Structural basis of Lassa fever nucleoprotein binding pathogen-associated pattern molecule dsRNA
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Lassa fever virus (LASV) infects thousands of people and produces more than 5,000 deaths each year in West Africa. This severe virus is a huge threat, as it transmits between human and rodents, and no effective vaccine or drug is available currently. One key of getting control of this disease lies in the nucleoprotein (NP) of LASV, which plays an essential role in viral replication, transcription and immune suppression. The full length NP crystal structure has been solved, showing a novel structural fold and multi-functions with unusual mechanisms in immune suppression and viral RNA transcription. The C-terminal domain of LAVS NP is a 3’-5’ exonuclease, whose activity is essential for viral immune suppression. This domain alone can suppress an immune response and can degrade dsRNAs with specific preference higher than for ssRNAs. However, the detail of the mechanism is unclear. To understand the mechanism while avoiding another domain’s effect (the N-terminal domain), the C-terminal domain of LASV NP was expressed and purified, and pathogen-associated pattern molecular RNAs were synthesized chemically and biologically to carry on crystallization and functional testing. The C-domain crystals in complex with a pathogen-associated pattern molecule, triphosphate 8 nucleotide dsRNA were obtained. The crystal belongs to the space group P3 with unit cell dimension a=b=177.6 Å, c=56.49Å, α =β=90°, γ=120°. This crystal structure showed that the dsRNA binds in the 3’-5’ exonuclease active site with one 3’ end of the dsRNA perfectly sitting for cleavage. We are trying to figure out the detailed mechanism by mutagenesis, fluorescence-labeled RNA gel scan and band shift assays.
Thesis, MPhil Master of Philosophy
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