The Untranslated 3'End of SARS-CoV-2 RNA as a Determinant of Obesity-Accelerated Infectivity

Project Summary/Abstract Obesity is an epidemic-scale problem in the U.S. affecting about 35% of the adult population, and is a major risk factor for the ongoing pandemic, COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA betacoronavirus that is the causative agent of COVID-19. Despite rapid progress in developing effective vaccines, progress in devising improved therapeutics has been slow, and there is an urgent need for anti-viral therapeutics for treatment of infected patients not protected by vaccines. Certain demographics are variably resistant to vaccination, for example, obesity markedly reduces effectiveness of several vaccines. Therapeutics can also be critical in the eventuality that mutations in the virus render the vaccine ineffective. The molecular events responsible for expression of SARS-CoV-2 proteins are known from studies of other betacoronaviruses; however, the regulatory pathways and pathological conditions determining their expression are poorly understood. The translation of viral RNA utilizes the host mRNA translation machinery, primarily regulated by specific RNA-binding proteins or complexes that bind sequence or structural elements in terminal non-coding regions. Importantly, the coding regions of SARS-CoV-2 genomic RNA and the ten subgenomic mRNAs (sgmRNAs) are likewise bordered by non-coding upstream and downstream regions, termed the 5'- leader and 3'-end sequences, respectively. A critical feature of the genome and the sgmRNAs of SARS-CoV-2 is that identical 5'-leader and 3'-end sequences are present in all. Thus, the terminal sequences represent novel, unexplored targets for interference with virus assembly and function. We have discovered a 39-nt sequence in the 3'-end of SARS-CoV-2 bearing structural similarity to the GAIT (interferon-gamma-activated inhibitor of translation) RNA element previously described by us. We show that glutamyl-prolyl tRNA synthetase (EPRS) a protein that binds the human GAIT element also binds the vGLE. Moreover, IFN-γ, a pro-inflammatory cytokine, and insulin, an obesity-induced hormone, markedly increases expression of a luciferase reporter bearing the intact vGLE. These results are the first to show a functional consequence of an RNA element in the 3'-end sequence of SARS-CoV-2. We hypothesize that binding of EPRS to the vGLE stimulates sgmRNA translation required for expression of structural and other SARS-CoV-2 proteins, and for programmed ribosomal frameshifting required for genome replication. We will test this hypothesis by pursuing two Specific Aims: In Aim 1 we elucidate the role of EPRS binding to the SARS-CoV-2 3'UTR vGLE in regulating viral replication and sgmRNA translation. In Aim 2 we develop RNA inhibitors that target EPRS/vGLE interactions and block viral protein expression. We anticipate these fundamental studies will provide the first information on the function of the 3'-end of SARS-CoV-2, and will provide a foundation for development of therapeutic agents to be used in combination with mechanistically distinct anti-viral agents targeting the vGLE and possibly emerging betacoronaviruses.