Structural basis of transition from initiation to elongation in de novo viral RNA-dependent RNA polymerases

Figures

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  Fig. 1.. Crystal structures of DENV2 NS5 EC reveal PE refolding upon transition to elongation. (A) The T30/P2 RNA construct used for EC assembly, and the 9-nt product (P9) formed during the assembly. (B) Global view of the native EC as a combination of surface and cartoon representations. (C) Nucleic acids resolved in EC structures. CTP and magnesium ions are shown as sticks and cyan spheres respectively. (D) A comparison of PE conformation in apo DENV2 NS5 (Left) and EC–CTP (Right) structures. For clarity, the RdRP fingers domain and RNA downstream of +1 are not shown. PE is shown as a combination of cartoon and semitransparent surface representations. CTP is shown as spheres. (E) HCV RdRP–RNA complex structures for comparison. Left: HCV IC structure obtained using a dinucleotide primer and an NDP; Right: template–primer–NDP structure obtained using HCV RdRP with a truncated PEN. Coloring scheme: palm, gray; thumb, slate; index, dark green; middle, orange; ring, yellow; pinkie, pink; PE, purple; active site XGDD residues, magenta; template, cyan (+1 nucleotide in orange); and product, green. HCV structures were superimposed to DENV2 NS5 EC structure by the traditional least-square method…

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  Fig. 2.. The details of PE interactions with the upstream RNA duplex in EC and its refolding from apo state to elongation state. (A) Stereopair images of PE interactions with the upstream RNA duplex. EC–CTP complex structure is shown as cartoon representations and with 3,500-K simulated-annealing omit 2Fo–Fc electron density maps overlaid (contoured at 1.2 σ). Side chains of key PE residues involved in RNA contacts are shown as sticks. (B) PE–RNA backbone interactions with the −7/−8 positions of the template (Left) and the −5/−6 positions of the product (Right). (C) Stereopair images for an illustration of PE refolding using the apo JEV NS5 (brown PE) and DENV2 NS5 EC–CTP (purple PE) structures. The JEV structure is used because it has relatively intact side chain models if compared with DENV2 apo structures. The α-carbon atoms of four hallmark residues (large spheres) and a reference residue (small spheres) are shown with the corresponding moving distances for illustration of the refolding scheme. Initiating nucleotides were modeled from the EC–CTP structure and are shown as semitransparent spheres. (D) Structure-based PE mutation design. Left: sequence logos generated using a…

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  Fig. 3.. Enzymology characterizations of DENV2 WT NS5 and its PE mutants. (A) Four types of RdRP assays based on the T30/P2 construct were also used for structure determination of EC. Initiation kinetics was assessed by a combination of P2-to-P3 and P2-to-P9 conversions. EC reactivity was assessed by P9-to-P10 conversion rate under high-salt condition (300 mM NaCl) using a quench-flow instrument. EC stability was assessed by P9-to-P10 conversion after an incubation under a challenging condition (525 mM NaCl and 30 °C) for various periods. (B) Gel images of representative NS5 constructs for P2-to-P3 conversion. Chemically synthesized P2 and P3 were used as migration markers, and P3 was also used as standard samples (M1 lanes) for relative P3 intensity quantitation. For every separated gel image, there were WT samples loaded (but not shown for the right three images) for quantitation of P3 relative intensities. (C) Analysis of P2-to-P9 conversion. Top: Gel images of representative NS5 constructs for P2-to-P9 conversion under 1,000 μM ATP concentration. Bottom Left: Fitting of conversion rates of P2 to P9 for representative NS5 constructs. Bottom Right: Bar chart showing conversion rates of…

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  Fig. 4.. A working model of the transition toward elongation in classical de novo RdRPs. (A) Two RNA constructs used for dissection of transition toward elongation in DENV2 and HCV RdRPs. (B) Multiple-nucleotide incorporation assays (P2 to P4 and P2 to P5 for the T30-7/P2 construct and P2 to P7 for the T28/P2 construct) were used to estimate the critical step during transition toward elongation for DENV2 RdRP. Chemically synthesized P2/P3/P4 was used as a migration marker (M lanes). The same 8-mer in Fig. 3C was also used as a migration marker (M lanes) and as standard samples (M lane, STD) for relative 4- to 5-mer, 5-mer, and 7-mer intensity quantitation. The P2-to-P7 gel images for DENV2 (Top) and HCV (Bottom) were cropped from different regions of the same gel, and lane 30 was included in both images for correlation. The STD samples were provided at the same molar amount as the RNA template, and the average intensity of P8 band on both sides of each gel is set to 1.0. Based on Stains-All staining nature, equal molar amount of shorter RNA (e.g., P7, P5, or P4) should have lower band intensity than that of P8 standard. Therefore, the relative intensity values in combination with time…

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  Fig. 5.. Structure diversity of de novo RdRP PEs and a structural comparison of DENV2 RdRP PE and T7 RNA polymerase specificity loop in transition toward elongation. (A) A structural comparison of representative PEs in classical de novo RdRPs. From Left to Right, JEV NS5 apo structure, HCV NS5B IC structure, CSFV NS5B apo structure, and phi6 RdRP IC structure. For clarity, only thumb (surface), PE (cartoon), XXDD-containing motif C (cartoon), and initiation nucleotides (cartoon or sticks) are shown. CSFV, HCV, and phi6 structures were superimposed to JEV NS5 apo structure by the traditional least-square method using RdRP motif C as the target region and were shown as separated structures. (B) A structural comparison of specificity loop conformations in different stages of T7 RNA polymerase transcription. Left: IC structure; Middle: structure of an intermediate complex between initiation and elongation; and Right: EC structure. The polymerase core module is shown as surface representations; the specificity loop, NTD, and nucleic acids are shown as cartoon representations. Coloring scheme: polymerase core, gray; NTD, olive; specificity loop, purple; template, cyan; product, green; and…