Specific Residues in the 2009 H1N1 Swine-Origin Influenza Matrix Protein Influence Virion Morphology and Efficiency of Viral Spread In Vitro
Specific Residues in the 2009 H1N1 Swine-Origin Influenza Matrix Protein Influence Virion Morphology and Efficiency of Viral Spread In Vitro
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DOI：
10.1371/journal.pone.0050595
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发表时间：
2012-11-27
期刊：
影响因子：
3.7
通讯作者：
Takimoto, Toru
中科院分区：
文献类型：
--
作者：
Bialas, Kristy M.;Desmet, Emily A.;Takimoto, Toru
2009年4月，一种新的流感病毒出现，这是两个先前存在的猪病毒株之间基因重新组合的结果。这种高度传染性的H1N1重组病毒(PH1N1)除了NA和M片段来自欧亚猪血统之外，包含与北美三重组体(TR)病毒相同的基因组背景。然而，尽管它们在遗传上高度相似，但通过免疫荧光和电子显微镜分析，我们发现由PH1N1分离株A/California/04/09(Acal-04/09)产生的病毒粒子在人肺和猪肾上皮细胞中以球形为主，而tr株则主要为丝状。此外，从鼻拭子标本中收集的9份临床PH1N1样本显示出与Acal-04/09毒株相似的球形形态。TR和PH1N1病毒之间的序列分析显示，病毒基质蛋白(M1)在第30、142、207和209位存在四个氨基酸差异，M1是流感形态的已知决定因素。为了测试这些氨基酸在病毒形态中的作用，我们拯救了突变的PH1N1病毒，在这些病毒中，四个M1残基中的每一个都被相应的tR残基取代。含有30、207和209位取代的PH1N1表现出向丝状形态的转变，表明这些残基在病毒粒子形态中的作用。与野生型相比，这些残基的替换导致病毒滴度较低，生长动力学降低，斑块表型较小，表明流感形态与体外有效的细胞到细胞传播之间存在相关性。此外，我们观察到表达野生型PH1N1 M1的细胞产生有效的病毒样颗粒，但不表达包含30、207和209位替换的M1或来自其他毒株的M1。这些数据表明，PH1N1特异性M1残留物在球形后代的产生和释放中发挥了直接作用，这可能有助于大流行病毒的快速传播。
In April 2009, a novel influenza virus emerged as a result of genetic reassortment between two pre-existing swine strains. This highly contagious H1N1 recombinant (pH1N1) contains the same genomic background as North American triple reassortant (TR) viruses except for the NA and M segments which were acquired from the Eurasian swine lineage. Yet, despite their high degree of genetic similarity, we found the morphology of virions produced by the pH1N1 isolate, A/California/04/09 (ACal-04/09), to be predominantly spherical by immunufluorescence and electron microscopy analysis in human lung and swine kidney epithelial cells, whereas TR strains were observed to be mostly filamentous. In addition, nine clinical pH1N1 samples collected from nasal swab specimens showed similar spherical morphology as the ACal-04/09 strain. Sequence analysis between TR and pH1N1 viruses revealed four amino acid differences in the viral matrix protein (M1), a known determinant of influenza morphology, at positions 30, 142, 207, and 209. To test the role of these amino acids in virus morphology, we rescued mutant pH1N1 viruses in which each of the four M1 residues were replaced with the corresponding TR residue. pH1N1 containing substitutions at positions 30, 207 and 209 exhibited a switch to filamentous morphology, indicating a role for these residues in virion morphology. Substitutions at these residues resulted in lower viral titers, reduced growth kinetics, and small plaque phenotypes compared to wild-type, suggesting a correlation between influenza morphology and efficient cell-to-cell spread in vitro. Furthermore, we observed efficient virus-like particle production from cells expressing wild-type pH1N1 M1, but not M1 containing substitutions at positions 30, 207, and 209, or M1 from other strains. These data suggest a direct role for pH1N1 specific M1 residues in the production and release of spherical progeny, which may contribute to the rapid spread of the pandemic virus.