The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases

Abstract

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.

Co‐localization of Middle East respiratory syndrome coronavirus ( MERS ‐CoV) and dipeptidyl peptidase‐4 in the respiratory tract and lymphoid tissues of pigs and llamas

“…() estimated that carcinoembryonic antigen‐related cell adhesion molecule 5 (CEACAM5) is an important surface attachment factor that facilitates entry of MERS‐CoV in vitro . More recent evidence suggests that the speed and efficiency of viral antigen to attach the host cell is accelerated by an entry complex that includes DPP4, a CoV‐activating transmembrane protease serine 2 (TMPRSS2) and the tetraspanin Cd9 (Earnest et al., ). In their study, mice transfected with the human DPP4 gene became significantly less susceptible to MERS‐CoV infection after silencing Cd9 or TMPRSS2 with small RNAs.…”

Structural insights into coronavirus entry

“…The former mechanism has been proposed to be the route of MERS-CoV entry into cell types relevant to lung infection, and therefore a significant determinant of MERS-CoV virulence . Moreover, tetraspanin CD9 has been implicated in clustering DPP4 and transmembrane serine proteases to promote early entry of MERS-CoV (Earnest et al, 2017(Earnest et al, , 2015. PEDV, which replicates in the epithelial cells of the small intestine, undergoes S proteolytic activation by trypsin, which is highly abundant in the intestinal lumen .…”

GRP78: A cell's response to stress

“…The MERS-CoV spike protein utilizes dipeptidyl peptidase 4 (DPP4) as its functional receptor for host cell entry [66]. In addition to its functional receptor, MERS-CoV can recognize other molecules to facilitate their attachment and entry, for example, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), and tetraspanin CD9 are identified as a factor promoting the virus entry in permissive cells [67,68]. Recently GRP78 was recognized as an attachment factor for MERS-CoV spike protein that enhances the virus entry in the presence of DPP4 [65].…”