The Impact of Mutations on the Functions of Nonstructural Protein 1 of SARS Coronavirus

Abstract

Introduction/BackgroundSevere acute respiratory syndrome coronavirus (SARS‐CoV) encoded nonstructural protein 1 (nsp1) orchestrates a multi‐pronged mechanism to shut down host gene expression, also known as the host shutoff. Nsp1 is a 180 amino acid long protein with a primarily flexible structure that allows nsp1 to interact with various cellular factors during host shutoff to inhibit translation and promote mRNA decay.Hypothesis/Goal of StudyMutations of the surface residues of nsp1 either attenuate or augment its host shutoff function prompting the hypothesis that these residues bind cellular proteins that facilitate nsp1 during the host shutoff. To identify cellular proteins that interact with nsp1 and assist its function, we isolated host proteins proximal to nsp1 in human epithelial kidney cells by using a fusion protein, nsp1‐BioID2. These isolated proteins are identified using mass spectroscopy and verified by western blot. Also, we are using computer‐based modeling to predict the binding sites of these nsp1‐interacting proteins.Methods and ResultsBy comparing protein complexes, we identified a large group of proteins including ribosomal proteins that are likely involved in host shutoff. In addition to the ribosomal proteins, factors implicated in the stress granule formation and RNA processing are identified and verified. We have tested these interactions in multiple mutants that are known to affect the host shutoff activity of nsp1. Surprisingly, none of the mutations in the structured region of nsp1 disrupts any of these mutations.ConclusionsBioID‐mediated tagging of the host proteins proximal to nsp1 allows us to identify multiple pathways modulated by nsp1.