A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS-and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.

The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensinconverting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.

Using chemical inhibitors and small interfering RNA (siRNA), we have confirmed roles for cathepsin B (CatB) and cathepsin L (CatL) in Ebola virus glycoprotein (GP)-mediated infection. Treatment of Ebola virus GP pseudovirions with CatB and CatL converts GP1 from a 130-kDa to a 19-kDa species. Virus with 19-kDa GP1 displays significantly enhanced infection and is largely resistant to the effects of the CatB inhibitor and siRNA, but it still requires a low-pH-dependent endosomal/lysosomal function. These and other results support a model in which CatB and CatL prime GP by generating a 19-kDa intermediate that can be acted upon by an as yet unidentified endosomal/lysosomal enzyme to trigger fusion.

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The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERSCoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lungderived cell line.

A unique coronavirus severe acute respiratory syndrome-coronavirus (SARS-CoV) was revealed to be a causative agent of a lifethreatening SARS. Although this virus grows in a variety of tissues that express its receptor, the mechanism of the severe respiratory illness caused by this virus is not well understood. Here, we report a possible mechanism for the extensive damage seen in the major target organs for this disease. A recent study of the cell entry mechanism of SARS-CoV reveals that it takes an endosomal pathway. We found that proteases such as trypsin and thermolysin enabled SARS-CoV adsorbed onto the cell surface to enter cells directly from that site. This finding shows that SARS-CoV has the potential to take two distinct pathways for cell entry, depending on the presence of proteases in the environment. Moreover, the protease-mediated entry facilitated a 100-to 1,000-fold higher efficient infection than did the endosomal pathway used in the absence of proteases. These results suggest that the proteases produced in the lungs by inflammatory cells are responsible for high multiplication of SARS-CoV, which results in severe lung tissue damage. Likewise, elastase, a major protease produced in the lungs during inflammation, also enhanced SARS-CoV infection in cultured cells.cell entry ͉ protease ͉ spike protein ͉ SARS S evere acute respiratory syndrome (SARS) is caused by a SARS-associated coronavirus (SARS-CoV), a newly emergent member in a family of Coronaviridae (1-6). Unlike other human coronaviruses, SARS-CoV causes a fatal respiratory disease in humans (1-6). Coronavirus is an enveloped virus with a positive-stranded large genomic RNA with Ϸ30 kb (7). Spikes exist on the virion surface and resemble solar corona, each of which is composed of a trimer of the spike (S) protein (7,8). The S protein is a type I fusion protein of an approximate molecular weight of 180 kDa. The prototypical coronavirus mouse hepatitis virus enters into cells via the cell surface, although a variant isolated from persistent infection enters from an endosome, the low pH of which induces its fusion activity (9). However, the entry pathway of SARS-CoV appears to be distinct from that of the other coronaviruses. Simmons et al. (10) hypothesized that SARS-CoV enters cells by an endosomal pathway, and S protein is activated for fusion by trypsin-like protease in an acidic environment. This idea is based on the following two findings: (i) SARS-CoV infection can be blocked by lysosomotropic agents, and (ii) S protein expressed on cells is activated for fusion by trypsin. These results were obtained by studies using pseudotype retroviruses harboring SARS-CoV S protein on the envelope and those using S protein expressed on cells by expression vectors (10).In the present study, we show that various proteases, as well as trypsin, are effective in inducing the fusion of SARS-CoVinfected VeroE6 cells. These proteases facilitated SARS-CoV entry from the cell surface, which indicates that SARS-CoV has the potential to enter cells via two diff...

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e Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro. Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.