Fig. 2: SARS-CoV-2 induced glycolysis and one-carbon metabolism supports viral RNA and protein expression, replication, and cytopathic effect.

a +strand gRNA FISH, Np IF, and merge with Hoechst stained nuclei of Vero-E6 TMPRSS2 + cells cultured in media with 25 mM glucose versus galactose as the sugar source and infected with SARS-CoV-2. b Mean ± SD fold change of live Vero E6 TMPRSS2 + cell number and median tissue culture infectious dose (TCID50) presented as fluorescent-focus units (FFU) per ml of culture supernatant at 48 h post infection of cells cultured in glucose versus galactose from n = 3 biologically independent replicates. c FISH analysis of +strand gRNA, IF for Np, and merge with Hoechst stained nuclei in SARS-CoV-2-infected Vero E6 TMPRSS2 + cells treated with DMSO or 100 nM piericidin A (PierA). d Mean ± SD fold change live cell number from n = 3 biologically independent replicates, as in c. e Mean ±  TCID50 from n = 3 biologically independent replicates in Vero E6 TMPRSS2 + , as in b. f Phase microscopic images of SARS-CoV-2 versus mock-infected Vero E6 TMPRSS + cells cultured for 48 h with DMSO, 1 μM of methotrexate (MTX), 30 μM hypoxanthine (hypo), 100 μM thymidine, or 1 mM formate, as indicated. Yellow scale bar indicates 100 μm. g Mean ± fold change live cell # and TCID50/ml from samples collected as in f from three biologically independent replicates. h FISH microscopic analysis of viral gRNA, IF of Np, and merge with Hoechst stained nuclei in SARS-CoV-2-infected Vero E6 TMPRSS2 + cells treated for 48 h with the indicated conditions. Yellow arrows indicate representative cells with high gRNA (red) but low Np (green) signal. i Ratios of +strand gRNA FISH versus Np IF signals from 500 Vero E6 TMPRSS2 + cells from 20 random fields for each condition in h are shown. j FISH microscopic analysis of viral gRNA, IF of Np, and merge with Hoecshst stained nuclei in SARS-CoV-2-infected A549 ACE2 + cells treated with the indicated conditions. Yellow arrows indicate representative cells with high gRNA (red) but low Np (green) signal. k Fold change mean ± SD live cell # and TCID50/ml from A549 ACE2 + samples collected as in j from n = 3 biologically independent replicates. l Flow-FISH analysis of Np subgenomic RNA in SARS-CoV-2-infected A549 ACE2 + cells treated with the indicated conditions. Of note, the leftmost peak in each row indicates uninfected cells. m Mean ± SD of Np subgenomic RNA mean fluorescence intensity (MFI) values from n = 3 biologically independent replicates, as in l. In all panels, cells were infected at MOI = 0.1 for 48 h. Microscopy images are representative of at least n = 3 biologically independent values. P-values in this figure were calculated by one-way ANOVA with multiple comparisons using Sidak method. Source data are provided as a Source Data file.