Geometric mismatches within the concentric layers of rotavirus particles: a potential regulatory switch of viral particle transcription activity

Abstract

Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T = 13 to T = 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.

FIG. 1.

Images and 3-D reconstructions of rotavirus particles. DLPs and TLPs were visualized by cryo-EM (a and b, respectively). The 3-D reconstructions at a resolution of about 2.5 nm show the layered structure of the particles (c and d). The DLP reconstruction shows two layers, made of VP2 (T = 1) and VP6 (T = 13) for the inner (yellow in panel c) and external (red in panel c) ones. The TLP reconstruction shows three layers made of VP2 (not visible on the figure, inside the red VP6 layer), VP6 (red in panel d), and VP7 (T = 13) (blue in panel d)-VP4 (T = 1) (green in panel d). The bars represent 100 nm (1,000 Å).

FIG. 2.

Images of VLP6 (a), VLP6/7 (b), VLP2/6 (c), and VLP2/6/7 (d) visualized by cryo-EM. The inset shows two VLP6 particles having different diameters.

FIG. 3.

3-D reconstructions of VLPs. VLP6 (a, upper reconstruction) and VLP6/7 (a, lower reconstruction) show a T = 19 icosahedral lattice. The resolution of the VLP6 and VLP6/7 reconstructions is equal to about 4 nm (Table 1). VLP2/6 (b, upper reconstruction) and VLP2/6/7 (b, lower reconstruction) show T = 13 icosahedral symmetry. The resolution of the VLP2/6 and VLP2/6/7 reconstructions has a value close to 2.5 nm. Above the reconstructions is a schematic of the hexagonal lattice and the relative positioning of the fivefold axes. For T = 19 (blue), a walk from a fivefold axis to another one requires three and two steps along each lattice vector. For T = 13 (red), a walk from a fivefold axis to another one still requires three steps along one lattice vector but requires only one step along other one. The VP2, VP6, and VP7 layers are colored yellow, red, and blue, respectively.

FIG. 4.

Central section in the reconstructions of rotavirus particles DLP (a), TLP (b), VLP6 (d), VLP6/7 (e), VLP2/6 (g), and VLP2/6/7 (h). The section of a VP6 trimer is circled in all reconstruction. VP6 appears to be formed by two domains: the head and the base. A low-density area is present in the center of the base, in agreement with the X-ray model (31). The arrows indicate fivefold axes of the particle; the areas located near these axes shown in panels c, f, and i. When the reconstructions are scaled so that VP6 has same radial position in corresponding maps, the VP6 trimers around the fivefold axis appear to have different orientations and the type I channel to have different diameters, depending upon the presence of VP7. The fivefold axis areas are shown in panel c for DLP and TLP, in panel f for VLP6 and VLP6/7, and in panel i for VLP2/6 and VLP2/6/7. The reconstructions of particles containing VP7 are drawn in blue (c [TLP], f [VLP6/7], and i [VLP2/6/7]), while others are shown in red (c [DLP], f [VLP6], and i [VLP6/7]).

FIG. 5.

Fitting of the X-ray model of VP6 into the cryo-EM maps for VLP6 (a), VLP6/7 (b), VLP2/6 (c), VLP2/6/7 (d), DLP (e), and TLP (f). Only the areas surrounding the type I channel are shown along the fivefold axis. The presence of VP7 results in a systematic reorientation of the VP6 trimers defining the type I channels. Here and in Fig. 6 and 7, the maps are shown in gray and the fitted models are arbitrarily colored, with the trimer closer to the fivefold axis in red.

FIG. 6.

Fitting of the VP6 atomic model into the cryo-EM maps for VLP6 (a), VLP6/7 (b), VLP2/6 (c), VLP2/6/7 (d), DLP (e), and TLP (f). A portion of the type I channel seen in a direction perpendicular to the fivefold axis. The layers above and below VP6 are VP7 and VP2, respectively.

FIG. 7.

Pseudo-atomic model of the VP6 layer for VLP6 (a), VLP6/7 (b), VLP2/6 (c), VLP2/6/7 (d), DLP (e), and TLP (f). VP6 trimers other than those located around fivefold axes display contacts that do not depend on the presence of VP7. The maps have been oriented so that the VP6 trimers near the twofold axis have similar orientations.