bioRxiv preprint doi: https://doi.org/10.1101/2020.04.17.047480; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Dysregulation of NAD Metabolism by Coronaviruses and PARPs Coronavirus infection and PARP expression dysregulate the NAD Metabolome: an actionable component of innate immunity Collin D. Heera,b, Daniel J. Sandersonc,&, Lynden S. Vothd,&, Yousef M.O. Alhammadd,&, Mark S. Schmidtb,&, Samuel A.J. Trammellb,*, Stanley Perlmane, Michael S. Cohenc, Anthony R. Fehrd,#, Charles Brennerb,**,# aFree Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City, IA, USA bDepartment of Biochemistry, University of Iowa, Iowa City, IA, USA cDepartment of Chemical Physiology & Biochemistry, Oregon Health Sciences University, Portland, OR, USA dDepartment of Molecular Biosciences, University of Kansas, Lawrence, KS, USA eDepartment of Microbiology & Immunology, University of Iowa, Iowa City, IA, USA *current address: Novo Nordisk Foundation, Center for Basic Metabolic Research, University of Copenhagen, Denmark **current address: Department of Diabetes & Cancer Metabolism, City of Hope National Medical Center, Duarte, CA USA &These authors contributed equally to the work Running title: Dysregulation of NAD metabolism by coronaviruses and PARPs #Address correspondence to: [email protected] and [email protected] Keywords: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19, transcriptomics, interferon, poly(ADP-ribose) polymerase (PARP), ADP-ribosylation, nicotinamide adenine dinucleotide (NAD) ABSTRACT are limited by and can be enhanced by pharmacological activation of NAM salvage. Innate immune responses are critical to We further showed that infection with the b- control of viruses. Coronaviruses (CoVs) are coronavirus murine hepatitis virus (MHV) positive strand RNA viruses with double- induces a severe attack on host cell NAD+ and stranded RNA replication intermediates that NADP+. Finally we show that NAMPT are recognized by the innate immune system. activation, NAM and NR dramatically Upon recognition, infected cells secrete decrease replication in a MHV infection model interferon, which induces a set of interferon- that is sensitive to PARP activity. The data stimulated genes that inhibit viral replication. show that the antiviral activities of Here we show that SARS-CoV-2 infection noncanonical PARP isozyme activities are strikingly dysregulates the set of genes limited by their own consumption of cellular involved in consumption and biosynthesis of NAD and that nutritional and nicotinamide adenine dinucleotide (NAD). pharmacological interventions to enhance Highly induced genes include those encoding NAD-based defenses may boost innate noncanonical poly(ADP-ribose) polymerase immunity. (PARP) family members known to function as mono ADP-ribosyltransferases but not known to deplete cellular NAD and genes encoding enzymes for salvage NAD synthesis from Disease attributed to the current novel nicotinamide (NAM) and nicotinamide riboside (NR). We demonstrate that coronavirus (CoV) outbreak (COVID-19) has overexpression of PARP10 is sufficient to rapidly spread globally, infecting more than 11 depress NAD levels and that the enzymatic million people and killing > 500,000 as of early activities of PARP10, PARP12 and PARP14 July, 2020 (1). The causative agent, severe 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.17.047480; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Dysregulation of NAD Metabolism by Coronaviruses and PARPs acquired respiratory syndrome coronavirus 2, PARP10, PARP11, PARP12, PARP13 and SARS-CoV-2, is transmitted largely by aerosol PARP14 (15). To determine whether these gene and liquid droplets that infect cells of the lung expression changes are incidental to MHV epithelium (2). Like other positive strand RNA infection, facilitate MHV infection, or are part of genome viruses, SARS-CoV-2 replication an innate immune response against MHV, we proceeds through formation of double-stranded treated cells with siRNAs to knock down (ds) RNA (3), which elicits an interferon (IFN) expression of these genes and then analyzed the response (4,5). IFN proteins are secreted by impact on MHV replication. Our data showed that infected cells to engage IFN receptors and, in an PARP7 plays a role in facilitating replication. In autocrine and paracrine manner, induce contrast, PARP14 is reQuired for full induction of expression of a set of ISGs that collectively IFN-β expression (15,16), suggesting PARP14 is reorient cellular metabolism toward infection directly involved in establishing the innate control by interference with transcription, immune response in CoV infected cells. translation and lipid biosynthesis (6,7). Most CoV genomes encode 16 non- CoV genomes do not encode enzymes structural proteins (nsps) (3,8,9). Nsp3 contains a needed for ATP generation, nucleotide, amino macrodomain, herein termed the CoV ADP- acid, lipid or protein synthesis, and therefore ribosylhydrolase (CARH) that removes ADP- depend on exploitation of host functions to ribosyl modifications from acidic amino acids on synthesize and assemble more viruses (3,8,9). protein targets. Thus, CARH reverses the Cellular and viral energy generation and modification that is installed by the IFN-induced biosynthetic programs depend on the four activities of MARylating PARP family members nicotinamide adenine dinucleotide (NAD) (17). CARH activity is reQuired for virulence in coenzymes, NAD+, NADH, NADP+ and vivo using mouse models of both MHV and NADPH, which are the central catalysts of SARS-CoV (17-19). Moreover, an active site metabolism (10). These coenzymes accept and mutation that ablates the ADP-ribosylhydrolase donate electrons in essential, ubiquitous activity of CARH in MHV resulted in a virus that processes of fuel oxidation, lipid, nucleotide and replicates poorly in primary bone-marrow amino acid biosynthesis, and the generation and derived macrophages (BMDMs) (15). We further detoxification of reactive oxygen species. The identified PARP12 and PARP14 as CoV-induced specific roles of these coenzymes in viral ISGs that are reQuired for the depressed replication and antiviral defenses remain largely replication of CARH mutant viruses, indicating unexplored. that their activity is opposed by CARH-mediated reversal of ADP-ribosylation (15). Among the ISGs are several members of the poly(ADP-ribose) polymerase (PARP) In support of the antiviral roles of IFN- superfamily. These enzymes have been induced MARylating PARP isozymes, PARP12 implicated in restriction of viral replication was shown to promote the degradation of nsp1 through mechanisms that are not well understood and nsp3 in Zika virus infection (20). PARP12 (11,12). PARP isozymes, except for the has also been shown to inhibit a wide variety of enzymatically inactive PARP13 protein, have an RNA viruses, including several alphaviruses, absolute requirement for NAD+ (13,14). which also contain nsp3-encoded ADP- However, rather than using NAD+ as an electron ribosylhydrolase activities (21,22). These acceptor, PARPs consume NAD+ in order to observations suggest that key events in the innate transfer the ADP-ribose moiety to protein side immune response to viral infections are played chains and/or to form ADP-ribose polymers (10). out in the infected cell’s NAD metabolome. Indeed, the canonical and best characterized To determine whether COVID-19 PARP superfamily members, PARP1 and disturbs NAD metabolism, we analyzed PARP2, form poly(ADP-ribose) (PAR) largely in transcriptomic data from SARS-CoV-2-infected response to DNA damage. However, most other human cell lines and organoids, SARS-CoV-2- members of the PARP superfamily possess infected ferrets, and a lung biopsy from a mono-ADP-ribosyltransfer (MARylation) deceased human victim of COVID-19. These data activities on target proteins (13). We previously indicate that the same noncanonical PARPs showed that murine hepatitis virus (MHV) induced by MHV are induced by SARS-CoV-2 infection strongly induces expression of and that in vivo infection with SARS-CoV-2 noncanonical PARP isozymes PARP7, PARP9, 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.17.047480; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Dysregulation of NAD Metabolism by Coronaviruses and PARPs down-regulates synthesis of NAD from cADP-ribose formation, nicotinamide tryptophan and nicotinic acid (NA) while methylation/oxidation, and other related upregulating synthesis capacity from functions in transport, binding, redox and nicotinamide (NAM) and nicotinamide riboside regulation (Supplementary Material 1). We (NR). We further show that disturbances to the then analyzed RNAseq data from SARS-CoV-2 NAD transcriptome scale with viral load. Though infection of three human lung cell lines, A549, noncanonical PARP isozymes are known to use normal