Small Molecule Inhibition of Mononegavirales RNA Polymerases

Figures

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  Figure 1. The genome organization and RNA synthesis of mononegaviruses. (A) Representative genomes of human‐pathogenic mononegaviruses are depicted from the 3′ end to the 5′ end. The 3′ leader region is shown in yellow, and the 5′ trailer region is shown in purple. The ORFs encoding viral structural proteins are colored as follows: nucleoprotein (N, light gray), phosphoprotein (P or VP35, light orange), matrix protein (M or VP40, orange), glycoprotein (G, light green), fusion protein (F, dark gray), hemagglutinin or hemagglutinin‐neuraminidase (H or HN, pink), and the polymerase (L, light blue). The ORFs encoding viral non‐structural proteins, including NS1, NS2, SH, M2‐1, M2‐2, VP30 and VP24 are shown in blue. Additional P‐cistron encoded proteins such as V, W, and C are indicated within the P ORF box. The complete viral genome sequences from GenBank are as follows: RABV: NC_001542.1; RSV: FJ614813.1; HMPV: PV218189.1; EBOV: MG572235.1; MARV: NC_001608.4; HPIV3: NC_001796.2; MeV: NC_001498.1; MuV: JN012242.1; NiV: NC_002728.1. (B) Schematic diagrams illustrating how RNA polymerase catalyzes mRNA transcription and genome replication, thus facilitating nucleocapsid formation and subsequent…

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  Figure 2. Domain organization and overall structure of Mononegavirales polymerases. RNA polymerases from representative virus families in Mononegavirales are shown as L proteins in complex with their corresponding cofactors (P, VP35, or C proteins). The domain architecture of the L protein is divided into six parts: the N‐terminal domain (NTD, gray), RNA‐dependent RNA polymerase (RdRp, light blue), polyribonucleotidyltransferase (PRNTase, green), connector domain (CD, yellow), methyltransferase (MTase, orange), and C‐terminal domain (CTD, red). The P protein is shown in pink, and the C protein is shown in brown. For RSV, HMPV, EBOV, and MARV, only the NTD‐RdRp‐PRNTase regions are resolved in the available structures. RABV L is shown bound to a single P protomer, whereas the other structures show L bound to tetrameric P. MeV L is additionally shown in complex with dimeric C proteins. The models used here in PDB code are RABV (6UEB), RSV (6PZK), HMPV (6U5O), EBOV (7YER), MARV (9IP2), HPIV3 (8KDC), MeV (9DUT), MuV (8IZL), NiV (9GJU).

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  Figure 3. The conserved catalytic cores of the RSV polymerase. (A) The RdRp domain contains three subdomains: fingers (residues 433–689 and 709–772, blue), palm (residues 690–708 and 773–877, red), and thumb (residues 878–968, green). The active site is indicated by an orange asterisk. (B) The conserved motifs (A–F) in the RdRp domain are as follows: motif A (residues 690–708, red), motif B (residues 773‐801, orange), motif C (residues 802–827, yellow), motif D (residues 828–865, lime), motif E (residues 866–876, royal blue), and motif F (residues 613–632, purple). (C) The conserved motifs (A′–E′) in PRNTase domain are as follows: motif A′ (residues 1207‐1222, red), motif B′ (residues 1262‐1267, orange), motif C′ (residues 1297, yellow), motif D′ (residues 1338–1339, lime), and motif E′ (residues 1380–1389, medium blue). Two loops, including priming loop (residues 1256‐1276, purple) and intrusion loop (residues 1329–1353, cyan), are critical for PRNTase activity. (D) The MTase domain contains the conserved catalytic K‐D‐K‐E motif (K1673‐D1779‐K1817‐E1848), which is common to 2′‐O methyltransferases, and the G[‐]GxG motif (1696‐GEGAG‐1700) forms part of the SAM binding site. The RSV RdRp…

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  Figure 4. The binding mode of the GTP analogue GMPPNP in the early elongating NiV L‐P complex. (A) Slice‐through view of the early elongating NiV L–P complex with an incoming GMPPNP. The structure is shown as a surface representation with a partially clipped surface. The RNA template and GMPPNP are shown as cartoons and stick, respectively. The template entrance and exit, the NTP entrance and product exit are highlighted. (B) Schematic of key residues involved in GMPPNP binding and the associated active‐site conformational changes in the early elongation NiV L–P complex. The quadruple solid line indicates a π–π interaction between C10 of the RNA template and F553. The double solid line indicates a π–cation interaction between the ribose of GMPPNP and R551. The triple dashed line indicates base‐pair formation between GMPPNP and C10. The purple dashed line indicates magnesium coordination. Superposition of the NiV L–P–RNA complex with the apo NiV L–P complex reveals movement of the active site in the elongation state, indicated by the black arrow. The models used here correspond to NiV L–P (PDB: 9GJT) and NiV L–P–RNA (PDB: 9GJU).

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  Figure 5. Structural basis and conformational changes induced by NNI binding to Mononegavirales polymerases. Structures of the RSV polymerase complex with MRK‐1 (A) and compound 22 (B), the MeV polymerase complex with ERDRP‐0519 (C), and the EBOV polymerase complex with suramin (D). The NNI‐bound polymerase structures are shown on the left in cartoon representation with different subunit coloring. The NNIs (MRK‐1, orange; Compound 22, magenta; ERDRP‐0519, pink; Suramin, purple) are shown as sticks. Zoom‐in: Superimposition of viral RNA polymerases based on the L protein in NNI‐bound and apo states highlights the conformational changes induced upon inhibitor binding. The models used here in PDB code are RSV L‐P‐MRK‐1 (8FPI), RSV L‐P‐compound 22 (9N36), MeV L‐P‐ERDRP‐0519 (9OCF), EBOV L‐P‐suramin (7YET).