A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages.
A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages.
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作者：
Bedi S;Noda T;Kawaoka Y;Ono A
通过分析对病毒复制施加固有阻断的宿主细胞，有助于鉴定促进或抑制病毒复制的宿主细胞决定簇。在这项研究中，我们表明，主要的人MDM，这是不允许IAV复制，不能支持病毒颗粒的形成。这种缺陷对原代人巨噬细胞是特异性的，因为分化为巨噬细胞样细胞的人单核细胞系支持IAV颗粒形成。我们进一步鉴定了两种病毒跨膜蛋白HA和M2在细胞表面上作为离散组装步骤之间的关联，这在MDM中是有缺陷的。缺陷HA-M2协会和颗粒出芽，但不是病毒释放，在MDM中被拯救的破坏肌动蛋白细胞骨架，揭示了一个以前未知的，负作用的肌动蛋白，它专门针对早期的一个步骤，在多步骤IAV生产。总的来说，我们的研究揭示了宿主介导的限制之间的关联病毒跨膜组件在IAV组装。甲型流感病毒（IAV）在上皮细胞中有效地繁殖，上皮细胞是其在呼吸道中的主要靶标。相反，大多数IAV株的生产性感染在巨噬细胞中被阻断或效率极低。IAV在人类巨噬细胞中复制缺陷的确切性质仍然未知。在这项研究中，我们表明，即使与分化成巨噬细胞样细胞的单核细胞系相比，原代人单核细胞衍生的巨噬细胞（MDM）在IAV生产中效率低下，尽管在质膜上的病毒糖蛋白表达水平相当。相关的荧光扫描电子显微镜显示，在细胞表面的出芽结构的形成是低效的MDM，即使观察到的病毒糖蛋白，血凝素（HA）的集群，这表明在IAV颗粒组装的一个步骤被阻止在MDM。使用原位邻位连接试验，我们进一步确定HA与神经氨酸酶（NA）相关，但不能与MDM质膜上的另一种病毒跨膜蛋白M2相关。值得注意的是，MDM中HA-M2缔合和颗粒组装的缺陷在细胞松弛素D处理后被逆转，细胞松弛素D抑制肌动蛋白聚合。这些结果表明，HA-M2的质膜上的协会是一个独立的步骤，在IAV的生产，这是容易受到抑制的肌动蛋白细胞骨架在MDM。在细胞松弛素D处理后，MDM中的病毒释放仍然无效，表明在该细胞类型中存在病毒释放的额外缺陷。总体而言，我们的研究揭示了存在多种细胞类型特异性机制负调控IAV生产在质膜MDM。
Identification of host cell determinants promoting or suppressing replication of viruses has been aided by analyses of host cells that impose inherent blocks on viral replication. In this study, we show that primary human MDM, which are not permissive to IAV replication, fail to support virus particle formation. This defect is specific to primary human macrophages, since a human monocytic cell line differentiated to macrophage-like cells supports IAV particle formation. We further identified association between two viral transmembrane proteins, HA and M2, on the cell surface as a discrete assembly step, which is defective in MDM. Defective HA-M2 association and particle budding, but not virus release, in MDM are rescued by disruption of actin cytoskeleton, revealing a previously unknown, negative role for actin, which specifically targets an early step in the multistep IAV production. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly. Influenza A virus (IAV) propagates efficiently in epithelial cells, its primary target in the respiratory tract. In contrast, productive infection of most IAV strains is either blocked or highly inefficient in macrophages. The exact nature of the defect in IAV replication in human macrophages remains unknown. In this study, we showed that even compared to a monocytic cell line differentiated to macrophage-like cells, primary human monocyte-derived macrophages (MDM) are inefficient in IAV production, despite comparable levels of expression of viral glycoproteins at the plasma membrane. Correlative fluorescence scanning electron microscopy revealed that formation of budding structures at the cell surface is inefficient in MDM even though clustering of a viral glycoprotein, hemagglutinin (HA), is observed, suggesting that a step in IAV particle assembly is blocked in MDM. Using an in situ proximity ligation assay, we further determined that HA associates with neuraminidase (NA) but fails to associate with another viral transmembrane protein, M2, at the MDM plasma membrane. Notably, the defects in HA-M2 association and particle assembly in MDM were reversed upon cytochalasin D treatment that inhibits actin polymerization. These results suggest that HA-M2 association on the plasma membrane is a discrete step in IAV production, which is susceptible to suppression by actin cytoskeleton in MDM. Virus release remained inefficient in MDM upon cytochalasin D treatment, suggesting the presence of an additional defect(s) in virus release in this cell type. Overall, our study revealed the presence of multiple cell-type-specific mechanisms negatively regulating IAV production at the plasma membrane in MDM.