Highly conserved residues of coronavirus nsp1 skew translational equilibrium toward viral protein production

Coronaviruses cause diseases in many species of animal including humans and have been a major cause of lethal human disease outbreaks for the past twenty years. There have been three major outbreaks of human coronaviruses in 2002, 2012, and 2019 by SARS-CoV, MERS-CoV, and SARS-CoV-2 respectively that cause severe disease and have a high rate of mortality. Beyond the severe pathogenesis of these diseases, their rapid spread and unpredictable spillover into the human population is equally worrisome. This underscores the need for therapeutics to treat and prevent current and future coronavirus outbreaks. To this end, it is paramount to understand the basic biochemistry of coronavirus replication and innate immune evasion. The coronavirus non- structural protein 1 (nsp1) is of particular interest to this work due to its ability to inhibit host protein synthesis. There are two important functional domains of nsp1: the ribosome-binding domain (KH/KY motif) and the genome-recognition domain (LLRK motif). The KH/KY motif confers binding to the 40S subunit of the ribosome and inhibits host translation. It has been hypothesized that a conserved, structured region of the 5’UTR interacts with the LLRK motif, releasing nsp1 from the 40S subunit, and thereby allows preferential translation of viral genome and mRNAs. Conversely, various cellular stressors active the integrated stress response (ISR), which phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2a) to reduce translation during times of stress. We hypothesized that the viruses evade the ISR by competitively inhibiting host protein synthesis, impeding expression of key stress and innate immune genes. A reverse genetic approach was employed to generate viruses with point mutations in either of these motifs to investigate their influence on the ISR. ; Master of Chemical Sciences