Figure 2.
C-terminal Truncations Increase the IFN-Antagonistic Activity of ORF3b
(A) Maximum likelihood phylogenetic tree of Sarbecovirus ORF3b. The ORF3b sequences of SARS-CoV-2 (Wuhan-Hu-1), SARS-CoV-2-related viruses from bats (RmYN02, RaTG13, ZXC21, and ZC45) and pangolins (P5L, P1E, P4L, and Pangolin-coV), SARS-CoV (Tor2, GZ0402, GZ02, Urbani, BJ02, BJ01, BJ182-4, P3pp1, and P3pp46), SARS-CoV-related viruses from civets (civet007) and bats (Rs7327, YN2013, Rs4231, YNLF34C, Shaanxi2011, Rm1, F46, HKU3-2, GX2013, and Yunnan2011), and two outgroup viruses (BM48-31 and BtKY72) were analyzed. The ORF3b sequences of all SARS-CoV-related viruses are summarized in Table S2, and the ORF3b sequences used in this study are summarized in Table S3. Strain name and host of each virus are indicated for each branch. ∗Bootstrap value > 70%.
(B) Proportion of the ORF3b lengths in each Sarbecovirus. The distribution of different lengths of ORF3b in each viral group is summarized in pie charts. The number in parentheses (n) indicates the number of sequences used in this analysis. The numbers at the pie charts give the protein length indicated, and the numbers in bold indicate the most prevalent protein length for each viral group.
(C) Anti-IFN-I activities of different Sarbecovirus ORF3b proteins. (Top) Illustration of protein lengths of 25 Sarbecovirus ORF3b isolates used in this study. The information of the 25 Sarbecovirus ORF3b isolates is summarized in Table S3. (Middle and bottom) HEK293 cells were cotransfected with a plasmid expressing one of 25 HA-tagged Sarbecovirus ORF3b proteins (summarized in B) (100 ng) and p125Luc (500 ng). 24 h post-transfection, SeV was inoculated at MOI 10. 24 h post-infection, cells were harvested for western blotting and dot blotting (middle) and for luciferase assay (bottom). The amino acid sequences of ZXC21 and ZC45 are identical. An uncropped dot blot is shown in Figure S2B.
(D) Subcellular localization of Sarbecovirus ORF3b. Cell lysates of the HEK293 cells transfected with a plasmid expressing HA-tagged Sarbecovirus ORF3b were separated into cytosolic and nuclear fractions as described in the STAR Methods section. The percentage of ORF3b protein localized in the nucleus (top, n = 4) and a representative western blot (bottom) are shown. TUBA and anti-lamin A/C (LMNA) were used as controls for cytosolic and nuclear proteins. ORF3b proteins of HKU3-2, GX2013, and Yunnan2011 were not used in this experiment because these ORF3b proteins were only poorly expressed.
(E–G) Anti-IFN-I activity of C-terminally truncated SARS-CoV ORF3b. (E) Illustration of the ORF3b mutants of SARS-CoV (Tor2) and a SARS-CoV-related bat virus (Rs4231). NLS, nuclear localization signal of c-Myc (PAAKRVKLD). (F) Subcellular localization of the ORF3b mutants. Cell lysates of the HEK293 cells transfected with a plasmid expressing HA-tagged ORF3b mutants were separated into cytosol or nuclear fractions as described in the STAR Methods section. The percentage of ORF3b protein localized in the nucleus (top, n = 4) and a representative western blot (bottom) are shown. TUBA and LMNA were used as controls for cytosolic and nuclear proteins. (G) HEK293 cells were cotransfected with a plasmid expressing the indicated Sarbecovirus ORF3b proteins (50 or 100 ng) and p125Luc (500 ng). 24 h post-transfection, SeV was inoculated at MOI 10. 24 h post-infection, cells were harvested for western blotting (top) and luciferase assay (bottom).
(H) Subcellular localization of ORF3b and IRF3. HeLa cells were transfected with the indicated plasmids expressing HA-ORF3b and were infected with SeV as described in the STAR Methods section. Representative figures are shown. Scale bar, 10 μm. The white circles in IRF3 and DAPI panels indicate the nuclear rims of cells expressing HA-ORF3b.
For western blotting, the input of cell lysate was normalized to TUBA. One representative blot of three independent experiments is shown. For the luciferase assay, the value of the SeV-infected empty-vector-transfected cells was set to 100%. The 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 (#) are shown. In (C), red asterisks indicate statistically significant differences (p < 0.05) compared SARS-CoV-2 Wuhan-Hu-1 ORF3b-transfected cells. In (G), blue and green asterisks indicate statistically significant differences (p < 0.05) compared the same amount of either Tor2 ORF3b L115∗-transfected cells or Rs4231 ORF3b WT-transfected cells. E, empty vector.