Investigating host protein binding to SARS coronavirus untranslated region using immobilized RNA

Abstract

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) encoded nonstructural protein 1 (Nsp1) is a key factor in dampening host gene expression, also known as host shut‐off. Nsp1 binds to the 40S ribosome, stalls ribosome assembly, and results cleavage of cellular messenger RNA (mRNA). Surprisingly, SCoV mRNA is resistant to cleavage under the same condition, allowing us to investigate host protein binding by SCoV mRNA that confers resistant to Nsp1‐initiated cleavage. The untranslated region of SCoV mRNA is about 100 nucleotides long and contain a RNA stem‐loop necessary for resisting endonucleolytic cleavage. We used T7 polymerase driven transcription and T4 RNA ligase to synthesize biotinylated RNA containing untranslated region of SCoV mRNA. The RNA was then incubated with cellular proteins in the presence of Nsp1 followed by UV crosslinking. RNA‐protein complexes were isolated using streptavidin‐coated magnetic beads. Proteins were separated by gel electrophoresis followed by their identification using LC‐MS/MS. Identification of these proteins will allow us to decipher the mechanism of host shutoff by Nsp1.Support or Funding InformationSC INBREFurman AdvantageThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.