Batishchev O V, Shilova L A, Kachala M V, Tashkin V Y, Sokolov V S, Fedorova N V, Baratova L A, Knyazev D G, Zimmerberg J, Chizmadzhev Y A
A. N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia Moscow Institute of Physics and Technology, Dolgoprudniy, Russia
A. N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia Moscow Institute of Physics and Technology, Dolgoprudniy, Russia.
J Virol. 2015 Oct 14;90(1):575-85. doi: 10.1128/JVI.01539-15. Print 2016 Jan 1.
Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ~6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ~5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release.
Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In particular, we need to know how the protective coat of protein inside the viral surface reacts to the changes in acid that come soon after internalization. We found that acid makes the molecules of the protein coat push each other while they are still stuck to the virus, so that they would like to rip the membrane apart. This ripping force is known to promote membrane fusion, the process by which infection actually occurs.
流感病毒从pH值中性的细胞外环境被摄取到内体中，内体内容物随后酸化，导致覆盖病毒脂质包膜内层单分子层的病毒基质蛋白（M1）发生变化。在pH值约为6时，M1在一个假定的引发阶段与病毒核糖核蛋白（RNP）相互作用；在此阶段，覆盖脂质包膜的M1支架的相互作用是完整的。随着酸化持续至pH值约为5，M1外壳解体，为病毒基因组从病毒内部转运至细胞质清除物理通道。在此，我们研究了M1的pH依赖性解体的物理化学机制。在中性介质中，M1蛋白在脂质双层上的吸附本质上是静电性的且可逆。M1二聚体中M1分子彼此之间的相互作用能量约为M1分子与脂质双层相互作用能量的十分之一。酸化由于M1电荷的变化驱动M1分子发生构象变化，导致其静电相互作用改变。将pH值从7.1降至6.0不会扰乱M1层；将pH值降得更低会使部分M1解吸，这是因为仍附着在膜上的M1单体之间的排斥力增加。包被有M1的脂质囊泡表现出pH依赖性的囊泡膜破裂，推测是由于这种排斥力产生的张力所致。因此，在pH值为5时囊泡的破裂与M1蛋白支架解体同时发生，可能会将脂质膜拉伸至破裂点，促进融合孔扩大以释放RNP。
流感仍然是全球人类的主要杀手之一，部分原因是流感病毒能迅速与细胞结合并摄取到免疫系统难以触及的区室中。为了在病毒隐藏期间攻击流感病毒，我们需要了解使内化病毒能够感染细胞的物理力。特别是，我们需要知道病毒表面内部的蛋白质保护壳如何应对内化后不久出现的酸性变化。我们发现，酸性会使仍附着在病毒上的蛋白质外壳分子相互推挤，从而想要撕开膜。已知这种撕裂力会促进膜融合，而膜融合是实际发生感染的过程。
