NAD metabolism plays multiple roles in influenza A virus replication in a novel ex vivo model of mouse lung infection

Abstract Despite the availability of prophylactic and therapeutic measures, Influenza A viruses (IAV) remain a major public health concern, causing an estimated 650 000 deaths annually. In this context, the identification of metabolic vulnerabilities of IAV replication could help develop complements to existing antiviral strategies. Here, we used nicotinamide phosphoribosyltransferase (NAMPT) inhibitors to demonstrate that nicotinamide adenine dinucleotide (NAD) is essential for IAV replication and infectious particles production both in vitro and ex vivo . We established an innovant ex vivo model of murine organotypic lung cultures (mOLC) that is pertinent to analyze cell metabolic alterations triggered by IAV infection. Using untargeted metabolomic profiling and spatial transcriptomic analyses, our research revealed that IAV-infection decreased NAD + levels in mOLCs, while PARPs, a group of NAD + -consuming enzymes, were upregulated. Pharmacological inhibition of the mono-ADP-ribosyl-transferase (mono-ART) activity of PARPs restricted IAV infection ex vivo , suggesting that NAD + sustains the proviral activity of these enzymes. Overall, our study identifies NAD metabolism as a central regulator of IAV infection, providing redox cofactor to host cell biosynthetic processes and substrate for mono-ART activity. Our results highlight NAMPT and PARP mono-ART activity as promising antiviral targets. Significance Statement Viruses are intracellular parasites that rely on the host cellular metabolism for their replication. Our results demonstrate that Influenza A virus (IAV) replication is critically dependent on NAD availability, an enzyme cofactor essential for redox metabolic reactions and a substrate for NAD+-consuming enzymes. Using a novel ex vivo infection model of mouse lung tissue, we found that IAV infection results in NAD depletion and enhanced expression of NAD-consuming PARP enzymes. Inhibitors of NAD biosynthesis and mono(ADP-ribosyl) transferase activity of PARP are restricting viral replication and infectious particles production. These findings highlight the proviral role for PARP mono-ART activity and identify NAMPT and PARP inhibitors as potential host-directed antivirals against IAV.