Multifunctional Protein A Is the Only Viral Protein Required for Nodavirus RNA Replication Crown Formation

Abstract

Positive-strand RNA virus RNA genome replication occurs in membrane-associated RNA replication complexes (RCs). Nodavirus RCs are outer mitochondrial membrane invaginations whose necked openings to the cytosol are “crowned” by a 12-fold symmetrical proteinaceous ring that functions as the main engine of RNA replication. Similar protein crowns recently visualized at the openings of alphavirus and coronavirus RCs highlight their broad conservation and functional importance. Using cryo-EM tomography, we earlier showed that the major nodavirus crown constituent is viral protein A, whose polymerase, RNA capping, membrane interaction and multimerization domains drive RC formation and function. Other viral proteins are strong candidates for unassigned EM density in the crown. RNA-binding RNAi inhibitor protein B2 co-immunoprecipitates with protein A and could form crown subdomains that protect nascent viral RNA and dsRNA templates. Capsid protein may interact with the crown since nodavirus virion assembly has spatial and other links to RNA replication. Using cryoelectron tomography and complementary approaches, we show that, even when formed in mammalian cells, nodavirus RC crowns generated without B2 and capsid proteins are functional and structurally indistinguishable from mature crowns in infected Drosophila cells expressing all viral proteins. Thus, the only nodaviral factors essential to form functional RCs and crowns are RNA replication protein A and an RNA template. We also resolve apparent conflicts in prior results on B2 localization in infected cells, revealing at least two distinguishable pools of B2. The results have significant implications for crown structure, assembly, function and control as an antiviral target.

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

“…Rather, the requirements of (+)RNA synthesis appear to demand an alternate state of protein A near the 7 nm central channel of the crown floor to access the viral dsRNA template inside the spherule (1, 10). In keeping with this, strong electron density spanning the central channel of the mature crown floor has been consistently observed in all cryo-EM studies of replication-active nodavirus crowns ((9–11) and this paper). To better examine this density, we locally refined the inner central ring region of the floor of cryo-ET-imaged crowns from FHV-infected cells without imposed symmetry.…”

“…9 shows that in mature nodavirus crowns active in (+)RNA synthesis, the crown floor’s central channel is occupied by an asymmetric electron density with a volume similar to the protein A Pol domain. This central density has been visualized in all previous cryo-ET reconstructions of mature crowns from FHV-infected cells (9–11), and was present in all 3D classes of mature crowns obtained in the current analysis. This is of considerable interest since the 24 Pol domains of the central turret (Fig.…”

“…1D) including an ~ 19 nm diameter central turret composed of 12 pairs of stacked apical and basal lobes, an inner toroidal floor, and legs projecting outward from the basal lobes, and showed by genetic tagging that each apical domain represents a protein A Pol domain (10). Protein A is the sole FHV protein required to form full crowns indistinguishable from those in FHV-infected cells (11).…”

Nodavirus RNA replication crown architecture reveals proto-crown precursor and viral protein A conformational switching

“…These extensive matching features and the absence of any alternate, unaccounted crown density, in combination with the fact that protein A is the only viral protein required to reproduce the full structure of mature crowns from full FHV infections ( 11 ), strongly implicate the legs as the A N segments that must be connected with the apical Pol segments. Due to the flexibility and potential length of the linker sequence between A N and Pol, more study is needed to determine if each leg connects with the apical lobe Pol immediately above it or to one side ( Fig.…”

“…1 D ) including an ~19 nm diameter central turret composed of 12 pairs of stacked apical and basal lobes, an inner toroidal floor, and legs projecting outward from the basal lobes and showed by genetic tagging that each apical domain represents a protein A Pol domain ( 2 ). Protein A is the sole FHV protein required to form full crowns indistinguishable from those in FHV-infected cells ( 11 ).…”