Reovirus polymerase lambda 3 localized by cryo-electron microscopy of virions at a resolution of 7.6 A

Abstract

Reovirus is an icosahedral, double-stranded (ds) RNA virus that uses viral polymerases packaged within the viral core to transcribe its ten distinct plus-strand RNAs. To localize these polymerases, the structure of the reovirion was refined to a resolution of 7.6 A by cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image reconstruction. X-ray crystal models of reovirus proteins, including polymerase lambda 3, were then fitted into the density map. Each copy of lambda 3 was found anchored to the inner surface of the icosahedral core shell, making major contacts with three molecules of shell protein lambda 1 and overlapping, but not centering on, a five-fold axis. The overlap explains why only one copy of lambda 3 is bound per vertex. lambda 3 is furthermore oriented with its transcript exit channel facing a small channel through the lambda 1 shell, suggesting how the nascent RNA is passed into the large external cavity of the pentameric capping enzyme complex formed by protein lambda 2.

Figure 1

Assessment of the resolution limit in reovirion reconstruction. The set of 7,939 particle images was subdivided randomly into two subsets, and independent reconstructions were computed from these data. Fourier shell correlation (solid curve) and phase difference (dashed curve) criteria demonstrated that the resolution of the final reconstruction was at least 7.6 Å.

Figure 2

Reovirion cryo-EM reconstructions. (a) Shaded-surface representation of complete virion, rendered at a resolution of 20 Å and viewed along a five-fold axis with a λ2 pentamer (blue) and a μ1₃σ3₃ heterohexamer (μ1, green; σ3, red) highlighted. The outlined region (dashed box) is shown at higher resolution in b. (b) Close-up view of the virion map rendered at a resolution of 7.6 Å illustrates fine details such as rodlike densities and also shows the interlocking arrangements of the five λ2 subunits in a pentamer (purple, green, magenta, greenish yellow and dark orange) and the three copies each of μ1 (red, yellow and light green) and σ3 (orange, dark blue and turquoise) that comprise a heterohexamer. (c) Equatorial section (2.2 Å thick) from the 20-Å rendering shows one quadrant of the view along a two-fold axis and identifies the approximate locations of viral components. (d) Same as c for the 7.6-Å rendering with locations of five-fold and three-fold axes indicated (the right and bottom edges of the panel coincide with two-fold axes). Numerous punctate features arise from α-helices viewed end on. (e) Stereo view of a small portion (N-terminal methyltransferase domain) of the λ2 X-ray model (ribbon) fitted into the corresponding density (wire cage) in the 7.6-Å EM map with α-helices in green and β-strands in yellow. Scale bars are labeled; bar in c also applies to d.

Figure 3

Fits of λ1 and λ3 X-ray models into reovirion cryo-EM map. (a) Slab through the cryo-EM map (10 Å thick), encompassing a five-fold axis and including core densities from low radii (~172 Å at bottom of panel) to the outer surface of the λ1 shell (radius ≈ 277 Å at top). (b) Same as a but with X-ray models of λ1 (black Cα backbone) and λ3 (dark orange, blue, orange, magenta and green Cα backbones) fit into the cryo-EM map. (c) Slab through the cryo-EM map (10 Å thick), viewed along a five-fold axis and toward the virion center, with the X-ray model of the λ3 pentamer fitted into the map. Thin dashed lines in b and c mark the boundaries of the slabbed regions shown, respectively, in c and a,b. Scale bar for a–c is labeled. (d) Plot of Laplacian correlation coefficients (LCC) computed by SITUS, which quantify the fit of the λ3 monomer X-ray model into a defined search volume within the reovirion cryo-EM density map. For this calculation, the cryo-EM map was rotated to make a five-fold axis coincide with the z-axis of a Cartesian coordinate system, and a region encompassing more than one asymmetric unit was selected as the search volume, as described in Methods and Supplementary Methods online. The fit of the λ3 X-ray model into this volume was based on an exhaustive six-dimensional search with angular and translational sampling intervals of 5° and 2.2 Å, respectively. Here the LCCs are only plotted for the z-plane, yielding the best fit, and the value at each x and y position in the plane corresponds to the orientation of the model at that position that gives the highest LCC. The x- and y-axes of the plot identify the pixel coordinates within the search volume, with each pixel representing a step size of 2.2 Å. The value of the maximum peak (0.047) rose only to 0.052 upon Powell minimization refinement. (e) Plot of the LCC for monomer and pentamer X-ray models of λ3 and λ2 fitted into the reovirion cryo-EM density search volume and for different rotation angles about the icosahedral five-fold axis. The 0° rotation angle for each model corresponds to the orientation and position of that model that gives the best fit as obtained in the experiments illustrated in d. (f) Stereo view of a small portion of the λ3 X-ray pentamer model (blue and orange ribbons distinguish adjacent monomers) fitted into the corresponding density (wire cage) in the 7.6-Å EM map. This region of the map includes minimal overlap between neighboring λ3 models.

Figure 4

Location of λ3 inside reovirus λ1 shell. (a) Space-filling representation of the inner surface of the λ1 shell viewed along a five-fold axis (white star). The five identical λ1-A subunits (those closest to the five-fold axis) are highlighted in yellow (A1), blue (A2), brown (A3), green (A4) and cyan (A5), and all five λ1-B subunits are shown in violet. Potential pores at the interfaces between neighboring λ1-A subunits are indicated by small red circles (shown also in c). The red line marks the position of a plane, ~6 Å from the five-fold axis, to one side of which over half of the pseudo atomic model of the reovirion was removed for the illustrations in e and f. (b) Same as a but with space-filling model of λ3 (red) added to show its position relative to the λ1 shell’s inner surface. The white arrow indicates the view direction in the cutaway illustrations in e and f. (c) Same as a but rendered to show the electrostatic surface potential of the λ1 shell inner wall. The λ3 footprint (black lines) outlines regions in λ1 within 5 Å of λ3 residues. (d) Electrostatic surface potential of λ3 overlaid with the footprint shown in c. The λ3 molecule (at right) and an enantiomeric version of it (at left) are viewed in a direction facing the mRNA exit channel and the surface that contacts the λ1 shell. The λ3 contact surface mainly consists of basic and neutral patches that seem complementary to the neutral and slightly acidic λ1 footprint seen in c. The λ3 enantiomer is included to help visualize the contact surface complementarity. (e) Shaded-surface, cutaway view of the pseudo atomic model of the reovirion, oriented with a five-fold axis vertical (red line) shows the arrangement of the globular λ3 molecules (red) on the inner surface of the λ1 shell. Most or all of the λ1-A1, -B2, -A2, -B3 and -A3 subunits, and small portions of the λ1-B1, -A5, -A4 and -B4 subunits, have been cut away. (f) Enlarged view of region outlined in e with λ2 in yellow, λ1 in blue and λ3 in red. Scale bars are labeled; bar in d also applies to a–c.

Figure 5

Stereo view of λ3 X-ray ribbon model with α-helices color-coded to signify position and correspondence with features in the cryo-EM map. The λ3 molecule is oriented with the mRNA exit channel facing the observer, a view opposite that for λ3 depicted in Figure 4b. Of the 48 total α-helices in λ3 (Table 1), the 33 that were well represented in the cryo-EM map are blue. The remaining 15 helices are distributed mainly at the periphery of the λ3 molecule, with three occurring in the N-terminal domain (green), seven in the polymerase domain (red) and five in the C-terminal domain (orange). A red asterisk marks the position of the nearest five-fold axis. Ball-and-stick figures highlight active site residues Asp585, Asp734 and Asp735 in orange and the priming NTP platform (Ser561) in magenta. A dotted line identifies the horizontal plane used to segment the λ3 molecule (the approximate one-half above the arrows was extracted) to reveal some of its internal features in Figure 4f.

Figure 6

Space-filling, cutaway view of the reovirus core, showing a proposed exit pathway for newly synthesized plus-strand (+) RNA transcripts leading from λ3 through the λ1 shell to the λ2 cavity. The view direction and region depicted are approximately the same as shown in Figure 4f, but additional portions of λ2 were included to help identify the large exit cavity. The approximate locations of four RNA capping guanylyltransferases (GTase1–4), three N-terminal methyltransferases (N-MTase1–3) and three C-terminal methyltransferases (C-MTase1–3) in the λ2 pentamer subunits are shown. The minus and plus strands of the template dsRNA and the nascent plus-strand transcript are orange, black and yellow, respectively. The transcript is shown exiting the channel at the top of λ3, traversing a putative channel formed at the interfaces between two λ1-A subunits (A1 and A5) and between λ2 and the λ1-A5 subunit, and finally into the large λ2 channel. The cap structure (^m7GpppG ^m2′) at the 5′ end of the dsRNA plus strand is shown tethered to the cap-binding region (green) on the λ3 molecule as previously proposed. The λ1 subunits are colored according to the same scheme as in Figure 4a,b. The nascent transcript is shown as having already been modified with a 5′ cap (green) by the RNA 5′ triphosphatase (position unknown) and λ2 capping domains.