6 research outputs found
<i>SIRT5</i>-KO cells express a higher basal level of viral restriction factors.
A. Expression heatmap of interferon-stimulated genes and other restriction factors, showing that mock-infected SIRT5-KO cells express higher basal levels of restriction factors, and that antiviral responses are stronger in SIRT5-KO cells. Data show mean log2 fold-change, compared to mock-infected WT, and the q-value between mock-infected WT and SIRT5-KO cells. Only genes differentially expressed between at least two conditions were included in the analysis (qB. RT-qPCR confirmation of restriction factors upregulated in non-infected SIRT5-KO cells (n = 8). Data show fold-changes compared to WT levels after normalization with ACTIN. Data show mean and SEM between replicates. p-values after unpaired two-tailed t-test are shown and asterisks summarize the results. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.</p
SARS-CoV-2 Nsp14 interacts with human SIRT5.
A. Cartoon representation of the protein structure of Nsp14/Nsp10 (PDB 7N0B) and SIRT5 (PDB 3YIR) shows the Nsp14 N-terminal ExoN domain and C-terminal MTase domain. B. Affinity-purification of Nsp14-strep and co-purification of endogenous SIRT5 after transfection in HEK293T cells, as shown by western blot. C. Immunofluorescence of transfected Nsp14-Strep and endogenous SIRT5 in A549 cells. D. CETSA in HEK293T cells transfected with Nsp14-step and/or SIRT5, showing an increase in the stability of SIRT5 and Nsp14 by western blot. E. Western blot showing the absence of SIRT5 in SIRT5-KD HEK293T cells. F. Strep-tag affinity-purification or Flag-tag immunoprecipitation, followed by western blot, after transfection with Nsp14-strep, Nsp10-flag and SIRT5 expression constructs. SIRT5 does not interact with Nsp10. 0.5 μg of each construct or of empty control plasmids were transfected in SIRT5-KD HEK293T cells in a six-well plate. G. Strep-tag affinity-purification and western blot after transfection of Nsp14-strep, SIRT5 and increasing concentrations of Nsp10-tag indicate competitive binding of SIRT5 and Nsp10. 0.5 μg of Nsp14-strep and SIRT5 plasmid were used in a 6-well plate, with 0, 0.5, 1 or 2 μg of Nsp10-Flag.</p
<i>SIRT5</i>-KO cells mount a stronger innate immune response.
RNA-seq analysis of WT and SIRT5-KO A549-ACE2 cells infected or mock-infected for 3 days with SARS-CoV-2 at MOI = 1. n = 4. A. Volcano plots showing differentially expressed genes between the different conditions. Highlighted genes display a q-value q1 (orange), or both (purple). Left panel: SIRT5-KO vs WT in mock-infected cells. Middle: Infected vs mock-infected WT cells. Right: Infected vs mock-infected SIRT5-KO cells. B. Normalized gene count of SARS-CoV-2. C-D. Unsupervised clustering of the 3221 genes differentially expressed between at least two of the four conditions (qC: heatmap of normalized expression. D. Z-scores of differentially expressed genes as grouped by clustering. Colored lines represent the quantification of an individual gene whereas solid black lines show the cluster Tukey boxplot. E. Enrichment analysis of biological gene sets in the identified gene clusters (C and D).</p
SIRT5 catalytic activity is necessary to interact with Nsp14.
A. Cartoon representation of the protein structure of SIRT5 catalytic site in complex with cofactor NAD and succinylated lysine substrate (SuK), showing conserved residues mutated in panel B. B. Strep-tag affinity-purification and western blot after transfection of Sirt5-KD HEK293T cells with Nsp14-strep and SIRT5 catalytic mutants, showing that the interaction with Nsp14 is lost in several mutants. C. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT5, in SIRT5-KD HEK293T cells incubated with increasing concentrations of SIRT5 inhibitor Sirt5-i. High concentrations of Sirt5-i prevent the interaction. D. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT5, in SIRT5-KD HEK293T cells incubated with NAMPT FK866 inhibitor (low cellular NAD), FK866 and NMN, or NMN alone (high cellular NAD). SIRT5 binding strength correlated with NAD levels. E. Pan-acetylation, malonylation and succinylation in SIRT5-KD HEK293T total or Strep-purified proteins, after transfection with Nsp14-Strep, GFP-strep control and/or SIRT5. No specific lysine modifications could be detected. F. Summary of mass spectrometry experiments. Nsp14-strep proteins purified from SIRT5-KD HEK293T, with or without co-transfection with SIRT5, were analyzed by mass spectrometry. No acetylation, malonylation, succinylation or glutarylation modifications could be detected.</p
Levels of viral restriction factors.
A. Principal component analysis of RNA-seq samples, showing that replicates are well separated based on knockout and infection status. B. Normalized gene count of interferon-stimulated genes and restriction factors, from Fig 7. (TIF)</p
SARS-CoV-2 Nsp14 interacts with human SIRT1.
A. Co-purification of endogenous sirtuins SIRT1, 2, 3, 6 and 7 after transfection of Nsp14-strep in HEK293T cells, as shown by western blot. Loading and purification controls are the same as in Fig 1B. B. Strep-tag affinity-purification and western blot after transfection of HEK293T cells with Nsp14-strep and SIRT1 WT and H355Y catalytic mutant, showing that the interaction with Nsp14 is lost in H355Y mutant. C. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT1, in HEK293T cells incubated with increasing concentrations of SIRT1 inhibitor Ex-527. High concentrations of Ex-527 prevent the interaction. D-E. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT1, in HEK293T cells incubated with increasing concentrations of SIRT1 specific activator SRT1720 (D) or non-specific activator resveratrol (E). Both drugs were cytotoxic at high concentrations and the apparent decrease in SIRT1 binding correlated with a similar decrease in the input lanes. F. In vitro desuccinylation activity of purified SIRT5 incubated with increasing concentrations of Nsp14, showing no effect. G. In vitro methyltransferase activity of purified Nsp14 incubated with increasing concentrations of SIRT5, showing no specific effect. Unmethylated GpppG cap-analog was used as a substrate.</p