An evolutionarily unique viral RdRP suggests a common dual-function feature of the priming element

Figures

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  Fig. 1.. Global views of the AfuPmV-1 RdRP in its apo state. (A) A color-coded bar showing structural or functional elements of AfuPmV-1 RdRP. Residue ranges of each element are defined by the numbers. Regions present in the WT construct but not resolved in the structure are indicated by gray bars at the top. (B) Stereo-pair images of the AfuPmV-1 RdRP crystal structure (NTP entry channel view). The coloring scheme is consistent with that in (A). Green spheres: viral RdRP invariant residues [the same for (C)]. (C) A comparison of representative viral RdRP structures. The coloring scheme is the same as in (A), except that the fingers domain is in pink, the N-terminal additional region (not including NE) of DENV2 is in cyan, and the C-terminal additional region of IAV is in light blue. All structures were superimposed using motif C residues by the least-square method (dsRNA binding channel view). (D) Structure-based sequence alignment of motifs A to G in representative viral RdRPs. Family/species assignments are listed on the left. Highly conserved residues, including nucleic acid polymerase–invariant (labeled by asterisks) or RdRP-invariant residues (green triangles), are shown in red.…

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  Fig. 2.. A crystal structure of AfuPmV-1 RdRP EC reveals PE refolding upon transition to elongation. (A) Stereo-pair images of the AfuPmV-1 RdRP EC. (B) Top: A comparison of PE conformations in the apo (left) and EC (right) states. For clarity, RdRP fingers and palm are not shown except that the motif C in the palm is shown as a reference. The lowercase letters indicate three PEN helices. The arrow in the right panel indicates the extension of a-helix upon transition to elongation. Bottom: Conformation comparison of PEN (left) and PEC (right) in the apo (brown PE) and EC (purple PE) states. The α-carbon atoms of hallmark residues (spheres) are shown with the corresponding moving distances. Initiating nucleotides were modeled from the EC structure and are shown as spheres. The PE-excluded thumb of the apo (blue) and EC (slate) is shown in surface representations. (C) Stereo-pair images showing PE interactions with template-product dsRNA in the EC structure. Side chains of key PE residues involved in RNA contacts are shown as sticks. Key water molecules are shown as red spheres. (D) PE interactions with dsRNA. (E) Alignment of representative Polymycoviridae RdRP PE sequences. The disordered…

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  Fig. 3.. Enzymology characterizations of AfuPmV-1 RdRP and its PE mutants. (A) Three types of RdRP assays based on the T30-2/P2 construct. The initiation kinetics was assessed by P2-to-P3 conversion, EC reactivity was assessed by P9-to-P10 conversion rate, and EC stability was assessed by P9-to-P10 conversion in a challenge-and-chase format. (B) Quantitative analysis of P2-to-P3 conversion. Relative 3-mer intensities at 30, 60, and 90 s were shown with average values and SDs based on three individual experimental sets (N = 3). (C) Assessment of EC reactivity by quench-flow kinetics. Top: Gel images of representative RdRP constructs for P9-to-P10 conversion, and DENV2 NS5 was used for comparison. Bottom: Fitted reaction rates and fitting errors are listed. Black boxes indicate the time points with fraction of 10-mer intensity of approximately 0.5. (D) Assessment of EC stability. Top: Fitting of EC inactivation (dissociation) rate constant (kina). Error bars show SDs of fraction of 10-mer intensity values measured in three individual experimental sets (N = 3).

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  Fig. 4.. Assessment of terminal initiation properties of AfuPmV-1 RdRP and its PE mutants. (A) Four RNA constructs used for terminal preference assessment. (B) Quantitative analysis of P2-to-P3 conversion on all four RNA constructs. Relative 3-mer intensities at 1 and 4 min (AfuPmV-1) or 45 and 120 min (DENV2) were shown with average values and SDs based on three individual experimental sets (N = 3). Terminal initiation index (Iterm) values are indicated for each RdRP constructs. The underlined values indicate higher-than-WT terminal initiation preferences among AfuPmV-1 RdRP mutants. (C) Six RNA constructs generated by combinations of the T30-2 template, GTP (p1), and five primers for assessment of primer-length effect for representative AfuPmV-1 RdRP constructs. (D) Gel images of p1/P2/P3/P4/P5/P7-to-P9 conversion by four representative RdRP constructs. The average 8-mer intensity in the marker lanes was set to 1 for calculation of relative 9-mer intensities. The chemically synthesized P8 (marker lanes) with 5′-hydroxyl migrates slower than the 9-mer bearing a 5′-phosphate (P9), while the p1-derived p9 (lanes 2 to 3/22 to 24/42 to 44/62 to 64) with 5′-triphosphate migrates faster than…

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  Fig. 5.. A working model of the transition toward elongation in de novo–only viral RdRPs. (A) Four RNA templates used for stability assessment for complexes with product length from 4 to 8 nucleotides. Red letters indicate incorporated nucleotides for complex assembly, and blue letters indicate the possibly incorporated nucleotides in the challenge-and-chase step. (B) Representative gel images of complex stability assessment of AfuPmV-1 and DENV2 NS5 RdRPs. Dashed boxes indicate time point(s) with about 50% complex dissociated/inactivated under the challenging condition. For DENV2 RdRP, P6- and P7-containing complexes were not assayed, because the P5-containing complex is already as stable as the P8-containing AfuPmV-1 RdRP complex, indicating the completion of the critical transition. (C) A comparison of inactivation (dissociation) rate constant (kina) of AfuPmV-1 and DENV2 RdRP complexes. Data were derived from three individual experimental sets (N = 3). The data of AfuPmV-1 RdRP T30-2/P9 were taken from Fig. 3, and those of DENV2 NS5 RdRP T30-2/P9 were published in our recent DENV2 study. N/D, values not determined; N/A, not assayed. (D) A working model of AfuPmV-1 and DENV2 RdRP PE…

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  Fig. 6.. A structural comparison of putative de novo–only viral RdRPs from +ssRNA and dsRNA virus groups. (A) Experimentally solved (with PDB entry labeled) or AF3-predicted (https://alphafoldserver.com) structures of putative de novo–only RdRPs from dsRNA (top half) and +ssRNA (bottom half) virus groups. Structures are shown as a combination of surface and cartoon representations. Order/family/genus/species assignments are based on the ICTV 2022_MSL38.V3 collection and are listed on top of each structural model. Coloring scheme: RdRP palm in gray, thumb in slate, fingers in pinky, PE in purple, and signature sequence XXDD/N of motif C in magenta. The N- and C-terminal additional regions, if present, are shown in cyan and green, respectively. All structures were superimposed using the RdRP motif C residues by the traditional least-square method. LtRV1, lactarius tabidus RNA virus 1; BCV1, beet cryptic virus 1; XdV-L1A, xanthophyllomyces dendrorhous virus L1A; MpRV, Micromonas pusilla reovirus; CPV, cypovirus 1; IBDV, infectious bursal disease virus; NoV, nodamura virus; OuMV, ourmia melon virus; IFV, infectious flacherie virus; CPMV, cowpea mosaic virus; PrV, providence virus; BYDV-PAV,…