ABSTRACT
Background: Respiratory epithelial cells (RECs) lining the upper airways are primary entry-point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID19). Managing the overwhelming inflammatory response and mucus hypersecretion are among the major challenges faced in achieving effective treatment. The present study analyzes the acute inflammatory and mucous responses following SARS-CoV-2 infection. Methods: Nasal swabs from COVID19 patients with mild and severe pathologies were analyzed for the expression of viral RNA (vRNA) for nucleocapsid (N) and spike (S) proteins, viral entry regulating host factors (ACE-2 and TMPRSS-2), epithelial inflammatory factors (IL-6, ICAM-1 and CXCL-8), respiratory mucins (MUC1, 2, 4, 5AC, 5B, 7, 16 and 19), mucin regulatory transcription factors (SPDEF and FOXA3) and select long noncoding RNAs (lncRNAs) by qRT-PCR. Sub-cellular localization and association of lncRNAs and SARS-CoV-2 vRNA was depicted by Dual-fluorescence in situ hybridization (FISH), and immunostaining for epithelial cell markers. A 3D in-vitro cell culture model was developed using primary human RECs differentiated on transwells at air-liquid interface and were infected with a SARS-CoV-2 clinical isolate (USA-WI1/2020) via apical as well as basal regions. Samples were collected at 0, 1, 4, 24 and 48 h postinfection (p.i.) and the expression of aforementioned factors were analyzed in cell lysates and media. Results: Severely affected patients showed significantly higher expression of IL-6, ICAM-1, and CXCL-8 along with the respiratory mucins, MUC4, 5AC, 16, and 19 and the transcriptional regulators, SPDEF and FOXA3 compared to the mild COVID19 patients. Our recently identified novel lncRNAs, LASI (lncRNA on antisense strand to ICAM-1), and TOSL (TNFAIP3-opposite strand lncRNA) were significantly higher in severe patients whereas NEAT1 and MALAT1 levels were lower as were the ACE2 and TMPRSS2. SARS-CoV-2 clinical isolate productively infected 3D human REC model with highest expression of SARS-CoV-2 N vRNA at 24 h p.i., and showed increased expression of inflammatory factors and LASI and TOSL at 1 h p.i. The dual-FISH staining of LASI and SARS-CoV-2 N1 vRNA validated that both the transcripts were enriched in nuclear/perinuclear region of RECs and, club cells and MUC5AC+ cells of severe COVID19.Conclusions: Together, these data indicate that severely infected COVID19 patients are impacted by high respiratory mucin expression and the associated airway inflammation. Interestingly, the lncRNAs, LASI and TOSL showed associated increased expression suggesting a possible role for these innate immunomodulators in SARS-CoV-2 induced innate airway mucosal responses.
ABSTRACT
Background: Since the outbreak of COVID-19, globally, more than 63 million people have been infected and 1.46 million people succumbed to death, and the number is still growing. It is well-established that attachment of spike glycoprotein of SARS-CoV-2 with ACE-2 is crucial for initiating infection. While a vaccine is awaited, alternate strategies can be adopted for blocking viral entry to host cells. Herein, we report two lead compounds that block the attachment of spike with ACE2 using lung epithelial cells. Methods: Methods: To identify the lead compounds, we conducted virtual screening of ∼3 million compounds that had potential to bind a site at ACE2/ Spike interface (PDB file 6M0J) using 'Glide' program of Schrödinger Suite. Then the combination of visual inspection and redocking with AutoDock Vina (to determine binding energy)was used to select 5 potential inhibitors of ACE2/ Spike interaction. These five compounds were then tested for their inhibitory activity in virological and biophysical assays. The inhibitory activity of these five compounds was measured using Vero-STAT1 knockout cells and a human bronchial epithelial cell line (UNCN1T). Results: Results: Of the five, two compounds, MU-UNMC-1 and MU-UNMC-2 with binding energy of-6.9kcal/mol and-7.8kcal/mol respectively, showed antiviral activity in two cell lines. In Vero-STAT1 cells, MU-UNMC-1 had IC50 of 5.35μM and 2.94μM, whereas MU-UNMC-2 had IC50 of 1.63μM and 0.54μM, after 24 and 48 hrs post infection (hpi), respectively. In UNCN1T cells, both compounds had significantly better efficacy. MU-UNMC-1 had an IC50 of 0.67μM and 1.16μM and at 24 and 48 hpi, respectively. MU-UNMC-2 had IC50 of 1.72μM and 0.89μM after 24 and 48 hpi, respectively. In Vero-STAT1 cells, the selectivity index (SI) (defined as CC50/IC50) of the compounds was 2.11(MU-UNMC-1) and 13.22(MU-UNMC-2), whereas in UNCN1T cells, the SI of the compounds was 9.27(MU-UNMC-1) and 4.15(MU-UNMC-2). Conclusion: Conclusion: We report the identification of two lead compounds (MU-UNMC-1 and MU-UNMC-2) that block the entry of SARS-CoV-2 in submicromolar concentration in biologically relevant human bronchial epithelial cells. Further, using structure-based similarity searches, we identified three additional chemotypes of these two compounds. These chemotypes of MUUNMC-3, MU-UNMC-4 and MU-UNMC-5) showed ∼2-fold better binding affinity with ACE2/Spike complex. These compounds are under investigation for their inhibitory effect in virological and biophysical assays.
ABSTRACT
The commonly used laboratory cell lines are the first line of experimental models to study the pathogenicity and performing antiviral assays for emerging viruses. Here, we assessed the tropism and cytopathogenicity of the first Swedish isolate of SARS-CoV-2 in six different human cell lines, compared their growth characteristics, and performed quantitative proteomics for the susceptible cell lines. Overall, Calu-3, Caco2, Huh7, and 293FT cell lines showed a high-to-moderate level of susceptibility to SARS-CoV-2. In Caco2 cells, the virus can achieve high titers in the absence of any prominent cytopathic effect. The protein abundance profile during SARS-CoV-2 infection revealed cell-type-specific regulation of cellular pathways. Type-I interferon signaling was identified as the common dysregulated cellular response in Caco2, Calu-3, and Huh7 cells. Together, our data show cell-type specific variability for cytopathogenicity, susceptibility, and cellular response to SARS-CoV-2 and provide important clues to guide future studies.