N overexpression in Vero E6 cells
The N-protein cassette was subcloned from pLVX-EF1a-SARS-CoV-2-N-2xStrep-IRES-Puro (Gordon et al., 2020) into pLVX-TetOne-Puro (Takara) using the restriction enzymes EcoRI and BamHI to create pLVX-TetOne-Puro-SARS-CoV-2-N-2xStrep. Sequence integrity was confirmed by Sanger sequencing (GeneWiz). To produce lentiviruses, HEK293T cells (50% confluent in a T175 flask) were transfected with 5 μg of each of the pLVX-TetOne-Puro lentiviral plasmids, 3.33 μg Gag-Pol packaging construct, and 1.66 μg VSV-G envelope (pMD2.G, Addgene) using PolyJet (SignaGen). Culture supernatant was precipitated with a final concentration of 8.5% PEG-6000 and 0.3M sodium chloride (NaCl), incubated at 4°C for 2-4 hours. Virions were pelleted at 3500 rpm for 20 minutes in a spin bucket rotor, suspended in 0.5 mL 1xPBS, and aliquoted for storage at 80°C. Vero E6 cells were seeded in T75 flasks at 50% confluence and transduced with 200 μL precipitated lentivirus derived from pLVX-TetOne-Puro-SARS-CoV-2-N-2xStrep or pLVX-TetOne-Puro empty vector. 48 hours post transduction, 10 μg/mL Puromycin was added to cultures to select transduced cells. Polyclonal stable cell lines were seeded into 15 cm dishes in triplicate and treated with 1 μg/mL doxycycline for 48 hours. Cells were washed in ice cold PBS and harvested by scraping in cold PBS. Cell pellets were lysed directly (8 M urea, 100 mM ammonium bicarbonate (ABC), 150 mM NaCl, protease inhibitor (mini-cOmplete, Roche) and phosphatase inhibitors (phosSTOP, Roche), and protein digestion and phosphopeptide enrichment was performed as described above for SARS-CoV-2 infected Vero E6 cells. During the analysis, one of three replicates of the N-overexpressed samples was found to be an outlier based on principal component analysis (as in Figures S1A and S1B) and was removed. N-overexpression (n = 2) was compared to empty vector (EV) controls (n = 3) during analysis (full data available in Table S1). Kinase activity predictions were also performed the same as for viral infection phosphoproteomics (full data available in Table S4).