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.

Coronaviruses (CoVs) are common human and animal pathogens that can transmit zoonotically and cause severe respiratory disease syndromes. CoV infection requires spike proteins, which bind viruses to host cell receptors and catalyze virus-cell membrane fusion. Several CoV strains have spike proteins with two receptor-binding domains, an S1A that engages host sialic acids and an S1B that recognizes host transmembrane proteins. As this bivalent binding may enable broad zoonotic CoV infection, we aimed to identify roles for each receptor in distinct infection stages. Focusing on two betacoronaviruses, murine JHM-CoV and human Middle East respiratory syndrome coronavirus (MERS-CoV), we found that virus particle binding to cells was mediated by sialic acids; however, the transmembrane protein receptors were required for a subsequent virus infection. These results favored a two-step process in which viruses first adhere to sialic acids and then require subsequent engagement with protein receptors during infectious cell entry. However, sialic acids sufficiently facilitated the later stages of virus spread through cell-cell membrane fusion, without requiring protein receptors. This virus spread in the absence of the prototype protein receptors was increased by adaptive S1A mutations. Overall, these findings reveal roles for sialic acids in virus-cell binding, viral spike protein-directed cell-cell fusion, and resultant spread of CoV infections. IMPORTANCE CoVs can transmit from animals to humans to cause serious disease. This zoonotic transmission uses spike proteins, which bind CoVs to cells with two receptor-binding domains. Here, we identified the roles for the two binding processes in the CoV infection process. Binding to sialic acids promoted infection and also supported the intercellular expansion of CoV infections through syncytial development. Adaptive mutations in the sialic acid-binding spike domains increased the intercellular expansion process. These findings raise the possibility that the lectin-like properties of many CoVs contribute to facile zoonotic transmission and intercellular spread within infected organisms.

Coronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and viruspriming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases. IMPORTANCEEnveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus priming with greater precision. Implications of these findings extend to the use of virus entry antagonists, such as protease inhibitors, which might be most effective when localized to these microdomains. Enveloped viruses require fusion with host cell membranes to deliver viral genetic material and initiate infection. This process is catalyzed by fusion glycoproteins, which project from virion membranes and operate by bringing virion and host cell membranes into proximity, ultimately stimulating their coalescence. Among the host cell factors required for this membrane fusion are receptors and proteases. Receptors tether viruses to host cell membranes, and proteases cleave fusion protein precursors to form the domains that catalyze membrane melding. This proteolytic step is termed "priming," and depending on the virus type, it may take place in virus-producing cells (1), in extracellular environments (2), or in virus target cells (3). Notably, several protease inhibitors prevent viral fusion protein cleavages, and as such, are antiviral age...

Host factors render cells susceptible to viral infection. One family of susceptibility factors, the tetraspanin proteins, facilitate enveloped virus entry by promoting virus-cell membrane fusion. They also facilitate viral egress from infected cells. In this Gem, we discuss recent insights into how tetraspanins assemble viral entry and exit platforms on cell membranes, and we speculate that tetraspanins contribute to nonviral membrane fusions by similar mechanisms.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging zoonotic pathogen with a broad host range. The extent of MERS-CoV in nature can be traced to its adaptable cell entry steps. The virus can bind host-cell carbohydrates as well as proteinaceous receptors. Following receptor interaction, the virus can utilize diverse host proteases for cleavage activation of virus-host cell membrane fusion and subsequent genome delivery. The fusion and genome delivery steps can be completed at variable times and places, either at or near cell surfaces or deep within endosomes. Investigators focusing on the CoVs have developed several methodologies that effectively distinguish these different cell entry pathways. Here we describe these methods, highlighting virus-cell entry factors, entry inhibitors, and viral determinants that specify the cell entry routes. While the specific methods described herein were utilized to reveal MERS-CoV entry pathways, they are equally suited for other CoVs, as well as other protease-dependent viral species.