key: cord-0895629-no7n99cg
authors: Luu, Ross; Valdebenito, Silvana; Scemes, Eliana; Cibelli, Antonio; Spray, David; Rovegno, Maximiliano; Tichauer, Juan; Cottignies-Calamarte, Andrea; Rosenberg, Arielle; Capron, Calude; Belouzard, Sandrine; Dubuisson, Jean; Annane, Djallali; Lorin de la Grandmaison, Geoffroy; Cramer-Bordé, Elisabeth; Bomsel, Morgane; Eugenin, Eliseo
title: Pannexin-1 channel opening is critical for COVID-19 pathogenesis
date: 2021-11-19
journal: iScience
DOI: 10.1016/j.isci.2021.103478
sha: 3db0eb6760ebafc0290dc0a58c388c1f8f892846
doc_id: 895629
cord_uid: no7n99cg

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly rampaged worldwide, causing a pandemic of coronavirus disease (COVID -19), but the biology of SARS-CoV-2 is under investigation. We demonstrate that both SARS-CoV-2 spike protein and human coronavirus 229E (hCoV-229E) or its purified S protein, one of the main viruses responsible for the common cold, induce the transient opening of Pannexin-1 (Panx-1) channels in human lung epithelial cells. However, the Panx-1 channel opening induced by SARS-CoV-2 is greater and more prolonged than hCoV-229E/S protein, resulting in ATP, PGE2, and IL-1β release. Analysis of lung lavage and tissues indicate that Panx-1 mRNA expression is associated with increased ATP, PGE2, and IL-1β levels. Panx-1 channel opening induced by SARS-CoV-2 spike protein is angiotensin-converting enzyme 2 (ACE-2), endocytosis, and furin dependent. Overall, we demonstrated that Panx-1 is a critical contributor to SARS-CoV-2 infection and should be considered an alternative therapy.

Viruses have evolved to use host-encoded proteins to facilitate infection, replication, and associated pathogenesis (Konig et al., 2008; Krupovic et al., 2018; Pillay, 2020; Walsh and Mohr, 2011; Zhou et al., 2017) . In severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), several host proteins have been described, but far more research is required to fully understand the mechanism of viral entry, replication, and pathogenesis, as well as vaccine evaluation (Bao et al., 2020; Hartenian et al., 2020; Tay et al., 2020; V'Kovski et al., 2021; Yuki et al., 2020) . SARS-CoV-2 is a positive sense, single-stranded RNA-based betacoronavirus with an envelope decorated with a notable surface glycoprotein, the Spike (S) protein. Viral entry occurs through the interaction of the S protein with the cellular receptor, angiotensin-converting enzyme 2 (ACE-2), and the processing of the S protein facilitates it by proteases such as trypsin/furin (Shang et al., 2020; Wu et al., 2020) , transmembrane serine protease 2 (TMPRSS2) (Hoffmann et al., 2020a; Hoffmann et al., 2020b) , and endosomal cathepsins (Shang et al., 2020) . The trypsin/furin pre-activation of the S protein is a key difference among coronaviruses, resulting in an increased binding affinity to the receptor and a widespread infectious cycle (Shang et al., 2020) . However, additional host factors contributing to the viral cell cycle and associated inflammation are still under investigation. Our data demonstrated that SARS-CoV-2 uses Pannexin-1 (Panx-1) channels to mediate infection and associated inflammation.

Panx-1 is widely expressed and forms oligomeric plasma membrane channels (Baranova et al., 2004; Michalski et al., 2020; Penuela et al., 2013; Qu et al., 2020; Ruan et al., 2020; Spagnol et al., 2014; Wang and Liu, 2021) . Panx-1 channels have J o u r n a l P r e -p r o o f unique characteristics; they contain one of the largest mammalian pores enabling the release of small ions, nucleotides, lipids, and small RNA into the extracellular space (MacVicar and Thompson, 2010; Pelegrin and Surprenant, 2006; Qu et al., 2011) . In healthy conditions, Panx-1 channels are generally in a closed state. However, during disease conditions such as stress, cancer, neurodegeneration, and HIV, the channel becomes open, resulting in the release of multiple pro-inflammatory factors (Gajardo-Gomez et al., 2020a; Orellana et al., 2011a; Penuela et al., 2014; Seror et al., 2011; Velasquez and Eugenin, 2014a; Velasquez et al., 2016) .

Our laboratory previously demonstrated that HIV induces Panx-1 channel opening to accelerate viral entry, replication, cell-to-cell spread, and inflammation (Gorska et al., 2021a; Malik and Eugenin, 2019; Orellana et al., 2013; Valdebenito et al., 2018; Velasquez and Eugenin, 2014a; Velasquez et al., 2016) . Furthermore, we identified that ATP secreted through the Panx-1 channel pore activates purinergic receptors that initiate signaling, immune recruitment, and inflammation (Eugenin, 2014; Gajardo-Gomez et al., 2020b; Gorska et al., 2021b; Velasquez et al., 2020) . Similar interactions between Panx-1 and purinergic receptors have been described in multiple lung functions such as blood pressure control, vasodilation/constriction, airway defense, and viral infection (Haywood et al., 2021; Ledderose et al., 2018; Martinez-Calle et al., 2018; Riteau et al., 2010; Swayne et al., 2020; Wirsching et al., 2020) . In addition, an active role of Panx-1 channels has been described in mucin hypersecretion (Seminario-Vidal et al., 2011; van Heusden et al., 2021) , hydrostatic pressure (Richter et al., 2014) , and general lung inflammation (Adamson and Leitinger, 2014) . More importantly, blocking Panx-1 channel opening reduces Pseudomonas aeruginosa infection and J o u r n a l P r e -p r o o f associated inflammation (Maier-Begandt et al., 2021; Wonnenberg et al., 2014) , the deleterious effects of smoking (Baxter et al., 2014) , cystic fibrosis (Higgins et al., 2015) , lung associated heart failure (Dahl et al., 2016) , blood vessel compromise (Luo et al., 2017) , ischemia-reperfusion (Kirby et al., 2021; Sharma et al., 2018) , coagulation , and ventilator-associated damage (Jia et al., 2018) suggesting a potential therapeutic approach for COVID-19. All these conditions are present and exacerbated in COVID-19 patients (Barnes et al., 2020; Calabrese et al., 2020; Gupta et al., 2020; Kempuraj et al., 2020; Lo et al., 2020) , suggesting that preventing Panx-1 opening could reduce infection and associated inflammation. However, whether Panx-1 channels and ATP are involved in COVID-19 pathogenesis was unknown.

Here, we show that Panx-1 channels open in response to SARS-CoV-2 or hCoV-229E. Our data indicate that SARS-CoV-2 S protein opens Panx-1 channels aggressively and for a prolonged time compared to hCoV-229E or its purified S protein, suggesting a different opening mechanism. Furthermore, we identify that the SARS-CoV-2-induced Panx-1 opening is ACE-2, furin, and endocytosis dependent. In agreement, upon Panx-1 channel opening induced by SARS-CoV-2 S protein, ATP, PGE2, and IL-1β are released into the extracellular space. Lung tissue and nasal swab analysis indicate that Panx-1 mRNA and protein are increased in immune and lung cell populations supporting the essential role of the Panx-1 channel in pathogenesis. In vivo analysis of lung lavage confirmed elevated ATP, PGE2, and IL-1β concentrations compared to other lung-related diseases such as chronic obstructive pulmonary disease (COPD), indicating a strong and acute physical response to SARS-CoV-2. We propose that blocking Panx-1 channels or associated purinergic signaling J o u r n a l P r e -p r o o f could prevent the devastating acute and long-term consequences of COVID-19 or other emerging coronaviruses.

SARS-CoV-2 and hCoV-229E induced the opening of Panx-1 channels. Primary cultures of human lung epithelial cells were treated with S Protein (recombinant S protein of SARS-CoV-2, the agent of COVID-19, 1 µg/ml) or 229E (hCoV-229E, the common cold virus, 0.1 MOI or recombinant S protein, 1-5 µg/ml) to determine the Ethidium uptake rate (Etd, 5 µM), by live-cell imaging, as a measure of Panx-1 channel opening. The Etd dye only crosses the plasma membrane in healthy cells by passing through specific large channels such as connexin (Cx) hemichannels and Panx-1 channels. Therefore, as a function of time, its intracellular fluorescence reflects channel opening as determined by the treatment with specific channel blockers (Contreras et al., 2002; Orellana et al., 2011b; Sanchez et al., 2009 ).

Nonspecific Etd uptake was not observed in the untreated or BSA, furin, or trypsin treated (2.5 to 5 µg/ml) human primary lung epithelial cells ( (1 to 0.01 MOI, data shown in Fig. 1N represents 0.1 MOI) or purified S protein (1) (2) (3) (4) (5) µg/ml) induced a smaller and more transient Panx-1 channel opening than SARS-CoV-2 S protein. The time course of Panx-1 opening induced by 229E reached a peak after 5-7 min post-treatment to return to baseline after 10 min (Fig. 1N , red lines, 229E, p≤1.67x10 -4 ). Therefore, SARS-CoV-2 S protein and hCoV-229E or its S proteininduced Panx-1 channel opening with a different time course and intensity. No cell death or loss was observed in all the cultures analyzed (data not shown).

To ensure whether Etd uptake induced by coronaviruses is solely a consequence of Panx-1 channel opening and no other large channels, we used Probenecid (Prob, 500 µM) and a Panx-1 blocking mimetic peptide (Panx-1 pep, 200 µM) to prevent Panx-1 channel opening in response to SARS-CoV-2 S protein or hCoV-229E (whole virus or its S protein). These treatments have previously been demonstrated to specifically block Panx-1 channels (Orellana et al., 2011a; Silverman et al., 2008; Silverman et al., 2009a; Velasquez et al., 2020) . Pre-incubation (10 min) of human lung epithelial cells with Probenecid ( Fig. 1M and N, blue lines, Prob+S protein or Prob+229E, respectively) or the Panx-1 mimetic blocking peptide (Fig. 1M and N, green lines, protein or Panx-1pep+229E, respectively) prevented the opening induced by the viral proteins (S protein, 2.5 µg/ml, or 229E, 0.1 MOI or its S protein, 1-5 µg/ml). Moreover, the pre-incubation of the epithelial cell cultures with the Panx-1 scrambled peptide (Scram, 200 µM) before treatment with S protein or 229E did not prevent the Etd uptake induced by the viral components (Fig. 1M and N, pink lines, 200 µM Scram+S protein or Scram+229E, respectively, p≤0.0002, compared to control conditions) . In J o u r n a l P r e -p r o o f contrast, Lanthanum (La 3+ ), a general connexin hemichannel blocker, or Cx43 E2 , an antibody that blocks Cx43 hemichannels at several concentrations as described previously (Evans et al., 2006; Orellana et al., 2011b; Siller-Jackson et al., 2008) , did not affect the S protein or 229E induced Etd uptake rate ( Fig. S1A and S1B), suggesting that Cx43 hemichannels do not participate in the dye uptake process. Furthermore, no toxic or nonspecific effects of these blockers alone were detected as determined by trypan blue exclusion, TUNEL staining, or cell detachment (See Fig. S1B ). Overall, coronaviruses induced the Panx-1 channel opening on primary lung epithelial cells

CoV-2 S-protein in combination with ATP increased Panx-1 channel opening. To expand our dye uptake analysis and ensure Panx-1 identity, we performed whole-cell patch-clamp recordings using human primary lung epithelial cells to examine the extent to which the Panx-1 channel is activated by SARS-CoV-2 S-protein (Fig. 2) . To that end, we measured changes in peak conductance induced by voltage ramps before and after adding either viral agent (in the presence of ATP) and normalized the conductance to those recorded in H-PBS alone. To verify that low doses of ATP (1 µM) do not induce Panx-1 channel opening but facilitate it, as occurs under several pathological conditions (Iglesias et al., 2008; Negoro et al., 2013; Silverman et al., 2009b) , fold changes in peak conductance were measured. As shown in Fig. 2A J o u r n a l P r e -p r o o f resulted in non-significant Panx-1 channel opening (Fig. 2D , hCoV-229E+ATP) as determined by the changes in peak conductance compared to untreated control conditions ( Fig. 2E -F, control: 1.05±0.01 folds, hCoV-229E+ATP: 1.00 ± 0.03 folds, n=10, paired t-test, p=0.341). In contrast, the application of SARS-CoV-2 S protein (1.0 µg/ml) plus ATP (1 µM) to lung epithelial cells resulted in a significant increase in the Panx-1 channel peak conductance, as indicated in the recordings (Fig. 2G ) and upon quantification of the fold changes in the peak of conductance ( Fig. 2H -I, control: 1.04 ±0.01 folds, SARS-CoV-2 S protein + ATP: 1.91 ± 0.01 folds, n=12, p<0.0001 paired ttest,). To confirm that changes in conductance were related to Panx-1 currents, Probenecid (1.0 mM) was applied, and fold changes in peak conductance were measured in the presence of S protein (Fig. 2 J) . Under this condition, no significant changes in peak conductance were recorded following application of S protein ( Fig. 2 K-L, control + Prob: 1.05 ± 0.01 folds, Prob + Prot S: 1.08 ± 0.05 folds, n=9, paired t-test, p=0.56). Thus, in accordance with the dye uptake data, our electrophysiological data confirmed that SARS-CoV-2 S protein induced a two-fold increase in Panx-1 channel opening in the presence of ATP.

Opening of Panx-1 channels in response to SARS-CoV-2 S protein or hCoV-229E or its S protein induces ATP, PGE2, and IL-1β release. Panx-1 channel opening has been associated with local and systemic inflammation as well as HIV entry into immune cells by a mechanism involving the local release of intracellular factors such as ATP, NAD + , prostaglandins, and other inflammatory lipids through the channel pore (Gajardo-Gomez et al., 2020b; Qu et al., 2011; Velasquez and Eugenin, 2014b; J o u r n a l P r e -p r o o f 2020). In addition, Panx-1 channel opening is indirectly associated with IL-1β processing and release (Lee et al., 2018; Pelegrin and Surprenant, 2006; 2009; Seil et al., 2010; Yang et al., 2019) , suggesting that Panx-1 amplifies inflammation, but its role in COVID-19 pathogenesis is unknown.

Human primary lung epithelial cells were treated with SARS-CoV-2 S Protein or 229E (whole virus or its S protein) for 1, 6, 12, and 24 h, and media was collected to determine ATP, PGE2, and IL1β release by ELISA (Fig. 3A , B, and C, respectively). In untreated control conditions (denoted by C in Fig. 3A -C), minimal baseline ATP, PGE2, and IL-1β release were observed (Fig. 3A , B, and C, respectively). Treatment with SARS-CoV-2 S protein (S protein) or 229E virus-induced ATP and PGE2 release, but treatment with SARS-CoV-2 S protein resulted in a greater than two-fold increase in the secretion of both ATP and PGE2 than 229E ( Fig. 3A and B, respectively, *p≤0.001, n=4 independent experiments, only 1 h is shown, but data were consistent after 6, 12, and 24 h). These differences in ATP, PGE2, and IL-1β release correlated with the extent and degree of Panx-1 channel opening observed by dye uptake between both viral components ( Fig. 1 ). In contrast, IL-1β release was similar for both S protein and 229E responses. Furthermore, we demonstrated that ATP, PGE2, and IL-1β release in response to S protein or 229E were Probenecid (Pro) and Panx-1 blocking mimetic peptide (pep) sensitive (Fig. 3A , B, and C; Pro and pep, respectively), supporting the idea that their secretion is Panx-1 dependent. Pre-incubation with Probenecid (Pro) or Panx-1 blocking peptide (pep) did not affect the baseline release of ATP, PGE2, and IL-1β (data not shown). In addition, pre-incubation with Cx43 hemichannels blockers such J o u r n a l P r e -p r o o f as Lanthanum (La 3+ ), a general connexin hemichannel blocker, or Cx43 E2 , an antibody that blocks Cx43 hemichannels, did not prevent the release of ATP, PGE2, and IL-1β ( Figure S2 ). Furthermore, a scrambled Panx-1 peptide (Sc) did not prevent the Panx-1 channel opening and ATP/PGE2/IL-1β release induced by S protein or 229E (Fig. 3 A, B, and C, respectively). Overall, we identified that Panx-1 channel opening induced by SARS-CoV-2 recombinant S protein or hCoV-229E (whole virus or its S protein) resulted in inflammation.

To determine the mechanism of Panx-1 channel opening induced by SARS-CoV-2 S protein and hCoV-229E (whole virus or its S protein), as well as the differences observed in dye uptake/electrophysiology/secretion of intracellular inflammatory factors between both coronaviruses, we focused on the differences in the cell receptor, endocytosis, and furin dependency described for both viruses (Shang et al., 2020; Walls et al., 2020c) .

To examine these mechanisms, we pre-treated human primary lung epithelial cell cultures with a human recombinant protein angiotensin-converting enzyme-2 (hrACE-2, 0.4 µg/ml) before treatment with SARS-CoV-2 S protein (S protein, 1μg/mL) or 229E (0.1 MOI) to assess the competitive binding of the virus to the endogenous ACE-2 protein as described (Lu and Sun, 2020) . To prevent endocytosis, we used the endocytosis inhibitors ammonium chloride (NH4Cl) (50 mmol/L), bafilomycin-A1 (50 nmol/L), and chloroquine (100 μg/ml). NH4Cl and chloroquine are lysosomotropic agents that selectively accumulate in endocytic compartments and increase the J o u r n a l P r e -p r o o f endosomal pH. Bafilomycin-A1 (50 nmol/L) is a specific blocker of v-type H + -ATPase (Eugenin et al., 2008) . Concentrations of these reagents were selected as such because they had been optimized to maximally inhibit the entry of avian leukosis virus into cells, a virus that requires endocytosis for infection as well as into smooth muscle cells (Eugenin et al., 2008) . Additionally, pre-incubation of S protein or 229E (whole virus or its S protein) infected cells with furin (0.86 µg/ml) to promote the furin cleavage at the S1-S2 junction present in the SARS-CoV-2 S protein, but not present in other coronaviruses was performed. SARS-CoV-2 S protein, therefore, has a furin cleavage site that strengthens the viral-host interaction through the ACE-2/S-protein interaction by ten-fold (Walls et al., 2020b ) as compared to other coronaviruses, including hCoV-229E (Ge et al., 2013; Lau and Peiris, 2005) , resulting in enhanced infectivity (Li et al., 2005) .

For this experiment, we confirmed that Etd uptake in control-untreated conditions was minimal ( Fig. 4 A-F, at 0-, 15-and 30-min. Dye uptake rate quantification is shown in Fig. 4S and T, black, Control). Treatment with S protein or 229E induced Panx-1 opening as detected by Etd uptake (Fig. 4G -L, for S protein at 0-, 15-and 30-min, data for 229E not shown). Dye uptake quantification is shown in Fig. 4S and T, S protein and 229E, respectively in red). The pre-incubation of primary lung epithelial cells with hrACE-2 to compete for the binding of SARS-CoV-2 S-protein to the host ACE-2 prevented the opening of Panx-1 channels induced by S protein (Fig. 4S , hrACE-2+S protein, blue). However, hrACE-2 did not affect the Panx-1 channel opening induced by 229E, suggesting a different opening mechanism than the S protein ( Fig. 4T , ACE-2+229E, blue). Pre-treatment (10 min) of the human primary lung epithelial cells with the endocytosis inhibitors, ammonium chloride (NH4Cl), bafilomycin-A1, or chloroquine, prevented the Panx-1 channel opening induced by the S protein and 229E virus, suggesting that endocytosis is essential to trigger Panx-1 channel opening ( Fig. 4S and T, green, Endo+ S protein or Endo+229E, respectively).

SARS-CoV-2 S protein harbors a furin cleavage site at the S1/S2 boundary not present in other coronavirus S proteins, suggesting higher transmissibility and pathogenesis are associated with this change (Belouzard et al., 2009; Millet and Whittaker, 2015; Walls et al., 2020a) . Thus, to determine whether furin processing of SARS-CoV-2 S protein participates in the Panx-1 channel opening, we preincubated the S protein and 229E with furin (0.86 µg/ml) for 10 min and then added them to the human lung primary epithelial cells to determine Panx-1 channel opening by dye uptake. Furin-treated S protein induced a greater and more prolonged Panx-1 channel opening than untreated S protein treatment (compare Fig pg/ml, p=0.563, n=4) compared to S protein without furin pre-treatment. In contrast, furin treatment of 229E did not significantly affect the secretion of ATP, PGE2, and IL1β

(compared to Fig. 3 ). 229E (the whole virus or 1-5 µg/ml of S protein) treatment for 1 h induced an ATP (18.5±7.78 nM), PGE2 (1.12±0.21 nM), and IL1β (21.02±6.08 pg/ml) release from human epithelial cells suggesting a different mechanism of cell activation compared to SARS-CoV-2. Further, experiments with 299E S protein indicated that ATP secretion was comparable to the whole virus (16.34±9.32 nM). The increase in MOI (10, 5, and 1) did not mimic the data obtained using SARS-CoV-2 S protein, indicating that protein concentration or viral titer do not account for the differences in the inflammatory profile. Furin, endocytic blockers, hrACE-2, or BSA alone, used as a control, did not affect the basal Panx-1 channel opening or ATP, PGE2, and IL1β secretion during the time course analyzed or triggered apoptosis (data not represented). Overall, our data demonstrate that SARS-CoV-2 has an inflammatory profile that is different from hCoV-229E. 

of Panx-1 protein expression. We next conducted immunofluorescence staining for Panx-1 and other cellular markers to determine the expression and distribution in human lung tissue samples using confocal microscopy under control and COVID-19

conditions. Tissue samples infected with SARS-CoV-2 were obtained from a rapid autopsy process from Assistance Publique-Hopitaux de Paris (AP-HP) at Raymond Poincaré Hospital, Garches, France, and UTMB. As a control, uninfected lung biopsy and tumor border tissue (tumor-healthy tissue) were obtained from the Anatomic Pathology Laboratory at UTMB. To ensure an unbiased assessment, patient personal information was not collected during data collection, and all samples were received and analyzed blindly. After all the data were acquired, clinical and COVID-19 status was requested to guarantee proper scientific rigor.

Staining was conducted for DAPI (a nuclear marker), Panx-1, EpCam (an epithelial marker) or Iba-1 (a macrophage marker), and SARSsense (a probe for SARS-CoV-2 mRNA using the RNAscope V-nCoV2019-sense probe). Analysis of control J o u r n a l P r e -p r o o f uninfected tissues indicates that Panx-1 protein was expressed in epithelial cells and macrophages at the alveolar wall ( Fig. 7A and a higher magnification of the alveolar wall is shown in Fig. 7B ). As predicted, no staining for the SARSsense probe was detected in the controls ( Fig. 7A and B Quantification of the total pixels per area (100 A.U. representing saturation, A.U.)

indicates that staining for EpCam was high for the control lung tissue in control conditions. All COVID-19 cases analyzed indicated that EpCam staining was at low to undetectable levels, demonstrating the large degree of lung damage ( (Meyer, 2007; Meyer and Zimmerman, 2002) . scRNAseq identified several cell types from the BAL, including immune cells (Berman et al., 2021; Stanczak et al., 2021) and lung cells (Berman et al., 2021) , supporting the concept of extensive lung damage within COVID-19 cases, as indicated in Fig. 6 . Most of these cells are released into the nasal scrape, and lung staining indicates a widespread expression of Panx-1 mRNA and protein that, in vitro, correlates with the secretion of ATP, PGE2, and IL-1β release.

Thus, to determine, in vivo, whether these products are concentrated in patient lung secretions, we quantified these inflammatory factors in BAL by ELISA. The BAL was collected from patients living with COPD for at least 8 years (10.02±5.04 years,

COVID-19 is an unprecedented pandemic that mainly affects the respiratory and immune systems. The rapid development and administration of vaccines have stymied its uncontrolled spread, resultant hospitalization, and death (Frederiksen et al., 2020; Hodgson et al., 2021; Ledford, 2020; Zhao et al., 2020) . However, the long-term consequences of the infection are still unknown. Therefore, understanding the pathogenesis of SARS-CoV-2 and progression into COVID-19 is urgent.

Here, we identified that Panx-1 channels become open upon binding the SARS-CoV-2 Spike protein, the hCoV-229E virus, or its S protein. The Panx-1 opening upon SARS-CoV-2 infection was dependent upon ACE-2, furin, and endocytosis, and that the Panx-1 opening resulted in the release of the pro-inflammatory biomolecules such as ATP, PGE2, and IL-1β into the extracellular space. Analysis of Panx-1 expression and distribution indicates that SARS-CoV-2 infection and COVID-19 disease are associated with enhanced inflammation and suggests that targeting Panx-1 channel opening or the subsequent release of intracellular inflammatory factors could provide alternative mechanisms of preventing COVID-19 associated damage or mitigating disease progression. Therefore, we propose that Panx-1 channels are a new host protein pathway required for SARS-CoV-2 infection and signaling.

Panx-1 channels participate in several inflammatory conditions exacerbated during COVID-19 pathogenesis, including hypoxia, coagulation, blood pressure, endothelial permeability, and apoptosis (Abdeen et al., 2021; AbdelMassih et al., 2021; Chang et al., 2020; Contoli et al., 2021; Hertzog et al., 2021; Li et al., 2020; J o u r n a l P r e -p r o o f 2020b; Montero et al., 2020; Page et al., 2021; Taz et al., 2021; Toldo et al., 2021; Wang et al., 2020) . We propose that upon Panx-1 channel opening, several biomolecules are released into the extracellular space and result in modulation of COVID-19 pathogenesis: first, ATP release and modification of local signaling enable viral entry through the activation of purinergic receptors; second, IL-1β release results in a pro-inflammatory response and recruitment of leukocytes into the area of infection (Kim et al., 2015) ; and third, the release of PGE2 into the extracellular matrix and its role in coagulation/vascular compromise (Friedman et al., 2015; Gross et al., 2007) highlights its importance in extensively vascularized regions that become damaged when challenged by conditions such as SARS-CoV-2 infection. These three mechanisms and their dysregulation during COVID-19, therefore, may partially or cumulatively contribute to the pathogenesis and progression of SARS-CoV-2 infection.

In HIV, we previously determined that the binding of gp120 to CD4 and CCR5/CXCR4 receptors and co-receptors induces opening of Panx-1 channels, ATP release, and subsequent purinergic receptor activation to enable HIV entry and subsequent undefined replication steps in macrophages and T-cells (Gajardo-Gomez et al., 2020a; Hazleton et al., 2012; Orellana et al., 2013; Velasquez and Eugenin, 2014a) .

We believe that this specific gp120 trigger results in intracellular calcium signaling and actin rearrangement that allows HIV to fuse with the host plasma membrane (Gajardo-Gomez et al., 2020a; Hazleton et al., 2012; Velasquez et al., 2020) . SARS-CoV-2 infectivity is initiated by binding of the virus to ACE-2, mainly expressed in the lung, kidney, and vascular endothelium (Zamorano Cuervo and Grandvaux, 2020; Zou et al., 2020) . A critical difference between SARS-CoV-2 and other coronaviruses is the furin J o u r n a l P r e -p r o o f (or protease)-dependent site that results in a 10-fold increase in the binding of ACE-2 to promote viral entry (Walls et al., 2020a) . This site is at the S1/S2 boundary of SARS-CoV-2 S protein (Walls et al., 2020c) . This difference has been associated with enhanced infectivity and potentially contributes to the high pathogenesis of SARS-CoV-2 over other coronaviruses.

Interestingly, this type of mutation or adaptation has been observed in several highly pathogenic viruses such as avian influenza and Newcastle virus (Klenk and Garten, 1994; Steinhauer, 1999; Walls et al., 2020c) . These changes could increase viral infectivity, transmissibility, and pathogenesis of viral infection. Subsequently, when we compared the effects of SARS-CoV-2 infection to hCoV-229E viral infection, Panx-1 opening and its sensitivity to furin, ACE-2, and endocytosis were more evident with SARS-CoV-2, suggesting that the S protein of SARS-CoV-2 is not only important for entry but also associated with inflammation and the pathogenesis of the virus.

Additionally, our ATP and PGE2 data demonstrated that the SARS-CoV-2 S protein, in contrast to the hCoV-229E virus, induced the release of these inflammatory factors. The differences in inflammation and Panx-1 channel opening between SARS-CoV-2 to hCoV-229E viruses cannot be explained due to MOI or protein concentrations due to the fact that IL1β secretion was similar. In the future, it will be interesting to determine whether the recent vaccines for COVID-19 can prevent viral entry and Panx-1associated inflammation or whether both effects could be separated.

Normally, Panx-1 channels exist in a closed state in healthy conditions (Pelegrin and Surprenant, 2006; Saez et al., 2010; Seror et al., 2011; Swayne and Boyce, 2017) .

Their focus as a target of therapies is potentially valuable since they are uniquely implicated in people compromised with the disease, such as HIV, and therefore expected to have minimal side effects in healthy conditions. For example, Probenecid is an FDA-approved drug to treat gout and is an excellent Panx-1 blocker, as second, prevention of the inflammasome activation; and lastly, maintenance of effective concentrations of drugs inside of the cells. This last one is of particular interest because when the Panx-1 channel opens to release ATP and other intracellular factors, drugs that need to reach an effective intracellular concentration can "leak" into the extracellular space through these same channels. Additionally, Panx-1 blockers also reduce the secretion of proteases into the extracellular space, such as cathepsins (Swayne et al., 2020) . Extracellular proteases, such as furin and cathepsins, promote SARS-CoV-2 infection over other coronaviruses, as demonstrated by others (Bestle et al., 2020; Dessie and Malik, 2021; Huffman et al., 2021; Murgolo et al., 2021; Raghav et al., 2021; Rossi et al., 2020; Watzky et al., 2021; Zhang et al., 2021) and our current data support these findings by identifying a novel mechanism of virus-mediated inflammation. Our data that ATP, PGE2, and IL1β are highly concentrated in the BAL J o u r n a l P r e -p r o o f obtained from COVID-19 individuals is exciting but needs to be considered carefully due to the fact that during the evolution of the disease, significant apoptosis of lung cells occurs . This apoptosis then contributes to the nonspecific release of ATP.

Glycyrrhizic acid derivates such as 18-α/β glycyrrhetinic acid and carbenoxolone are most likely the most common gap junction and Panx-1 channel blockers (Patel et al., 2014) . Glycyrrhizic acid inhibits SARS-CoV replication in Vero cells with high selectivity while conferring high protection with minimal adverse reactions (Hoever et al., 2005; Ming and Yin, 2013; Utsunomiya et al., 1997) . Simoes and Bagatini, 2021) . Our data in long-term HIV-infected individuals indicate that several chronic effects of HIV are correlated with ATP dysfunction and vascular disease, such as cognitive impairment (Velasquez et al., 2020) . A critical characteristic of the COVID-19 tissues analyzed is the localized infiltration of immune cells, fibrosis, hemorrhagic events, and intravascular coagulation, unlike other diseases.

Our data indicate that platelets are a key component of the SARS-CoV (Abbracchio and Ceruti, 2006; Hechler and Gachet, 2015) . Upon activation, platelets become prothrombotic and accelerate the release of pro-inflammatory factors to promote vasoconstriction and coagulation (Gachet and Hechler, 2020; Grenegard et al., 2008; Mahaut-Smith et al., 2016; Manica et al., 2018; Oury and Wera, 2021) . It is also known that purinergic and thrombin can synergize (Dorsam and Kunapuli, 2004; Nylander et al., 2003; 2004) to contribute to the extensive coagulation observed during COVID-19 pathogenesis. Platelet activation by ATP and thrombin further promote ADP release from dense granules (Woulfe et al., 2001) , potentially resulting in a vicious cycle observed in deadly cases of COVID-19. Other purinergic receptors, such as P2Y2, P2Y6, and P2X7, could also contribute to pathological effects in pulmonary cell types. P2Y2, activated by ATP but not ADP (Abbracchio and Ceruti, 2006) , regulates endothelial inflammation, most notably promoting the adhesion and chemotaxis of inflammatory cells (Alberto et al., 2016; Burnstock, 2017; Buscher et al., 2006; Gabl et al., 2015; Liu et al., 2016; Marcet et al., 2004; Muhleder et al., 2020; Sophocleous et al., 2020; van Heusden et al., 2020) .

P2Y12 has also been demonstrated to contribute to endothelial cell pathology (Sidiropoulou et al., 2021; Vemulapalli et al., 2020) . Purinergic receptor activation has also been implicated in the dysregulation of Ca 2+ signaling and wave propagation (MacVicar and Thompson, 2010) . Interestingly, other studies have also demonstrated that Ca 2+ signaling may result from direct calcium leakage through Panx-1-mediated calcium channels out to the extracellular space and affect intercellular communication, wave propagation, and homeostasis (Vanden Abeele et al., 2006) . Panx-1 channel J o u r n a l P r e -p r o o f permeability and opening potentially have far-reaching implications in several pathways that we have partially addressed in this manuscript; however, we still need to examine the distribution, function, and potential therapeutic use of these receptors to prevent or revert the early and long-term consequences of SARS-CoV-2 infection.

First, the mechanism of Panx-1 channels opening and ATP secretion induced by SARS-CoV-2 S protein but not by the hCoV-229E virus is unknown. However, it is dependent on furin activity, but how this cleavage alters channel gating and permeability is 

All authors declare no competing interests.

J o u r n a l P r e -p r o o f Alves, V.S., Leite-Aguiar, R., Silva, J.P.D., Coutinho-Silva, R., and Savio, L.E.B. (2020) . Purinergic signaling in infectious diseases of the central nervous system. Brain Behav Immun 89, 480-490. 10.1016/j.bbi.2020.07.026 . Anthonypillai, C., Sanderson, R.N., Gibbs, J.E., and Thomas, S.A. (2004) . The distribution of the HIV protease inhibitor, ritonavir, to the brain, cerebrospinal fluid, and choroid plexuses of the guinea pig. J Pharmacol Exp Ther 308, 912-920. 10.1124 /jpet.103.060210. Ballestar, E., Farber, D.L., Glover, S., Horwitz, B., Meyer, K., Nikolić, M., Ordovas-Montanes, J., Sims, P., Shalek, A., Vandamme, N., et al. (2020 . Single cell profiling of COVID-19 patients:

an international data resource from multiple tissues. medRxiv, .20227355. 10.1101 .11.20.20227355. Bao, L., Deng, W., Huang, B., Gao, H., Liu, J., Ren, L., Wei, Q., Yu, P., Xu, Y., Qi, F., et al. (2020 . The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice. Nature 583, 830-833. 10 .1038/s41586-020-2312-y. Baranova, A., Ivanov, D., Petrash, N., Pestova, A., Skoblov, M., Kelmanson, I., Shagin, D., Nazarenko, S., Geraymovych, E., Litvin, O., et al. (2004) . The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins. Genomics 83, 706-716. 10.1016/j.ygeno.2003.09.025 S0888754303003124 [pii] . Barnes, B.J., Adrover, J.M., Baxter-Stoltzfus, A., Borczuk, A., Cools-Lartigue, J., Crawford, J.M., Dassler-Plenker, J., Guerci, P., Huynh, C., Knight, J.S., et al. (2020) . Targeting Relative to the control (C), SARS-CoV-2 S protein and hCoV-229E treatment induced an ATP secretion that was strongly Panx-1 dependent. Treatment with the S proteininduced ATP secretion was ~2.5-fold more effective than treatment with 229E (*p≤0.001, n=4). Pre-treatment with Panx-1 blockers, Probenecid, or the Panx-1 mimetic peptide, before treatment with the S Prot or 229E, did not result in significantly elevated concentrations of ATP. Additionally, pre-treatment with Scram before treatment with S protein or 229E resulted in elevated concentrations of ATP comparable to cells treated with S protein or 229E alone, respectively (*p≤0.001, n=4). Each value corresponds to the mean ± SD (n=4). (B) Determination of PGE2 secretion from primary human airway epithelial cells for the control (C) and after S protein (S Prot) or hCov-229E virus (229E) treatment in the presence or absence of Probenecid (Pro), Panx-1 mimetic peptide (pep), or the scrambled peptide (Sc). Relative to the control (C), SARS-CoV-2 S protein J o u r n a l P r e -p r o o f and hCoV-229E treatment induced a PGE2 secretion that was also strongly Panx-1 dependent. Treatment with the S protein-induced PGE2 secretion was also ~2.5-fold more effective than treatment with 229E (*p≤0.001, n=4). Pre-treatment with Panx-1 blockers, Probenecid, or the Panx-1 mimetic peptide before treatment with the S Prot or 229E did not result in significantly elevated concentrations of ATP. Additionally, pretreatment with Scram before treatment with S protein or 229E resulted in elevated concentrations of PGE2 comparable to cells treated with S protein or 229E alone, respectively (*p≤0.001, n=4). Each value corresponds to the mean ± SD (n=4). (C)

Determination of IL-1β secretion from primary human airway epithelial cells for the control (C) and after S protein (S Prot) or hCoV-229E virus (229E) treatment in the presence or absence of Probenecid (Pro), Panx-1 mimetic peptide (pep), or the scrambled peptide (Sc). Relative to the control (C), SARS-CoV-2 S protein and hCoV-229E treatment-induced IL-1β secretion was also strongly Panx-1 dependent.

Treatment with the S protein-induced IL-1β secretion was similar to treatment with 229E (*p≤0.001, n=4). Pre-treatment with Panx-1 blockers, Probenecid, or the Panx-1 mimetic peptide before treatment with the S Prot or 229E, did not result in significantly elevated concentrations of ATP. Additionally, pre-treatment with Scram before treatment with S protein or 229E resulted in elevated concentrations of IL-1β comparable to cells treated with S protein or 229E alone, respectively (*p≤0.043, n=3, relative to control conditions).

Each value corresponds to the mean ± SD (n=3). It should be noted that although IL-1β secretion is correlated with Panx-1 channel activity, it is an indirect measure of cellular activation.

J o u r n a l P r e -p r o o f mmol/L; bafilomycin-A1, 50 nmol/L: and chloroquine, 100 μg/mL, green downward triangle), or furin (0.86 µg/ml, pink leftward triangle). Furin pre-treatment of S protein resulted in the most significant uptake of Etd even relative to treatment with S protein alone (p≤0.00025, n=3, between 4-24 min for S protein and 4-42 min for furin-treated S protein compared to control conditions. Each value corresponds to the mean ± SD (n=3). (T) Quantification of the time course of Etd uptake for the airway epithelial cells that were untreated (black square) or treated with the hCoV-229E (229E) alone (red circle) or 229E pre-treated with human recombinant ACE-2 protein (0.4 µg/ml, blue upward triangle), endocytotic inhibitors (NH4Cl, 50 mmol/L; bafilomycin-A1, 50 nmol/L: J o u r n a l P r e -p r o o f and chloroquine, 100 μg/mL) (green downward triangle), or furin (0.86 µg/ml, pink leftward triangle). For this experiment, Panx-1 channel opening induced by hCoV-229E was only dependent on endocytosis but independent of ACE-2 and furin pathways (p≤0.0012, n=3) compared to control conditions. Each value corresponds to the mean ± SD (n=3). 

 This study did not generate new unique reagents.

 Unique standardized data types were not generated. All other unique data will be shared upon reasonable request to the lead contact.

 This study did not generate unique original code.

 Any additional information required to re-analyze the data reported in this paper is available upon reasonable request to the lead contact. for 30 min as described (Loveday et al., 2021) . Additional methods are presented and described in detail in the STAR method section.

Single-cell RNA sequencing nasal epithelia: Sequencing data was obtained from publicly available information generated by the scientific community and characterized through the COVID-19 Cell Atlas Project (https://www.covid19cellatlas.org/). Public datasets used in this publication were specifically generated by the Vieira Braga and

Shalek labs and approved for both groups. Nasal epithelia were collected by nasopharyngeal brushes or swabs. The Vieira Braga dataset was collected from an upper airway nasal brush from patients not infected with COVID-19. The Shalek set was collected from nasal epithelial scrapes from uninfected and COVID-19 infected patients.

As previously described, samples were collected from patients (Delorey et al., 2021; Ziegler et al., 2021) . Representative cell populations were selected to display the relative expression of Panx-1 mRNA.

J o u r n a l P r e -p r o o f ATP Assay. ATP concentration was determined using the ATPlite luminescence assay system (PerkinElmer, MA) by combining 100 μL of the sample with 100 μL of ATPlite reagent. Luminescence was measured using a PerkinElmer EnVision Multilabel Plate

Reader. The extracellular concentration of ATP was determined by comparing sample luminescence to a standard curve generated using ATP standards provided by the manufacturer.

Analysis of IL-1β and PGE2 release. Tissue culture media and BAL were collected and inactivated at 65 o C for 30 min as described for SARS (Rabenau et al., 2005a; Rabenau et al., 2005b; Rabenau et al., 2005c) and stored at −80ºC. There were no freeze-thaw cycles before analysis. Samples were analyzed for IL-1β (Quantikine ELISA kit; R and D Systems, Minneapolis MN, USA) and PGE2 (Abcam, Cambridge, MA, USA) by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.

Quantification and Statistical Analysis. 

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CV) infected conditions was low but significant for EpCam (CV, p=0.00025, n=9 cases with 6 sections per individual). In contrast, Panx-1 expression was high and significant (CV, p=0.00153, n=9 cases with 6 sections per individual). SARSsense (Sense) staining for mRNA was low but significant

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Panx-1 expression was high and significant p=0.00102, n=9)

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Panx-1 staining in COVID-19-infected patients was ~2-fold higher relative to the control and indicated an increase in expression of Panx-1 in macrophages

Similarly, red blood cell (RBC) hemoglobin

Epithelial Cell Basal Medium (ATCC Cat#: PCS-300-030) supplemented with Bronchial Epithelial Cell Growth Kit (ATCC Cat#: PCS-300-040) and (Cat:11875-093), penicillin/streptomycin (Cat

Fetal bovine serum (Cat: S11150H) was purchased from R&D Systems (Minneapolis, MN). The SARS-229E isolate was obtained from the ATCC (ATCC Cat: VR-740). SARS-CoV-2 Spike-Membrane protein was purchased from

) unless otherwise designated. Methods SARS-CoV-2 and hCoV-229E-infection and S-protein treatment

Control (CDC) and the National Institute of Health (NIH)

Experiments were carried out 2 days post-transfection. Also, we used a siRNA to Cx43 was used as a control. The Panx-1 mimetic blocking peptide 10 Panx-1 (WRQAAFVDSY) and the scrambled peptide (FADRYWAQVS) were synthesized by Peprotech, NJ. Dye uptake and time-lapse fluorescence imaging. To characterize the functional state of Panx-1 channels, dye uptake experiments using ethidium (Etd) bromide were performed as described previously

Cells were washed twice in Hank´s balanced salt solution and then exposed to Locke's solution (containing: 154 mM NaCl, 5.4 mM KCl, 2.3 mM CaCl2, 5 mM HEPES

This calculation (F1-FB) -buffered saline (H-PBS) containing (mM): NaCl 147

SARS-CoV-2 detection by RT-qPCR. 500 µl of blood and processed for Reverse transcription and one-step quantitative polymerase chain reaction (RT-qPCR) as described (Real et al., 2020). Total RNA was extracted from 150 µl of PPP using

Mini kit for CE-certified purification of viral RNA/DNA (Macherey-Nagel) according to the manufacturer's recommendations. RT-qPCR was performed from 50 µl of eluted RNA, using TaqMan RNA-to-CT 1-step Kit

Briefly, 4 targets are enrolled in our system. ORF1 gene (specifically the region encoding RNA-dependent RNA polymerase, RdRp), S gene (encoding Spike protein), and N gene (encoding nucleocapsid protein) aim at SARSCoV-2, human RNase P RPPH1 gene runs in a duplex with each SARS-CoV-2 assay, serving as an internal positive control. TaqMan 2019nCoV Control Kit v1 (Thermo Scientific) was used together to monitor assayspecific amplification. For each RT-qPCR reaction

Lung tissue sections were cut and processed, as described recently

The lung tissue sections were treated with the RNAscope Multiplex Fluorescent Reagent Kit v2 Assay protocol (ACDbio), following manufacturer instructions. The procedure includes multiple steps of sample pre-treatment, including RNAscope target retrieval reagent for 15 min, RNAscope protease plus for 15 min, hybridization (RNA probe V-nCOV2019-S-sense, specific for SARS-CoV-2), and signals development (TSA Plus cyanine 5 fluorophore, suggested

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The authors acknowledge financial support from the National Institute of Health grant:MH096625/MH128082, The National Institute of Neurological Disorders and Stroke, NS105584, and UTMB internal funding (to E.A.E)