Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, infecting over 100 million people and claiming over 2 million lives globally. This drastically highlights the need for understanding the host-virus protein-protein interactions and immune evasion strategies governing SARS-CoV-2 pathogenesis. Our overall aim was to identify antiviral immune targeting during SARS-CoV-2 infection and protein overexpression. To understand if SARS-CoV-2 can block immune signaling, we first examined nuclear translocation of the transcription factors, IRF3 and NF-κB, as nuclear translocation is one step in interferon induction, during SARS-CoV-2 infection of the human airway epithelial cells. We found that in infected cells, there was limited IRF3 and NF-κB nuclear translocation, suggesting SARS-CoV-2 can limit immune activation. Additionally, through promoter luciferase signaling assays, the viral proteins NSP1, the host translation shutoff factor, and NSP13, the RNA helicase, can block signaling to the IFN-β and NF-κB promoters. To further investigate this reduction in promoter signaling, we examined nuclear translocation of NF-κB in the context of NSP1 and NSP13 plasmid overexpression. Surprisingly, we found that while NSP13 reduced NF-κB nuclear translocation, NSP1 was unable to despite its ability to limit promoter signaling. Instead, we found, utilizing Click-it chemistry, that NSP1 reduces host protein synthesis. Last, we found NSP13 targets TBK1 activation by limiting its phosphorylation in a dose-dependent manner. Collectively, our data illustrate that SARS-CoV-2 can block multiple innate immune signaling pathways through distinct mechanisms.

IMPACT: This research will promote understanding the role of the Type-I Interferon signaling pathway during embryo implantation, potentially leading to a new diagnostic or treatment target in early pregnancy failure. OBJECTIVES/GOALS: Studies suggest interferon signaling regulation is tightly balanced between physiologic and pathophysiologic growth in early pregnancy. We propose to determine the impact of interferon-mediated inflammation on embryo implantation and early pregnancy failure in normal conditions and chronic inflammatory diseases in a novel mixed-mouse model. METHODS/STUDY POPULATION: To probe the role of type-I interferons (IFNs) in implantation, we will utilize a mouse model and non-surgically transfer both Ifnar1-/-and Ifnar1-/+ embryos into an immune-competent pseudopregnant wildtype female recipient. This will allow analysis of a litter with distinct genotypes within the same, immune-competent, uterine environment. Type-I IFN stimulation will be systemically induced with Poly-(I:C) at various time points around implantation. A similar approach will be used in mouse models of chronic inflammatory disease states associated with early pregnancy loss (e.g. systemic lupous erythematous). With this model, we will be able to control for deficiencies in