Mechanisms of SARS-CoV-2 Nsp1 Mediated Host mRNA Nuclear Export Inhibition

Abstract

Non-structural protein 1 (Nsp1) is the major virulence factor and host gene expression inhibitor encoded by SARS-CoV-2. SARS-CoV-2 Nsp1 inhibits host gene expression through multiple mechanisms, including translational blockage and mRNA cleavage via the interaction with the ribosome and the inhibition of mRNA nuclear export by hijacking NXF1-NXT1. While the mechanism of SARS-CoV-2 Nsp1 induced translational blockage is well studied, the mechanism of how Nsp1 targets NXF1 to inhibit mRNA nuclear export was poorly understood. This dissertation provides mechanistic details for the Nsp1-NXF1 interaction and demonstrates the importance of Nsp1 mediated host mRNA export inhibition to SARS-CoV-2 virulence and pathogenesis using a separation-of-function mutation to Nsp1. Specifically, we performed a systematic structure-guided mutagenesis study on Nsp1, revealing the NXF1 binding sites on Nsp1. Using a combination of biochemical and structural approaches, we identified that an Nsp1 N-terminal acidic patch interfaces with a basic patch on the RRM domain of NXF1. This Nsp1-N acidic patch is crucial for the Nsp1-NXF1 interaction, but it is separated from the Nsp1 surfaces important for ribosome binding and mRNA cleavage functions. Indeed, we demonstrated that mutations to the Nsp1 N-terminal acidic patch are separation-of-function mutations, which largely alleviate the Nsp1 induced mRNA export inhibition but retain Nsp1’s ability to block translation. We showed the importance of the Nsp1 N-terminal acidic patch to SARS-CoV-2 virulence as the SARS-CoV-2 mutant virus with mutations to this acidic patch is attenuated. Collectively, our findings provide powerful tools to better understand the complex network of Nsp1 induced host gene expression inhibition. Antagonizing the Nsp1 N-terminal acidic patch represents a potential new way to facilitate host immunity against SARS-CoV-2.