The crossroads between the methionine cycle and the coronavirus RNA capping.

Metabolic assemblies (MAs) are complexes of enzymes that optimize metabolic processes transferring metabolites between enzymes in close proximity, preventing reverse reactions and enzymatic inhibition. Despite their importance, the spatiotemporal regulation and quaternary structures of enzymes forming MAs remain unclear, hindering the development of effective and targeted therapies to treat communicable and non- communicable diseases that rely on metabolic dysregulations, such as cancer and Coronavirus (CoV) infections. The methionine cycle enzymes form MAs localized in specific cellular compartments depending on the cell fate. This pathway is essential to supply metabolites for CoV RNA capping, which prevents triggering the cell's innate immune responses during CoV infection. The CoV RNA replication and capping enzymes form MAs in double-membrane vesicles (DMVs), and likely modify host MAs to locally produce metabolites for successful viral replication. Understanding the interactions between CoV RNA capping machinery and the methionine cycle in the lung could reveal novel and specific drug targets to inhibit viral replication, which is crucial for addressing current and future CoV outbreaks. Recent data showed that the methionine cycle enzymes MAT2A and MAT2B interact with SARS-CoV-2 capping protein nsp9 and that SARS-CoV-2-RNA interacts with MAT2A and AHCY. MAT2A/B complex produces SAM, the substrate and AHCY hydrolyzes SAH the product of the host and viral methyltransferases nsp14-nsp10 and nsp16-nsp10. Our preliminary data show that MAT2A co-localizes with double-stranded RNA in lung epithelial cells infected with mouse hepatitis virus (MHV-A59) and SARS-CoV-2, suggesting a conserved mechanism. Moreover, AHCY was found in the viral replication vesicles of the Zika virus, and its knockdown significantly reduced Zika virus RNA replication. Furthermore, our preliminary data that nsp16- nsp10 interacts with AHCY, indicating a possible conserved metabolic hub among positive-sense double-strand RNA viruses and potential drug targets. However, whether MAs between CoV RNA capping and the methionine cycle are formed, their spatiotemporal regulation and quaternary protein structures are unknown. Our central hypothesis is that CoVs recruit methionine cycle enzymes and other MAs to DMVs, forming CoV- host MAs that facilitate SAM production and prevent SAH-feedback inhibition during RNA capping. In this proposal, my laboratory will test this hypothesis using proteomics, metabolomics, and structural biology approaches to respond to these three main questions: 1) How MAT2A/B-nsp9 form a MA in the DMVs to enhance SAM production? 2) How AHCY forms a MA with the viral MTases to alleviate SAH inhibition in the DMVs? 3) Whether CoV-RNA supports the methionine cycle MAs in the DMVs? To determine whether these MAs are conserved across -coronavirus we will use adherent cells permissive to infection with mouse hepatitis virus and SARS-CoV-2 alongside purified proteins. Successful completion of this work will underline novel mechanisms of metabolic regulation that can inform specific structure-guided drug design.