Exploring the dynamics of nsp1 and RNA interaction in SARS-CoV with undergraduate researchers

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Project Summary Viral host shutoff proteins selectively hijack cellular machinery to aid viral propagation. In SARS coronaviruses (SARS-CoV-1 and SARS-CoV-2), nonstructural protein 1 (nsp1) serves as the host shutoff factor by dampening host gene expression through ribosomal stalling on host mRNAs followed by their cleavage and decay. However, how nsp1 selectively targets host mRNAs over the viral RNA remains an unsolved puzzle. The long-term goal of this research is to study the steps of nsp1-mediated host shutoff to clearly understand how it specifically selects host mRNAs but not capped and polyadenylated viral RNA that resembles host mRNAs. The rationale for this project is based on the observation that the deletion of stem-loop 1 (SL1) from the viral RNA leader sequence eliminates its ability to escape host shutoff. A critical factor in differentiating viral RNA from host mRNA lies in the interaction between nsp1 and SL1. Based on our previously published results that nsp1 associates with stress granule proteins and disengages G3BP1 protein from the stress granule, we propose to examine the protein and RNA composition of stress granules to determine if viral RNA is protected from decay because of its specific localization connected to the interaction between nsp1 and viral RNA. In Aim 1, we propose to conduct an in-depth study of the interaction between nsp1 and SL1 that will map the sequence responsible for binding, characterize the proteomic profile of the host proteins bound to SL1, and analyze the effect of nsp1 on host mRNA translation and stability. In Aim 2, we propose to examine the disassembly of stress granules in the presence of nsp1 and study its effect on selective cleavage and decay of host mRNA. This multidisciplinary collaborative research will engage undergraduate students to be trained in a highly transformative experience with a team of biochemists, computer scientists, and immunologists to explore a current and relevant topic in biomedical sciences. Overall, our research will connect nsp1’s ability to bind viral RNA sequence to RNA stability and localization and will allow us to examine the mechanism that leads to host mRNA cleavage and decay. Finally, this project will identify interactors of nsp1 and pave the way for designing anti-viral therapeutics.