Understanding Ribosomal Dependent Virulence Pathways Employed by Non-Structural Protein 1 in Emerging Human Beta-Coronaviruses

Project Summary/ Abstract Betacoronaviruses (β-CoVs) present a substantial threat to global health. Hence, determining viral targets for therapeutic intervention is an utmost priority. One attractive target is Non-structural protein 1 (Nsp1) which restricts the innate immune response by inhibiting host gene expression. Nsp1 is encoded by all β-CoVs and is hypothesized to function through three host-viral interaction pathways: two are in the cytosol requiring the ribosome, and one in the nucleus impeding host mRNA trafficking through the nuclear pore. First in the cytosol, Nsp1 restricts protein translation by binding inside the mRNA entry channel of the ribosome, sterically blocking proper host mRNA loading. Secondly, Nsp1 coordinates endonuclease cleavage of host mRNA using eukaryotic initiation factor 3g (eIF3g) as a co-factor on the ribosome. This cleaves host mRNA at the 5' end leaving the transcripts translationally inert. However, for Middle East Respiratory Virus (MERS), I present data showing the eIF3g alone is not sufficient as a co-factor for endonuclease cleavage. Recent evidence has emerged emphasizing the importance of the two ribosome-dependent mechanisms for Nsp1 function and viral replication. However, the endonuclease function of Nsp1 remains poorly understood. I hypothesize that the endonuclease function is a crucial aspect of Nsp1 biology and elucidating its molecular mechanism will pave the way for potent drug development which can restrict current and future β-CoVs. Currently, the FDA approved drug, Montelukast, has been shown to inhibit the SARS-CoV-2 endonuclease complex. Aim 1: Determine the structure of the SARS-CoV-2 Nsp1 endonuclease complex. I present a preliminary cryoEM structure of SARS-CoV-2 Nsp1 facilitating the endonuclease complex. I show Nsp1 binding eIF3g and mRNA on 40S subunit of the ribosome, however, my study was limited in scale and is currently at low resolution (~6Å local resolution). As such I propose collecting a large dataset on a 300kV microscope to obtain a high-resolution map, resolving the molecular details of the endonuclease function. Additionally, I propose binding studies to elucidate details of the mechanism of action for Montelukast inhibiting the endonuclease complex of SARS-CoV-2 Nsp1. Successful completion of this aim will determine the molecular mechanism of the endonuclease function of Nsp1 and significantly advance Nsp1 as a therapeutic target. Aim 2: Elucidate essential co-factors for MERS Nsp1 endonuclease function. My preliminary data strongly supports that eIF3g alone is not sufficient as a co-factor for MERS Nsp1 endonuclease function. Thus, I will employ proximity labeling with TurboID to identify what co-factors are needed for MERS Nsp1 to cleave mRNA by employing biochemical and molecular biology methods. Then I will recapitulate the minimal endonuclease complex for MERS Nsp1 in-vitro. Successful completion of this aim will show how the endonuclease function of Nsp1 is conserved across the divergent landscape of β-CoVs.