A Study of Nonstructural Protein 1 of SARS Coronavirus

Abstract

Severe acute respiratory syndrome coronavirus (SARS‐CoV) encoded nonstructural protein 1 (nsp1) orchestrates a multi‐pronged mechanism to down regulate host gene expression, also known as host shutoff. Nsp1 is a 180 amino acid long protein with primarily flexible structure that allows nsp1 to interact with various cellular factors during host shutoff to inhibit translation and promote mRNA decay. Mutations of the surface residues of nsp1 either attenuates or augments its host shutoff function prompting the hypothesis that these residues bind cellular proteins that facilitate nsp1 during host shutoff. To identify cellular proteins that interact with nsp1 and assist its function, we conducted a quantitative proteomics experiment by comparing protein complexes associated with nsp1 and its inactive mutant (K164AH165A) and computer modeling to predict structural changes. By comparing protein complexes, we identified a previously undetected mechanism by which nsp1 exploits the nuclear pore complex and disrupts nuclear‐cytoplasmic transport of biomolecules. Expression of nsp1 in 293 cells disrupts Nup93 localization around the nuclear envelope without triggering proteolytic degradation of the protein while other nucleoporins and the nuclear lamina remain unperturbed, suggesting a significant alteration of the nuclear pore complex in the presence of nsp1. However, expression of mutant nsp1, which does not bind to Nup93, does not alter the nuclear pore complex. To better understand the structural basis of this protein’s function, we have utilized computational modeling tools, more specifically, I‐TASSER (Iterative Threading ASSEmbly Refinement). Structural models of various mutants of nsp1 were obtained (by using I‐TASSER) in order to explore the structural impact of different mutations. Our preliminary explorations show significant topological changes on nsp1’s surface for specific mutations. These perturbations of surface properties can provide a steric‐based mechanism of interfering with nsp1’s binding or recognition function therefore, aggravating its host shutoff function.