Revisiting Viral RNA-Dependent RNA Polymerases: Insights from Recent Structural Studies

Figures

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  Figure 1. (A) Polymerization reaction catalyzed by RdRP. (B) The structure of poliovirus RdRP (PDB 1RA6) showing the fingers (magenta ribbon), palm (gray), and thumb (green) subdomains. The motifs A (red), B (orange), C (yellow), D (navy blue), E (cyan), F (blue), and G (brown) are indicated. The aspartates involved in catalysis from motifs A and C are shown. The conserved aspartates of the motif A (D-x(4,5)-D) and motif C (GDD) are represented as ball-and-stick, and the main channels are indicated.

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  Figure 2. The RdRP–VPg complexes of caliciviruses. (A) Multimerization of MNV RdRP–VPg complex. Six monomeric units of RdRP of MNV (PDB 5Y3D), shown in shades of blue, associate with each other and the VPg moiety (shown as orange ribbons) to form efficient ball-like replication machinery. (B) Zoomed-in view of a single MNV RdRP–VPg interaction unit. The close-up of the two molecules of MNV RdRP is shown in shades of blue with the interacting helices of VPg (α1 and α2) shown in orange. The residues of the RdRP participating in the interaction with the VPg are also shown in ball-and-stick representation and colored green.

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  Figure 3. RTCs of SARS-CoV-2. (A) RdRP (light blue), nsp7 (green), nsp8 (purple), nsp13 (Dodger blue), template (yellow), and product (orange) RNA are shown in surface representation. The positively charged tentacle-like amino-terminal extensions of the two nsp8 copies can be seen clasping the RdRP. The disposition of the nsp12 with respect to the other domains indicates its extensive interaction with the RTC. (B) The complex of the nsp9 with the RTC is shown. The interaction of the NiRAN domain (1-385, cyan) with the nsp9 (red surface) is implicated as one of the first steps in the capping of the 5′ genomic RNA. (C) The NTD of STAT2 (1-136, dark green) interacts with the residues 314-327 of the fingers subdomain (305-495, navy blue), while the coiled-coil domain of STAT2 (137-315, lime green) interacts with residues 847-855 of the thumb subdomain (721-888, Dodger blue). The NTD (273-307, purple), and the palm (496-720, cyan) subdomains of RdRP are indicated.

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  Figure 4. The association of the P domain of HPMV with the L. (A) The figure shows the interaction of the coiled-coil domain of the P (residues 169-195) with the L protein (PDB 6U5O). The four helices of the tetrameric P domain are shown as yellow ribbons, and the important domains of L are indicated in different colors in surface representation. (B) The superposed structure of HRSV (PDB 6PZK, blue ribbon), PIV5 (PDB 6V85, gray ribbon), and HPMV (PDB 7ORL, green ribbon) RdRPs and their corresponding P domains. The strong resemblance between the structural conformations of HPMV and HRSV and their association with P is observable. The large conformation shift of the MTase, CBD, and CTD of PIV5 with respect to the mononegaviral L–P complex is obvious, though the P interactions seem similar. (C) Cooperative binding of ANPP32 to two copies of FluC polymerase during replication (PDB 6XZQ). The structures of human FluC polymerases (RdRP: brown, PA: green, PB: orchid, RNA: yellow) bound to the amino-terminal leucine-rich repeat domain of ANP32A (blue) are shown. The ANPP32 plays a pivotal role during replication by stabilizing the replication complex.

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  Figure 5. Superposition of Junin, LACV, and MACV L–Z complexes. The Junin RdRP complex showing the PA-like (purple), RdRP (blue), and PB2-like (yellow) domains. The PA-like domain harbors the endonuclease (residues 1-196). The Z-protein that acts like a molecular switch is shown in red color while the loop-680 that binds to the host factors is shown in cyan. The superposed structures of the L and Z proteins of LASV (green ribbons) and MACV (pink ribbons) indicate variability at the N-terminus.

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  Figure 6. The contributions of the NTDs in replication and transcription. (A) NTDs of the peribunyaviral polymerases. The transcription initiation stage of the LACV polymerase (PDB 7ORL). The endonuclease and the CBD are shown in sea green and light orange colors, respectively. The other domains from N- to C-terminus are depicted in rainbow colors and represented as ribbons. The template and the product RNA are shown in yellow and gray colors, respectively. The domains at the amino terminus are involved in cap-snatching. (B) NTDs of the Flu polymerases. The structure of FluA RdRP (PDB 6EAJ, light gray) with the bound physiological RNA primer (maroon spheres) superposed on the apo state of FluB RdRP (PDB 5EPI, dark gray) with bound 5′cRNA (Tan). The CBDs (320-489) of FluA and FluB are colored in light blue and light green colors, respectively, while the midlink (251-319) regions of the two are indicated in dark blue and dark green colors, respectively. The CBD and the midlink regions have rotated by 60° and 77°, respectively, with respect to the corresponding domains in the apo state of FluB. (C) NTDs of Phenuiviridae RdRPs. The superposed ribbon representation of the L protein of SFTSV…

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  Figure 7. The RdRPs of Reoviridae. (A) The figure shows the RdRP domain of CPV in the quiescent stage with the bound RNA during transcription. The template (yellow), non-template (pink), and transcript (orange) RNA are shown as ladders. Modules A (912-1010, light blue) and B (1067-1140, green) from the polymerase (PDB 6TY8, gray surface) are indicated. The noticeable deflection of the module A α-helix (955-1007) of the C-terminal bracelet domain during the elongation stage (PDB 6TZ2, purple ribbon) is evident. (B) The in situ structure of rotaviral RdRP. The RdRP of Rotavirus at transcript-elongated state (blue ribbon, PDB 6OGZ) was superposed on the duplex open state (green ribbon, PDB 6OGY). The marked conformational changes around residues N31-A69, D923-L996, and N1072-D1088 can be observed. The transcript is shown in orange and the template strand in yellow. (C) The in situ structure of BTV RdRP. The BTV RdRP in its core (PDB 6PO2) shows the disposition of the VP3 (green ribbon) and the RdRP (blue surface). The close interaction of the N-terminal region (red ribbon) of the alternating VP3 with the bracelet domain of the RdRP (purple) around the five-fold axis is believed to be…

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  Figure 8. Motifs, pockets, and their role in replication. (A) The configuration of the motif F in the apo and the template-bound states of TAV RdRP. The reorganization of motif F in the bound (green, PDB 7OM6) and unbound (blue, PDB 7OM9) states is highlighted. The template and the product strands are shown in yellow and orange, respectively. (B) Picornaviral EV71 RdRPs at the translocation start stage (PDB 6LSE, light blue surface) with the template (yellow) and product (orange) RNAs represented as a ladder. The key interacting residues R277 and H44 of the nucleobase-binding surface pocket are shown in blue. The motif G (109-118), implicated in resisting the reverse translocation events, is colored in pink. (C) The structures of FluA RdRP showing the disposition of the template, product, and the capped mRNA during elongation (PDB 6T0V) and termination (PDB 6TW1). The PB (gray surface) and RdRP (light blue ribbon) domains are indicated, while the PA domain is masked for clarity. The template RNA is depicted in light yellow (elongation) and yellow (termination), the product in orange (elongation) and red (termination), and the capped mRNA in magenta (elongation) and purple (termination)…

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  Figure 9. The ligand–RdRP interaction diagram for the Enterovirus and SARS-CoV-2 RdRPs with their inhibitor compounds. (A) Enterovirus D68 RdRP–NADPH (PDB 5ZIT), (B) SARS-CoV-2 RdRP–suramin (site 1) (PDB 7D4F), (C) SARS-CoV-2 RdRP–suramin (site 2) (PDB 7D4F), (D) SARS-CoV-2 RdRP–favipiravir (PDB 7DFG), (E) SARS-CoV-2 RdRP–ribavarin (PDB 7DFH), (F) SARS-CoV-2 RdRP–remdesivir (PDB 7BV2), (G) SARS-CoV-2 RdRP–molnupiravir (PDB 7OZU).

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  Figure 10. Inhibitor binding sites of DENV2 and SARS-CoV-2 RdRPs. (A) The binding sites of suramin derivatives in SARS-CoV-2 RdRP (yellow ribbon). The first one is near the active site (magenta), and the other site is located at the template binding region (blue). The NSP7 and NSP8 that are bound to the RdRP are shown in shades of blue. (B) The structure of SARS-CoV-2 RdRP with the bound nucleoside-analog remdesivir (green ball and stick). The template and the product are shown in yellow and orange, respectively. Ser861 obstructs the cyano group of the inhibitor during translocation. (C) The structure of the DENV2 RdRP showing the interactions with the inhibitor RK-0404678 at the active site (magenta) and in the thumb subdomain (blue). The inhibitor is shown as spheres and the RdRP is depicted as a yellow ribbon.

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  Figure 11. The HCV RdRP–inhibitor complexes showing the interactions of different residues with the bound inhibitor: (A) compound 25 (PDB 6MVK), (B) compound 45 (PDB 6MVP), (C) compound 31 (PDB 6MVQ), (D) compound 49 (PDB 6MVO), (E) compound 5 (PDB 5QJ0), and (F) compound 13 (PDB 5QJ1).

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  Figure 12. The DENV and Zika RdRP inhibitor complexes showing the interactions of different residues with the bound inhibitor: (A) DENV3 RdRP–NITD-434 (PDB 6XD0), (B) DENV3 RdRP–NITD-640 (PDB 6XD1), (C) DENV2 RdRP–RK0404678 (PDB 6IZX), (D) DENV2 RdRP–RK0404678 (PDB 6IZZ), (E) Zika virus RdRP–compound 7 (PDB 6LD2), (F) Zika virus RdRP–compound 13 (PDB 6LD3), (G) Zika virus RdRP–compound 14 (PDB 6LD4), (H) Zika virus RdRP–compound 15 (PDB 6LD5).