Department of Bioengineering, University of California, Los Angeles , Los Angeles, California 90095, United States.
J Am Chem Soc. 2013 Sep 18;135(37):13710-9. doi: 10.1021/ja400146z. Epub 2013 Sep 6.
The M2 protein is a multifunctional protein, which plays several roles in the replication cycle of the influenza A virus. Here we focus on its ability to promote budding of the mature virus from the cell surface. Using high-resolution small-angle X-ray scattering we show that M2 can restructure lipid membranes into bicontinuous cubic phases which are rich in negative Gaussian curvature (NGC). The active generation of negative Gaussian membrane curvature by M2 is essential to influenza virus budding. M2 has been observed to colocalize with the region of high NGC at the neck of a bud. The structural requirements for scission are even more stringent than those for budding, as the neck must be considerably smaller than the virus during 'pinch off'. Consistent with this, the amount of NGC in the induced cubic phases suggests that M2 proteins can generate high curvatures comparable to those on a neck with size 10× smaller than a spherical influenza virus. Similar experiments on variant proteins containing different M2 domains show that the cytoplasmic amphipathic helix is necessary and sufficient for NGC generation. Mutations to the helix which reduce its amphiphilicity and are known to diminish budding attenuated NGC generation. An M2 construct comprising the membrane interactive domains, the transmembrane helix and the cytoplasmic helix, displayed enhanced ability to generate NGC, suggesting that other domains cooperatively promote membrane curvature. These studies establish the importance of M2-induced NGC during budding and suggest that antagonizing this curvature is a viable anti-influenza strategy.
M2 蛋白是一种多功能蛋白，在甲型流感病毒的复制周期中发挥多种作用。在这里，我们重点关注它促进成熟病毒从细胞表面出芽的能力。使用高分辨率小角 X 射线散射，我们表明 M2 可以将脂质膜重构为富含负高斯曲率（NGC）的双连续立方相。M2 主动产生负高斯膜曲率对于流感病毒出芽是必不可少的。已经观察到 M2 与芽颈部的高 NGC 区域共定位。与出芽相比，切口的结构要求甚至更为严格，因为在“挤压”过程中颈部必须比病毒小得多。与此一致的是，诱导立方相中的 NGC 量表明 M2 蛋白可以产生与大小为球形流感病毒小 10 倍的颈部相当的高曲率。对包含不同 M2 结构域的变体蛋白进行类似的实验表明，细胞质两亲性螺旋对于 NGC 的产生是必要且充分的。降低其两亲性并已知减弱出芽的螺旋突变会减弱 NGC 的产生。包含膜相互作用结构域、跨膜螺旋和细胞质螺旋的 M2 构建体显示出增强的产生 NGC 的能力，这表明其他结构域协同促进膜曲率。这些研究确立了 M2 诱导的 NGC 在出芽过程中的重要性，并表明拮抗这种曲率是一种可行的抗流感策略。
