A Transmembrane Serine Protease Is Linked to the Severe Acute Respiratory Syndrome Coronavirus Receptor and Activates Virus Entry

Abstract

Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors. The primary receptor for the human severe acute respiratory syndrome CoV (SARS) CoV is angiotensin-converting enzyme 2 (ACE2). ACE2 immunoprecipitation captured transmembrane protease/ serine subfamily member 2 (TMPRSS2), a known human airway and alveolar protease. ACE2 and TMPRSS2 colocalized on cell surfaces and enhanced the cell entry of both SARS S-pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell surface complex comprising a primary receptor and a separate endoprotease operates as a portal for activation of SARS-CoV cell entry.Viruses exit from infected cells embedded with the energy required to enter new host cells. When viruses encounter new host cells, energy stored within metastable virus surface proteins is dissipated through protein refoldings and used to open the viruses and allow viral genomes to access the cell. This conversion from high-energy metastable to low-energy end stages is spatially and temporally regulated by a variety of triggers that are incorporated into the surface proteins. Depending on the virus, one or a combination of cell receptor bindings, protonations in the endosome, disulfide reductions, and proteolytic cleavages triggers viral protein refolding and opening. Insights into these activating conditions have advanced our understanding of virus-host interactions and have revealed new approaches for antiviral therapeutics.These activating virus entry events can be further dissected through research with the human CoVs (HCoVs). The HCoVs are notable pathogens (27, 48), with one of them accounting for severe acute respiratory syndrome (SARS) (12, 24). Evolution of the CoVs in their protruding surface or spike (S) proteins can change virus-activating conditions and permit zoonoses (30, 40) and virulence changes. Unraveling S protein activations is therefore central to understanding HCoV tropism, ecology, and pathogenesis.The S proteins include cell receptor-binding domains (RBDs) and virus-cell membrane fusion domains. Like other class I viral fusion proteins, the HCoV spikes require proteolytic priming to be activated (7). Notably, the majority of pathogenic HCoVs exit producer cells with unprimed S proteins (2, 34) and thus rely on target cell proteases for activation. Therefore, the HCoV cell entry factors on target cells include virus-binding agents (cell receptors) and also virus protein-cleaving agents (cell proteases).SARS-CoV binds to its ectopeptidase receptor, angiotensin-converting enzyme 2 (ACE2), with very high affinit...

Differentiated phenotypes of primary murine alveolar epithelial cells and their susceptibility to infection by respiratory viruses

“…ATI-like cells had robust surface expression of ACE2, suggesting that ACE2 binding is not the limiting factor in susceptibility to v2163 infection. Other groups have recently demonstrated the importance of a cellular protease, TMPRSS2, in enhancing ACE2-dependent and -independent infection by SARS-CoV (Glowacka et al, 2011;Matsuyama et al, 2010;Shulla et al, 2011). It is not known whether TMPRSS2 is important for infection by mouse-adapted strains of SARS-CoV.…”

“…3B). In addition, these cells were evaluated for expression of TMPRSS2, a serine protease that interacts with ACE2 and enhances infection of cells by SARS-CoV (Shulla et al, 2011). The ATI-like cells also had robust surface expression of TMPRSS2 on day 5 after isolation (Fig.…”

Technical Considerations for the Generation of Novel Pseudotyped Viruses

“…The addition of exogenous tosyl phenylalanyl chloromethyl ketone (TPCK) trypsin has been demonstrated to facilitate activation of SARS-CoV [85,86] MERS-CoV [10,87] and human coronavirus 229E [88]. Exogenous TPCK trypsin is also used for influenza, but an additional step is required in order to deactivate the protease, using commercially available protease inhibitors, prior to inoculation of the PVs onto target cells [65,77].…”

Cell-based antiviral screening against coronaviruses: Developing virus-specific and broad-spectrum inhibitors

“…Endocytosis of the receptor-virus complex can occur, and upon acidification of the endosome, the host protease cathepsin L is activated and can cleave the viral spike protein to initiate viral fusion. The coronaviral spike can also be activated by extracellular proteases (trypsin) or proteases present on the cell surface (type II transmembrane serine protease or TMPRSS2), and this cleavage allows coronaviruses to enter cells in an cathepsin-independent manner (Glowacka et al, 2011; Matsuyama et al, 2010; Shulla et al, 2011 and reviewed in Simmons, Zmora, Gierer, Heurich, & Pohlmann, 2013). Upon viral entry and fusion of the viral and host cell membranes, the positive sense RNA genome, which is 5ʹ methyl-capped and poly-adenylated, is translated in the cytoplasm.…”

“…Cells expressing SARS-CoV S and a T7 polymerase are plated with cells expressing ACE2 and a luciferase or GFP reporter plasmid driven by a T7-dependent promoter. Upon receptor interaction and fusion, the cytoplasm from both cell types mixes, allowing for the transcription and translation of the luciferase or GFP (Shulla et al, 2011; Simmons et al, 2005). Pseudotyped virions were used by Kawase et al in their work identifying commercially available protease inhibitors targeting TMPRRS2 (Kawase, Shirato, van der Hoek, Taguchi, & Matsuyama, 2012).…”