SARS-CoV-2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions

Fig. 1. Expression of Nsp2 and its binding to eIF4E2. A Representative image of western blot analysis of expression of Strep-Nsp2 in HEK293T cells stably transfected with either Lipofectamine-3000 or LTX. B Biotin-Agarose pull-down. Left, western blot analyses of eIF4E2 (upper panel) and Nsp2 (lower panel). Right, graph represents densitometric ratio of eIF4E2 to Nsp2. C m7GTP-Sepharose pull-down. Left, western blot analyses of Nsp2 (upper panel) and eIF4E2 (lower). Right, graph represents densitometric ratio of Nsp2 to eIF4E2. C—control; N—normoxia; H—hypoxia. The blots shown are representative of three completely independent experiments

Fig. 2. Nsp2 co-localizes with eIF4E2 and RER under normal and hypoxic conditions. A Strep-Nsp2 was probed using anti-strep antibody (green) and eIF4E2 probed using anti-eIF4E2 antibody (red), DAPI (blue) stained nuclei. Merge channel from max intensity projections of z-stacks under hypoxia shows increased co-localization (yellow). B Strep-Nsp2 (green) and calnexin (red) probed. Under hypoxia, granular staining in merged channel (yellow) shows increased co-localization from best focal plane. C Representative image of Ribosomal marker (RbS3) probed using anti-RbS3 antibody (red) and Strep-Nsp2 (green). Colocalization shown in merged channel. DAPI stained nuclei

Fig. 3. Nsp2-cells demonstrate higher cap- and HCV-dependent translation under normal and hypoxic conditions. Expression of Renilla and Firefly luciferases from capped-Renilla-HCV-IRES-FF Luciferase mRNA in control (white bars) and Nsp2 cells (grey bars) grown under normal or hypoxic conditions. Graphs represents mean of Renilla units (expressed from the capped mRNA), set as one (A), and Firefly (expressed from the HCV IRES mRNA) (B) luciferase signals (± SD, n = 3). Statistical analysis of luciferase signals to one in control cells grown under normal or hypoxic conditions was performed using Student’s t test, with p < 0.05. C Nsp2-cells have higher translation rate. Graph represents the rate of H3-Leu incorporation in 0.1 mg of recovered protein extract. The difference in the slopes of linear protein synthesis between the two cell lines was highly significant (p = 0.0157 t-test)

Fig. 4. Polysomal analysis of control and Nsp2-cells in sucrose density gradients. A The representative of A254 profiles representing lysates distribution of cells grown under normal (left panel) and hypoxic (right panel) conditions in sucrose density gradients. Top of the gradient is on left side of each image. Position of 40S, 60S, and 80S along Ps profile are indicated. Arrow, 80S pick. L, light polysomes representing low translated mRNAs, fractions #1–6; H, heavy polysomes representing more efficiently translated mRNAs, fractions # 7–11. Ps/Ms, ratio of the 18S RNA signals in polysomes versus monosomes (fractions #4 and 5). B Real-time-PCR analysis of GAPDH, FGF2, and VEGF-C mRNAs distribution in sucrose density gradients. Left panels, distribution of the corresponding mRNA along sucrose density gradient similar to ones presented on (A). Graphs represent percentage of individual mRNA in each fraction. Filled circle, control cells; empty circle, Nsp2-cells. Right panels, graphs representing percentage of individual mRNA in light polysomal fractions #1–6 (white bars) and in heavy polysomal fractions #7–11 (grey bars) relative to the amount of this mRNA in all eleven fractions. The…

Fig. 5. Western blot analyses of FGF2 and VEGF proteins in lysates. A FGF2 CUG1 and AUG isoforms in Nsp2 expressing cells in normoxic and hypoxic conditions (Right and left panel represents three biologically independent experiments). GAPDH was used as a normalization control. B Densitometric ratio of CUG1 to GAPDH (left panel) and AUG to GAPDH (right panel). C Left, the representative images of western blots of VEGF-C, EGFR, and GAPDH. Right, graphs of the densitometric analysis of western blots. The graphs represent the mean ratio of signal of VEGF-C to GAPDH normalized to that in control cells grown under normal conditions, set as one (± SD). Western blot analysis was performed for each set of proteins using lysates from three separate experiments and repeated at least twice for each set of lysates

Fig. 6. Western-blot analysis of polysomal fractions. The representative images of A254 profiles (as in Fig. 4A) and western blots of 60S ribosomal protein L7a (RsL7a), eIG4G, eIF4A, Strep-Nsp2, eIF4E2, and 4EBP in corresponding fractions (Frs) along sucrose gradients in control and Nsp2 cells grown under normal (A) and hypoxic (B) conditions. Arrows on the A254 profiles pont to the picks representing 80S. The western blot analysis of proteins was investigated in three sucrose density gradients using lysates from separate experiments. Arrow, position of Nsp2 signal

Fig. 7. Nsp2 may affect composition of the cap-binding complexes. (A) Left, the representative images of western-blot analyses of proteins expressed in control and Nsp2-cells under normal and hypoxic conditions. Arrow indicates position of Strep-Nsp2. Right, ratio of densitometric signal of P-4EBP to total 4EBP in cells grown under normal or hypoxic conditions normalized to that in control cells grown under normal conditions, set as one (± SD, n = 3). (B) Left, the representative images of western blot analysis of proteins bound to m7GTP-Sepharose under normal and hypoxic conditions in control and Nsp2-cells. Arrow indicates position of Strep-Nsp2. Right, ratios of densitometric signals of eIF4G (upper graph) and eIF4E2 (lower graph) to eIF4E bound to m7GTP-Sepharose in experiments presented in lanes 1–4 (± SD, n = 3). Graph eIF4E/eIF4G: p = 0.014 for C–H versus C–N; p = 0.033 for Nsp2-H versus C–H. Graph eIF4E2/eIF4E: p = 0.03 for C–H versus C–N; p = 0.025 and 0.023 for Nsp2–H versus C–N and versus C–H, respectively