Marsh Glenn A, Rabadán Raúl, Levine Arnold J, Palese Peter
Department of Microbiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA.
J Virol. 2008 Mar;82(5):2295-304. doi: 10.1128/JVI.02267-07. Epub 2007 Dec 19.
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.
甲型流感病毒的基因组由八个不同的负链RNA片段组成。这八个片段通过一种尚未完全了解的机制以等摩尔比掺入出芽的病毒粒子中。关于将病毒核糖核蛋白包装到新组装的病毒颗粒中，已经提出了两种不同的模型：随机掺入模型和选择性掺入模型。在过去几年中，来自许多不同实验室的越来越多支持选择性掺入模型的证据不断积累。特别是，不同的研究小组表明，几乎每个片段5'和3'端编码序列内的一些大的病毒RNA区域足以包装外源RNA序列。如果包装区域对病毒的生存能力至关重要，我们预计它们会是保守的。通过对甲型流感病毒序列进行大规模分析，我们开发了一种识别保守RNA区域的方法，其保守性不能用群体结构或氨基酸保守性来解释。有趣的是，保守序列位于被确定为对有效包装很重要的区域内。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。使用通用引物通过逆转录和针对单个片段的定量PCR分析这些病毒中病毒RNA的包装情况。采用这种方法，我们在聚合酶基因片段中鉴定出了一些区域，如果这些区域发生突变，会导致该特定聚合酶病毒RNA的包装减少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，证明这些高度保守的区域对有效包装很重要。
