Date of Award

6-3-2025

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

First Advisor

Susan Baker

Abstract

Coronaviruses have evolved a breadth of mechanisms to antagonize host innate immune responses. One widely studied mechanism is the ability to deconjugate ISG15 from target proteins in a process called deISGylation, which is catalyzed by the papain-like protease (PLP) domain of viral non-structural protein (nsp) 3. Since PLP activity is required for viral replication, it has been challenging to generate an infectious virus with a PLP that maintains protease activity but has lost deISGylase activity. In this study, we generated a chimeric MHV-A59 with its PLP2 domain replaced with that of the human coronavirus HKU1, which has been documented to have minimal deISGylase activity in vitro. By using this virus, we determined that the deISGylase activity is critical for inhibiting the IFN-response in primary macrophages and promoting robust viral replication. Importantly, we also report that without deISGylase activity, the chimeric virus is highly attenuated in vivo and is more rapidly cleared from infected mice compared to MHV-A59. Accordingly, we found that at earlier stages of infection, MHV-HP2 induces a higher IFN response in the liver, providing a potential mechanism for the rapid clearance of the deISGylase-deficient coronavirus. These results document the critical role of viral deISGylase activity as a key contributor to innate immune evasion during coronavirus infection. We also investigated the ability of ISG15 to be secreted from the cell in response to coronavirus infection. The mechanism of ISG15 secretion was unknown. Here, we report that ISG15 is secreted from inflammasome-activated macrophages via gasdermin D (GSDMD) pores on the plasma membrane, mirroring the unconventional secretory pathway used by IL-1β and IL-18. Secretion of ISG15 is negated by deletion of GSDMD. It is also inhibited by treatment with the small-molecule pan-caspase inhibitor zVAD-fmk, the FDA-approved drug disulfiram, the caspase-1 inhibitor VX-765, and the NLRP3 inhibitor MCC-950, all of which block GSDMD pore formation. This study paves the way for investigating how extracellular ISG15 contributes to viral pathogenesis. Finally, we interrogated the effects of extracellular ISG15 on coronavirus replication and activation of the innate immune response through the addition of recombinant ISG15 (rISG15) to macrophages. By analyzing changes in gene expression and cytokine release in BMDMs treated with rISG15 and comparing them to IFN-driven responses, we determined that extracellular ISG15 has antiviral and proinflammatory effects during MHV-infection that is distinct from the IFN response. This study contributes to our understanding of how coronavirus infection drives inflammation within the infected host.

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