Earnest James T, Hantak Michael P, Li Kun, McCray Paul B, Perlman Stanley, Gallagher Tom
Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, IL, United States of America.
Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA, United States of America.
PLoS Pathog. 2017 Jul 31;13(7):e1006546. doi: 10.1371/journal.ppat.1006546. eCollection 2017 Jul.
Infection by enveloped coronaviruses (CoVs) initiates with viral spike (S) proteins binding to cellular receptors, and is followed by proteolytic cleavage of receptor-bound S proteins, which prompts S protein-mediated virus-cell membrane fusion. Infection therefore requires close proximity of receptors and proteases. We considered whether tetraspanins, scaffolding proteins known to facilitate CoV infections, hold receptors and proteases together on cell membranes. Using knockout cell lines, we found that the tetraspanin CD9, but not the tetraspanin CD81, formed cell-surface complexes of dipeptidyl peptidase 4 (DPP4), the MERS-CoV receptor, and the type II transmembrane serine protease (TTSP) member TMPRSS2, a CoV-activating protease. This CD9-facilitated condensation of receptors and proteases allowed MERS-CoV pseudoviruses to enter cells rapidly and efficiently. Without CD9, MERS-CoV viruses were not activated by TTSPs, and they trafficked into endosomes to be cleaved much later and less efficiently by cathepsins. Thus, we identified DPP4:CD9:TTSP as the protein complexes necessary for early, efficient MERS-CoV entry. To evaluate the importance of these complexes in an in vivo CoV infection model, we used recombinant Adenovirus 5 (rAd5) vectors to express human DPP4 in mouse lungs, thereby sensitizing the animals to MERS-CoV infection. When the rAd5-hDPP4 vectors co-expressed small RNAs silencing Cd9 or Tmprss2, the animals were significantly less susceptible, indicating that CD9 and TMPRSS2 facilitated robust in vivo MERS-CoV infection of mouse lungs. Furthermore, the S proteins of virulent mouse-adapted MERS-CoVs acquired a CD9-dependent cell entry character, suggesting that CD9 is a selective agent in the evolution of CoV virulence.
包膜冠状病毒（CoV）的感染始于病毒刺突（S）蛋白与细胞受体结合，随后受体结合的S蛋白发生蛋白水解切割，这促使S蛋白介导病毒与细胞膜融合。因此，感染需要受体和蛋白酶紧密靠近。我们考虑四跨膜蛋白（已知有助于CoV感染的支架蛋白）是否能将受体和蛋白酶聚集在细胞膜上。利用基因敲除细胞系，我们发现四跨膜蛋白CD9而非CD81形成了二肽基肽酶4（DPP4，中东呼吸综合征冠状病毒受体）与II型跨膜丝氨酸蛋白酶（TTSP）成员TMPRSS2（一种CoV激活蛋白酶）的细胞表面复合物。这种由CD9促进的受体和蛋白酶聚集使得中东呼吸综合征冠状病毒假病毒能够快速有效地进入细胞。没有CD9时，中东呼吸综合征冠状病毒未被TTSP激活，而是进入内体，很久之后才被组织蛋白酶切割，且切割效率较低。因此，我们确定DPP4:CD9:TTSP是中东呼吸综合征冠状病毒早期高效进入所必需的蛋白复合物。为了评估这些复合物在体内CoV感染模型中的重要性，我们使用重组腺病毒5（rAd5）载体在小鼠肺中表达人DPP4，从而使动物对中东呼吸综合征冠状病毒感染敏感。当rAd5-hDPP4载体共表达沉默Cd9或Tmprss2的小RNA时，动物的易感性显著降低，这表明CD9和TMPRSS2促进了小鼠肺中中东呼吸综合征冠状病毒在体内的强劲感染。此外，具有毒力的小鼠适应型中东呼吸综合征冠状病毒的S蛋白获得了依赖CD9的细胞进入特性，这表明CD9是CoV毒力进化中的一个选择因素。
