Freie Universität Berlin, Faculty of Veterinary Medicine, Institute of Immunology and Molecular Biology, Philippstraße 13, 10115 Berlin, Germany.
Robert Koch Institute, Nordufer 20, 13353 Berlin, Germany.
J Gen Virol. 2012 Feb;93(Pt 2):282-292. doi: 10.1099/vir.0.038554-0. Epub 2011 Oct 19.
Influenza virus assembly and budding occur in the 'budozone', a coalesced raft domain in the plasma membrane. The viral transmembrane protein M2 is implicated in virus particle scission, the ultimate step in virus budding, probably by wedge-like insertion of an amphiphilic helix into the membrane. In order to do this, M2 is hypothesized to be targeted to the edge of the budozone, mediated by acylation and cholesterol binding. It was recently shown that acylation and cholesterol binding affect the membrane association of the cytoplasmic tail of M2 and targeting of the protein to coalesced rafts. This study tested whether combined removal of the acylation site (C50) and the cholesterol recognition/interaction amino acid consensus motifs (key residues Y52 and Y57) in the amphiphilic helix of M2 influenced virus formation. Recombinant influenza viruses were generated in the influenza strain A/WSN/33 background with mutations in one or both of these features. In comparison with the wild-type, all mutant viruses showed very similar growth kinetics in various cell types. Wild-type and mutant viruses differed in their relative M2 content but not regarding the major structural proteins. The morphology of the viruses was not affected by mutating M2. Moreover, wild-type and mutant viruses showed comparable competitive fitness in infected cells. Lastly, a global comparison of M2 sequences revealed that there are natural virus strains with M2 devoid of both lipid-association motifs. Taken together, these results indicate that the acylation and cholesterol-binding motifs in M2 are not crucial for the replication of influenza virus in cell culture, indicating that other factors can target M2 to the budding site.
流感病毒的组装和出芽发生在“出芽区”，即质膜中的合并筏域。病毒跨膜蛋白 M2 参与病毒粒子的断裂，即出芽的最后一步，可能通过亲水头插入膜中形成楔形来实现。为了做到这一点，M2 被假设通过酰化和胆固醇结合靶向到出芽区的边缘。最近的研究表明，酰化和胆固醇结合会影响 M2 细胞质尾部的膜结合和蛋白靶向到合并筏的情况。本研究测试了 M2 中亲水头的酰化位点 (C50) 和胆固醇识别/相互作用氨基酸共识基序 (关键残基 Y52 和 Y57) 的联合去除是否会影响病毒的形成。在流感 A/WSN/33 株的背景下，用一个或两个特征的突变生成了重组流感病毒。与野生型相比，所有突变病毒在各种细胞类型中的生长动力学非常相似。野生型和突变病毒在 M2 的相对含量上有所不同，但主要结构蛋白则没有差异。突变 M2 不会影响病毒的形态。此外，野生型和突变病毒在感染细胞中的竞争适应性相当。最后，对 M2 序列的全局比较表明，存在天然病毒株，其 M2 缺失了两个脂质结合基序。总之，这些结果表明，M2 中的酰化和胆固醇结合基序对于流感病毒在细胞培养中的复制不是至关重要的，表明其他因素可以将 M2 靶向到出芽部位。
