Gog Julia R, Afonso Emmanuel Dos Santos, Dalton Rosa M, Leclercq India, Tiley Laurence, Elton Debra, von Kirchbach Johann C, Naffakh Nadia, Escriou Nicolas, Digard Paul
DAMTP, Centre for Mathematical Sciences, University of Cambridge, Cambridge, UK.
Nucleic Acids Res. 2007;35(6):1897-907. doi: 10.1093/nar/gkm087. Epub 2007 Mar 1.
Genome segmentation facilitates reassortment and rapid evolution of influenza A virus. However, segmentation complicates particle assembly as virions must contain all eight vRNA species to be infectious. Specific packaging signals exist that extend into the coding regions of most if not all segments, but these RNA motifs are poorly defined. We measured codon variability in a large dataset of sequences to identify areas of low nucleotide sequence variation independent of amino acid conservation in each segment. Most clusters of codons showing very little synonymous variation were located at segment termini, consistent with previous experimental data mapping packaging signals. Certain internal regions of conservation, most notably in the PA gene, may however signify previously unidentified functions in the virus genome. To experimentally test the bioinformatics analysis, we introduced synonymous mutations into conserved codons within known packaging signals and measured incorporation of the mutant segment into virus particles. Surprisingly, in most cases, single nucleotide changes dramatically reduced segment packaging. Thus our analysis identifies cis-acting sequences in the influenza virus genome at the nucleotide level. Furthermore, we propose that strain-specific differences exist in certain packaging signals, most notably the haemagglutinin gene; this finding has major implications for the evolution of pandemic viruses.
基因组片段化有助于甲型流感病毒的重配和快速进化。然而，片段化使病毒粒子组装变得复杂，因为病毒体必须包含所有八个vRNA种类才能具有感染性。存在延伸到大多数（如果不是全部）片段编码区域的特定包装信号，但这些RNA基序定义不明确。我们在一个大型序列数据集中测量密码子变异性，以识别每个片段中与氨基酸保守性无关的低核苷酸序列变异区域。大多数显示极少同义变异的密码子簇位于片段末端，这与之前绘制包装信号的实验数据一致。然而，某些保守的内部区域，最显著的是在PA基因中，可能表明病毒基因组中存在以前未识别的功能。为了通过实验验证生物信息学分析，我们在已知包装信号内的保守密码子中引入同义突变，并测量突变片段掺入病毒粒子的情况。令人惊讶的是，在大多数情况下，单核苷酸变化会显著降低片段包装。因此，我们的分析在核苷酸水平上鉴定了流感病毒基因组中的顺式作用序列。此外，我们提出在某些包装信号中存在毒株特异性差异，最显著的是血凝素基因；这一发现对大流行病毒的进化具有重要意义。
