. The respective conserved amino acids in NP may thus be critical for the assembly and/or incorporation of sets of eight vRNA segments.