Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells

Abstract

Stress granule formation is induced by numerous environmental stressors, including sodium arsenite treatment and viral infection. Accordingly, stress granules can inhibit viral propagation and function as part of the antiviral host response to numerous viral infections. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antagonizes stress granule formation, in part, via interaction between SARS-CoV-2 nucleocapsid (N) protein and Ras-GTPase-activating SH3-domain-binding protein 1 (G3BP1). However, it is unclear whether there are differential effects in different cell types. In this study, we assessed interaction between the N protein of SARS-CoV-2 S clade and G3BP1/2 in Vero and Calu-3 cells and investigated the effect of various SARS-CoV-2 strains on sodium arsenite-induced stress granule formation. Our data show that SARS-CoV-2 S clade N protein interacts with both G3BP1 and G3BP2 more strongly in Calu-3 vs. Vero cells. Consistent with this observation, infection with SARS-CoV-2 S clade induces stress granule formation in Vero but not in Calu-3 cells. However, infection with SARS-CoV-2 S clade, as well as other SARS-CoV-2 variants, inhibits sodium arsenite-induced stress granule formation in both cell lines. Taken together, our results show differential effects of SARS-CoV-2 infection on stress granule formation that is dependent on host cell type, rather than virus strain type.

Production of a Monoclonal Antibody to the Nucleocapsid Protein of SARS-CoV-2 and Its Application to ELISA-Based Detection Methods with Broad Specificity by Combined Use of Detector Antibodies

“…The cells were washed and infected with each SARS-CoV-2 variant at a multiplicity of infection (MOI) of 0.01 and incubated for 1 h at 37 °C with shaking every 20 min. After incubation, the medium was replaced with 2 mL of DMEM containing 2% FBS and cultured for 72 h. Thereafter, the cell culture supernatants were collected, and the viruses were quantified by plaque assay on Vero E6 cells as described previously [ 26 , 27 , 28 ]. After determining the pfu number, the virus stocks were stored at −70 °C for future use.…”

In vitro and in vivo suppression of SARS‐CoV‐2 replication by a modified, short, cell‐penetrating peptide targeting the C‐terminal domain of the viral spike protein

“…Virus titers were measured as described previously. 20,21 In brief, Vero E6 cells (7 × 10 5 cells/well) were cultured on six-well plates for 18 h and then infected with 10-fold serial dilutions of virus culture supernatants or tissue homogenates with shaking every 20 min at 37°C in a 5% CO 2 incubator. After 1 h incubation, the culture supernatants were aspirated, and the wells were overlaid with 3 mL DMEM/F12 medium (Thermo Fisher Scientific) containing 2% Oxoid agar and N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK, 1 µg/mL)-treated trypsin.…”

“…SARS-CoV-2 N protein expression was measured by confocal microscopy as described previously. 21 Vero cells or Calu-3 cells (5 × 10 4 cells/well) were plated on poly-L-lysine-coated cover glasses on 12-well culture plates, cultured in DMEM containing 10% FBS for 18 h, and then infected with SARS-CoV-2 (0.1 MOI) in phosphatebuffered saline (PBS) for 1 h. The plates were then replenished with DMEM containing 2% FBS. Three hours after infection, the cells were treated with the indicated concentrations of R-SARS-CoV-2 Spike CD, R-SARS-CoV-2 Spike CD(S), or R-t-Spike CD(D).…”

“…After 48 h, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% bovine serum albumin (BSA). The cells were then immunostained for 1 h with anti-SARS-CoV-2 N mAb (clone 1G10C4 mAb) 21,27 followed by Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific). Nuclei were stained with Hoechst 33258.…”

Multiple functions of stress granules in viral infection at a glance

“…The interaction between the N protein and G3BP1/2 supports SARS-CoV-2 infection. Some studies agree that the N protein could disrupt SG formation by sequestering G3BP1/2 from interacting with other proteins (Stukalov et al, 2021;Kim et al, 2022). The non-structural protein 1 (Nsp1) of the virus can decrease the level of G3BP1, which is associated with nuclear accumulation of the SG-nucleating protein TIAR (Dolliver et al, 2022).…”