Differential Signaling and Virus Production in Calu-3 Cells and Vero Cells upon SARS-CoV-2 Infection

Abstract

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19) pandemic. Signaling pathways that are essential for virus production have potential as therapeutic targets against COVID-19. In this study, we investigated cellular responses in two cell lines, Vero and Calu-3, upon SARS-CoV-2 infection and evaluated the effects of pathway-specific inhibitors on virus production. SARS-CoV-2 infection induced dephosphorylation of STAT1 and STAT3, high virus production, and apoptosis in Vero cells. However, in Calu-3 cells, SARS-CoV-2 infection induced long-lasting phosphorylation of STAT1 and STAT3, low virus production, and no prominent apoptosis. Inhibitors that target STAT3 phosphorylation and dimerization reduced SARS-CoV-2 production in Calu-3 cells, but not in Vero cells. These results suggest a necessity to evaluate cellular consequences upon SARS-CoV-2 infection using various model cell lines to find out more appropriate cells recapitulating relevant responses to SARS-CoV-2 infection in vitro.

Keywords: Apoptosis; COVID-19; SARS-CoV-2; STAT1; STAT3; STAT3 phosphorylation.

Fig. 1

Differential production of SARS-CoV-2 in Vero and Calu-3 cells. Vero and Calu-3 cells (5×10⁴ cells/well in 12-well plates) were cultured in DMEM containing 10% FBS. After overnight culture, cells were washed with PBS and SARS-CoV-2 in PBS at a MOI of 0.1 was added into each well (n=3). After 1 h of incubation, the medium was replenished with DMEM containing 2% FBS. (A) Supernatants of virus-infected cell cultures were collected at the indicated times after virus infection to isolate viral RNAs. Virus replication was quantified by real-time RT-PCR analysis of the SARS-CoV-2 RdRP gene. Copy numbers of the RdRP gene in 1 µL of the cDNA samples were calculated using a standard curve of RdRP cDNA. (B) Supernatants of virus-infected Vero and Calu-3 cells (12-well plates) were collected at the indicated times after virus infection. Virus production was quantified by plaque formation assay. *p<0.05, **p<0.01, ***p<0.001.

Fig. 2

Effect of SARS-CoV-2 on induction of apoptosis. (A, B) Vero (A) and Calu-3 (B) cells were infected with 0.5 MOI of SARS-CoV-2 or without virus for the indicated times. After 1 h of incubation, the medium was replenished with DMEM containing 2% FBS. After 72 h of incubation, cell lysates were prepared, and Western blot analysis was performed to detect PARP, cleaved Caspase-3, c-Myc, and cyclin D1. β-actin was used as the control.

Fig. 3

STAT1 and STAT3 phosphorylation in SARS-CoV-2-infected Vero and Calu-3 cells. (A) Vero cells were infected with 0.5 MOI of SARS-CoV-2 (left) or were mock-infected (right) for the indicated times. (B) Calu-3 cells were infected with 0.5 MOI of SARS-CoV-2 (left), indicated MOIs (middle), or were mock-infected cells (right) for the indicated times with the indicated MOIs. Western blotting of the cell lysates was performed with the indicated antibodies. β-actin was used as the control.

Fig. 4

Effects of pathway-specific inhibitors on SARS-CoV-2-induced STAT1 and STAT3 phosphorylation in Calu-3 cells. (A) Calu-3 cells were pretreated with 0.1% DMSO, 10 μM PD169316, 25 μM PD98059, 25 μM SP600125, 25 μM AG490, or 1 μM JAK inhibitor I for 30 min. (B) Calu-3 cells were pretreated with the indicated concentrations of S3I-201 inhibitor for 30 min. The cells were washed with PBS and then infected with SARS-CoV-2 in PBS at 0.5 MOI. After 1 h of incubation, the medium was replenished with DMEM containing 2% FBS. (C) Calu-3 cells were treated with 0.1% DMSO, 10 μM PD169316, 25 μM PD98059, 25 μM SP600125, 25 μM AG490, 1 μM JAK inhibitor I, or 20 μM S3I-201. (A-C) After 48 h of incubation, cell lysates were prepared, and Western blotting was performed with the indicated antibodies. (D) Calu-3 cells were pretreated with 0.1% DMSO, 25 μM AG490, 1 μM JAK inhibitor I, 20 μM S3I-201, or 10 μM STA-21 for 30 min. The cells were washed with PBS and then infected with SARS-CoV-2 in PBS at 0.5 MOI. After 1 h of incubation, the medium was replenished with DMEM containing 2% FBS. After 48 h of incubation, the cells were treated with 20 nM Leptomycin B for 3 h. To evaluate phosphorylation and localization of STAT3, cells were fixed with 4% paraformaldehyde, stained with phospho-STAT3 antibody (green), and observed by confocal microscopy. Hoechst 33258 was used for nuclei staining (blue).

Fig. 5

Production of SARS-CoV-2 in Vero and Calu-3 cells in the presence of pathway-specific inhibitors. (A, B) Vero cells were cultured overnight in 24-well plates and pretreated with PBS, 0.1% DMSO, 1 μM JAK inhibitor I, 20 μM S3I-201, or 10 μM STA-21 for 30 min. The cells were washed with PBS and infected with SARS-CoV-2 in PBS (0.1 MOI). After 1 h of incubation, the medium was replenished with DMEM containing 2% FBS. After 3 days of incubation, virus production was measured by the plaque formation assay (A) and real-time RT-PCR analysis of the SARS-CoV-2 RdRP gene (B). (C, D) Calu-3 cells in 12-well plates were pretreated with PBS, 0.1% DMSO, 1 μM JAK inhibitor I, 20 μM S3I-201, or 10 μM STA-21 for 30 min. The cells were washed with PBS and infected with SARS-CoV-2 in PBS at 0.1 MOI. After 72 h of incubation, virus production was measured by the plaque formation assay (C) and real-time RT-PCR analysis of the SARS-CoV-2 RdRP gene (D). *p<0.05, **p<0.01, ***p<0.001 compared to virus only controls.

Fig. 6

Possible STAT3 signal transduction pathways in SARS-CoV-2-infected Vero Cells and Calu-3 cells. SARS-CoV-2 enters host cells by binding to the cell surface receptor ACE2. Cellular responses to SARS-CoV-2 differ depending on the host cell type. In Vero cells, STAT1 and STAT3 are dephosphorylated within 2 days after virus infection and apoptosis occurs as observed by an increase in the amount of cleaved PARP and cleaved Caspase-3. In contrast, in Calu-3 cells, phosphorylation of STAT1 and STAT3 persists for days after virus infection and no prominent apoptosis occurs, but induction of proliferation-associated Cyclin D1 and c-Myc proteins occurs. Phosphorylation and dimerization of STAT3 are required for virus production in Calu-3 cells. Possible regulation of the STAT3 signaling pathways by SARS-CoV-2 proteins is shown.