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.

Keywords: RNA replication complex; crown complex; cryo-EM tomography; nodavirus; positive-strand RNA virus.

Figure 1

Nodavirus genome organization and replication. (A) The bipartite FHV RNA genome encodes the viral replicase protein A, the key protein for viral RNA replication, on RNA1 and the capsid protein precursor on RNA2. Subgenomic RNA3, transcribed from RNA1, encodes protein B1, identical to the C-terminus of protein A, and protein B2 which is an RNAi suppressor. (B) Cryo-EM tomogram of Drosophila S2 cells with nodavirus replication complexes: invaginations (spherules) of the outer mitochondrial membrane into a vastly dilated intermembrane space. Arrowheads: black = outer mitochondrial membrane; white = inner mitochondrial membrane; red = spherules, filled with dsRNA; yellow = crown-like structures at the spherule open necked connections to the cytoplasm. (C) Tomographic reconstruction of the 12-fold symmetrical nodavirus replication complex crown, from reference [24].

Figure 2

Subcellular localization of nodavirus proteins A and B2 in infected cells. Super-resolution Structural Illumination Microscopy (SIM) reveals that in nodavirus-infected cells, protein B2 localizes near the sites of protein A at mitochondria. Higher magnification insets show that protein A and B2’s localization is predominantly at distinct adjacent sites and that even at the resolution of light microscopy only small portions of their distributions overlap, as further demonstrated by fluorescence intensity measurements across a selected line profile (yellow arrow) in the lower right panels. Blue and red arrowheads indicate separate, distinct localization of proteins B2 and A, respectively; a black arrowhead indicates their occasional co-localization.

Figure 3

Small amounts of protein B2 interact with protein A and segregate with purified mitochondria. (A) Subsets of nodavirus proteins A and B2 physically interact. Antibodies directed against protein A co-immunoprecipitate protein B2 and antibodies directed against protein B2 co-immunoprecipitate protein A. The interaction is insensitive to micrococcal nuclease treatment, showing that it is not mediated through RNA. (B) Cell fractionation procedures to isolate mitochondria show that the majority of protein A but only a small amount of protein B2 (~8%) tracks with mitochondrial marker VDAC, while the great bulk of protein B2 tracks with cytoplasmic tubulin.

Figure 4

Analysis of the replication of wt FHV RNA1 and its mutant derivative RNA1-noB2 replication in insect and mammalian cells. (A) Schematic maps of wildtype RNA1 and an RNA1-derivative that lacks coding potential for B2 due to mutations of initiator and downstream methionine codons that are silent in the -1 frame encoding protein A sequence. (B) B2 is a strong suppressor of RNA-interference (RNAi), and RNA1-noB2 does not support stable accumulation of RNA1 and subgenomic RNA3 and protein A in Drosophila S2 cells that have a strong antiviral RNAi response (left lanes). By contrast, RNA1-noB2 launches RNA1 and RNA3 replication levels very similar to wildtype RNA1 in mammalian BSRT7/5 cells that do not display robust RNAi action (right lanes). (C) Fluorescence images of BSRT7/5 cells confirm accordingly that B2 is not needed and show that protein A and dsRNA replication intermediates are observed at the mitochondria of wildtype RNA1 and RNA1-noB2 transfected cells at similar levels and distribution.

Figure 5

Cryo-ET of nodavirus RNA replication complexes in mammalian cells. (A,F) Indistinguishable spherules are induced on the outer mitochondrial membrane in mammalian BSRT7/5 cells transfected with either wildtype nodavirus RNA1 (A) or with RNA1-noB2 (F). Arrowheads: black = outer mitochondrial membrane; white = inner mitochondrial membrane; red = spherules, filled with dsRNA; yellow = crown-like structures at the spherule open necked connections to the cytoplasm. (B,G) Close-up side views of spherules with crowns visible in the plane of sectioning. (C,H) Close-up top views of crowns. (D,I) Cross sections of EM density maps of subtomogram-averaged side views of crowns. Arrowheads: light blue = apical lobe; dark blue = basal lobe; green = leg; orange = floor. (E,J) Cross sections of EM density maps of subtomogram-averaged top views of crowns with blue arrowheads pointing to the central crown turret and green arrowheads to the extended legs. Tomogram movies corresponding to panels A and F and Fourier Shell Correlation (FSC) curves corresponding to panels (D,E,I,J) are provided in Supplementary Movies S1 and S2 and Supplementary Figure S1.

Figure 6

Comparison of nodavirus RNA replication complex crowns formed in nodavirus-infected insect cells to those formed in mammalian cells in the absence of Protein B2 and genomic RNA2. (A) On the right, light orange side and top view presentations of RNA1-noB2 replication-induced crowns in mammalian cells display all of the hallmark 12-fold symmetrical features of wildtype FHV infection-induced RC crowns shown in the published high-resolution crown image [24] on the left and the matched low-pass gaussian-filtered, slightly lower resolution version in the center. As shown, these common features include the apical and basal lobes of the central turret, inner floors with similar diameter central openings and central densities, and leg-like radial extensions. (B) Cross-sectioned comparison to further illustrate the matching shapes and membrane interactions of the high-resolution wildtype crown (leftmost images in panel (A)) and the RNA1-noB2 crown structures. OMM = outer mitochondrial membrane.