Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication

Figures

• 

  Fig. 1.. Genome organization and protein ORFs of FHV. The bipartite FHV genome, (+)RNA1 and (+)RNA2, express protein A and capsid protein, respectively. Protein A is the master FHV RNA replication protein that is involved in various functions such as membrane association (transmembrane domain and a possible membrane-interacting domain in the Iceberg region), replication complex formation, RNA 5′ capping, and RdRp enzymatic activities. The various functional domains of protein A are highlighted in the ORF and their amino acid coordinates are marked above the ORF. Note that both the indicated core RNA-capping domain and the adjacent Iceberg domain appear to contribute to RNA-capping functions (20). Protein A replicates the viral RNAs to form minus-sense products, likely as dsRNA. During minus-strand synthesis, a FHV RNA1 3′ end coterminal (−)RNA3 is synthesized, which in turn replicates to make the (+)RNA3. RNA3 codes for the B1 and B2 proteins. B1 is a preferentially nuclear protein with unknown function, whereas B2 is a well-established RNAi suppressor.

• 

  Fig. 2.. Improved cryo-ET procedures to visualize FHV replication complexes at the mitochondria. (A) Section of the reconstructed tomogram showing the side view of FHV replication complexes. Various spherules and the OMM are highlighted with black arrowheads. (B) Section of the reconstructed tomogram showing the top view of the OMM with FHV replication complex crowns. (C and D) Insets of A and B showing a zoomed-in view of a single spherule and three top views of the crown, respectively. (E) False-color representation of C highlighting the various densities and subcellular regions at the replication complex. Blue, crown side view; white, OMM; red, densely coiled internal fibril; translucent orange, cytoplasm; and translucent green, mitochondrial intermembrane space (IMS). (F) False-color representation of D highlighting the top view of the OMM (translucent white), and the crowns (blue).

• 

  Fig. 3.. Subtomogram averaging of the crown structure reveals important new structural details. (A) High-density threshold (contour level = 4.2) side view of the crown. The dotted gray line through the A, B, and C map the OMM for point of reference. (Inset) Region in A marking the three structurally distinct regions in the crown: apical lobe, basal lobe, and leg. (B) Cross-section view of the low-density threshold crown (from C) as a transparent white shell encasing the high-density crown (from A) in blue. Asterisks indicate unusual densities bridging between denser head group regions of the lipid bilayer, and the dashed lines below the crown depict the continuation of the reshaped OMM into the body of the spherule RNA replication vesicle. (C) Low-density threshold (contour level = 2) side view of the crown show the additional density contributions made by the membrane. (D) Top view of the high-density threshold crown highlights the 12-fold symmetry of the crown complex. (E) Bottom view of the high-density threshold crown.

• 

  Fig. 4.. Two-dimensional slices of the crown electron-density map display the higher-resolution substructure. (A and B) High-density threshold side view of the crown with dotted lines representing the slices shown in B after 90° rotation. (C and D) High-density threshold top-view of the crown with dotted lines representing the slices shown in D after 90° rotation. (Scale bars in B and D, 10 nm.)

• 

  Fig. 5.. C terminus of protein A is exposed at the mitochondria in cells replicating FHV. (A) RNA1-GFP11-ΔB1 construct incorporates three distinct changes: 1) M897L amino acid change to knock out B1 expression, 2) *999R amino acid change to extend protein A ORF, and 3) C-terminal extension of protein A ORF to express a 16-aa GFP11 sequence (amino acids 1,008 to 1,023). RNA1-His-ΔB1 disrupts the GFP11 sequence to incorporate the His-tag at amino acid positions 1010 to 1015 within protein A. (B) Northern blot analysis on RNA isolated from cells transfected with the indicated FHV RNA1 expression plasmids to compare in cis RNA replication capacity of the encoded protein A fusions. fs = RNA1fs, GFP11 = RNA1-GFP11-ΔB1, His = RNA1-His-ΔB1, and WT = RNA1-WT. Ribosomal RNA (rRNA) shown as a loading control. RNA1fs is a full-length RNA1 derivative in which an engineered frameshift blocks protein A expression (39). (C) Widefield fluorescence microscopy of S2 cells. (Upper) Cells transfected with expression plasmids RNA1-WT and GFP1-10 and (Lower) cells transfected with RNA1-GFP11- ΔB1 and GFP1-10 plasmids. Immunostained protein A is shown in red and GFP fluorescence shown in cyan. The Merge image…

• 

  Fig. 6.. Site-specific nanogold labeling maps the protein A polymerase domain to the distal lobe of the crown. (A) Experimental scheme for infection, isolation, and native labeling of His-tagged protein A with 5-nm Ni-NTA-nanogold for cryo-ET imaging. (B) Reconstructed tomograms of mitochondria isolated from FHV-WT (Left) or FHV-His-ΔB1 (Right) after nanogold-labeling. The white arrowheads indicate 10-nm fiducial gold used for alignment during tomogram reconstruction of both samples. The yellow arrowheads indicate the 5-nm Ni-NTA-nanogold. (Right Insets) Two individual spherules showing nanogold-labeling. (C) Representative nanogold-labeled subtomograms from a higher resolution cryo-ET dataset. (D) Subtomogram averaging of nanogold-labeled and unlabeled crowns. Unlabeled = 141 unlabeled crowns used for averaging. Unlabeled + nanogold labeled = 141 unlabeled crown + 184 gold labeled crowns included in averaging. Overlay = Unlabeled crown in blue overlayed with a mesh view of the unlabeled + nanogold-labeled crown in yellow. The black arrow highlights the major additional densities contributed by the nanogold appearing at the apex of the crown.

• 

  Fig. 7.. Protein A polymerase secondary structure prediction and fitting into the EM density map. (A) iTasser predicted secondary structure of protein A polymerase domain (Pol-Pred, amino acids 453 to 896) shown to the left with the product-exit face in the front. The top four iTasser structural analogs shown (high to low, left to right) in the same orientation as the FHV-predicted polymerase domains. (B) Manual C terminus up and down fit of Pol-Pred into the EM density map. The Pol-Pred structure is rainbow colored from the N to the C terminus going from blue to red. The N-to-C color key is also shown to the right. (C) Top two fits (based on correlation score) obtained from fitting Pol-Pred into the segmented apical lobe using the rigid-body docking tool in Segger.

• 

  Fig. 8.. Dodecameric arrangement of protein A to form the crown complex. (A) Tilted side view of the crown with alternate segments of the 12-fold symmetric crown colored in blue and white. The floor densities are shown in transparent white with black silhouette. (B) Single predicted protein A segment shown in two-color in side view with the mapped polymerase domain in yellow and remainder of protein A N-terminal densities in blue. The remaining crown segments are shown in transparent white with black silhouettes for unobstructed viewing of the highlighted crown segment. The asterisks mark the two separate membrane-interaction sites of each crown segment. A protein A linear map from the N terminus to the C terminus is shown to the right with the same color scheme as the density map. The asterisks in the protein A linear map indicate the well-characterized N-terminal transmembrane domain TM and the possible membrane-interaction domain within the Iceberg region.