Coronavirus genomes, where the blueprint for building and assembling the virus is stored, do not encode enzymes needed for energy generation and synthesis of biological building blocks. These viruses depend on the exploitation of infected host cell functions for their assembly. The viral programs responsible for energy generation and the formation of chemical compounds that are encoded in the viral genome depend on the host’s nicotinamide adenine dinucleotide (NAD+) system, which is required for the functioning of many enzymes in the infected cells, such as those needed for antiviral defense. How the host NAD+ system and the enzymes that depend on it are hijacked for viral replication or employed as antiviral defenses are largely unexplored.

Heer and colleagues from the University of Iowa show in an article published in the Journal of Biological Chemistry that SARS-CoV-2 infection strikingly upregulates a subset of antiviral enzymes that depend on NAD+ called non-canonical Poly-ADP-ribose polymerases (PARPs) and induces the expression of genes encoding enzymes for NAD+ synthesis from NAD+ precursors nicotinamide riboside (NR) and a form of vitamin B3 called nicotinamide (NAM) but also downregulates other NAD+ biosynthetic pathways. They then show that supplementation with NR and NAM dramatically decreases the replication of a mouse coronavirus that is sensitive to PARP activity. These data suggest that nutritional and pharmacological interventions to enhance NAD+ levels may boost innate immunity to coronaviruses.

To get a better sense of the relationship between NAD+ and coronaviruses investigators analyzed SARS-CoV-2-infected human cell lines, a lung biopsy from people living with or dead from COVID-19, and bronchoalveolar lavage fluid from healthy individuals as well as those infected with COVID-19. Heer and colleagues show that SARS-CoV-2 infection turns on the genes encoding enzymes for salvage NAD+ synthesis from nicotinamide (NAM) and nicotinamide riboside (NR) while reducing the production of enzymes for other NAD+ biosynthetic pathways, such as those that utilize nicotinic acid (NA). The investigators showed that non-canonical PARP enzymes consistently have increased protein production by SARS-CoV-2 infections.

The research team from the University of Iowa shows that the activity of these non-canonical PARP enzymes is limited by cellular NAD+ and can be enhanced by pharmacological activation of NAD+ synthesis. They further demonstrate that infection with a mouse version of coronavirus induces a severe attack on host cell NAD+. These results presented warrant the testing of NAD+ boosting agents in the context of CoV infections. In addition to animal trials, the safety of various forms of vitamin B3 should allow rapid clinical assessments of NAD boosters to be evaluated.

(Heer et al., 2020 | Journal of Biological Chemistry) Mouse coronavirus infection disturbs the NAD+ system. The researchers tested levels of NAD+ and related compounds in mouse cells derived from brain tumors (A) and bone marrow (B) infected with mouse coronavirus (MHV). The white bars depict non-infected mouse cells, and the black bars depict mouse cells infected with mouse coronavirus.

The investigators then established a system to test whether increased NAD+ status opposes mouse coronavirus infection using a mutant strain of the virus that replicates poorly and isn’t as detrimental to host cells (i.e., virulent) because it cannot block the antiviral PARP response. Consistent with their analysis that SARS-CoV-2 infection upregulates the pathways for NAD+ synthesis from NAM and NR, the authors found that NAM and a clinically tested preparation of NR strongly inhibited replication of the less-hostile mutant mouse coronavirus but not the non-mutated strain. The investigators speculate that NAD+ boosting approaches involving increased NA-dependent synthesis were unlikely to be strongly effective because their data showed that SARS-CoV-2 infection inhibited the levels of host enzymes that participate in that pathway for NAD+ generation.

(Heer et al., 2020 | Journal of Biological Chemistry) Boosting NAD+ levels with NAM and NR depresses the replication of a mutant mouse coronavirus with limited replication ability and virulence but does not affect unmutated mouse coronavirus. Mouse cells were infected with either unmutated (WT) or mutated (N1347A) mouse coronavirus with limited ability to replicate and harm the host cell and either mock-treated (DMSO or H2O) and treated with NA, NAM, NR, or an activator of an enzyme that participates in the generating NAD+ (SBI). These results show that NAM, NR, and SBI have anti-viral effects in a mutant form of mouse coronavirus that isn’t as harmful to host cells as naturally occurring mouse coronaviruses.

These data suggest that the antiviral activities of non-canonical PARP enzyme activities are limited by the availability of NAD+ and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses. The data justify the further analysis of how nutritional and therapeutic modulation of NAD+ status may potentially restrict viral infection by boosting antiviral PARP activity.

“While caution should be exercised with respect to any preventative measure, NAD+ boosting approaches have the potential to support the innate immune system and address the age-, smoking-, and comorbid conditions associated with worse SARS-CoV-2 outcomes,” said the investigators in the article.

As there is no standard of care for housemates or caretakers of infected people, the researchers suggest that NAD-boosting could be tested as a protective intervention against infection for people in the proximity of quarantined or hospitalized COVID-19 individuals. The potential societal benefit of a safe and readily available molecule to support prevention and public health is hard to overstate, especially as new outbreaks of COVID-19 emerge.