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 and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I pretreatment, while 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 (HAEC), we observe the absence of IFN-I stimulation by SARS-CoV-2 alone, but detect failure to counteract STAT1 phosphorylation upon IFN-I pretreatment resulting in near ablation of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment post infection and found 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 (ORF) 3b and changes to ORF6 suggest 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.

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

Figure 1.. SARS-CoV-2 sensitive to type I IFN pretreatment.

A) Vero E6 cells were treated with 1000 U/mL recombinant type I (hashed line) IFN or mock (solid line) for 18 hours prior to infection. Cells were subsequently infected with either SARS-CoV WT (black) or SARS-CoV-2 (blue) at an MOI of 0.01 as described above. Each point on the line graph represents the group mean, N ≥ 3. All error bars represent SD. The two tailed students t-test was used to determine P-values: *** P < 0.001. B) Vero cell protein lysates from IFN-I treated and untreated cells were probed 48 hours post infection by Western blotting for phosphorylated STAT1 (Y701), STAT1, IFITM1, SARS spike, and Actin.

Figure 2.. SARS-CoV-2 attenuated and IFN-I sensitive in Calu3 respiratory cells.

A) Calu3 2B4 cells were treated with 1000 U/mL recombinant type I (hashed line) IFN or mock (solid line) for 18 hours prior to infection. 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 SD. The two tailed students 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 48 hours post infection by Western blotting for phosphorylated STAT1 (Y701), STAT1, IFITM1, SARS spike, and Actin.

Figure 3.. Differential IFN-I sensitivity and pSTAT1 phosphorylation following SARS-CoV-2 or influenza A virus on polarized human airway epithelial cultures (HAEC).

A) HAEC were pretreated with 1000 U/mL IFN-α basolaterally for 2 hours prior to and during infection. 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 determined by plaque assay on MDCK cells (influenza virus) or Vero E6 cells (SARS-CoV-2). At the 48 h endpoint, cultures were lysed for western blot analysis. B) Western for total STAT1 or phospho-STAT1 at 48 hpi, and actin as loading control. C) Influenza A virus titers by plaque assay on MDCK cells. 0 h, virus inoculate; 2 h, virus in third apical wash; 8, 24, 48 h, virus in apical washes at these time points. D) 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.

Figure 4,. SARS-CoV-2 impacted by post IFN-I treatment.

A-B) Vero E6 and Calu3 2B4 cells were infected with either SARS-CoV WT (black) or SARS-CoV-2 (blue) at an MOI 0.01 (A, Vero cells) or MOI 1 (B, Calu3 cells). Cells were subsequently treated with 1000 U/mL recombinant type I IFN (hashed line) or mock (solid line) for 4 hours following infection. Each point on the line graph represents the group mean, N ≥ 3. All error bars represent SD. The two tailed student’s t-test was used to determine P-values: **P <0.01 *** P < 0.001.

Figure 5,. Conservation of SARS-CoV IFN antagonists.

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