Matsumoto Yusuke, Ohta Keisuke, Kolakofsky Daniel, Nishio Machiko
Department of Microbiology, School of Medicine, Wakayama Medical University, Wakayama, Japan
Department of Microbiology, School of Medicine, Wakayama Medical University, Wakayama, Japan.
J Virol. 2017 Apr 13;91(9). doi: 10.1128/JVI.02203-16. Print 2017 May 1.
The genome RNA of human parainfluenza virus type 2 (hPIV2) that acts as the template for the polymerase complex is entirely encapsidated by the nucleoprotein (NP). Recently, the crystal structure of NP of PIV5, a virus closely related to hPIV2, was resolved in association with RNA. Ten amino acids that contact the bound RNA were identified and are strictly conserved between PIV5 and hPIV2 NP. Mutation of hPIV2 NP Q202 (which contacts a base rather than the RNA backbone) to various amino acids resulted in an over 30-fold increase of polymerase activity as evidenced by a minireplicon assay, even though the RNA-binding affinity was unaltered. Using various modified minireplicons, we found that the enhanced reporter gene expression could be accounted for by increased minigenome replication, whereas mRNA synthesis itself was not affected by Q202 mutation. Moreover, the enhanced activities were still observed in minigenomes partially lacking the leader sequence and which were not of hexamer genome length. Unexpectedly, recombinant hPIV2 possessing the NP Q202A mutation could not be recovered from cDNA. We examined the importance of amino acids in the putative RNA-binding domain of hPIV2 NP for polymerase activity using minireplicons. Abnormally enhanced genome replication was observed upon substitution mutation of the NP Q202 position to various amino acids. Surprisingly, this mutation enabled polymerase to use minigenomes that were partially lacking the leader sequence and not of hexamer genome length. This mutation does not affect fundamental properties of NP, e.g., recognition of gene junctional and editing signals. However, the strongly enhanced polymerase activity may not be viable for the infectious life cycle. This report highlights the potential of the polymerase complex with point mutations in NP and helps our detailed understanding of the molecular basis of gene expression.
作为聚合酶复合体模板的人副流感病毒2型（hPIV2）的基因组RNA完全被核蛋白（NP）包裹。最近，与hPIV2密切相关的病毒PIV5的NP晶体结构与RNA结合解析出来。确定了与结合RNA接触的10个氨基酸，它们在PIV5和hPIV2 NP之间严格保守。将hPIV2 NP的Q202（其接触碱基而非RNA主链）突变为各种氨基酸，通过微型复制子试验证明聚合酶活性增加了30多倍，尽管RNA结合亲和力未改变。使用各种修饰的微型复制子，我们发现报告基因表达增强可归因于微型基因组复制增加，而mRNA合成本身不受Q202突变影响。此外，在部分缺乏前导序列且不是六聚体基因组长度的微型基因组中仍观察到增强的活性。出乎意料的是，具有NP Q202A突变的重组hPIV2无法从cDNA中回收。我们使用微型复制子研究了hPIV2 NP假定RNA结合域中氨基酸对聚合酶活性的重要性。将NP Q202位点突变为各种氨基酸后观察到异常增强的基因组复制。令人惊讶的是，这种突变使聚合酶能够使用部分缺乏前导序列且不是六聚体基因组长度的微型基因组。这种突变不影响NP的基本特性，例如对基因连接和编辑信号的识别。然而，强烈增强的聚合酶活性对于感染性生命周期可能不可行。本报告强调了NP中具有点突变的聚合酶复合体的潜力，并有助于我们详细了解基因表达的分子基础。
