The Impact of SARS-CoV-2 nsp5 Resistance Mutations on Antagonism of the Innate Immune Response Público
Edwards, Kristin (Spring 2024)
Abstract
Coronaviruses pose a significant threat to public health, with outbreaks such as SARS-CoV-1 and MERS-CoV underscoring the potential for severe respiratory illnesses. Since 2019, the emergence of SARS-CoV-2, causing COVID-19, has escalated into a global pandemic with devastating consequences. Understanding the molecular mechanisms of SARS-CoV-2 infection and immune evasion is crucial for developing effective therapeutic interventions. This paper presents an investigation into the impact of mutations in the SARS-CoV-2 non-structural protein 5 (nsp5) on the host innate immune response.
The study explored how the E166V mutation in nsp5 affects the ability of the virus to evade host immune surveillance. Using a SARS-CoV-2 replicon system, the project evaluated the interaction between nsp5 mutants and the retinoic acid-inducible gene I (RIG-I) pathway, a key component of the innate immune response. Luciferase assays demonstrated that while the E166V mutant has been linked to decreased replication fitness, there is no significant difference in interferon-beta (IFN-β) promoter activity between the two strains within a 24-hour timeframe. Additionally, the BA.1 E166V replicon demonstrated higher replication activity than the BA.1 WT mutant within the 24hr time course with equivalent activity at the 24hr time point. This suggests that decreased nsp5 activity may not significantly impact the antagonism of the RIG-I pathway within the first 24hr post-induction.
Future directions for research include extending the time course of both luciferase assays to capture prolonged immune responses, validating RIG-I expression and cleavage by nsp5 mutants through Western Blot analysis, and exploring the impact of mutations on the expression of interferon-stimulated genes (ISGs). Furthermore, the study proposes utilizing inhibitors such as nirmatrelvir to assess IFN-β stimulation in the context of antiviral resistance and exploring additional mutant strains to delineate key determinants of viral pathogenicity and immune evasion.
Overall, this investigation contributes to our understanding of how mutations in SARS-CoV-2 may impact host-virus interactions and informs the development of novel therapeutic strategies to combat COVID-19 and future coronavirus outbreaks. By elucidating the complex dynamics between viral genetics and host immune responses, this research aims to advance our ability to effectively manage and mitigate the impact of emerging infectious diseases.
Table of Contents
Introduction 1
Infection 2
Immune Response 4
Evasion of Immune Response 6
Current Treatments and Mutations 8
Experimental Aims 9
Methods 11
Cell Line 11
Replicon System 11
Plasmids 12
IFN-β Stimulation Assay 12
Replicon Fitness Assay 13
Results 14
Validation of IFN-β Assay Design using poly[I:C] 14
IFN-β Stimulation by SARS-CoV-2 Replicons 15
Replicon Fitness Comparison 16
Discussion & Future Directions 17
References 21
List of Figures
Figure 1 3
Figure 2 4
Figure 3 6
Figure 4 7
Figure 5 8
Figure 6 9
Figure 7 10
Figure 8 13
Figure 9 14
Figure 10 15
Figure 11 16
Figure 12 17
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