Elucidating the molecular nature and evolutionary conservation of RNA pathogen-associated molecular patterns (PAMPs) across the poxvirus family

PROJECT SUMMARY Poxviruses cause disease in disparate eukaryotic hosts, including insects and humans. However, the molecular identity and evolutionary conservation of the RNA pathogen-associated molecular patterns (PAMPs) they generate are poorly defined. Although poxviruses are DNA viruses, infection produces immunostimulatory RNAs sensed by host pattern recognition receptors (PRRs), activating innate immunity. The precise nature and conservation of these RNA PAMPs, and how host and viral factors regulate them, remain unknown. Addressing this is essential for understanding host-virus coevolution and could inform new antiviral or vaccine strategies. Our long-term goal is to define the PAMPs that trigger innate immune responses during poxvirus infection across animal hosts. The objective of this proposal is to define the nature and conservation of RNA PAMPs generated during poxvirus infection, focusing on the regulatory roles of host and viral DUSP11 RNA triphosphatases. We hypothesize that poxvirus infection generates or unveils RNA PAMPs of viral and/or host origin that are recognized by PRRs, and that DUSP11 enzymes mask their immunogenicity in both mammals and insects. We will test this hypothesis via two specific aims: Aim 1: Define the RNA PAMPs generated during mammalian poxvirus infection. We will infect mammalian cells with vaccinia virus, isolate infection-induced RNAs, and use biochemical, sequencing, and functional assays to identify and characterize immunostimulatory RNAs. We will determine their features, ability to activate RIG-I, and how host DUSP11 shapes their abundance and immunogenicity. Aim 2: Develop an insect poxvirus system to determine if RNA PAMPs are conserved across poxviruses. Using entomopoxviruses and mutants lacking vDUSP11, we will characterize RNA PAMPs generated during insect infection and compare their features and regulation to those in mammals. This project leverages advances in RNA biochemistry and sequencing, and our discovery that both vertebrate and insect poxviruses encode DUSP11 homologs that dephosphorylate RNA PAMPs to evade immune detection. Our expected contribution is a molecular map of RNA PAMPs in both mammals and insects, and new insight into how DUSP11 enzymes regulate their immunogenicity. This research is significant because it will provide the first systematic identification of RNA PAMPs in poxvirus infection, reveal evolutionary conservation of immune evasion mechanisms, and enable targeting of conserved immune pathways for vaccine and antiviral development. The project is innovative in that it directly interrogates the identity, structure, and conservation of RNA PAMPs across anciently diverged poxviruses using viral and host DUSP11 mutants as tools essential to map immunogenic RNAs. These results will be important because by resolving how poxviruses generate and mask immunogenic RNAs, we will advance understanding of host-pathogen coevolution and PRR function and possibly reveal strategies for broad-spectrum antiviral intervention and vaccine adjuvant design.