tetano
Editor, Senior Moderator
J Virol
. 2022 Mar 28;e0012822.
doi: 10.1128/jvi.00128-22. Online ahead of print.
Distinctive Roles of Furin and TMPRSS2 in SARS-CoV-2 Infectivity
Rachid Essalmani[SUP] 1 [/SUP], Jaspreet Jain[SUP] #[/SUP][SUP] 2 [/SUP], Delia Susan-Resiga[SUP] #[/SUP][SUP] 1 [/SUP], Ursula Andréo[SUP] #[/SUP][SUP] 1 2 [/SUP], Alexandra Evagelidis[SUP] 1 [/SUP], Rabeb Mouna Derbali[SUP] 1 [/SUP], David N Huynh[SUP] 1 [/SUP], Frédéric Dallaire[SUP] 2 [/SUP], Mélanie Laporte[SUP] 2 [/SUP], Adrien Delpal[SUP] 3 [/SUP], Priscila Sutto-Ortiz[SUP] 3 [/SUP], Bruno Coutard[SUP] 4 [/SUP], Claudine Mapa[SUP] 5 [/SUP], Keith Wilcoxen[SUP] 5 [/SUP], Etienne Decroly[SUP] 3 [/SUP], Tram Nq Pham[SUP] 2 [/SUP], Éric A Cohen[SUP] 2 6 [/SUP], Nabil G Seidah[SUP] 1 [/SUP]
Affiliations
Abstract
The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (PRRAR[SUB]685[/SUB]↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2' as KPSKR[SUB]815[/SUB]↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2' cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.
Keywords: Calu-3 cells; HeLa cells; SARS-CoV-2; TMPRSS2; cell-to-cell fusion; furin; spike; viral entry; viral infection.
. 2022 Mar 28;e0012822.
doi: 10.1128/jvi.00128-22. Online ahead of print.
Distinctive Roles of Furin and TMPRSS2 in SARS-CoV-2 Infectivity
Rachid Essalmani[SUP] 1 [/SUP], Jaspreet Jain[SUP] #[/SUP][SUP] 2 [/SUP], Delia Susan-Resiga[SUP] #[/SUP][SUP] 1 [/SUP], Ursula Andréo[SUP] #[/SUP][SUP] 1 2 [/SUP], Alexandra Evagelidis[SUP] 1 [/SUP], Rabeb Mouna Derbali[SUP] 1 [/SUP], David N Huynh[SUP] 1 [/SUP], Frédéric Dallaire[SUP] 2 [/SUP], Mélanie Laporte[SUP] 2 [/SUP], Adrien Delpal[SUP] 3 [/SUP], Priscila Sutto-Ortiz[SUP] 3 [/SUP], Bruno Coutard[SUP] 4 [/SUP], Claudine Mapa[SUP] 5 [/SUP], Keith Wilcoxen[SUP] 5 [/SUP], Etienne Decroly[SUP] 3 [/SUP], Tram Nq Pham[SUP] 2 [/SUP], Éric A Cohen[SUP] 2 6 [/SUP], Nabil G Seidah[SUP] 1 [/SUP]
Affiliations
- PMID: 35343766
- DOI: 10.1128/jvi.00128-22
Abstract
The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (PRRAR[SUB]685[/SUB]↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2' as KPSKR[SUB]815[/SUB]↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2' cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.
Keywords: Calu-3 cells; HeLa cells; SARS-CoV-2; TMPRSS2; cell-to-cell fusion; furin; spike; viral entry; viral infection.
