Genetic complementation reveals structure-function links in nodavirus RNA replication complex crowns

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 A's 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 A's multiple conformations likely differentially contribute to crown assembly, RNA template recruitment, (-) and (+) strand synthesis, RNA capping, and progeny RNA release. Protein A's 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 A's 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 A's C-terminal intrinsically disordered region than polymerase or capping activity. These and other results reveal and illuminate the cooperative, interdependent nature of protein A's diverse functions.