Highly conserved regions of influenza A virus polymerase gene segments are critical for efficient viral RNA packaging
Highly conserved regions of influenza A virus polymerase gene segments are critical for efficient viral RNA packaging
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DOI：
10.1128/jvi.02267-07
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发表时间：
2008-03-01
影响因子：
5.4
通讯作者：
Palese, Peter
中科院分区：
文献类型：
--
作者：
Marsh, Glenn A.;Rabadan, Raul;Palese, Peter
甲型流感病毒的基因组由八个不同的负义RNA片段组成。这八个片段通过一种尚未完全理解的机制以等摩尔比并入出芽病毒体中。已经提出了两种不同的模型包装病毒核糖核蛋白到新组装的病毒颗粒：随机掺入模型和选择性掺入模型。在过去的几年里，越来越多的证据，从许多不同的实验室，支持选择性纳入模型已经积累。特别是，不同的研究小组已经表明，几乎每个片段的5'和3'端的编码序列内的一些大的病毒RNA区域足以包装外源RNA序列。如果包装区对病毒的生存能力至关重要，我们希望它们能够被保存下来。通过对甲型流感病毒序列的大规模分析，我们开发了一种鉴定保守RNA区域的方法，这些区域的保守性不能用种群结构或氨基酸保守性来解释。有趣的是，保守序列位于被鉴定为对有效包装重要的区域内。通过利用流感病毒反向遗传学，我们拯救了在这些高度保守区域内含有同义突变的突变病毒。这些病毒中病毒RNA的包装通过使用通用引物的逆转录和单个片段的定量PCR进行分析。采用这种方法，我们已经确定了聚合酶基因片段中的区域，如果突变，会导致该特定聚合酶病毒RNA包装减少90%以上。聚合酶病毒RNA包装水平的降低通常也会导致其他病毒RNA的减少，结果形成了片段相互作用的层次结构模式。这项工作为甲型流感病毒的选择性包装机制提供了进一步的证据，表明这些高度保守的区域对于有效包装很重要。
The genome of the influenza A virus is composed of eight different segments of negative-sense RNA. These eight segments are incorporated into budding virions in an equimolar ratio through a mechanism that is not fully understood. Two different models have been proposed for packaging the viral ribonucleoproteins into newly assembling virus particles: the random-incorporation model and the selective-incorporation model. In the last few years, increasing evidence from many different laboratories that supports the selective-incorporation model has been accumulated. In particular, different groups have shown that some large viral RNA regions within the coding sequences at both the 5' and 3' ends of almost every segment are sufficient for packaging foreign RNA sequences. If the packaging regions are crucial for the viability of the virus, we would expect them to be conserved. Using large-scale analysis of influenza A virus sequences, we developed a method of identifying conserved RNA regions whose conservation cannot be explained by population structure or amino acid conservation. Interestingly, the conserved sequences are located within the regions identified as important for efficient packaging. By utilizing influenza virus reverse genetics, we have rescued mutant viruses containing synonymous mutations within these highly conserved regions. Packaging of viral RNAs in these viruses was analyzed by reverse transcription using a universal primer and quantitative PCR for individual segments. Employing this approach, we have identified regions in the polymerase gene segments that, if mutated, result in reductions of more than 90% in the packaging of that particular polymerase viral RNA. Reductions in the level of packaging of a polymerase viral RNA frequently resulted in reductions of other viral RNAs as well, and the results form a pattern of hierarchy of segment interactions. This work provides further evidence for a selective packaging mechanism for influenza A viruses, demonstrating that these highly conserved regions are important for efficient packaging.