mRNA Nuclear Export Machinery as Target of SARS-CoV-2 Nsp1 Protein - The global COVID-19 pandemic caused by the SARS-CoV-2 outbreak underscores the need for understanding viral-host interactions that impact host gene expression and antiviral responses. A critical virulence factor of coronaviruses is the nonstructural protein 1 (Nsp1). We uncovered a previously unknown mechanism of Nsp1- mediated inhibition of host antiviral responses: Nsp1 targets the cellular mRNA nuclear export pathway. Nuclear export of cellular mRNAs through the nuclear pore complex (NPC) is obligatory for eukaryotic gene expression including those genes encoding antiviral factors. By contrast, SARS-CoV-2 mRNA synthesis takes place at viral transcriptional factories in the cytoplasm. We have demonstrated that SARS-CoV-2 Nsp1 protein inhibits host mRNA nuclear export by targeting the cellular NXF1-NXT1 complex, a receptor required for mRNA translocation through the NPC to the cytoplasm. We identified mutations in Nsp1 that specifically reduce its binding to NXF1- NXT1. Importantly, a SARS-CoV-2 recombinant virus carrying a mutant Nsp1 deficient in NXF1-NXT1 binding is unable to inhibit mRNA nuclear export and is attenuated in cells and in mice. Together, our data indicate that Nsp1 is a regulator of host mRNA nuclear export via blocking NXF1 function, overcoming cellular antiviral responses. This proposal combines the synergistic expertise between the principal investigators in biochemistry, cell biology, structural biology, and viral pathogenesis to investigate in depth the molecular mechanisms of SARS-CoV-2 Nsp1 protein function as inhibitor of mRNA nuclear export and its impact on the virus life cycle and on virulence. In Aim 1, we will use biochemical, genetic, and imaging approaches to determine the interactions within the mRNA export machinery that lead to Nsp1 inhibition of mRNA export. In addition, we will use a combination of biophysical and structural biology approaches to determine the molecular basis for the recognition between Nsp1 and NXF1-NXT1. These findings will uncover new mechanisms that mediate or regulate cellular mRNA nuclear export and how they are targeted by SARS-CoV-2. In Aim 2, we will assess the functions of the Nsp1-NXF1 interaction during virus replication and pathogenesis. We will generate loss-of-function recombinant viruses carrying Nsp1 mutants defective in NXF1 binding and investigate the impact of these mutations on host mRNA export, viral replication, induction of host responses in infected cells, and on viral pathogenesis in two animal models, mice and hamsters. Additionally, Nsp1 polymorphisms associated with new variants of concern have been found. We will therefore address the functional significance of these polymorphisms for the Nsp1- NXF1 interaction and for viral pathogenesis. Moreover, we will identify the host mRNAs whose nuclear export are impacted by Nsp1 and determine their role in antiviral response. These extensive studies will likely not only define novel virulence function(s) of SARS-CoV-2 Nsp1, but also shed light on novel antiviral defense mechanisms. Since antagonizing Nsp1 function results in expression of mRNAs encoding antiviral factors, our proposed studies may uncover new strategies for designing antivirals against SARS-CoV-2.
