Dissection of functional 5' UTR elements that repress SARS-CoV-2 Nsp1 activity

SARS-CoV-2 protein nonstructural protein 1 (Nsp1) induces a global translation shutdown in host cells upon infection. Irrespective of mechanism, the Nsp1 imparted translation shutdown is required for efficient viral replication and to suppress the host immune response. Thus, fully understanding this protein involves understanding how it interacts with viral components and host machinery. The viral genome can escape the translational shutdown via secondary structure in its 5' untranslated region (UTR). The first hairpin structure, stem-loop 1 (SL1), has been identified as necessary and sufficient to evade Nsp1-mediated translation shutdown. Previous reports show that host genes are suppressed differently by Nsp1. For example, translation- related genes, especially those with terminal oligopyrimidine (TOP) motifs, are translated more efficiently in the presence of Nsp1, while immune response genes are suppressed. Despite proven significant impacts of the 5' UTR of SARS-CoV-2 and host genes, other elements remain understudied in this interaction with Nsp1. Therefore, this work examines if the 5' UTR has other functional regions that might influence translational control and evasion of the translational shutdown. This research utilizes a recently developed method called direct analysis of ribosome targeting (DART), a high throughput method that tests the ribosome recruitment ability of thousands of 5' UTRs. To analyze RNA features of SARS-CoV-2 and host genes that impact ribosome recruitment, a diverse pool of viral and host sequences was generated to allow thorough examination of each region of the 5' UTR and its role in translation and evasion of host shutdown. The pool includes all known natural mutations reported in the NCBI virus sequence repository, along with systematic scanning, structural disrupting and compensatory mutations. Completing DART with and without Nsp1 will elucidate what elements facilitate translation and the evasion of Nsp1-mediated translational shutdown. The translation shutdown mechanism is thought to function through a two-pronged approach where the C-terminal domain binds the ribosome at the mRNA entry channel and sterically blocks RNAs from loading onto the ribosome, and the N-terminal domain (NTD) cleaves RNAs while bound to the ribosome, both activities preventing RNAs from being translated. However, it is unclear whether channel exclusion and cleavage are linked activities of Nsp1, or if different RNA features can mediate mRNA channel entry or escape of RNA cleavage. To address this, a high throughput cleavage experiment will be completed on pools of diverse RNAs to examine what host or viral features mediate resistance or susceptibility to cleavage, in ribosome-containing or depleted lysate. These cleavage experiments will be completed using the previously described RNA pool containing SARS-CoV-2 and immune related genes, along with another RNA pool of 24,000 sequences comprised of human genes, including translation-related sequences. This work will be instrumental for understanding the role of Nsp1 in coronavirus pathogenesis and to inform the design of future therapeutics.