Structure of a rabies virus polymerase complex from electron cryo-microscopy

Abstract

Nonsegmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS RNA viruses carry all of the enzymatic functions required for viral messenger RNA (mRNA) transcription and replication: RNA polymerization, mRNA capping, and cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 Å resolution. The complex closely resembles the vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L-protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a preinitiation conformation. Comparison with the likely elongation state, seen in two structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.

Keywords: NNS RNA viruses; rabies lyssavirus; replication; transcription; vesicular stomatitis virus.

Fig. 1.

Effect of P on conformation of L. (A) Electron micrograph of negatively stained RABV L. (Scale bar, 25 nm.) (B) Representative 2D class averages of L alone or in complex with P_11–50. (Scale bar, 5 nm.) Arrowheads indicate flexible conformations of the three C-terminal globular domains of L. (C) Three-dimensional reconstructions of negatively stained L alone or in complex with the indicated fragment of P. Red dashed circle indicates poor density for the disordered C-terminal globular domains in the absence of any P. (Right) The VSV L atomic model fit into the RABV L-P_FL reconstruction.

Fig. 2.

Structure of the RABV L-P complex. (A) A 3.3-Å cryo-EM map of RABV L-P_1–91 and model fit into the density with clear side-chains even on exterior residues. (B) Domain organization of RABV L, with key catalytic residues listed. Numbers give amino acid positions at domain boundaries. (Below) Locations of conserved regions (CRI-VI) throughout NNS RNA virus L proteins and amino acid similarity score with VSV L (Indiana strain); black lines indicate identical residues (∼35%). (C) Structure of RABV L-P_1–91 (Left) and the model of VSV L-P_35–106 from a 3.0-Å cryo-EM map (Right) (7). Domains are colored as in B. P for both structures is shown in surface representation, colored in plum (RABV) and light pink (VSV). The five unassigned residues of RABV P are represented in plum ribbon. (D) Domain organization of RABV P (Top) amino acid positions (red asterisk, position of Ser63P, typically phosphorylated). (Below) Alignment with VSV P (purple lines, modeled RABV P residues; boldface letters, modeled VSV P residues; asterisks, shared residues; colons, chemically similar residues; periods, weakly similar residues; red, phosphorylation sites).

Fig. 3.

Association of RABV P_1–91 with L. (A, Left) RABV L-P_1–91 as in Fig. 2C. Bubbled letters indicate the regions shown in greater detail in i–iv on the Right. (A, Right) (i) Unassigned residues of P at the top of the CTD with the 3.3-Å map in black mesh. (ii–iv) Indicated residues of L (black) and P (purple) in stick representation. (B) CTDs of RABV and VSV L; P is in plum for RABV and light pink for VSV. Black arrows indicate the C-terminal residues of each L. (C) Superposition of RABV L (colored cartoon, as in Fig. 2) and VSV L (gray cartoon). (Left) Different paths taken by RABV P (plum, surface) and VSV P (light pink, surface) through L. (Right) RABV P (plum, surface) is partially coincident with the VSV C-tail (red, surface) in the interdomain cavity of L, while VSV P occupies far less of the same cavity.

Fig. 4.

Analysis of negatively stained L-GFP-P_1–91 and L-P dimers. (A, Left) Two-dimensional class averages of negatively stained L-GFP-P_1–91 and matched projections of the RABV L atomic model with GFP position approximated (SI Appendix, Figs. S5 and S6). (A, Right) GFP trace from a 3D reconstruction of negatively stained RABV L-GFP-P_1–91 (SI Appendix, Fig. S6) superimposed on the RABV L atomic model. (B) Two-dimensional classes of negatively stained RABV L-P_FL dimers and matched projections of the RABV L atomic model (SI Appendix, Figs. S7–S9), as described in legend to SI Appendix, Fig. S7. Square boxes in A and B are 336 Å on a side. (C) Minimal distances between Cα atoms of either P₈₇ (C:C) or the N terminus of the P segment bound at the CTD apex (N:N) for each projection-matched dimer. (D) Approximate locations of P₁ and P₉₁ with respect to L determined from the projection-matching analysis.

Fig. 5.

Proposed RNA transit through L. (A–C) Cutaways showing continuous internal cavities (gray, translucent surface) through L (colored, as in Fig. 2C) when the priming loop residues (green ribbon) are deleted from the model. Black arrows indicate nucleotide and template entrance channels and the presumed RNA product exit channel. (B) Cavity into which the priming loop may retract during elongation (white dashed arrow). Nascent RNA may continue toward the CAP active site (HR motif) following internal initiation for mRNA transcription (solid orange arrow) or directly upward toward the MT domain following terminal de novo initiation for genomic RNA replication (dashed orange arrow). (C) The presumed template exit channel at the back of the CAP domain (black dashed circle) is occupied by small loops, which we propose move aside as polymerization progresses.