Fig 1: SIRT5 proviral activity is independent of the MAVS signaling pathway.A. Western blot showing 90% reduction of MAVS in A549-A/T cells transduced with a CRISPR lentivirus against MAVS. A549-A/T cells stably co-express ACE2 and TMPRSS2. B. Viral titers in MAVS-KD cells treated with DMSO or SIRT5 inhibitor Sirt5-i, after infection with SARS-Cov-2 for 3 days at MOI = 0.1, as shown by plaque assay. Sirt5-i had a similar effect in WT and MAVS-KO, suggesting that SIRT5 function is independent of MAVS. n = 9. C. RT-qPCR of GFP or Nsp14 after doxycycline induction. WT and SIRT5-KO A549-ACE2 cells were stably transduced with doxycycline-inducible constructs for GFP and Nsp14. After doxycycline treatment for 48 hours at 100ng/mL, GFP was strongly overexpressed, but Nsp14 failed to be expressed. Data show fold-changes compared to the first column (WT cells transduced with GFP without doxycycline), after normalization with ACTIN. n = 4. D. Hypotheses for the role of the SIRT5/Nsp14 interaction during SARS-CoV-2 infection. In model 1, Nsp14 could enhance SIRT5 activity, which would decrease innate immune responses and favor viral replication. In model 2, Nsp14 could redirect SIRT5 to novel targets, potentially in the replication-transcription complex, where SIRT5 could deacylate other viral proteins. In model 3, the Nsp14/SIRT5 complex could be primarily involved in mRNA cap methylation. Absence or inhibition of SIRT5 would lead to incomplete cap methylation and stronger immune recognition of viral mRNA. B-C. Data show mean and SEM between biological replicates. Asterisks summarize the results of one-way ANOVAs followed by Holm–Šidák multiple comparisons test (on log-transformed data for plaque assays). *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.
Fig 2: SIRT5-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 (q<0.01). B. 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.
Fig 3: SIRT5 proviral activity is partially independent from the interaction with Nsp14.A. Strep-tag affinity-purification and western blot after transfection of SIRT5-KD HEK293T cells with SIRT5 and Nsp14-strep from different coronaviruses, showing that the interaction with SIRT5 is specific to SARS-like coronaviruses. B. Decrease in supernatant-associated viral mRNA levels in HCT-8 cells infected with HCoV-OC43 for 5 days at MOI = 0.1, and treated with SIRT5 inhibitor Sirt5-i, as shown by RT-qPCR. Data show fold-change compared to DMSO-treated levels. n = 4. C. Decrease in viral titers in HCT-8 cells infected with HCoV-OC43 for 5 days at MOI = 0.1, and treated with SIRT5 inhibitors Sirt5-i, as shown by plaque assay. n = 4. B-C. Data show mean and SEM between biological replicates. Asterisks summarize the results of one-way ANOVAs followed by Holm–Šidák multiple comparisons test (on log-transformed data for plaque assays) *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.
Fig 4: SIRT5-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 q<0.05 (green), log2 fold-change >1 (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 (q<0.01). C: 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).
Fig 5: SIRT5 is a proviral factor.A. Western blot showing the absence of SIRT5 and SIRT1 in SIRT5- and SIRT1-KO A549-ACE2 cells, after CRISPR knockout. B. Decrease in cell-associated viral mRNA levels in SIRT5- and SIRT1-KO cells infected with SARS-Cov-2 for 3 days at MOI = 0.1 or MOI = 1, as shown by RT-qPCR. Data show fold-changes compared to WT levels at MOI = 0.1. n = 4. C. Decrease in viral titers in SIRT5- and SIRT1-KO cells infected with SARS-Cov-2 for 3 days at MOI = 1, as shown by plaque assay. n = 4. D. Absence of cytotoxicity in A549-ACE2 cells treated with Sirt5-i and Ex-527 inhibitor, as measured by flow cytometry. n = 4. E. Decrease in cell-associated viral mRNA levels in A549-ACE2 cells infected with SARS-Cov-2 for 3 days at MOI = 0.1, and treated with SIRT5 and SIRT1 inhibitors Sirt5-i and Ex-527, as shown by RT-qPCR. Data show fold-change compared to DMSO-treated levels. n = 6. F. Decrease in viral titers in A549-ACE2 cells infected with SARS-Cov-2 for 3 days at MOI = 0.1, and treated with SIRT5 and SIRT1 inhibitors Sirt5-i and Ex-527, as shown by plaque assay. n = 9. G/H. Same as E. (with n = 4), and F. (n = 6), using Calu3 cells. B-H. Data show mean and standard error of the mean (SEM) between biological replicates. RT-qPCR results were internally normalized with GAPDH and ACTIN reference genes. Viral titers after plaque assay are expressed in log-transformed PFU (plaque-forming unit) per mL of supernatant. Asterisks summarize the results of one-way ANOVAs followed by Holm–Šidák multiple comparisons test (on log-transformed data for plaque assays) *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.
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