Chlanda Petr, Mekhedov Elena, Waters Hang, Sodt Alexander, Schwartz Cindi, Nair Vinod, Blank Paul S, Zimmerberg Joshua
Section on Integrative Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, Maryland, USA
Section on Integrative Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, Maryland, USA.
J Virol. 2017 Oct 13;91(21). doi: 10.1128/JVI.00947-17. Print 2017 Nov 1.
The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion. Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion.
流感病毒糖蛋白血凝素（HA）高度保守的细胞质尾部含有三个半胱氨酸，在翻译后会被共价结合的脂肪酸修饰。虽然病毒HA酰化在病毒复制中至关重要，但其物理化学作用尚不清楚。我们使用病毒样颗粒（VLP）来研究酰化对形态、蛋白质掺入、脂质组成和膜融合的影响。去酰化中断了HA与M1的相互作用，因为去酰化的突变型HA未能在球状VLP内掺入M1层，而丝状颗粒掺入了更多的神经氨酸酶（NA）。虽然HA酰化不影响VLP的形状、脂质组成或HA的侧向间距，但酰化显著影响包膜曲率。与野生型HA相比，在没有基质（M1）蛋白层的情况下，去酰化的HA与包膜曲率平坦的释放颗粒相关。通过分子动力学模拟计算了棕榈酸酯的自发曲率，发现其与从VLP大小分布得出的曲率值相当。细胞-细胞融合试验表明，H2（A/日本/305/57）、H3（A/爱知/2/68）和H3（A/乌隆/72）病毒之间存在与毒株无关的融合孔扩大失败。相反，酰化对分离的VLP与脂质体的低pH依赖性融合没有影响：融合孔形成并扩大，如使用冷冻电子断层扫描（cryo-ET）观察到的完全融合产物所示。我们提出，酰化的主要作用机制是控制膜曲率并改变HA与M1蛋白的相互作用，而单独作用的去酰化HA对融合的阻碍可能会被其他有助于降低孔扩张能量障碍的病毒蛋白所平衡。甲型流感病毒是一种空气传播病原体，可引起季节性流行和偶尔的大流行。血凝素（HA）是病毒粒子表面丰富的一种糖蛋白，在甲型流感病毒的组装和进入中都很重要。HA通过酰化进行修饰，去除酰化会废除病毒复制。在这里，我们使用冷冻电子断层扫描获得三维图像，以阐明HA酰化在VLP组装中的作用。我们的数据表明，HA酰化有助于HA在病毒组装过程中弯曲膜的能力以及HA与M1支架蛋白的相互作用。此外，我们关于VLP的数据以及基于假设的病毒数据表明，HA酰化虽然对融合孔形成并不关键，但以靶标依赖的方式有助于孔的扩张。
