Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry
Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry
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DOI：
10.1128/jvi.00725-07
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发表时间：
2007-10-01
影响因子：
5.4
通讯作者：
Herrmann, Andreas
中科院分区：
文献类型：
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作者：
Eifart, Patricia;Ludwig, Kai;Herrmann, Andreas
由冠状病毒小鼠肝炎病毒株A59(MHV-A59)感染需要通过与宿主细胞各自的靶膜融合来释放病毒基因组。融合是由病毒S蛋白介导的。在这里，我们用独立的方法研究了MHV-A59进入小鼠成纤维细胞的途径。通过空斑减少试验评估的细胞感染被溶酶体化合物和干扰依赖于笼蛋白的内吞作用的物质强烈地抑制，这表明MHV-A59是通过内吞作用被携带并被输送到酸性内体隔室的。在内体隔室存在抑制蛋白水解酶的物质时，感染仅略有减少，排除了内吞摄取通过切割激活S蛋白的融合潜力所必需的。标记MHV-A59的荧光共聚焦显微镜证实病毒是通过内吞作用携带的。细胞内间隔的明亮标记表明它们与病毒包膜融合。在中性pH条件下，未观察到与质膜融合。然而，当病毒与细胞结合并将pH降至5.0时，我们观察到质膜的强烈标记。电子显微镜显示低pH引发S胞外结构域的构象变化。这些变化很可能是不可逆转的，因为在没有靶膜的情况下对病毒进行低pH处理会导致不可逆转的融合活性丧失。这些结果表明，内吞作用在MRVA59感染中起主要作用，内膜室的酸性pH触发了介导融合的S蛋白的构象变化。
Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MRV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.