Virus alteration of triphosphate balance spanning 700 million years of evolution

Viruses are experts at outsmarting the defenses of the animals they infect, but scientists are still uncovering exactly how they do this. This project investigates a group of viral proteins that help viruses hide from the immune systems of birds, reptiles, and insects. Studying how these proteins work across such a wide range of animals will reveal whether viruses have used the same tricks for millions of years—long before humans ever existed. This would unveil that different types of animals have been using a common virus defense that will help the biotechnology industry design better ways to fight infections in people, animals, crop pathogens, and the environment. The research will also create new laboratory tools and methods that can be used by other scientists, and it will train and provide research opportunities for the next generation of American students and young researchers. By supporting this work, federal funding will help uncover fundamental secrets of how viruses and their hosts have been locked in a battle for survival, key for any real understanding of how hosts and microbes interact. This proposal investigates the evolutionary conservation of viral DUSP11 (vDUSP11) activity across poxviruses infecting hosts from insects to vertebrates, focusing on 5’ triphosphate (5’PPP) RNA balance during infection. Preliminary studies identified 16 distinct vDUSP11s, with evidence that at least two avipox variants act on RNA polymerase III (pol III) transcripts to attenuate RIG-I-mediated antiviral responses. We hypothesize this proviral function is conserved among avipox and entomopox vDUSP11s, reflecting an ancient immune evasion mechanism targeting 5’PPP-RNAs, a known pathogen-associated molecular pattern (PAMP) in vertebrates and possibly broader phyla including insects. To test this, we will: 1.Determine if diverse vDUSP11s modify pol III RNAs in mammalian cells via northern blot; 2.Evaluate effects of vDUSP11 expression on RIG-I activation and gene induction using RT-qPCR; 3.Test whether vDUSP11s enhance vesicular stomatitis virus (VSV) infection in mammalian models; and 4.Assess whether vDUSP11s reduce 5'PPP-RNA PAMP-ogenicity in insect cells to evaluate proviral conservation. Stable cell lines expressing various vDUSP11s will enable transcript, gene expression, and viral replication assays. Anticipated outcomes include identifying universally or selectively conserved vDUSP11 activities, revealing convergent viral immune evasion strategies. These findings will advance understanding of triphosphate balance and reveal new paradigms for conserved host-pathogen interactions. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.