bioRxiv2025
Positive-strand RNA viruses replicate their RNA genomes in virus-induced, membrane-bounded organelles. As first found for nodaviruses, the necked cytosolic portals of these organelles bear ringed "crown" complexes of viral RNA replication proteins that drive synthesis, capping and release of new RNA genomes. Nodavirus crowns contain two 12-mer rings of viral protein A with C-proximal polymerase domains stacked. In the basal ring, protein As N-proximal RNA capping domains form a central, toroidal floor, while in the apical ring these domains extend radially outward. A third protein A conformation provides a putative central Pol domain interacting with the viral dsRNA replication intermediate in a vesicle beneath the crown. Protein As multiple conformations likely differentially contribute to crown assembly, RNA template recruitment, (-) and (+) strand synthesis, RNA capping, and progeny RNA release. Protein As high copy numbers may provide robustness to these processes. To test such concepts, we combined mutational, complementation and functional analyses. Strong complementation between null mutants in protein As polymerase and RNA capping active sites showed that they operate in independent protein A copies, likely at distinct sites. Thus, neither function is required in all protein A copies, nor are both required in any single copy. Lack of complementation between mutants in distinct RNA capping steps implied that major RNA capping steps must be performed in the same protein. Although RNA polymerase and capping activity were not required in all protein A subunits, none of a series of deletions across these domains were complementable, showing the importance of structural and other requirements for crown assembly, etc.. Surprisingly, RNA replication was more sensitive to depleting the fraction of subunits retaining protein As C-terminal intrinsically disordered region than polymerase or capping activity. These and other results reveal and illuminate the cooperative, interdependent nature of protein As diverse functions. Author summaryPositive-strand RNA viruses represent the largest genetic class of viruses and include human, animal, and plant pathogens causing major agricultural, economic, and environmental consequences. Using no DNA intermediates to multiply their RNA genomes, these viruses modify cellular membranes into novel, infection-specific RNA replication organelles. Emerging results show that RNA replication proteins encoded by many or most of these viruses assemble into ringed, crown-like viral protein complexes gating portals to these compartments. We previously revealed that nodavirus crowns contain two stacked 12-mer rings of viral replicase protein A, which contains polymerase, RNA capping and other domains. The nodavirus experiments reported here are among the earliest explorations in cells to illuminate the functions and interactions of such multi-domain RNA replication proteins in the context of their highly multimeric crowns. Critical questions include whether all domains and interactions are required in all protein A conformations, whether protein A multiplicity might provide dose-responsive redundancy for any crown functions, or whether defects in individual protein copies might inhibit or even poison operation of the entire crown. The results have significant implications for positive-strand RNA virus biology and thus for efforts toward virus control and beneficial uses.