Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology
Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology
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DOI：
10.1128/jvi.00592-15
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发表时间：
2015-09-01
影响因子：
5.4
通讯作者：
Briggs, John A. G.
中科院分区：
文献类型：
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作者：
Chlanda, Petr;Schraidt, Oliver;Briggs, John A. G.
甲型流感病毒在感染细胞的质膜上的组装导致包膜病毒粒子的释放，这些包膜病毒粒子在适应组织培养的菌株中通常是圆形的，而在从患者分离的菌株中则是丝状的。病毒蛋白血凝素（HA）、神经氨酸酶（NA）、基质蛋白1 （M1）和M2离子通道都有助于病毒的组装。当它们在细胞中单独或联合表达时，在一定条件下，它们都能介导膜包裹颗粒的释放，但它们在病毒组装、释放和形态中的相对作用尚不清楚。为了研究这些作用，我们通过质粒衍生的HA、NA和M蛋白（M1和M2）的组合表达或感染甲型流感病毒来生产膜包裹颗粒。我们通过生化方法、电子显微镜、电子断层扫描和低温电子断层扫描来监测颗粒释放、颗粒形态和质膜形态。我们的数据表明，HA、NA或HANA （HA + NA）的表达通过非特异性诱导膜曲率导致颗粒释放。相反，与M蛋白的共表达将糖蛋白聚集成丝状膜突起，这些突起可以通过在底部形成收缩的颈部作为颗粒释放出来。HA和NA优先分布在这些颗粒内不同弯曲的膜上。出芽的中间产物和释放的颗粒在形态上与感染甲型流感病毒时产生的产物相似。总之，我们的数据为流感病毒组装提供了新的见解，并表明M段与任何一种糖蛋白一起是组装和释放真正类似病毒的膜包裹颗粒的最低要求。甲型流感病毒是一种主要的呼吸道病原体。它组装被膜包裹的病毒颗粒，其形状从球形到丝状不等。在这里，我们研究了单个病毒蛋白在介导病毒组装和决定病毒形状中的作用。为了做到这一点，我们使用了一系列电子显微镜技术来获得和比较病毒颗粒和病毒样颗粒在组装期间和组装后的二维和三维图像。病毒样颗粒是用不同的病毒蛋白组合产生的。在我们的研究结果中，我们发现一种或两种病毒表面蛋白（血凝素和神经氨酸酶）与由该片段编码的病毒膜相关蛋白的共表达导致丝状病毒样颗粒的组装和释放，其方式与流感病毒粒子的出芽和释放非常相似。这些数据为单个病毒蛋白在甲型流感病毒组装中所起的作用提供了新的见解。
The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like.IMPORTANCEInfluenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two-and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by theMsegment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.