Research ArticleVIROLOGY

The novel asymmetric entry intermediate of a picornavirus captured with nanodiscs

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Science Advances  24 Aug 2016:
Vol. 2, no. 8, e1501929
DOI: 10.1126/sciadv.1501929
  • Fig. 1 Icosahedrally averaged A-particle formed asymmetrically.

    (A) The surface-rendered map of the CVB3 A-particle is colored radially according to the scale bar (numbers in Å), with the symmetry axes indicated. (B) The central section of the cryo-EM reconstruction shows strong density corresponding to the retained remodeled RNA genome (red). (C) Rendering the map at a higher contour (3σ above the mean) shows that the RNA genome is packed in layers inside the virus, as seen in CV-A16 (11).

  • Fig. 2 Sharpened asymmetrically formed A-particle map.

    (A) The sharpened A-particle map is surface-rendered and radially colored according to the scale bar, with the icosahedral symmetry axes labeled. (B) The magnified square area shows no openings at the twofold axis and the quasi-threefold axis, as highlighted by the lower and upper black circles, respectively. (C) The quality of the map (gray mesh) is illustrated by the fit of the Cα backbone in the separated β strands of the VP1 protein (blue wire), as well as the bulky side chains of Tyr1157, Trp2078, and Phe3220 (using the naming convention where residues in VP1, VP2, VP3, and VP4 are numbered sequentially, starting with 1001, 2001, 3001, and 4001, respectively).

  • Fig. 3 Rigid body movements of virus proteins to form the CVB3 A-particle.

    (A) The structure of the CVB3 A-particle is shown as a ribbon diagram with VP1, VP2, VP3, and VP4 colored according to convention (blue, green, red, and yellow, respectively), with pocket factor rendered as an orange surface. (B) The four structural proteins of the A-particle (blue, green, red, and yellow) are aligned with the structure of CVB3 (dark gray) to illustrate the outward movements. (C to E) The structures of the pocket factor (orange wire), the N terminus of VP1 (blue wire), and the VP4 of CVB3 are shown fitted into the corresponding A-particle densities.

  • Fig. 4 Extending densities from the in situ CVB3 A-particle.

    (A) The asymmetric reconstruction is radially colored according to the key and surface-rendered at low contour to show the connection extruding from a single threefold to a nanodisc density. (B) A thin slice through the center of the map (gray density with symmetry axes marked) shows how the extruding density is visualized as a unique threefold pore (blue arrow) through the capsid shell at 2σ contour. (C) When the map with the same radial coloring as (A) is surface-rendered at 1.5σ, the unique site on the capsid adjacent to the nanodisc (black square outline) shows distinct weak density resulting from different protein extrusions becoming flexible upon extension. (D) The fitted capsid structure rendered in blue, red, and green wire for VP1, VP2, and VP3, respectively, shows how the protein loops extend through the weak capsid density at this asymmetric location only. (E) Left: A thin slice oriented the same as (B) but rendered at 2.75σ shows asymmetrically disordered genome densities. Right: The map was rotated 90° around the threefold axis (black arrow). Both slices are radially colored according to the key.

  • Fig. 5 Residues interacting with RNA genome.

    Top: Slabs (25 Å thick) of the icosahedral (left) and asymmetric (right) 3D maps viewed along an icosahedral twofold axis, with the newly built atomic model of the CAR-nanodisc A-particle. The viral proteins are colored accordingly, and the two-, three-, and fivefold icosahedral symmetric axes are indicated by arrows. (A to C) Subregions of the reconstructions (indicated in black rectangular outline) are shown in close-ups with the indicated symmetric axes. The asymmetric, remodeled region of the map (C) is distinguished by loss of density, especially notable in the threefold view. The slabs are parallel to but displaced ~15 Å closer (two- and fivefold) or farther (threefold) from the central section. Envelopes of protein shell and RNA density (gray mesh) are shown, with the virus structures fitted. Residues interacting with RNA are shown (spheres) from left to right and indicated with a one letter code in (B). The four residues in twofold close-ups are Pro1022, His1052, Ser2045, and Trp2038. The residues in threefold close-ups are Gln4004, Ala4012, Trp2038, [Tyr2009 only in (B)], Trp2038′, Ser2045, and His1052. The five residues in fivefold close-ups are His1052, Arg1013, Gln4004, Ala4012, and Arg1013′. The prime symbol (′) indicates a symmetry-related residue.

  • Fig. 6 Subclasses that have stronger threefold and propeller tip protein extensions can be classified.

    (A to C) Reconstructions surface-rendered in gray show the three maps from the subpopulations of asymmetric A-particles oriented to illustrate the densities. The upper panels show densities protruding downward away from the capsid, and the lower panels show each map that was cut where indicated (red dotted lines in the upper panels) and rotated 90°, with the protruding densities highlighted (red). The asymmetric units are indicated (blue lines and symbols). The subclasses consisted of (A) particles that have two density protrusions across the twofold at each propeller tip, (B) proteins exiting the threefold and the propeller tip, and (C) a strong rope-like density only at the threefold.

  • Fig. 7 Two A-particles resulting from different stimulations.

    (A and B) Schematic representation of the locally stimulated (A) and globally stimulated A-particle (B). Locally stimulated A-particle has pores only at the site of receptor engagement and asymmetrically externalizes the VP4s and the VP1 N termini around the receptor binding site, whereas the globally stimulated A-particle has global twofold openings and externalizes all VP4s and the VP1 N termini.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/8/e1501929/DC1

    fig. S1. Negative-stain transmission electron microscopy images of CAR-nanodiscs bound to CVB3.

    fig. S2. Central sections and FSC curves of the icosahedral and asymmetric 3D maps.

    fig. S3. Local resolution of the icosahedral 3D reconstruction.

    fig. S4. Local resolution of the asymmetric 3D reconstruction.

    fig. S5. Quantification of density.

    table S1. Superimposing CVB3 (PDB ID: 1COV) with CAR-nanodisc A-particle structure illustrates rigid body movement of the protomer and describes the expansion of about 2 Å.

    table S2. Classification of the CAR-nanodisc particles.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Negative-stain transmission electron microscopy images of CAR-nanodiscs bound to CVB3.
    • fig. S2. Central sections and FSC curves of the icosahedral and asymmetric 3D maps.
    • fig. S3. Local resolution of the icosahedral 3D reconstruction.
    • fig. S4. Local resolution of the asymmetric 3D reconstruction.
    • fig. S5. Quantification of density.
    • table S1. Superimposing CVB3 (PDB ID: 1COV) with CAR-nanodisc A-particle structure illustrates rigid body movement of the protomer and describes the expansion of about 2 Å.
    • table S2. Classification of the CAR-nanodisc particles.

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