Research ArticleVIROLOGY

Viral evolution identifies a regulatory interface between paramyxovirus polymerase complex and nucleocapsid that controls replication dynamics

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Science Advances  04 Mar 2020:
Vol. 6, no. 10, eaaz1590
DOI: 10.1126/sciadv.aaz1590
  • Fig. 1 Recovery of recCDVs with tail-truncated N proteins.

    (A) Schematic of the RdRP complex showing N (blue, cyan, and green) and N-tail binding P-XD (green) through N-MoRE (orange). L protein docks to the P C terminus (14, 26). (B) CDV N is organized as N-core (amino acids 1 to 391) and N-tail (amino acids 392 to 523) domains. N-tail contains modules of conserved residues: boxes 1 to 3 (yellow, orange, and red). Box 2 harbors N-MoRE. The [425–479] junction is represented below. (C) Minigenome reporter assays to determine RdRP bioactivity. Relative luciferase units were normalized to the peak activity in the presence of full-length N (n = 12) [nonlinear regression using a log(normal) model]. Error bars: Geometric SD. Two-way analysis of variance (ANOVA) with Sidak’s post hoc test. ns, not statistically significant. (D) Enumeration of progeny syncytia after transfer of initial infectious centers. Mean (n = 9) represented as black bar. Kruskal-Wallis test with post hoc Dunn’s test. (E) Representative microphotographs (×100 magnification) of recCDV CPE at 35 hours post-infection (h.p.i.). (F) Mean syncytium extension of three independently recovered recombinant viruses (14, 22, and 35 hours post-infection). (G) Substitutions reported for N and P ORFs of recombinant CDV encoding full-length (n = 3) or Δ[425–479] (n = 10) N after 10 passages. (−): no substitution.

  • Fig. 2 Identification of specific mutations compensating for N-tail truncation.

    (A) Minigenome activity profiles of full-length (blue) and tail-truncated (red) N. Normalized activities of selected mutations in full-length (blue line and symbols) and tail-truncated (red line and symbols) N background (n = 3) are overlaid. A natural allele A/T allele variation exists at residue 410 between CDV strains 5804PeH and Onderstepoort. Statistical analysis and regression modeling as in Fig. 1C. (B and C) Peak minigenome activities after rebuilding of candidate substitutions, analyzed as in (A). Each set of peak activity in the context of either N or Δ[425–479] was compared with one-way ANOVA with Dunnett’s multiple comparisons post hoc test. Mutations associated with statistically significant polymerase activity differences in either N background or showing the greatest increase in the presence of tail-truncated N are highlighted with yellow shading. (D) Localization of compensatory mutation candidates in N-core. Surface representation (left) and ribbon model enlargements (right) of mutation sites are shown [Protein Data Bank (PDB): 4UFT]. Substituted side chains as sticks, color-coded by positive (green), neutral (blue), or negative (red) effect on RdRP activity in the distinct N-tail background. (E) Localization of compensatory mutation candidates in P-XD. Ribbon models of P-XD (PDB: 1T6O) are shown, color coding as in (D).

  • Fig. 3 Recovery of recCDV with confirmed compensatory mutations.

    (A) Schematic of recCDV genome organization [red star: N-E156Q + A410D (CDV 5804PeH background); green star: N-S415P; blue star: P-R469G; mutant color coding is maintained in all subpanels of this figure]. (B) Enumeration of progeny syncytia after transfer of initial infectious centers after recovery. (C) Mean syncytium extension of three independently recovered recombinants each, analyzed at 14, 22, and 35 hours post-infection. (D) Genetic stability of recCDV with full-length (n = 3) or Δ[425–479] (n = 10) N and engineered substitutions as specified after five passages. N and P genes were analyzed. (E) Multicycle growth profiles of recCDV as specified. Symbols represent mean viral titers (n = 3), and error bars denote SD to the mean. Two-way ANOVA with Dunnett’s multiple comparisons post hoc test.

  • Fig. 4 Electronic properties of compensatory microdomains in N-core and P-XD.

    (A to D) Ribbon (A and C) and electrostatic surface (B and D) representations of compensatory microdomains in CDV N-core (A and B) and P-XD (C and D). Residues subjected to mutagenesis are labeled, and negative (red) and positive (blue) charges are indicated. (E to H) Peak minigenome activities in the distinct N-tail backgrounds after charge-reversal or charge-neutralization mutagenesis as specified in N (E and F) or P (G and H). Statistical analysis and regression modeling as in Fig. 2 (B and C). One-way ANOVA with Holm-Sidak’s multiple comparisons post hoc test.

  • Fig. 5 Biochemical interaction of compensatory microdomains in N-core and P-XD.

    (A) Top: Ribbon representation of P-XD (pink)/N-MoRE (orange) (PDB: 1T6O). Bottom: Triangular prism configuration of P-XD. (B) Affinity coprecipitation with Glutathione Sepharose beads. Coomassie blue–stained gels (left) and anti-CDV N immunoblots (right) of eluted protein fractions after SDS–polyacrylamide gel electrophoresis are shown. *Copurified cellular contaminants. (C) Densitometry of coprecipitations. Values denote precipitation efficiencies relative to input material (n ≥ 3 ± SD). One-way ANOVA with Holm-Sidak’s multiple comparisons post hoc test. (D) Biolayer interferometry showing association and dissociation of N[1–419] ± E156K substitution with biosensors loaded with indicated GST fusions. (E) Affinity coprecipitation as in (B) with natural P-XD compensatory mutations A465V and A469G and engineered A465R. Coomassie blue–stained gels. (F) Densitometry of coprecipitations as in (C) (n = 5 ± SD). One-way ANOVA with Holm-Sidak’s multiple comparisons post hoc test. (G) Phenotypic effects of compensatory and engineered mutations at P positions 465 and 469. (H) Molecular docking of P-XD:N-MoRE complexes (PDB: 1T6O) into N-core (PDB: 4UFT). Top: Surface of N protomers (blue, purple, and green) and P-XD (pink) with N-MoRE (orange). Bottom: Predicted contacts between P-XD and N-core acidic loop and N-MoRE with N-core loop (residues 133 to 142; yellow). Right: Docking scores and PRODIGY predictions (36).

  • Fig. 6 Structural conservation of interaction sites and mechanistic hypothesis.

    (A) Surface representation of three protomers of morbillivirus RNP juxtaposed to RSV RNP (9) and a prediction of MeV RNP with N-MoRE relocated into N-core (12). N-core acidic loop and RSV N residues involved in P binding (24) are shown in red, and relocated MeV N-MoRE is in orange. (B) Mechanistic hypothesis of dynamic control of RdRP processivity through induced N-tail refolding and three-way interface formation between N-core and P-XD:N-MoRE. Compensatory mutations are proposed to restore proper kinetic control through reduced affinity of the interacting P-XD (green) and N-core (cyan, light marine blue) microdomains.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaz1590/DC1

    Fig. S1. Multisequence alignments of N and P proteins of representative members of the paramyxovirus family.

    Fig. S2. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.

    Fig. S3. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.

    Fig. S4. Genetic stability of recombinant viruses harboring rebuilt compensatory mutations.

    Fig. S5. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.

    Fig. S6. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.

    Fig. S7. Expression and purification of recombinant N and P protein subsets.

    Fig. S8. Molecular docking of P-XD:N-MoRE complexes into N-core.

    Fig. S9. Position of the acidic N-core loop in distinct paramyxovirus N proteins and activity associations.

    Fig. S10. Spatial organization and electronic properties of P-XD in crystal structures solved for MeV, SeV, HeV, and MuV and corresponding homology models generated for CDV, HPIV-3, NiV, and PIV-5.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Multisequence alignments of N and P proteins of representative members of the paramyxovirus family.
    • Fig. S2. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.
    • Fig. S3. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.
    • Fig. S4. Genetic stability of recombinant viruses harboring rebuilt compensatory mutations.
    • Fig. S5. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.
    • Fig. S6. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.
    • Fig. S7. Expression and purification of recombinant N and P protein subsets.
    • Fig. S8. Molecular docking of P-XD:N-MoRE complexes into N-core.
    • Fig. S9. Position of the acidic N-core loop in distinct paramyxovirus N proteins and activity associations.
    • Fig. S10. Spatial organization and electronic properties of P-XD in crystal structures solved for MeV, SeV, HeV, and MuV and corresponding homology models generated for CDV, HPIV-3, NiV, and PIV-5.

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