BC-73 Investigating the role of nonstructural protein 1 in the selective translation control of SARS-CoV viral RNA over host mRNAs

Nonstructural protein 1 (nsp1) from recent zoonotic coronaviruses (SARS-CoV-1, SARS-CoV-2, MERS-CoV) functions as a host shutoff protein that stalls host mRNA translation and triggers their degradation, successfully suppressing host gene expression. In contrast, viral RNA selectively escapes the effects of the two-pronged suppression mechanism and continues the synthesis of viral proteins due to the presence of stem-loop 1 (SL1) in the viral leader sequence. Recent structural analysis of nsp1 bound to the 40S ribosomal subunit suggests that the flexible C-terminal region of nsp1 binds to the 40S ribosome at the mRNA binding site by folding into two defined structures (alpha-helices); this interaction blocks host mRNAs from binding, stalling host mRNA translation. Meanwhile, the interaction between nsp1 and the viral leader sequence is thought to counteract that of nsp1-mediated translation suppression for the viral RNA. The exact nature of the selective control of translation has yet to be fully elucidated. Understanding these mechanisms more accurately is crucial to advance the current knowledge of similar host shutoff mechanisms in other viruses and uncover new anti-viral targets to control the spread of such viruses. Research in our laboratory is based on the hypothesis that the selective protection and preferential translation of viral RNA over host mRNA is based on nsp1’s ability to interact with RNA in both the nucleus and cytoplasm. We have previously established that nsp1 directly binds to viral SL1 sequence leading to two separate complexes, a small complex when nsp1 concentration is lower and a large complex when nsp1 concentration is significantly higher. These experiments were conducted by gel-shift assay using purified RNA and nsp1. By introducing mutations in the viral SL1, we mapped the interaction between nsp1 and viral RNA to understand the complex formation. However, when ribosomes were present, nsp1-SL1 complex formation changed into an even larger assembly.The objective of this project is to isolate and characterize the complexes formed between viral RNA, nsp1, and cellular extract (containing ribosomes) by mass spectrometry with a focus on clarifying the steps of viral RNA translation in the presence of nsp1. Our current results indicate that multiple cytoplasmic proteins bind the nsp1-SL1 complex but mutations in SL1 change its composition.