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. 2020 Dec 25;295(52):17986-17996.
doi: 10.1074/jbc.RA120.015138. Epub 2020 Oct 13.

Coronavirus infection and PARP expression dysregulate the NAD metabolome: An actionable component of innate immunity

Collin D Heer  1 , Daniel J Sanderson  2 , Lynden S Voth  3 , Yousef M O Alhammad  3 , Mark S Schmidt  4 , Samuel A J Trammell  4 , Stanley Perlman  5 , Michael S Cohen  2 , Anthony R Fehr  6 , Charles Brenner  7
Affiliations

VSports在线直播 - Coronavirus infection and PARP expression dysregulate the NAD metabolome: An actionable component of innate immunity

Collin D Heer et al. J Biol Chem. .

Abstract

Poly(ADP-ribose) polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using NAD as the source of ADPR. Although the well-known poly(ADP-ribosylating) (PARylating) PARPs primarily function in the DNA damage response, many noncanonical mono(ADP-ribosylating) (MARylating) PARPs are associated with cellular antiviral responses VSports手机版. We recently demonstrated robust up-regulation of several PARPs following infection with murine hepatitis virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly up-regulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while down-regulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+ Finally, we show that NAMPT activation, NAM, and NR dramatically decrease the replication of an MHV that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses. .

Keywords: ADP-ribosylation; COVID-19; NAD biosynthesis; PARP; RNA-Seq; SARS-CoV-2; Severe acute respiratory syndrome coronavirus 2; gene transcription; interferon; nicotinamide adenine dinucleotide (NAD); plus-stranded RNA virus; poly(ADP-ribose) polymerase; post-translational modification (PTM); transcriptomics V体育安卓版. .

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Conflict of interest statement

Conflict of interest—C. B. is chief scientific adviser of ChromaDex and owns shares of ChromaDex stock. C. B. , S. A. J. T. , S. P. , and A. R. F. filed an invention disclosure on uses of NAD-boosting with respect to protection against coronavirus infection V体育ios版.

"VSports手机版" Figures

Figure 1
Figure 1
SARS-CoV-2 dysregulates the NAD gene set in vitro as a function of viral load. Differential expression analysis was performed on RNA-Seq data with respect to a 71 gene set representing the NAD transcriptome (Table S1). A–F, depicted are volcano plots representing normalized relative expression versus −log(P) with respect to mock infected in (A) human Calu3 lung cancer cells (MOI = 2), (B) NHBE cells (MOI = 2), (C and D), A549 cells at low MOI = 0.2 without and with introduction of ACE2 expression, respectively, and (E and F) A549 cells at high MOI = 2 without and with introduction of ACE2 expression, respectively. Further information is available in supporting material 2–7.
Figure 2
Figure 2
SARS-CoV-2 dysregulates the NAD gene set in vivo.A–D, differential expression analysis was performed on RNA-Seq data with respect to mock infected in (A) expanding enterocytes (MOI = 1), (B) ferret trachea infected with SARS-CoV-2, (C) lung of a diseased COVID-19 patient versus a control lung sample, and (D) BALF from SARS-CoV-2 infected versus healthy control human patients. Further information is available in supporting material 8–11.
Figure 3
Figure 3
PARP10 overexpression is sufficient to depress cellular NAD+ levels whereas SBI enhances activities of overexpressed PARP7, PARP10, PARP12, and PARP14.A, HEK 293T cells were grown with the indicated expression plasmids for GFP or PARP10 and treated with NAMPT activator (10 μm SBI). n = 3 for each group. Error bars represent S.E., p-values are from an unpaired two-tailed t test. See also supporting information 13. B, GFP, PARP7, PARP10, PARP12, and PARP14-expressing HEK293 cells were treated with 10 μm SBI and cells were collected 18 h later. Western blotting using indicated antibodies indicates that SBI promotes PARP7, PARP10, PARP12, and PARP14 activity. n = 3. Representative blots of three independent experiments are shown. **, p≤ 0.01; ***, p≤ 0.001.
Figure 4
Figure 4
MHV infection disturbs the NAD metabolome.A and B, DBT cells (A) and BMDM cells (B) were mock infected or infected with MHV at a MOI of 3 pfu/cell and cells were collected at 12 h post infection. n = 3–4 mock; n = 4 MHV. Error bars represent S.E., p-values are from unpaired two-tailed t test. **, p ≤ 0.01; ***, p ≤0.001. See also supporting material 14 and 15.
Figure 5
Figure 5
Boosting NAD+ levels depresses replication of CARH mutant MHV.A, NAD biosynthetic pathways. Red arrows depict gene expression that is depressed by SARS-CoV-2. Green arrows depict gene expression that is increased by SARS-CoV-2. B, 17Cl-1 cells were infected with 0.1 pfu/cell WT or N1347A MHV and either mock treated (DMSO or H2O) or treated with NA, NAM, SBI, or NR as described in “Experimental procedures.” DMSO served as a solvent control for NAM, SBI, and NA, whereas H2O served as a solvent control for NR. Cells were collected at 18 hours post-infection (hpi) and analyzed for virus replication by plaque assay. Data are representative of two independent experiments, n = 3 biological replicates. C, BMDMs were infected with 0.1 pfu/cell WT or N1347A MHV and treated with H2O or treated with NR as described in “Experimental procedures.” Cells were collected at 18 hpi and analyzed for virus replication by plaque assay. Data are representative of two independent experiments. n = 4 biological replicates. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
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