Park Jung-Eun, Li Kun, Barlan Arlene, Fehr Anthony R, Perlman Stanley, McCray Paul B, Gallagher Tom
Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL 60153.
Department of Pediatrics, University of Iowa, Iowa City, IA 52242.
Proc Natl Acad Sci U S A. 2016 Oct 25;113(43):12262-12267. doi: 10.1073/pnas.1608147113. Epub 2016 Oct 10.
Middle East respiratory syndrome coronavirus (MERS-CoV) infects humans from zoonotic sources and causes severe pulmonary disease. Virions require spike (S) glycoproteins for binding to cell receptors and for catalyzing virus-cell membrane fusion. Fusion occurs only after S proteins are cleaved sequentially, first during their secretion through the exocytic organelles of virus-producing cells, and second after virus binding to target-cell receptors. To more precisely determine how sequential proteolysis contributes to CoV infection, we introduced S mutations obstructing the first cleavages. These mutations severely compromised MERS-CoV infection into human lung-derived cells, but had little effect on infection into several other cell types. These cell type-specific requirements for proteolysis correlated with S conformations during cell entry. Without the first cleavages, S proteins resisted cell receptor-induced conformational changes, which restricted the second, fusion-activating cleavages. Consistent with these findings, precleaved MERS viruses used receptor-proximal, cell-surface proteases to effect the second fusion-activating cleavages during cell entry, whereas the more rigid uncleaved MERS viruses trafficked past these cell-surface proteases and into endosomes. Uncleaved viruses were less infectious to human airway epithelial and Calu3 cell cultures because they lacked sufficient endosomal fusion-activating proteases. Thus, by sensitizing viruses to receptor-induced conformational changes, the first S cleavages expand virus tropism to cell types that are relevant to lung infection, and therefore may be significant determinants of MERS-CoV virulence.
中东呼吸综合征冠状病毒（MERS-CoV）从动物源感染人类并引发严重肺部疾病。病毒粒子需要刺突（S）糖蛋白来结合细胞受体并催化病毒与细胞膜融合。融合仅在S蛋白依次被切割后发生，第一次切割发生在其通过病毒产生细胞的胞吐细胞器分泌过程中，第二次切割发生在病毒与靶细胞受体结合之后。为了更精确地确定顺序性蛋白水解如何促进冠状病毒感染，我们引入了阻碍首次切割的S突变。这些突变严重损害了MERS-CoV对人肺源性细胞的感染，但对几种其他细胞类型的感染影响很小。这些细胞类型对蛋白水解的特异性需求与细胞进入过程中的S构象相关。没有首次切割，S蛋白会抵抗细胞受体诱导的构象变化，这限制了第二次融合激活切割。与这些发现一致，预切割的MERS病毒在细胞进入过程中利用受体近端的细胞表面蛋白酶来进行第二次融合激活切割，而更刚性的未切割MERS病毒则绕过这些细胞表面蛋白酶进入内体。未切割的病毒对人气道上皮细胞和Calu3细胞培养物的感染性较低，因为它们缺乏足够的内体融合激活蛋白酶。因此，通过使病毒对受体诱导的构象变化敏感，首次S切割将病毒嗜性扩展到与肺部感染相关的细胞类型，因此可能是MERS-CoV毒力的重要决定因素。
