Fig. 4
From: SARS-CoV-2 hijacks cellular kinase CDK2 to promote viral RNA synthesis
Phosphorylation of nsp12 by CDK2 enhances the RdRp activity. a Flag-nsp12, Myc-CDK2, and HA-CyclinA were co-transfected in HEK293T cells for 48 h. Flag-tagged nsp12 protein was immunoprecipitated according to the previous experimental method and then treated with/without λ-PPase, followed by immunoblotting with antibodies against Pho-CDK2 substrates, Flag, HA, and Myc. b CDK2/CyclinA Kinase Enzyme Systems with the ADP-GloTM Assay. Purified nsp12 or Histone H1 incubated in a kinase reaction mixture containing 50 μM ATP and 6.4 ng active CDK2/CyclinA. The experiments were performed in quadruplicate. Data are shown as mean ± SD, two-sided Mann–Whitney test, *p < 0.05. c In vitro CDK2/CyclinA kinase assay. Purified CDK2/CyclinA was incubated with recombinant proteins nsp12 in vitro phosphorylation system. The reaction mixture was collected and examined using immunoblotting. d Phosphorylation site in nsp12 was identified by mass spectrometry (MS). e, f CoV-Gluc, Flag-nsp7, Flag-nsp8, Flag-nsp12, or T20 mutant nsp12 were co-transfected in HEK293T cells for 48 h. The minus-strand Gluc-RNA was quantified by RT-qPCR. Cell lysates were analyzed by immunoblot with the indicated antibodies. g HEK293T cells were transfected with Flag-nsp7, Flag-nsp8, Flag-nsp12 mutant T20E, Gluc-RNA plasmids and individual siRNAs for 48 h. The minus-strand Gluc-RNA was quantified by RT-qPCR and protein expression was detected by Western blot analysis. The experiment was performed in triplicate. Data are shown as mean ± SD, two-tailed unpaired Student’s t-test in e–g, ***P < 0.001, ns not significant
