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. Author summary Enveloped viruses rank among the most dangerous zoonotically emerging pathogens. Their cell entry often requires multiple transmembrane proteins in the target cell, which may interact with each other to promote viral-cell membrane fusion. Susceptibility to virus infection may correlate with these transmembrane protein interactions. Here we report that the scaffolding tetraspanin protein CD9 links the receptor for MERS-CoV to a membrane fusion-activating protease called TMPRSS2, forming a complex that promotes rapid and efficient infection. The related human CoV strain 229E was also facilitated by CD9, indicating that multiple CoVs depend on tetraspanin-directed clustering of receptors and proteases for efficient cell entry. Reliance on CD9 specifically applied to virulent, in vivo mouse lung-adapted MERS-CoVs, suggesting that the most efficient virus entry pathways in natural respiratory CoV infections are facilitated by tetraspanins. This suggestion was reinforced by selectively regulating gene expression in vivo , using recombinant adenovirus transducing vectors. The findings demonstrated that CD9 facilitated MERS-CoV infections in mice.
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