Research ArticleVIROLOGY

An ultraweak interaction in the intrinsically disordered replication machinery is essential for measles virus function

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Science Advances  22 Aug 2018:
Vol. 4, no. 8, eaat7778
DOI: 10.1126/sciadv.aat7778
  • Fig. 1 P1–304 structural dynamics.

    (A) Population of αR conformation of P1–304 as calculated from an ASTEROIDS ensemble based on chemical shifts and SAXS (gray bars) compared to those expected from a statistical coil ensemble (flexible-meccano, black lines). (B) NHN RDCs (1DNH-N) of P1–304 obtained from alignment in a liquid crystal made from polyethylene glycol (PEG) and 1-hexanol (gray bars). Red line shows NHN RDCs back-calculated from the ASTEROIDS ensemble selected against chemical shifts only. (C) Heteronuclear {1H}-15N Overhauser effects (nOes) of P1–304, measured at a 1H frequency of 600 MHz and 25°C. (D) Representation of the differential flexibility along the sequence of PNTD. The image shows the color and width of the ribbon as a function of the heteronuclear nOe in (C) (thick, red ribbon corresponds to more flexible regions; thin, blue ribbon to less flexible regions). Annotations indicate the presence of helical elements. The protein is highly dynamic, and the actual conformation is not representative of the ensemble of conformers.

  • Fig. 2 P1–304 interaction with N.

    (A) R2 of P1–304 (gray bars) and P1–304N1–525 (red lines) as purified from a Superdex 200 column (fig. S3) measured at a 1H frequency of 950 MHz. (B) Interaction profile of P1–304 with P1–50N1–525. Titration admixtures included 25 (gray), 50 (red), 100 (green), and 150 μM (blue) final concentration of P1–50N1–525 and P1–304 at a final concentration of 50 μM. Shown are normalized peak intensities (I/I0). (C) Interaction profile of P1–304,HELL→AAAA with P1–50N1–525. Concentrations and colors are the same as in (B). (D) 1H-15N heteronuclear single-quantum coherence (HSQC) spectrum of P1–304 in absence (blue) and presence (red) of P1–50N1–525. ppm, parts per million. (E) 1H-15N HSQC spectra of P1–304 (red), P1–50N1–525 (green), and P1–304N1–525 (blue).

  • Fig. 3 δα4 interaction with N.

    (A) Intensity ratios of 15N P140–304 peaks extracted from 1H-15N HSQC spectra in the presence of 5% (gray bars), 10% (red), or 20% (blue) P1–50N1–525 with respect to the unbound P140–304. Concentration of P140–304 remained constant at 200 μM throughout the titration. (B) R of P140–304 alone (gray bars) and with 20% P1–50N1–525 (blue) recorded at 16.5 T. (C) CPMG relaxation dispersion of 15N P140–304 (200 μM) in the presence of 20% P1–50N1–525: ΔR2,eff was determined at 14 T as the difference in effective R2 at CPMG frequencies of 31 and 1000 Hz. (D) CPMG relaxation dispersion of 15N P140–304 (200 μM) in the presence of 20% P50N525 at 22.3 T and 14 T. Data from eight sites throughout δ and α4 were fitted simultaneously, assuming a two-site exchange process giving kex = 624 ± 88 s and population, 4.7 ± 0.5%. Example sites from residues S180 (δ) and L193 (α4) are shown.

  • Fig. 4 Conservation and functional impact of the δα4 motif.

    (A) Alignment of phosphoproteins from different related viruses (respective UniProt identifiers are shown on the left, and morbilliviruses are shown above the dashed line). Full PNTD sequences aligned with MUSCLE (35). Positions of the helical elements predicted using PSIPRED (57) are shown as boxes (red, negatively charged; blue, positively charged). (B) Conservation of MeV sequences of PNTD. Comparison of 259 nonredundant sequences curated in Genbank (60). The position of α helices, additional interaction sites δ and α, STAT interaction site, and the RNA editing site defining the start position of the unique domain of V are indicated. Image obtained using WebLogo software (http://weblogo.berkeley.edu/logo.cgi). (C and D) Functional impact of HELL→AAAA mutation in cellulo. (C) Ability of MeV P constructs to associate with MeV N protein in cultured human cells, as determined by Gaussia luciferase–based protein complementation assay. Gray zone indicates the threshold NLR (normalized luminescence ratio) value. (D) Inability of HELL→AAAA P mutant to support the expression of two reporter genes from either a (+) or (−) strand MeV minigenome when coexpressed with MeV N and L proteins using a reverse genetic assay [relative light units (RLU): luciferase reporter activity]. NanoLuc, nanoluciferase.

  • Fig. 5 N-δα4 binding site and N0P model.

    (A) Ratio of peak intensities from 1H-15N HSQC spectra of free 15N P140–304 and 15N P140–304 bound to unlabeled N1-261 (gray bars) superimposed with an intensity ratio of 15N P140–304 unbound and bound to P1–50N1–525. (B) Transverse relaxation of 15N13C2DN1–261. (C) Secondary structure propensities (SSPs) for N1–261. Red bars indicate interaction regions with P140–304. (D) 1H-15N HSQC spectra of 15N, 2DN1–261 alone (red) and in the presence of 50 μM (blue) and 170 μM (cyan) unlabeled P140–304. (E) Cartoon of interaction of P1–304 (yellow and blue) with NCORE (gray). Red surface denotes the interaction region on N. Dashed red line indicates the dynamic region of NCORE. IDRs are marked as dotted lines. Yellow indicates transient helices. (F) Representation of conformational space available to P1–304 (blue) when both α1/2 (yellow spheres) and δα4 (yellow spheres) are bound to NCORE (gray surface; red shading shows the α4 interaction region on NCORE). All conformations shown have both interaction sites in proximity to the respective N binding sites. Gray indicates NTAIL.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/8/eaat7778/DC1

    Fig. S1. Scheme showing the different domains of N and P proteins referred to in the main text.

    Fig. S2. NMR of P1–304.

    Fig. S3. Purification of P1–304N1–525 complexes.

    Fig. S4. Interaction of N with α1/2 of P.

    Fig. S5. Analysis of P interaction dynamics with N1–525.

    Fig. S6. Interaction of P1–160 with N.

    Fig. S7. P140–304 interaction with P1–50N1–525.

    Fig. S8. Defining the conformational ensemble of P1–304N1–525 from SAXS curves.

    Fig. S9A. Expression of MeV P protein constructs used for functional studies.

    Fig. S9B. Functional assays using MeV minireplicon.

    Fig. S10. NMR spectroscopy of N-terminal domain of NCORE in interaction with P1–304.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Scheme showing the different domains of N and P proteins referred to in the main text.
    • Fig. S2. NMR of P1–304.
    • Fig. S3. Purification of P1–304N1–525 complexes.
    • Fig. S4. Interaction of N with α1/2 of P.
    • Fig. S5. Analysis of P interaction dynamics with N1–525.
    • Fig. S6. Interaction of P1–160 with N.
    • Fig. S7. P140–304 interaction with P1–50N1–525.
    • Fig. S8. Defining the conformational ensemble of P1–304N1–525 from SAXS curves.
    • Fig. S9A. Expression of MeV P protein constructs used for functional studies.
    • Fig. S9B. Functional assays using MeV minireplicon.
    • Fig. S10. NMR spectroscopy of N-terminal domain of NCORE in interaction with P1–304.

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