Mutational analyses of the influenza A virus polymerase subunit PA reveal distinct functions related and unrelated to RNA polymerase activity.
Mutational analyses of the influenza A virus polymerase subunit PA reveal distinct functions related and unrelated to RNA polymerase activity.
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DOI：
10.1371/journal.pone.0029485
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Liang Y
中科院分区：
文献类型：
--
作者：
Liang Y;Danzy S;Dao LD;Parslow TG;Liang Y
甲型流感病毒聚合酶是由PA、PB1和PB2亚基组成的异源三聚体复合体。我们先前报道了PA C末端的一个双密码子替换突变(G507A-R508A)，记为J10，对病毒RNA的合成没有明显的影响，但阻止了感染性病毒的产生，表明PA可能具有不依赖于其聚合酶活性的新作用。为了进一步研究PA在病毒生命周期中的作用，我们现在已经在J10位点两侧从残基497到518的区域产生并表征了额外的突变。所有测试的双密码子突变都完全消除或显著降低了病毒的传染性，但它们通过不同的机制做到了这一点。一些病毒表现出与J10相似的效果，因为突变的聚合酶支持正常水平的病毒RNA合成，但仍然无法产生具有感染性的病毒颗粒。其他人则通过扰乱PA蛋白在细胞中的正常核定位来消除聚合酶活性。我们还设计了单密码子突变，这些突变预计会聚集在PA晶体结构中J10位点附近，并发现改变残基K378或D478每个都会产生类似J10的表型。在对J10本身的进一步研究中，我们发现这种突变本身并不影响类病毒粒子的形成和释放，而是削弱了这些粒子整合组成病毒基因组的八个基本RNA片段(VRNAs)的能力。综上所述，我们的分析确定了PA C末端区域的突变，这些突变至少影响了三个不同的活性：蛋白质核定位、病毒RNA合成和有效包装所有八个vRNA所需的反式作用功能。
Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs.
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影响因子：
5.4
作者：
Huet, Sebastien;Avilov, Sergiy V.;Ellenberg, Jan
通讯作者：
Ellenberg, Jan
影响因子：
5
作者：
NIETO, A;DELALUNA, S;ORTIN, J
通讯作者：
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影响因子：
5.4
作者：
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通讯作者：
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DOI：
10.1073/pnas.0507415102
发表时间：
2005-12-20
影响因子：
11.1
作者：
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通讯作者：
Stech, J
影响因子：
5.4
作者：
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通讯作者：
Nieto, A