Structural basis and dynamics of Chikungunya alphavirus RNA capping by the nsP1 capping pores

Abstract

Alphaviruses are emerging positive stranded RNA virus which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The non-structural protein 1 (nsP1) is responsible for RNA capping and the gating of replication organelles by assembling into monotopic membrane-associated dodecameric pores (Jones R. et al. Nature 2021). The capping path is unique for Alphavirus; beginning with the N7 methylation of a GTP molecule, followed by the covalent linkage of a m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA (Ahola T. et al. PNAS 1995). Here we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and SAM, how it reaches a metastable post-methylation state with SAH and m7GTP in the active site, the subsequent covalent transfer of m7GMP to nsP1 and post-reaction conformational changes triggering the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of intermediates of the reaction. Our data identify the molecular determinants allowing each pathway transition, provide explanation for the need for the SAM methyl donor all along the pathway and new clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together our results set new ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals. Significance statementHere we present biochemical and structural characterization of the capping pathway carried out by the Chikungunya virus nsP1 capping pores. We provide five Cryo-EM structures representative of the different steps of the reaction. These structures reveal the molecular determinants and dynamics associated with the alphavirus capping process. In addition, we biochemically show the RNA capping specificity and the reversibility of the reaction which allow nsP1 to cap and decap RNAs and to release intermediates of the reaction. These data provide a new biochemical clues on the enzymatic activity of nsP1 capping pores and a new structural landscape that will be instrumental for the design of effective antivirals targeting the viral RNA capping for blocking the infection.