Skip to main content
. 2020 Sep 4;32(12):108185. doi: 10.1016/j.celrep.2020.108185

Figure 1.

Figure 1

SARS-CoV-2 ORF3b Is a Potent IFN-I Antagonist

(A) Maximum likelihood phylogenetic tree of full-length Sarbecovirus sequences. The full-length sequences (~30,000 bp) of SARS-CoV-2 (Wuhan-Hu-1 as a representative), SARS-CoV-2-related viruses from bats (n = 4) and pangolins (n = 4), SARS-CoV (n = 190), SARS-CoV-related viruses from civets (n = 3) and bats (n = 54), and outgroup viruses (n = 2; BM48-31 and BtKY72) were analyzed. Accession number, strain name, and host of each virus are indicated for each branch. Branches including SARS-CoV (n = 190) and SARS-CoV-related viruses from civets (n = 3) were collapsed for better visualization. The uncollapsed tree is shown in Figure S1, and the sequences used are summarized in Table S1. A scale bar indicates 0.1 nucleotide substitutions per site. NA, not applicable.

(B) Comparison of the protein lengths of Sarbecovirus ORFs. The amino acid numbers of ORF1a, S (ORF2), ORF3a, ORF3b, E (ORF4), M (ORF5), ORF6, ORF7a, and N (ORF9a) of sarbecoviruses are shown. The viral sequences used correspond to those in (A). Bars indicate average values, and each dot represents one viral strain. ORFs with low similarity (e.g., ORF8 and ORF9b) were excluded from this analysis.

(C and D) Potent anti-IFN-I activity of SARS-CoV-2 ORF3b. (C) HEK293 cells were cotransfected with five amounts of plasmids expressing hemagglutinin (HA)-tagged SARS-CoV-2 ORF3b, SARS-CoV ORF3b, and IAV NS1 (50, 100, 200, 300, or 500 ng) and p125Luc, a plasmid encoding firefly luciferase under the control of the human IFNB1 promoter (500 ng). 24 h post-transfection, SeV was inoculated at MOI 10. 24 h post-infection, the cells were harvested for western blotting (top), dot blotting (middle), and luciferase assay (bottom). For western blotting, the input of cell lysate was normalized to anti-alpha-tubulin (TUBA), and one representative result of three independent experiments is shown. The band of each viral protein is indicated by a white arrowhead. kDa, kilodalton. In the luciferase assay, the value of the SeV-infected empty-vector-transfected cells was set to 100%. (D) A549 cells were electroporated with a plasmid expressing HA-tagged SARS-CoV-2 ORF3b, SARS-CoV ORF3b, or IAV NS1 (500 ng). 24 h post-transfection, SeV was inoculated at MOI 10. 24 h post-infection, the cells were harvested for western blotting (top) and real-time RT-PCR (bottom). For western blotting, the input of cell lysate was normalized to TUBA, and one representative result of three independent experiments is shown. ORF3b and NS1 were run on separate blots for better visualization. Figure S2A shows them on the same blot with high and low exposure. For qRT-PCR, the expression levels of endogenous IFNB1 and GAPDH were quantified. For the luciferase assay (C) and real-time RT-PCR (D), mean values of three independent experiments with SEM are shown, and statistically significant differences (p < 0.05) compared with the SeV-infected empty-vector-transfected cells (#) and the same amount of the SARS-CoV-2 ORF3b-transfected cells (∗) are shown. E, empty vector.

See also Figures S1 and S2 and Table S1.