Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter

Abstract

Segmented negative-strand RNA virus (sNSV) polymerases transcribe and replicate the viral RNA (vRNA) within a ribonucleoprotein particle (RNP). We present cryo-EM and X-ray structures of, respectively, apo- and vRNA bound La Crosse orthobunyavirus (LACV) polymerase that give atomic-resolution insight into how such RNPs perform RNA synthesis. The complementary 3' and 5' vRNA extremities are sequence specifically bound in separate sites on the polymerase. The 5' end binds as a stem-loop, allosterically structuring functionally important polymerase active site loops. Identification of distinct template and product exit tunnels allows proposal of a detailed model for template-directed replication with minimal disruption to the circularised RNP. The similar overall architecture and vRNA binding of monomeric LACV to heterotrimeric influenza polymerase, despite high sequence divergence, suggests that all sNSV polymerases have a common evolutionary origin and mechanism of RNA synthesis. These results will aid development of replication inhibitors of diverse, serious human pathogenic viruses.

Graphical abstract

Figure 1

Overall Structure of LACV Polymerase (A) Schematic representation of the domain structure of the monomeric LACV polymerase (top) aligned to that of heterotrimeric (PA-PB1-PB2) influenza polymerase (bottom). Structurally or functionally equivalent domains are similarly colored. A notable difference with the influenza polymerase is the clamp (magenta), involved in 3′ vRNA end binding, which is inserted into the LACV PA-C like domain. The LACV α-ribbon (orange) is structurally equivalent to the influenza β-ribbon despite being inserted in a different loop of the fingers domain. The LACV palm domain has an insertion specific for the California serogroup of orthobunyaviruses (salmon). The LACV fingernode (gray) is functionally equivalent to the influenza β-hairpin. The PB1 C-ext/PB2-Nterm interface is replaced by the LACV bridge domain. The LACV thumb ring domain (yellow) is structurally homologous to the influenza PB2 N1 and N2 domains. L₁₇₅₀ lacks the last 518 residues of the L protein currently of unknown structure (black stripes). (B) Illustrated representation of two views of the crystal structure of L₁₇₅₀ in complex with the 3′ (cyan) and 5′ vRNA (yellow). Protein domains are colored as in (A). (C) Structural comparison between L₁₇₅₀ and influenza (FluB2 structure, PDB: 4WRT) polymerases with equivalent PA-like, PB1-like and PB2-N like regions colored green, blue, and red, respectively. The 3′ (cyan) and 5′ vRNA (yellow) vRNAs are indicated. A more detailed structural comparison is in Figure S4. See also Figures S1 and S4.

Figure 2

Cryo-EM Reconstruction of Apo-L₁₇₅₀ (A) 3D reconstruction of the apo-L₁₇₅₀ containing the entire dataset of cryo-EM imaged particles, determined at 8.3 Å resolution. The dataset can be separated into three distinct states: (B) A 3D class displaying only partial density for the endonuclease (9.7 Å resolution). (C) A 3D class displaying density for all regions of the polymerase (9.7 Å resolution). (D) A 3D class lacking density for most of the vRBL domain and California insertion (9.3 Å resolution). Flexible regions are indicated with dotted lines. The domains are colored as in Figure 1. See also Figure S2.

Figure 3

3′ vRNA End Binding to LACV Polymerase (A) Overview of the 3′ vRNA (cyan sticks) binding site showing the clamp (magenta) and other interacting loops colored as in Figure 1A. The distal short complementary strand is in gray sticks. The RNA electron density is from the final 2Fo-Fc map contoured at 1.5 σ. K368 on helix α16 is protected from trypsin cleavage upon 3′ end binding. (B) Protein-RNA interactions of nucleotides 1–6 of the 3′ vRNA extremity. Hydrogen bonds are shown as green dotted lines. (C) Protein-RNA interactions of the clamp with 3′ vRNA nucleotides 6–9. See also Figures S3 and S5.

Figure 4

5′ vRNA End Binding and Induced Structural Changes (A) Overview of the 5′ vRNA stem-loop (yellow sticks) binding site with interacting loops colored as in Figure 1A. The RNA electron density is from the final 2Fo-Fc map contoured at 1.5 σ. K430 on the arch is protected from trypsin cleavage upon 5′ end binding. (B) Protein-RNA interactions in the 5′ vRNA stem region with hydrogen bonds as green dotted lines. (C) Protein-RNA interactions of the fingernode loop with 5′ vRNA loop bases G7 and U8. (D) Superposition of the L₁₇₅₀ −3′ vRNA structure without (light green ribbons) and with (colored as in Figure 1A) soaking of nucleotides 1–10 of the 5′ vRNA. Upon 5′ vRNA binding (yellow) the backbone interaction with His760 and His761 pulls helix α30 up allowing stabilization of an ordered configuration of the fingertips residues 949–958 (blue sticks). Multiple new contacts are formed, including hydrophobic interactions with α30 residues V762 and L766 and hydrogen bonds (dashed green lines) with residues from the linker region between PA-C like domain α29 and fingers domain α30, notably His757. Hydrogen bonds between Arg958 and Glu959 to Gln1145 stabilize polymerase active site motif B (dark red). See also Figures S3, S5 and S6.

Figure 5

The LACV Polymerase Active Site and Entrance and Exit Tunnels. (A) The arrangement of the conserved RdRp motifs in the LACV active site colored gold, light blue, green, red, brown, and blue for motifs A–F, respectively. Additional sNSV specific motifs G (from the PA-C like domain) and H are shown in pink and gray (see Figure S7). Superposition of the polio virus elongation complex structure (PDB: 3OLB, 3OL8) shows the positions of the catalytic divalent cations (black spheres), the priming nucleotide (N+1, gray) and incoming NTP (N+2, magenta) and template strand (light gray sticks). (B) The LACV polymerase structure (gray cartoon) with the 5′ and 3′ vRNA in, respectively, yellow and cyan is shown with the tunnels (green) marked with arrows as template entry, NTP entry, product, and template exit, as calculated with MOLE 2.0 (Sehnal et al., 2013). The endonuclease, bridge, thumb-ring and lid are, respectively, in forest green, blue, gold, and brown. (C) The same representation and orientation as (B) for the influenza A polymerase structure (PDB: 4WSB) with additionally the PB2 cap-binding domain in orange, the putative priming loop in magenta and the PB1 C-extension in dark gray. (D) Diagram showing the conserved residues forming the template entrance in LACV polymerase which is partially occluded by the flexible α-ribbon (orange). Colors are as in Figure 1A. (E) As (D) but showing the putative template exit channel in LACV polymerase. See also Figure S7.

Figure 6

Model of RNA Synthesis by LACV Polymerase (A) Illustrated representation of the LACV polymerase (gray) looking down the template entry channel showing the disposition of key structural elements (arch, clamp, α-ribbon, fingertips, fingernode) colored as in Figure 1A. The 5′ and 3′ vRNA extremities are, respectively, yellow and cyan tubes, except that nucleotide 11 in each case is in red highlighting their wide separation (>20 Å). The figure shows the impossibility of formation of a distal 5′ and 3′ duplex between nucleotides 12–15 of each strand, while maintaining the single-stranded ends bound as in the observed conformation. (B) Model for the initiation conformation of LACV based on superposition with the influenza polymerase (PDB: 4WSB) and the poliovirus elongation complex (PDB: 3OLB, 3OL8) structures. The observed 5′ and 3′ vRNAs are, respectively, red and blue for influenza and yellow and cyan for LACV and numbered accordingly. The LACV clamp binding to the 3′ end is in magenta. The poliovirus template strand is in gray and the active site is indicated by motif C (green), the catalytic divalent cations (black) and the priming and incoming NTPs (gray and magenta, respectively). The influenza vRNA distal duplex starts with the 3′-5′ 10:11 base pair (labeled). The template nucleotide numbering in outline white counts back from the active site, assuming initiation at position 1. The template nucleotide numbering in black numbers counts along the LACV template assuming the first LACV 3′-5′ base pair 12:12 aligns with the influenza 10:11 base pair. This would allow for connectivity between the distal LACV duplex and the observed 5′ end hook binding but imply an overshoot of the active site by 5 nucleotides. This is discussed further in the text. (C) Model of the elongation state showing trajectories of template RNA (cyan) and product RNA (orange) and NTPs through the polymerase tunnels (green). The observed positions of the 3′ and 5′ ends are shown as well as the position of the active site. After a short template-product duplex, which is accommodated in the interior cavity, each strand exits separately along different tunnels, the template back to the front of the polymerase where it can re-integrate into the RNP and the nascent strand to the rear where product processing occurs i.e., progeny cRNP assembly in the case of replication or mRNP assembly or translation coupling in the case of transcription.

Figure 7

Schematic Model of vRNA Replication An LACV RNP is schematically represented with the polymerase (purple or green), with template entrance (TEn), template exit (TEx), NTP entry and nascent RNA exit channels as marked, interacting with the viral RNA (black or yellow) and proximal NPs (ellipses colored with a blue-to-red gradient). The complementary 5′ and 3′ vRNA ends are, respectively, cyan and red. The NPs form a chain linked together by flexible NP-NP interactions involving the N-terminal arm (blue) and the C-terminal arm (red) and each NP sequesters 11 nucleotides RNA (Reguera et al., 2014; Reguera et al., 2013). Small circles mark consecutive 11 nucleotide segments of the vRNA. The polymerase itself can sequester around 20–22 template nucleotides. (1) In the inactive state, whether after vRNP assembly or in virions, both ends of the genomic RNA are sequestered into the specific 5′ and 3′ RNA binding sites of the polymerase, thus circularizing the RNP. (2) For de novo RNA synthesis or cap-dependent transcription (not shown) the 3′ end is relocated into the polymerase active site for initiation, by an unknown mechanism. Distal 3′-5′ duplex formation may occur before or after initiation depending on whether initiation is internal (followed by prime and align) or at position 1 (see Figure 6B and main text). Duplex formation could bring the NPs at the 5′ and 3′ (NPa and NPz) closer enhancing the circularization of the NP scaffold but would need to be dissociated to proceed with elongation. (3) With the 5′ end bound to the allosteric site for the activation of the RNA synthesis, a nascent cRNA begins to be synthesized. (4) As elongation proceeds, the template dissociates from the proximal NP and is channelled into the active site. Because of the proximity of the entrance and exit channels the disruption of the RNA-NP assembly may only affect one NP. Early on, the 5′ end is detached from its specific binding site on the polymerase and enters the RNP by loading onto NPz. As incoming template is released from NPy on one side, the outgoing 3′ end is loaded on it from the other side. More generally, the RNA being pulled into the cavity by the polymerase motor detaches from the proximal NP which is pulled to the left thus pushing the NP-RNA array in the direction of the arrow. This model would imply that 5′ end binding is only required to activate initiation. This would be a difference from the influenza situation where the maintenance of 5′ end binding is required, at least during transcription, for self-polyadenylation to occur. (5) Once the nascent c5′ end emerges from the exit channel it can recruit an incoming apo-polymerase as the first step in encapsidating the progeny cRNP with incoming apo-NPs. This may be facilitated by polymerase dimer formation (see main text). (6) Approaching termination the template 5′ end would be copied and the template 3′ end (bound to NPy) would approach its starting point. (7a) At termination the template 3′ end rebinds to its specific binding site on the polymerase to avoid base pairing with the emerging template 5′ end which subsequently rebinds to its polymerase binding site, thus completing the replication cycle. (7b) Due to polymerase dimer formation, the nascent c3′ end, which emerges last from the product exit channel, can easily find and bind to specific 3′ binding site on the green polymerase, thus completing progeny cRNP formation. Without polymerase dimer formation being maintained throughout replication (or other mechanism for keeping the polymerases in close proximity), it is unclear how the c3′ could find and bind to the correct polymerase which may have diffused far away.

Figure S1

Purification and X-Ray Structure Solution, Related to Figure 1 (A) SDS PAGE of purified L₁₇₅₀ after a S200 gel filtration run. (B) Scheme for X-ray crystal structure solution by the MIRAS methods using tantalum cluster, platinum, and selenomethionine derivatives. (C) X-ray energy scan of selenomethionine derivative at Se absorption edge. (D) X-ray energy scan of tantalum derivative at Ta absorption edge. (E) Experimentally phased map at 2.85 Å resolution contoured at 1 σ (cyan mesh) with superposed final model showing a number of selenomethionines (anomalous difference peaks contoured at 4.0 σ, orange mesh) and one tantalum cluster position (anomalous difference peaks contoured at 4.0 σ, green mesh).

Figure S2

Electron Microscopy of Apo-L₁₇₅₀, Related to Figure 2 (A) Negative stain EM micrograph of purified L₁₇₅₀. Scale bar is indicated. (B) Representative cryo-EM micrograph collected at 80 kV on a Krios microscope using a Falcon II direct detector (defocus of the chosen micrograph: 1.4 μm). (C) Comparison between ab initio 2D class averages (top) and reprojections of the 3D reconstruction (bottom), showing the reliability of the 3D reconstruction. Below each comparison, the corresponding view of the 3D reconstruction with the fitted pseudo-atomic model is displayed. (D) Fourier Shell Correlation (FSC) based on the gold standard FSC = 0.143 criterion shows a resolution of 8.3 Å. (E) Separated helices are clearly visible inside the map, consistent with the 8.3 Å resolution identified by the FSC. (F) Local resolution identifies flexible parts of the polymerase. A cut-away view is displayed with the pseudo-atomic model on the left and the cryo-EM reconstruction colored by resolution on the right. A bar indicates the color code corresponding to the local resolution. The most flexible parts, which correspond to the vRBL domain and the α-ribbon, are highlighted with a dotted ellipse.

Figure S3

Biophysical and Biochemical Analysis of vRNA Binding, Related to Figures 3 and 4 (A) Measurements of Kd by fluorescence anisotropy. (B) Binding of duplexes with different length 3′ overhangs. (C) Sequence analysis of 3′ end binding. (D) Protection from trypsin cleavage by 3′ or 5′ end vRNA binding. (A) 3′ and 5′ vRNA affinity to L₁₇₅₀ measured by fluorescence anisotropy. 5 nM of 25-mer RNAs corresponding to 3′ (red) or 5′ (blue) vRNA and labeled with fluorescein were titrated with L₁₇₅₀ over the protein concentration range from 3 nM to 1 μM (left). Comparison of ssRNA (red) and dsRNA (green) binding to L₁₇₅₀ upon titration with a broader protein concentration range (right). The calculated affinity values are indicated in the offset tables. (B) Mobility shift assay of radiolabelled, artificial panhandle RNAs with different lengths of 3′ overhang bound to L₁₇₅₀. The RNAs used are schematically represented showing the overhangs and coloring the polymerase interacting nucleotides in red, the distal duplex nucleotides in green, the up/downstream nucleotides in black and guanine linker in white. The radioactive signals belonging to shifted bands were recorded with a Typhoon and quantified with ImageQuant. Amounts of bound RNAs were normalized against the amount of bound 22 nt long 3′ ssRNA used as a reference. The graphic plots the average values and Sd from four independent experiments. Radiography for one MSA experiment is shown in the offset maintaining the same order of RNAs as in the graphic but shifting the ssRNA control to the right lane. (C) Sequence specificity of the 3′ vRNA end binding to L₁₇₅₀. Mobility shift assay of the wild-type radiolabelled 22 nt long 3′ vRNA (WT) (top-left, same as in B) and all possible 33 point mutants within the first 3′ 11 nucleotides. As in (B) amounts of bound RNAs were normalized against the amount of bound WT 3′ vRNA used as a reference. The graphic plots the average values and SD from four independent experiments. The RNA variants tested are loaded in the gel lanes maintaining the same order as in the graphic. (D) Protection from trypsin cleavage by 3′ or 5′ end vRNA binding. Limited trypsination of L₁₇₅₀ without RNA, with 3′ vRNA and with 5′ vRNA was visualized by SDS-PAGE. The cleavage products were identified by ESI-TOF mass spectrometry to determine their molecular mass, tryptic MALDI-TOF to identify the tryptic peptides belonging to each, and Edman degradation to sequence the N-term of each band. The resulting molecular weight, peptide boundaries, and methods used to cross-validate the identification on each experiment are shown in the table. The results are schematically summarized on the bottom right of the panel showing the L₁₇₅₀ construct as a colored bar where the regions before and after the trypsin cleavage site are colored in red and blue, respectively, the RdRp region in green and the L₁₇₅₀ C-term region in yellow. The differentially cleaved region is represented by the dashed red lines box. The protein fragments belonging to each region are highlighted with dashed lines squares on the SDS-PAGE.

Figure S4

Structure Comparison between LACV and Influenza Polymerase, Related to Figure 1 In each panel equivalent structural features are highlighted for LACV (left) and influenza A (PDB: 4WSB) (right) polymerases after superposition. The root mean square structural similarity for Cα positions is 3.18 Å for PA, 3.24 Å for PB1 and 4.33 Å for the first third of PB2 (PB2-N). (A) PA like region. (B) PB1 like region. (C) PB2-N like region.

Figure S5

Details of Protein-RNA Interactions, Related to Figures 3 and 4 (A) Residues (color coded as shown, according to Figure 1A) interacting with the 3′ vRNA and short duplex region. Based on data from CONTACT (Table S2) (B) As (A) but for the 5′ vRNA.

Figure S6

Comparison of 5′ vRNA Binding in LACV and Influenza and Induced Structural Changes in the Arch and Fingernode, Related to Figure 4 (A) Stick model of the LACV (left) and influenza (right) 5′ vRNA stem loops. The base paired residues are in pink, the additional stacked residues in gray and the flipped out bases of the loop in light cyan. Hydrogen bonds are shown as green dashed lines. Nucleotide U11, which is partially ordered in the LACV structure, is shown in yellow. (B) The structure of L₁₇₅₀ before (light green) and after (colored as in Figure 1A) soaking with 10 nucleotides 5′ vRNA (yellow tube). 5′ vRNA binding prompts a reorientation of arch residues Val437 and Ser438 and induces a significant reconfiguration of the arch. (C) The interaction with 5′ vRNA loop bases G7 and U8 radically changes the configuration of the fingernode loop (light green before to gray after) allowing the stacking of Tyr1120 onto G7 which makes base-specific hydrogen bonds with Gln1116 and Asp1123 and base-specific recognition of U8 by main-chain interactions. (D) Influenza 5′ vRNA nucleotides 1–16 (red tube) and 3′ vRNA nucleotides 10–14 (blue tube) are superposed on the L₁₇₅₀ 5′ vRNA soaked structure after superposition of bat influenza FluA structure (PDB: 4WSB) and LACV polymerases. LACV polymerase is shown as a gray cartoon with 5′ vRNA nucleotides 1–10 in yellow. Partially ordered LACV U11 is highlighted in yellow sticks and corresponds roughly to A10 in influenza. The LACV α-ribbon (orange) would clash with a distal, base paired region positioned as in influenza, but could rotate (arrow) to play a role similar to the PB1 β-ribbon (green) in binding the duplex region.

Figure S7

Newly Identified Conserved Motifs in all sNSV Polymerases and Details of the Polymerase Tunnels, Related to Figure 5 (A) The conservation of motif G (RYφφ, bold absolutely conserved, φ denoting a hydrophobic residue) is shown in an alignment (left) including two strains each from Orthomyxoviruses (Influenza A and B), Bunyaviruses (Ortho: LACV/Bunyamwera, Hanta: Hantaan/Imgin, Phlebo: Rift Valley fever/Uukuniemi, Nairo: Crimean-Congo haemorrhagic fever/Kupe, Tospo: Tomato spotted wilt/ Peanut bud necrosis) and Arenavirus (Lassa/Junin). The environment of motif G is shown for LACV (middle) and influenza (right) with the neighboring motifs A, C, D (including conserved Lys1228/481) and E. Structurally similar elements are colored according to Figure 1A. (B) The same for motif H (K)xφxφ) showing for LACV (middle) and influenza (right) the interactions of the conserved lysine with the backbone of the motif B loop. Neighboring LACV fingernode (gray) is highlighted (middle) as well as the analogous β-hairpin (gray) in influenza polymerase (right). In both cases neighboring motifs A, B, and F are marked. Structurally similar elements are colored according to Figure 1A. (C) Section through the L₁₇₅₀ polymerase structure in van der Waals surface representation colored according to surface electrostatic potential showing that the four conserved channels and central cavity are positively charged. The 3′ and 5′ RNAs are shown as blue and yellow ribbons, respectively. Green arrows indicate the NTP and RNA traffic following the homology with influenza and other similar RNA polymerases. (D) As C but solvent accessible residues are colored according to their degree of conservation among orthobunyaviruses (violet > 90%, 90% < pink > 60%, white < 60%). (E) The NTP entry channel is lined by conserved basic residues R287, K673 (PA-C like), K956, R958 (fingertips), K1063 (motif A), K1227, K1228 (motif D) some of which are only positioned correctly upon 5′ vRNA binding. Coloring of domains is as in Figure 1A. (F) View into the product exit channel showing contributing conserved residues from the fingers, bridge thumb ring and lid (colored as in Figure 1A).