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Role of LGP2 in the Innate Immune System upon Viral Infections

Gillich, Nadine

[thumbnail of Dissertation Nadine Gillich.pdf] PDF, English
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Abstract

RIG-I-Iike receptors (RLRs) are a family of pattern recognition receptors that play an important role in the induction of cellular antiviral responses. RLRs comprise LGP2, RIG-I and MDA5. The latter two initiate antiviral signaling upon binding of viral cytoplasmic double-stranded (ds) RNA, resulting in the expression of interferons (IFNs) and IFN stimulated genes. LGP2 enhances MDA5- and represses RIG-I-mediated signaling even though in the latter case the physiological implication is less clear. Whether posttranslational modifications of LGP2 are involved in its diverse functions remains obscure. Hepatitis delta virus (HDV), a small RNA virus with a circular genome, is an important human pathogen responsible for the most severe form of viral hepatitis. HDV was shown to be sensed by MDA5 but the contribution of LGP2 to induction of the IFN response has not been explored. Hence, the aim of my thesis work was (i) to gain a deeper understanding of the role of LGP2 in regulating RLR signaling and (ii) to determine the contribution of LGP2 and its natural polymorphisms (encoding Q425R, N461S, R523Q) to sensing of HDV and other human viral pathogens. Using knockout and overexpression systems, immunocompetent lung A549 and hepatic HepaRGNTCP cells were measured for their IFN response upon viral infection and synthetic dsRNA stimulation. Mass spectrometry (MS) was performed to elucidate the impact of phosphorylation on the regulatory function of LGP2. Studies in A549 cells indicated faster RIG-I and delayed MDA5 signaling. LGP2 inhibited RIGI and strongly enhanced MDA5 signaling. RNA binding but not ATP hydrolysis was important for both LGP2 functions upon synthetic dsRNA stimulation. In HDV infected HepaRGNTCP cells LGP2 was shown to directly bind HDV RNA. Moreover, LGP2 RNA binding and ATP hydrolysis function were essential to fully activate an IFN response that impaired HDV replication. MS identified S169, S365 and S464 as differentially regulated LGP2 phosphorylation sites. Follow-up functional assays revealed enhanced RIG-I inhibition by the phosphoablative S169A substitution in LGP2. Preliminary data with an S365A/S464D LGP2 double mutation, mimicking steady-state phosphorylation at those sites, indicated delayed responsiveness of this LGP2 mutant towards HDV sensing. Investigation of the Q425R, N461S and R523Q LGP2 variants identified Q425R LGP2, which predominates in the African population, as a gain-of-function version. Q425R LGP2 enhanced basal and accelerated HDV-induced IFN signaling, thus lowering viral replication. This variant also enhanced MDA5-mediated antiviral signaling upon severe acute respiratory syndrome coronavirus type 2 infection. Mechanistically, Q425R LGP2 enhanced MDA5-RNA binding compared to wild-type LGP2. In conclusion, the results obtained during my thesis work broaden our understanding of the regulation of RLR signaling by LGP2. In the future, the gained knowledge might facilitate the development of new antiviral interventions by targeting RLRs for disease control.

Document type: Dissertation
Supervisor: Bartenschlager, Prof. Dr. Ralf
Place of Publication: Heidelberg
Date of thesis defense: 18 November 2021
Date Deposited: 17 Feb 2022 13:42
Date: 2023
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
DDC-classification: 570 Life sciences
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