Type I Interferon Susceptibility Distinguishes SARS-CoV-2 from SARS-CoV

Abstract

SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While distinct from SARS-CoV, both group 2B CoVs share similar genome organization, origins to bat CoVs, and an arsenal of immune antagonists. In this report, we evaluate type I interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication to SARS-CoV, the novel CoV is much more sensitive to IFN-I. In Vero E6 and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I pretreatment, whereas SARS-CoV is not. In line with these findings, SARS-CoV-2 fails to counteract phosphorylation of STAT1 and expression of ISG proteins, while SARS-CoV is able to suppress both. Comparing SARS-CoV-2 and influenza A virus in human airway epithelial cultures, we observe the absence of IFN-I stimulation by SARS-CoV-2 alone but detect the failure to counteract STAT1 phosphorylation upon IFN-I pretreatment, resulting in near ablation of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment postinfection and found that SARS-CoV-2 was sensitive even after establishing infection. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. The absence of an equivalent open reading frame 3b (ORF3b) and genetic differences versus ORF6 suggest that the two key IFN-I antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to IFN-I responses between SARS-CoV and SARS-CoV-2 that may help inform disease progression, treatment options, and animal model development.IMPORTANCE With the ongoing outbreak of COVID-19, differences between SARS-CoV-2 and the original SARS-CoV could be leveraged to inform disease progression and eventual treatment options. In addition, these findings could have key implications for animal model development as well as further research into how SARS-CoV-2 modulates the type I IFN response early during infection.

Keywords: 2019-nCoV; COVID-19; IFN; SARS-CoV; SARS-CoV-2; coronavirus; interferon; type I interferon.

FIG 1

SARS-CoV-2 is sensitive to type I IFN pretreatment. (A) Vero E6 cells were treated with 1,000 U/ml recombinant type I (hashed line) IFN or mock (solid line) for 18 h prior to infection. The cells were subsequently infected with either SARS-CoV wild type (WT; black) or SARS-CoV-2 (blue) at an MOI of 0.01, as described in the text. Each point on the line graph represents the group mean (n ≥ 3). All error bars represent the standard deviations (SD). A two-tailed Student t test was used to determine P values (***, P < 0.001). (B) Vero E6 cell protein lysates from IFN-I-treated and untreated cells were probed at 48 h postinfection by Western blotting for phosphorylated STAT1 (Y701), STAT1, IFITM1, SARS spike, and actin.

FIG 2

SARS-CoV-2-attenuated and IFN-I-sensitive in Calu3 respiratory cells. (A) Calu3 2B4 cells were treated with 1,000 U/ml recombinant type I (hashed line) IFN or mock treated (solid line) for 18 h prior to infection. The cells were subsequently infected with either SARS-CoV WT (black) or SARS-CoV-2 (blue) at an MOI of 1. Each point on the line graph represents the group mean (n ≥ 3). All error bars represent the SD. A two-tailed Student t test was used to determine P values (***, P < 0.001). (B) Calu3 cell protein lysates from IFN-I-treated and untreated cells were probed at 48 h postinfection by Western blotting for phosphorylated STAT1 (Y701), STAT1, IFITM1, SARS spike, and actin. (C) Western blot quantification for phosphorylated STAT1, total STAT1, and IFITM1. A two-tailed Student t test was used to determine P values (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 3

Differential IFN-I sensitivity and pSTAT1 phosphorylation after SARS-CoV-2 or influenza A virus infection on polarized HAEC cultures. (A) HAEC were pretreated with 1,000 U/ml IFN-α basolaterally for 2 h prior to and during infection. The cultures were then infected apically with influenza A/California/09 H1N1 virus or SARS-CoV-2. Apical washes were collected at the indicated times, and progeny titers were determined by plaque assay on MDCK cells (influenza virus) or Vero E6 cells (SARS-CoV-2). At the endpoint (48 h), the cultures were lysed for Western blot analysis. (B) Western blotting for total STAT1 or phospho-STAT1 at 48 hpi with actin as loading control. (C to E) Western blot analyses were performed for total STAT1, phospho-STAT1, or their ratios at 48 hpi, and protein levels were quantified by densitometry and normalized to the actin control (n = 3). (F) Influenza A virus titers by plaque assay on MDCK cells. 0 h, virus inoculate; 2 h, virus in third apical wash; 8, 24, and 48 h, virus in apical washes at these time points (n = 3). (G) SARS-CoV-2 titers by plaque assay on Vero E6 cells. 0 h, virus inoculate; 2 h, virus in second apical wash; 8, 24, 48 h, virus in apical washes at these time points (n = 3). A two-tailed Student t test was used to determine P values (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 4

SARS-CoV-2 impacted by after IFN-I treatment. (A and B) Vero E6 and Calu3 2B4 cells were infected with either SARS-CoV WT (black) or SARS-CoV-2 (blue) at an MOI of 0.01 (A; Vero cells) or at an MOI of 1 (B; Calu3 cells). The cells were subsequently treated with 1,000 U/ml recombinant type I IFN (hashed line) or mock treated (solid line) for 4 h after infection. Each point on the line graph represents the group mean (n ≥ 3). All error bars represent the SD. A two-tailed Student t test was used to determine P values (**, P < 0.01; ***, P < 0.001).

FIG 5

Conservation of SARS-CoV IFN antagonists. The viral protein sequences of the indicated viruses were aligned according to the bounds of the SARS-CoV ORFs for each viral protein. The sequence identities were extracted from the alignments for each viral protein, and a heat map of the percent sequence identity was constructed using EvolView (www.evolgenius.info/evolview) with SARS-CoV as the reference sequence. TR, truncated protein.