Identification of Ancient Antiviral Defenses Using Viral Ubiquitination Modulators

Arthropod-borne viruses or “arboviruses” are insect-transmitted viral pathogens that pose a significant threat to human and animal health worldwide. However, we still lack effective therapeutics to combat most arbovirus infections. A deeper understanding of the conserved innate immune mechanisms that restrict arbovirus replication in insect and mammalian hosts may reveal strategies for preventing or treating arboviral disease. Thus, we sought to develop an innovative, yet simplistic, approach to identify conserved antiviral host factors affecting arbovirus replication that, at the same time, inherently provides insight into virally-encoded strategies that evade these host defenses. Here, we propose to use a novel arbovirus "rescue" assay wherein candidate immune evasion proteins (IEPs) encoded by unrelated mammalian viruses can be transiently expressed in insect cells and assayed for their ability to sensitize insect cells to arbovirus infection. The rationale behind this approach is that mammalian virus-encoded IEPs that enhance arbovirus replication in insect cells likely do so because they happen to inhibit key antiviral factors/pathways that are conserved between insect and mammalian hosts. One can then use these IEPs as “tools” to identify and characterize the conserved immunity factors these IEPs target. Thus, this screening methodology provides a mechanism to both identify novel IEPs encoded by mammalian pathogens and the functionally-relevant components of the eukaryotic innate immune response these IEPs inhibit. To discover IEPs that promote arbovirus replication in insect cells, we will screen an expression library encoding 93 “viral ubiquitination modulators (VUMs)” derived from 33 different mammalian viruses. VUMs are virally-encoded proteins that inhibit, usurp, and/or re-direct the host ubiquitination system to promote viral replication. Given that ubiquitination is a key post-translational modification that alters the stability and/or function of cellular proteins, VUMs often hijack the ubiquitination system to inhibit or degrade host proteins that are critical for antiviral defense. We hypothesize that some VUMs may target cellular antiviral factors that are conserved between insect and mammalian hosts and that are critical for restricting arboviruses. Indeed, our initial screens identified VUMs from disparate mammalian viruses that enhance arbovirus replication in insect cells. These results, along with our prior successes in using this system to identify novel IEPs and conserved antiviral responses (Rex et al., 2024, Nat. Microbiol.; Embry et al., 2024, PLoS Pathog.), suggest that we can use VUMs as tools to both inhibit, and discover, conserved antiviral immunity factors. Our study will: 1) Identify VUMs encoded by mammalian viruses that promote arbovirus replication in insect cells; 2) Identify conserved insect and mammalian factors interacting with VUM “hits” from our arbovirus rescue assays; and 3) Identify the conserved host factors interacting with VUMs that normally block arbovirus replication. Our long-term goal is to use this system to define the critical eukaryotic innate immune mechanisms that restrict arbovirus replication. Project Number: 1R21AI196664-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Don Gammon | Institution: UT SOUTHWESTERN MEDICAL CENTER, DALLAS, TX | Award Amount: $249,000 | Activity Code: R21 | Study Section: Viral Pathogenesis and Immunity Study Section [VPI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19666401