Coronavirus Infection and PARP Expression Dysregulate the NAD Metabolome: A Potentially Actionable Component of Innate Immunity

Collin D. Heer; Daniel J. Sanderson; Yousef M.O. Alhammad; Mark S. Schmidt; Samuel A.J. Trammell; Stanley Perlman; Michael S. Cohen; Anthony R. Fehr; Charles Brenner

Given that divergent CoVs consistently induce members of the PARP superfamily at the mRNA level, we asked whether MHV infection alters the NAD metabolome. We infected delayed brain tumor (DBT) cells with MHV-A59 at a multiplicity of infection (MOI) of 3 PFU/cell and subjected the cells to quantitative targeted analysis of the NAD metabolome by LC-MS 12 hours after infection (Trammell & Brenner, 2013) . As shown in Figure 2 , infection led to a significant > 3-fold depression of cellular NAD + and NADP + with respect to control cells at 12 hours after a mock infection. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.17.047480 doi: bioRxiv preprint It is well known that PARP1 activation by DNA damage greatly increases its catalytic activity, leading to depression of cellular NAD + and ATP (Cohen, 2020; Gupte, Liu, & Kraus, 2017) . It is less clear whether transcriptional induction of the MARylating enzymes such as PARP10-that are induced substantially by viruses-might disturb cellular NAD + . To address, this, we overexpressed PARP10 in HEK 293T cells and measured changes in NAD metabolites using LC-MS/MS. We found that overexpression of GFP-PARP10 significantly depressed NAD + compared to overexpression of GFP alone (Figure 3) . We next determined if the PARP10mediated loss in NAD + could be restored by boosting NAD + levels, either by increasing synthesis or decreasing consumption. To increase NAD + synthesis we treated cells with SBI-797812 (Gardell et al., 2019) , a small molecule allosteric activator of NAMPT, which promotes NAM salvage. To decrease NAD + consumption, we treated cells with veliparib (Donawho et al., 2007) , a selective inhibitor of PARP1 Figure 3 . PARP10 overexpression is sufficient to reduce cellular NAD + levels and this can be overcome pharmacologically. HEK 293T cells were grown with the indicated expression plasmids for GFP or PARP10 and treated with small molecules to inhibit NAMPT (200 nM FK866), activate NAMPT (10 µM SBI-797812) or inhibit PARP1 and PARP2 (300 nM Veliparib). Normalized data show that cellular NAD + is substantively depressed by PARP10 expression and that stimulating biosynthetic activity by NAMPT activation or inhibiting NAD + consuming activity with PARP1,2 inhibition can overcome the PARP10-mediated depression of NAD + levels. n = 2 for each sample. Error bars represent SEM, p-values are from t test. See also Supplementary Information 8. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.17.047480 doi: bioRxiv preprint and PARP2, which are major NAD + consumers in cells. We found that both NAMPT activation and PARP1/PARP2 inhibition restored cellular NAD + under these conditions.