Nodavirus RNA Replication Crown Architecture Reveals Proto-Crown Precursor and Viral Protein A Conformational Switching

Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric “crown” at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryo-electron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multi-domain viral RNA replication protein A. Each ring contains an ~ 19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a “proto-crown” precursor that assembles prior to RNA template recruitment, RNA synthesis and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding “legs.” The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and non-symmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility and evolutionary plasticity and new insights into (+)RNA virus replication and control. Significance Positive-strand RNA viruses - including coronaviruses, alphaviruses, flaviviruses and many other medically and economically important pathogens - replicate their RNA genomes by virus-encoded machinery that has been poorly characterized. Using an advanced nodavirus model, we identify a major precursor in RNA replication complex assembly and show it to be a 12-mer ring of viral RNA replication protein A, whose single particle cryo-EM structure reveals functional features of its membrane interaction, assembly, polymerase and RNA capping domains. We further show that fully functional RNA replication complexes acquire a second 12-mer ring of protein A in alternate conformation atop the first, and a central density likely to represent another polymerase conformation. These findings provide strong foundations for understanding, controlling and beneficially using such viruses.