Structure, function and dynamics of alphavirus capping pores.

Abstract

Positive-sense single stranded RNA viruses such as coronaviruses, flaviviruses or alphaviruses, carry out transcription and replication inside virus-induced membranous organelles within host cells. Remodelling of the host cell membranes for formation of these organelles is coupled to the membrane association of the viral replication complexes and RNA synthesis. These viral niches allow for concentration of metabolites and proteins for the synthesis of viral RNA, also preventing its detection by the cellular innate immune system. I will present the cryo-EM structural characterization of the chikungunya virus non-structural protein 1, the viral protein responsible for RNA capping and membrane binding of the viral replication machinery, and the capping mechanism. The structures show the enzyme in its active form assembled in a monotopic membrane-associated dodecameric rings. It provides the structural basis for the coupling between membrane binding and allosteric activation of the capping enzyme. We also show different stages of the reaction pathway carried out by the enzyme, 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 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.