Fig. 1.

SARS-CoV-2 non-structural protein 13 (nsp13) hijacks host deubiquitinase USP13 to stabilize itself and counteracts host antiviral immune response. a HEK293T cells were transfected with empty vector (EV) or Flag-nsp13. The cells were then lysed and blotted with indicated antibodies. b The IFN-β RNA levels in control and nsp13 expressing cells transfected with Poly: IC for 8–12 h were analyzed by qRT-PCR. c Control and nsp13 expressing cells were transfected with 3p-hpRNA for 8–12 h, and then harvested to analyze the IFN-β RNA levels by qRT-PCR. d HEK293T cells were transfected with EV or Flag-nsp13. The cells were then lysed and immunoprecipitated with anti-Flag agarose beads. The beads were boiled and probed with indicated antibodies. e Control and nsp13 expressing cells were transfected with HA-TBK1 and treated with Poly: IC. The cells were then lysed and immunoprecipitated with anti-HA agarose beads. The beads were boiled and analyzed with indicated antibodies. f A549 cells were transfected with Flag-nsp13 followed by immunoprecipitation assay. The interaction of nsp13 and USP13 was detected by Western blot. g nsp13 expression in control or USP13 knockdown A549 cells was detected by Western blot. h Western blot analysis of nsp13 expression in A549 cells treated with spautin-1. i nsp13 expression in USP13 knockdown A549 cells re-expressing indicated constructs was detected by Western blot. j, k nsp13 expressing A549 cells stably expressing USP13 shRNA were infected with influenza virus A/PR/8/34. The M2 RNA (j) and protein (k) levels in cells were analyzed by qRT-PCR and Western blot, respectively. l, m A549 cells expressing indicated constructs were infected with influenza virus A/PR/8/34, and then harvested to detect the RNA (l) and protein (m) levels of M2 by qRT-PCR and immunoblotting, respectively. n–o Control and nsp13 expressing A549 cells were treated with spautin-1 and then infected with influenza virus A/PR/8/34. The M2 RNA (n) and protein (o) levels in cells were examined by qRT-PCR and Western blot, respectively. p, q nsp13 expressing A549 cells stably expressing USP13 shRNA were infected with influenza virus A/PR/8/34. The RNA (p) and protein (q) levels of IFN-β in cells were detected by qRT-PCR and ELISA, respectively. r, s Control and nsp13 expressing A549 cells treated with spautin-1 were infected with influenza virus A/PR/8/34. The IFN-β RNA (r) and protein (s) levels in cells were examined by qRT-PCR and ELISA, respectively. Data are shown as mean ± SEM from three independent experiments. p value was determined by two-tailed unpaired t test (*p < 0.05; **p < 0.01; ***p < 0.001)