NAD+ Suppresses EV-D68 Infection by Enhancing Anti-Viral Effect of SIRT1

Figures

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  Figure 1. EV-D68 infection influences cellular metabolism. RD cells cultured in 10 cm dishes infected with EV-D68 Fermon at an MOI of 1 and non-infected as control. After 18 h infection, cells were harvested for LC-MS analysis (n = 3 samples per group). (a) Principal component analysis of metabolomics data in the mock and EV-D68-infected group. (b) Volcano map of differentially expressed metabolites in EV-D68 compared with that in the mock group. (c) VIP analysis of the top 30 differentially expressed metabolites between the mock and EV-D68 group. The color of each rectangle in the right panel represents the relative level of the metabolites. The metabolites with VIP > 1 and p < 0.05 were determined as significantly different metabolites. The full name of metabolites were shown in in Supplementary Tables S1–S4. (d) Pie charts showing the HMDB classifications of the 93 differential metabolites. (e) KEGG pathway enrichment analysis of the significantly altered metabolites between the mock and EV-D68 group.

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  Figure 2. NAD+ suppresses EV-D68 proliferation in vitro. (a,b) RD cells were incubated with different concentrations of flavin adenine dinucleotide (FAD), L-Cystathionine (L-Cth), 8-Hydroxyguanosine (8-OHG), L-Acetylcarnitine (ALCAR), or nicotinamide adenine dinucleotide (NAD+) as indicated for 48 h. Then the cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. RD cells were treated with different concentrations of exogenous FAD (0, 2.5, 5, and 10 µM), 8-OHG (0, 5, 10, and 20 nM), L-Cth (0, 0.1, 0.5, and 1 mM), ALCAR (0, 0.5, 1, and 2 mM), and NAD+ (0, 0.5, 1, and 2 mM) 6 h prior to infection with EV-D68 Fermon at an MOI of 1 for 18 h. Viral VP1 protein levels were assessed by Western blot. (c) As with the cell line and experiment mentioned above, RD cells were treated with different concentrations of exogenous NAD+ (0, 0.5, 1, and 2 mM) 6 h prior to infection with EV-D68 Fermon at an MOI of 1, and after 24 h the supernatant of the cells was collected. Viral titers were determined using TCID50. Data in all quantitative panels are normalized based on β-actin presented as the mean ± SD of n = 3 replicates. Error bars indicate SD (n = 3). “−” represents absence, and “+”…

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  Figure 3. NAD+ suppresses EV-D68 proliferation in vivo. (a) Schematic representation of the experimental design for NAD+ treatment in neonatal mice infected with EV-D68(ATCC VR-1824). Mice were intracranially inoculated with EV-D68 (1 × 105 PFU/mouse) and treated with continuous NAD+ therapy until the seventh day. Viral loads in major organs and tissues were evaluated using RT-qPCR on the 3rd day, and survival rates were monitored until the 14th day. (b) Representative images showing symptoms of healthy mice in the mock group and the mice with limb paralysis in the EV-D68(ATCC VR-1824) infection group on 7 dpi. (c) Bar graph showing the percentage of mice with paralysis between the EV-D68-infected and NAD+ treatment groups. (d,e) Levels of EV-D68 viral nucleic acids and VP1 protein in major organs and muscle tissues were quantified using Western blot and RT-qPCR on the third day. (f) Histopathological changes in tissues from mock, EV-D68-infected, and EV-D68-infected with NAD+ treatment groups (n = 3) were analyzed. Pathological images of spine muscles and hind limb muscles, scale bar = 100 μm. Yellow arrow: Vacuolar degeneration of muscle cells. Brown arrow: Fibrosis of interstitial…

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  Figure 4. Viral infection regulated the expression of enzymes in the NAD+ salvage pathway, leading to a reduction in NAD+ level. (a) RD cells were cultured in 6-well plates and infected with EV-D68 Fermon at an MOI of 0 or 1 for 24 h. WST—detection of NAD+ in cells by WST-8 reaction colorimetry. (b–e) RD and A549 cells were infected with EV-D68 Fermon at an MOI of 1. Then, 18 h later, the expression changes of NAD+ synthesis genes (Nmnat1 to 3, Nampt, and Nmrk2) and NAD+ consuming genes (Sirt1 to 7, Parp1 to 6, Parp9, 10, 12, 14, and CD38) were measured using RT-qPCR. (f) A549 cells were infected with EV-D68 Fermon at an MOI of 1. Then, 18 h later, the protein levels of VP1, SIRT1, SIRT2, NAMPT, NMNAT2, and CD38, compared to the control β-actin, were determined using Western blot. Data in all quantitative panels are normalized based on β-actin or GAPDH presented as the mean ± SD of n = 3 replicates. Error bars indicate SD (n = 3). “−” represents absence, and “+” represents presence. * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 5. NAD+-consuming enzyme SIRT1 inhibits EV-D68 replication. (a) A549 cells were infected with EV-D68 Fermon at a multiplicity of infection (MOI) of 0, 0.5, and 1. Samples were collected after 24 h. Another group were infected with EV-D68 Fermon at an MOI of 1; samples were collected at 0, 8, and 16 h post-infection (hpi). Protein levels of SIRT1 and VP1 were detected using Western blot. (b) RD cells infected with EV-D68 Fermon at an MOI of 1 for 24 h. Cytoplasm extracts (CEs) and nuclear extracts (NEs) were prepared. SIRT1, β-actin, and lamin A expression levels were detected by Western blot analyses using the corresponding antibodies. (c) RD cells were infected with EV-D68 Fermon at an MOI of 1, and SIRT1 localization was assessed by immunofluorescence at 24 hpi. The SIRT1 was stained with the preliminary antibody rabbit anti-SIRT1 and secondary florescent affiliate antibody goat anti-rabbit AlexaFluor488. The nuclei were counterstained with DAPI. Scale bar represents 50 μm. (d–g) 293T cells were transfected with pBudCE4.1-Sirt1 or pBudCE4.1-mtSirt1 at 2, 4 μg, with pBudCE4.1 as the control. After 48 h, cells were infected with EV-D68 Fermon at an MOI of 1 for 24 h. Protein levels…

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  Figure 6. NAD+ augments the antiviral effect of SIRT1. (a,b) 293T cells were transfected with si-Sirt1-1 and si-NC for 30 nM; 48 h later, cells were treated with NAD+ for 8 h and then infected with EV-D68 Fermon at an MOI of 1 for 24 h. Protein levels of SIRT1 and viral VP1 were detected by Western blot. The intensity of Western blot band signals was quantified behind by Image J (a). And viral titers were measured using TCID50 (b). (c,d) 293T cells were transfected with si-Sirt1-3 and si-NC for 30 nM. Then, 48 h later, followed the same experiment mentioned before (a,b). (e,f) 293T cells were transfected with pBudCE4.1-Sirt1 and pBudCE4.1 for 2 μg. Then, followed the same experiment mentioned before (a,b). (g) RD cells were treated with NAD+ for 8 h, and SIRT1 localization was assessed by immunofluorescence. The SIRT1 was stained with the preliminary antibody rabbit anti-SIRT1 and secondary florescent affiliate antibody goat anti-rabbit AlexaFluor488. Thr nuclei were counterstained with DAPI. Scale bar represents 10 μm. (h) Schematic presentation of the luciferase reporter plasmid, containing a pol I promoter, the 5′ UTR region of EV-D68, and the Renilla luciferase gene (Rluc) infused with…

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  Figure 7. Schematic diagram illustrating the regulatory mechanism of NAD+ and SIRT1 during EV-D68 infection. EV-D68 infection decreases the level of cellular NAD+ (Black arrow: decrease), which promotes the expression of SIRT1. SIRT1 has an inhibitory effect on EV-D68, and it is strengthened by NAD+.