SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

https://www.biorxiv.org/content/biorxiv/early/2020/02/20/2020.02.19.956581.full.pdf

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein.

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic coronavirus that emerged in late 2019 and has caused a pandemic of acute respiratory disease, named ‘coronavirus disease 2019’ (COVID-19), which threatens human health and public safety. In this Review, we describe the basic virology of SARS-CoV-2, including genomic characteristics and receptor use, highlighting its key difference from previously known coronaviruses. We summarize current knowledge of clinical, epidemiological and pathological features of COVID-19, as well as recent progress in animal models and antiviral treatment approaches for SARS-CoV-2 infection. We also discuss the potential wildlife hosts and zoonotic origin of this emerging virus in detail. In this Review, Shi and colleagues summarize the exceptional amount of research that has characterized acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19) since this virus has swept around the globe. They discuss what we know so far about the emergence and virology of SARS-CoV-2 and the pathogenesis and treatment of COVID-19.

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Characteristics of SARS-CoV-2 and COVID-19

Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.

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Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV.

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 TMPRSS2-expressing 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.

https://www.pnas.org/content/pnas/117/13/7001.full.pdf

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting 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.

Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2

The type II transmembrane protease TMPRSS2 activates the spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) on the cell surface following receptor binding during viral entry into cells. In the absence of TMPRSS2, SARS-CoV achieves cell entry via an endosomal pathway in which cathepsin L may play an important role, i.e., the activation of spike protein fusogenicity. This study shows that a commercial serine protease inhibitor (camostat) partially blocked infection by SARS-CoV and human coronavirus NL63 (HCoV-NL63) in HeLa cells expressing the receptor angiotensin-converting enzyme 2 (ACE2) and TMPRSS2. Simultaneous treatment of the cells with camostat and EST [(23,25)trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester], a cathepsin inhibitor, efficiently prevented both cell entry and the multistep growth of SARS-CoV in human Calu-3 airway epithelial cells. This efficient inhibition could be attributed to the dual blockade of entry from the cell surface and through the endosomal pathway. These observations suggest camostat as a candidate antiviral drug to prevent or depress TMPRSS2-dependent infection by SARS-CoV.

/pdf/simultaneous-treatment-of-human-bronchial-epithelial-cells-4bs9fu865k.pdf

Simultaneous Treatment of Human Bronchial Epithelial Cells with Serine and Cysteine Protease Inhibitors Prevents Severe Acute Respiratory Syndrome Coronavirus Entry

During the emergence of novel coronavirus 2019 (nCoV) outbreak in Wuhan city, China at the end of 2019, there was movement of many airline travelers between Wuhan and Japan, suggesting that the Japanese population was at high risk of infection by the virus. Hence, we urgently developed diagnostic systems for detection of 2019 nCoV. Two nested RT-PCR and two real-time RT-PCR assays were adapted for use in Japan. As of February 8, 2020, these assays have successfully detected 25 positive cases of infection in Japan.

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Development of Genetic Diagnostic Methods for Detection for Novel Coronavirus 2019(nCoV-2019) in Japan.

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 MERS-CoV 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 lung-derived cell line.

Kazuya Shirato

Papers

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2

Simultaneous Treatment of Human Bronchial Epithelial Cells with Serine and Cysteine Protease Inhibitors Prevents Severe Acute Respiratory Syndrome Coronavirus Entry

Development of Genetic Diagnostic Methods for Detection for Novel Coronavirus 2019(nCoV-2019) in Japan.

Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2