Structure of the measles virus ternary polymerase complex

Figures

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  Fig. 1. Biochemical characterization and overall structure of MeV polymerase complex. a Elution profile of the purified MeV RNA polymerase complex on a Superose 6 Increase size-exclusion column. SDS-PAGE was performed to assess the expression of L, P, and C proteins (repeated ≥ 3 times). b RNA synthesis activity as determined in a fluorescence-based primer extension assay. The bars show the mean of relative fluorescence units and standard deviation (SD) for three independent biological replicates. Statistical significance was calculated with a two-tailed unpaired t-test (***P ≤ 0.001). c Schematic diagram of the domain architecture of MeV L protein divided into six parts: N-terminal domain (NTD), RdRp, PRNTase, connector domain (CD), MTase, and C-terminal domain (CTD). MeV P protein comprises three parts: NTD, oligomerization domain (OD), and C-terminal X domain (XD). MeV C protein consists of two parts: N-terminal (N) and C-terminal (C) domains, with labeled domain boundary. d, e Cryo-EM density maps of MeV Lcore-P complex and MeV Lfull-P-C complex. All maps are displayed using DeepEMhancer sharpened ones with a contour level of 0.03 in ChimeraX. f, g Atomic models of MeV Lcore-P…

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  Fig. 2. The catalytic cores of the MeV L protein. a The RdRp catalytic core is shown in cylinders. The NTD and three subdomains of the RdRp domain (left) are shown in light blue, dark blue (fingers: residues 362-574, 672-742), green (palm: residues 575-671, 743-843), and purple (thumb: residues 844-934), respectively. The active site is marked with a red asterisk. The regions surrounding the active site (middle) are superimposed with those of RSV (PDB: 6PZK) and EBOV (PDB: 7YER). The conserved motifs (A-F) are shown on the right: motif A (residues 654-671, green), motif B (residues 743-763, dark blue), motif C (residues 764-787, yellow), motif D (residues 788-822, light blue), motif E (residues 823-840, purple), and motif F (528-550, brown). b The PRNTase catalytic core (light pink) is displayed by cylinders with the highlighted conserved motifs (motifs A’–E’): motif A’ (residues 1141-1160, light blue), motif B’ (residues 1214-1217, lime), motif C’ (residue 1247, dark blue), motif D’ (residues 1288-1289, yellow), and motif E’ (residues 1328-1337, brown). The NTD and RdRp domains are gray. Motif B’ in the priming loop (residues 1191-1232, green) and Motif D’ in the intrusion loop…

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  Fig. 3. The L–P and L–C interfaces. a The MeV Lcore is shown as a surface representation with P protomers depicted in cartoon. The color scheme matches that of Fig. 1c. The interaction interfaces between MeV L and P are distributed across five main regions, as highlighted in the five inserts. In the close-up views, atomic interactions between key residues of L and P, shown as sticks for salt bridges and hydrogen bonds, are indicated by black dashed lines (see Supplementary Fig. 8 for detailed interactions). Main chain or side chain atoms are marked based on their involvement in interactions. b The MeV L and P proteins in Lfull-P-C complex are shown as a surface representation with C proteins depicted in cartoon. P tetramer is omitted for clear presentation in the bottom. The color scheme matches that of Fig. 1c. The interaction interfaces between MeV L and C are distributed across three main regions, as highlighted in the three inserts. In the close-up views, atomic interactions between key residues of L and C, shown as sticks for salt bridges and hydrogen bonds, are indicated by black dashed lines (see Supplementary Fig. S10 for detailed interactions). Main chain or side chain atoms are…

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  Fig. 4. The MeV C dimer adjusts CD-MTase-CTD spatial organizations. a The MeV Lfull-P-C complex is superimposed with those of HIPV3, NDV, MuV and HPIV5. The Lcore, P protomers, and C dimer are shown as cartoon with CD-MTase-CTD domain depicted in surface representations. b A schematic comparison of CD-MTase-CTD spatial organizations among MeV, HIPV3, NDV, MuV and HPIV5. The NTD, RdRp, and PRNTase of all five structures are aligned and colored in dark gray. The CD, MTase and CTD domains are colored in yellow, orange, and red, respectively. The C1 and C2 of MeV are colored in brown and light brown, respectively. The outlines of HPIV3/NDV/MuV and HPIV5 MTase-CTD are depicted in black dashed lines on the MeV diagraph. The up green dash lines show the angle shifted between the different MTase-CTD domains, while the bottom ones indicate the distances between the active sites of cap and methylation. The models used here are 8KDC (HPIV3), 7YOU (NDV), 8IZL (MuV), 6V85 (HPIV5), and 9DUT (MeV). c The potential RNA tunnels of MuV, HPIV5, and MeV nascent mRNAs for RNA capping and methylation. The red curve indicates the possible transcribed mRNA path.

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  Fig. 5. The model of elongation complex. a The potential new nascent RNA exits in Lfull-P-C complex are indicated by dashed circles with L shown in Coulombic electrostatic potential and surface representations and P displayed in cartoon. b The Coulombic electrostatic potential (left) and the surface representation (right) with the modeled RNA duplex are presented. The RNA duplex was manually modeled by superimposing the RdRp domains of Lfull-P-C complex and the Influenza B polymerase elongation complex (PDB: 6QCT). The clip surfaces on the active site display the inside channels for template RNA, complementary RNA, nascent replicated RNA, mRNA and NTP. The color scheme is the same as in Fig. 1.

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  Fig. 6. C protein regulates MeV polymerase activity. a Schematic representation of the MeV full-length genome and single-step replication minigenome. b, Th antisense NanoLuc sequence is flanked by a T7 promoter, the 5′ and 3′ untranslated regions (UTRs) of the MeV genome without 5’ trailer, and two RNA endonucleases. HHRz: hammerhead ribozyme; HDVrz: hepatitis delta ribozyme. The pCAGGS-minigenome, pCAGGS-N, pCAGGS-L, pCAGGS-PΔC, and pCAGGS-C plasmids are co-transfected into 293T cells, leading to NanoLuc mRNA transcription and protein expression. c The C protein downregulates the transcriptional activity of RNA polymerase, as assessed using the MeV NanoLuc single-step replication minigenome system. A full set of MeV minigenome plasmids, consisting of pCAGGS-minigenome-NanoLuc, pCAGGS-N, pCAGGS-L, and pCAGGS-PΔC, was transfected into 293T cells along with increasing amounts of a plasmid expressing Flag-C. A luciferase assay was performed 48 hours post-transfection. Relative luciferase activity was determined by normalizing NanoLuc luciferase activity to firefly luciferase activity. Expression levels of Flag-C, P-His, HA-N, Strep-L, and GAPDH in the minigenome assay were assessed by…