Translational Regulation of SARS-CoV-2 in response to viral S protein-induced signaling

Adopted folding of the RNA elements within the genomes of RNA viruses allows binding of host and viral proteins and plays an essential role in host-virus interactions. However, the mechanistic insights on how these RNA elements could sense the transduced signals from host cells are lacking for SARS-CoV-2. This project is based on our recent discovery of a novel and structurally conserved RNA element within the ORF1a RNA and S mRNA of SARS-CoV-2. Our studies showed that this element regulates the translation of viral proteins in response to viral S protein-induced signaling in human lung cells. In prior studies, we identified a translational silencing-dependent mechanism of controlling inflammation in myeloid cells. These studies showed that the assembly of an L13a-dependent multi-protein RNA-binding complex (IFN-gamma-activated-inhibitor of translation) or GAIT complex on the GAIT elements found in the 3' untranslated region (UTR) of target mRNAs causing translational inhibition. Our studies also showed the physiological importance of this mechanism as an endogenous defense against inflammation. Importance of the GAIT-like elements in human responses to coronavirus and other respiratory pathogens is emerging from the published studies by us and others. Our preliminary work presented in this proposal shows that treatment of human lung cells with SARS- CoV-2 S protein activates a DAP kinase-dependent signaling mechanism causing the release of L13a from large ribosomal subunit followed by the formation of L13a-dependent RNA-binding complex on "GAIT-like" viral RNA elements of SARS-CoV-2, thus inhibiting translation. This led us to name these elements virus activated inhibitor of translation (VAIT) elements. Together, these studies lead us to hypothesize that ribosome-released form of L13a upon SARS-CoV-2 infection promotes the assembly of RNA- binding complex on VAIT element of ORF1a RNA and S mRNA, which is biochemically and functionally distinct from IFN-g-induced GAIT complex. The role of translational inhibition caused by the VAIT complex is testable. We will test this hypothesis by pursuing the following two aims. Aim 1. To elucidate the composition of the translation inhibitory VAIT RNP complex and identification of the upstream kinase in signaling. In this aim we will use the combined approach of Gel filtration, RNA-affinity chromatography and mass spectrometry to identify the subunits of the RNA-binding complex. Small molecule inhibitors and siRNA of the potential kinases will be used to dissect the signaling aspect. Aim 2. To test the virological significance of VAIT element mediated translational silencing. In this aim we will use extensive mutation in the VAIT element of the S gene within the viral genomic RNA of the trans-complementation system to test the role of VAIT element-mediated translational silencing in the intracellular expression of viral proteins and nucleic acid. Mechanistic insights on VAIT element mediated translational silencing of SARS-CoV-2 RNAs may provide novel targets of small molecule intervention and facilitate the development of next-generation mRNA vaccines.