Determination of immunomodulatory effects of SARS-COV-2 structural, non-structural, and accessory proteins on macrophage-like cells in the context of type I IFN antagonism and inflammasome activation

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2022-2-02

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Aydın, Yağmur

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Infection with SARS-CoV-2 inhibits early type I interferon response but activates inflammasome signaling and pro-inflammatory cytokine production. The virus accomplishes these opposing effects by manipulating the host cell immunity through a multitude of encoded viral proteins of structural, non-structural, or accessory origin. In this thesis, to investigate the immunomodulatory effects of SARS-CoV-2 encoded proteins on type I IFN antagonism and inflammasome activation, five SARS-CoV-2 structural, nonstructural, or accessory protein-expressing stable THP1-Dual cell lines (NSP9, NSP10, ORF3a, ORF8 or N) and a MOCK control was generated using a 3rd generation lentiviral gene transduction approach. The cell lines were then exposed to virus-mimetic pattern recognition ligands or to recombinant IFN-β. Their type I IFN as well as type I IFN-dependent ISG15 responses were assessed, respectively. Our findings indicate that ORF3a and ORF8 accessory proteins significantly antagonized type I IFN production and ORF3a downregulated ISG15 production. NSP9 and N were found to antagonize type I IFN production only when compared to WT, but not to mock, suggestive of a minor impact. To measure the impact of SARS-CoV-2-related protein expression on inflammasome activation, and pyroptosis, NLRP3, NLRC4, AIM2, or non-canonical inflammasomes were stimulated via corresponding activating ligands. Our results confirmed that ORF3a accessory protein significantly over-activated NLRP3 and NLRC4 inflammasome-mediated IL-1β production and lactate dehydrogenase enzyme release, indicative of pyroptotic cell death. NPS9 expression in the cell line possibly blocked cell membrane trafficking in response to nigericin, thereby preventing nigericin-mediated cell death. Furthermore, N structural protein hindered pyroptosis but induced pyroptosis-independent cell death after non-canonical and NLRC4 inflammasome activation, suggesting that Nucleocapsid interfered with gasdermin-D-mediated pore formation, but triggered a pyroptosis-independent cell death mechanism.

Subject Keywords

SARS-COV-2, Antiviral Immunity, Type I IFN Antagonism, Inflammasome

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https://hdl.handle.net/11511/96222

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

Y. Aydın, “Determination of immunomodulatory effects of SARS-COV-2 structural, non-structural, and accessory proteins on macrophage-like cells in the context of type I IFN antagonism and inflammasome activation,” M.S. - Master of Science, Middle East Technical University, 2022.