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Decoding toll-like receptors and adaptors TRIF and MyD88 in macrophage responses to murine betacoronaviruses of differing neurovirulence: identification of interferon-stimulated ISG15 as a potential antiviral restriction factor
Dissertation   Open access

Decoding toll-like receptors and adaptors TRIF and MyD88 in macrophage responses to murine betacoronaviruses of differing neurovirulence: identification of interferon-stimulated ISG15 as a potential antiviral restriction factor

Adam Michael Glass
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2025
DOI:
https://doi.org/10.17918/00010926
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Abstract

Betacoronavirus ISG15 MyD88 Toll-like receptor TRIF Macrophage Microbiology
In the 21st century, three highly pathogenic Betacoronaviruses ([beta]-CoVs) (SARS-, MERS-, and SARS-CoV-2) have emerged within approximately ten-year intervals (2002, 2012, and 2019). Among these [beta]-CoVs, only SARS-CoV-2 has been linked to neurologic complications, manifesting during acute COVID-19 or in affected individuals currently living with "Long Covid" neuropsychiatric syndromes. There is an urgent need to understand the pathophysiology of CoV disorders and develop disease-modifying therapies. [beta]-CoV that naturally infect the mouse (mouse hepatitis viruses, MHVs) are well-established models to dissect pathogenesis and test intervention strategies. Disease severity during [beta]-CoV infection increases with viral clearance, suggesting an immune response that is both protective and pathogenic. Strikingly, [beta]-CoV infections in humans and mice are associated with exacerbated infiltration of inflammatory macrophages to infected tissues, including the lungs and brain. However, there yet remains a gap in knowledge surrounding the role of innate immune sensors, particularly toll-like receptors (TLRs) and their adaptor proteins, involved in macrophage sensing of [beta]-CoV infection. Our overall hypothesis is that elucidating the signaling pathways that dictate macrophage responses to [beta]-CoVs is crucial for developing novel therapeutics that limit macrophage-driven hyperinflammation. Using a combinatorial approach, we investigate the role of TLRs and their adaptors TIR-domain-containing adaptor-inducing interferon-[beta] (TRIF) and myeloid differentiation primary response protein 88 (MyD88) in macrophage antiviral and inflammatory responses to the neurovirulent MHV-JHM and the neuroattenuated MHV-A59 [beta]-CoVs. ELISA quantification of pro-inflammatory cytokines from MHV-infected immunocompetent (WT) or adaptor-deficient macrophages revealed significantly higher production of IL-6, TNF[alpha], and IL-1[beta] following MHV-JHM infection compared to MHV-A59, which associates with their divergent neurovirulence in vivo. While cytokine secretion was dependent on MyD88, TLR2-mediated IL-6 production was negatively regulated by TRIF. To assess MHV replication, viral titers, nucleocapsid production, and dsRNA synthesis were evaluated via plaque assay, immunoblotting, and immunofluorescence, respectively. Surprisingly, replication of both MHV strains was impaired in the absence of TRIF or TLR2 despite increased expression of the dsRNA sensor melanoma differentiation-associated protein 5 (MDA5) in macrophages deficient in either adaptor. IFN-[beta] neutralization further revealed that MHV-A59, but not -JHM, restriction was IFN-[beta]-dependent. Inhibitors targeting pan-caspase activity or the NLRP3 inflammasome were unable to rescue MHV-JHM replication, however analysis following one hour of viral adsorption suggested a potential restriction mechanism during the entry stages of infection. Our assessment of macrophage antiviral responses led us to investigate the ubiquitin-like interferon stimulated gene 15 (ISG15), which is among the most abundantly expressed ISGs and can function freely or as a conjugated protein modification. ISG15 expression and/or conjugation was induced following TLR ligand stimulation or MHV infection and negatively correlated with viral replication. Immunofluorescence and proximity ligation analysis identified the MHV nucleocapsid and a dsRNA-associated protein as potential targets of ISG15 conjugation, however both MHV strains antagonize this process. Remarkably, our data revealed a novel role for TRIF and MyD88 as negative regulators of ISG15. Together, these studies underscore the complexity of innate immune signaling in macrophage responses to [beta]-CoV infection, indicating distinct roles for the TLR adaptors TRIF and MyD88, and highlight ISG15 as a potential pan-CoV restriction factor.

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