Coronavirus endoribonuclease antagonizes ZBP1-mediated necroptosis and delays multiple cell death pathways
Monika Evdokimova1, Shuchen Feng1, Allen Caobi2,3
1Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153.
Abstract:
Identifying conserved mechanisms used by viruses to delay host innate responses can reveal potential targets for antiviral therapeutics. Here, we investigated coronavirus nonstructural protein 15 (nsp15), which encodes a highly conserved endoribonuclease (EndoU). EndoU functions as an immune antagonist by limiting the accumulation of viral replication intermediates that would otherwise be sensed by the host. Despite being a promising antiviral target, it has been difficult to develop small-molecule inhibitors that target the EndoU active site. We generated nsp15 mutants of the coronaviruses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mouse hepatitis virus (MHV)-A59 and identified conserved residues within the amino-terminal domain that are required for EndoU activity. Loss of EndoU activity caused the activation of host sensors, which limited viral replication in interferon-responsive cells and attenuated disease in MHV-infected mice. Using transcriptional profiling, we found that MHV EndoU mutant viruses upregulate multiple host sensors, including Z-form nucleic acid-binding protein 1 (ZBP1). We found that nsp15 mutants induced early, robust ZBP1-mediated necroptosis. EndoU mutant viruses also induced ZBP1-independent apoptosis and pyroptosis pathways, causing early, robust cell death that limits virus replication and pathogenesis. Overall, we document the importance of the amino-terminal domain for EndoU function. We also highlight the importance of nsp15/EndoU activity for evading host sensors, delaying cell death, and promoting pathogenesis.
Insights
Coronaviruses use nsp15/EndoU to suppress host immunity and delay cell death. Inhibiting this enzyme activates immune sensors, limiting viral spread and disease.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Coronaviruses possess conserved mechanisms to evade host innate immune responses.
- Nonstructural protein 15 (nsp15) is a conserved endoribonuclease (EndoU) crucial for viral replication and immune evasion.
- Targeting viral EndoU is a promising antiviral strategy, but active site inhibitors are challenging to develop.
Purpose of the Study:
- Investigate the role of coronavirus nsp15/EndoU in antagonizing host innate immunity.
- Identify conserved residues essential for nsp15/EndoU activity.
- Determine the impact of nsp15/EndoU loss on host sensor activation and viral pathogenesis.
Main Methods:
- Generated nsp15 mutants for SARS-CoV-2 and MHV-A59.
- Assessed nsp15/EndoU activity and identified essential residues in the amino-terminal domain.
- Utilized transcriptional profiling to analyze host sensor activation.
- Evaluated viral replication, cell death pathways (necroptosis, apoptosis, pyroptosis), and disease attenuation in vivo.
Main Results:
- Conserved residues in the amino-terminal domain of nsp15 are critical for EndoU activity.
- Loss of nsp15/EndoU activity triggers host sensors, including Z-form nucleic acid-binding protein 1 (ZBP1).
- nsp15/EndoU deficient viruses induce ZBP1-mediated necroptosis, ZBP1-independent apoptosis, and pyroptosis, leading to early cell death and limited viral replication.
- Disease severity was attenuated in mice infected with MHV nsp15/EndoU mutants.
Conclusions:
- The amino-terminal domain of nsp15 is essential for its endoribonuclease function.
- nsp15/EndoU activity is critical for coronaviruses to evade host immune sensors and delay cell death.
- Disrupting nsp15/EndoU function represents a viable strategy for developing novel antiviral therapeutics against coronaviruses.
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