3.88 A structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy

Abstract

Cytoplasmic polyhedrosis virus (CPV) is unique within the Reoviridae family in having a turreted single-layer capsid contained within polyhedrin inclusion bodies, yet being fully capable of cell entry and endogenous RNA transcription. Biochemical data have shown that the amino-terminal 79 residues of the CPV turret protein (TP) is sufficient to bring CPV or engineered proteins into the polyhedrin matrix for micro-encapsulation. Here we report the three-dimensional structure of CPV at 3.88 A resolution using single-particle cryo-electron microscopy. Our map clearly shows the turns and deep grooves of alpha-helices, the strand separation in beta-sheets, and densities for loops and many bulky side chains; thus permitting atomic model-building effort from cryo-electron microscopy maps. We observed a helix-to-beta-hairpin conformational change between the two conformational states of the capsid shell protein in the region directly interacting with genomic RNA. We have also discovered a messenger RNA release hole coupled with the mRNA capping machinery unique to CPV. Furthermore, we have identified the polyhedrin-binding domain, a structure that has potential in nanobiotechnology applications.

Figure 1. Overall structure of the CPV capsid

a, Radially coloured, shaded surface view of the CPV reconstruction as viewed along a two-fold axis. b, An extracted asymmetric unit, colour coded by protein subunits, including one turret protein (TP) (green), two copies of the capsid shell proteins (CSP-A in blue and CSP-B in purple), and two copies of the large protrusion protein (LPP-5, near the five-fold axis, in yellow; LPP-3, near the three-fold axis, in brown). c, d, Views of density maps of one α-helix (c) and four β-strands (d) for CSP-B superimposed with the corresponding Cα model, showing the clear turn and deep groove of the α-helix with a pitch of ~5.8Å and the density for the bulky side chain of residue 520 in c, and the clear separation of β-strands in d (note also the densities for the bulky side chains of amino acids 341 and 343). e, Cα model of a β-strand of TP in the density map, showing zigzagging of the backbone and the clear densities for side chains. All the inter-consecutive Cα distances are ~3.8Å , which is a well established distance in polypeptide chains.

Figure 2. A conformational change between CSP-A and CSP-B: implication for packing and sliding of the dsRNA genome

a, Atomic model of CSP-A, coloured from blue at the N terminus to red at the C terminus. b, Close-up view of the CSP-A (blue) and CSP-B (purple) density map, showing that an α-helix (the upper helix within the green density) in CSP-A transforms into part of a β-hairpin in CSP-B (the upper part in the yellow density). c, Density maps with Cα models, showing the conformational change between CSP-A (left) and CSP-B (right). One α-helix in CSP-A transforms into part of the β-hairpin in CSP-B (indicated by curved arrow). The changing path is also indicated by an empty arrow. d, A 10Å slab extracted from the two-fold map showing the ordered dsRNA genome with a ~27Å distance between the adjacent dsRNA strands (arrow). The dotted box indicates the A-spike plug all the way in the central chamber of the turret.

Figure 3. Nascent mRNA release hole coupled with the GTase active site of TP in a way unique to CPV

a, Close-up view of the atomic model of CSP-A (two copies) and CSP-B. The mRNA release hole comprises loops contributed by the two copies of CSP-A (red and orange) and one CSP-B (cyan). b, c, Cα models of one CSP-B (cyan), two copies of CSP-A (red and orange) and two GTase domains (blue and light blue), showing the mRNA releasing and capping pathway, which is illustrated by green arrows in b (viewed from outside of the capsid) and c (viewed from inside of the capsid). d, Cα models of the GTase domains in a turret, showing that the active-site cleft (yellow stars) of GTase rotates away from the central chamber, facing sideways, thus coupled with the mRNA release hole (white circle).

Figure 4. Location and structure of the unique polyhedrin-binding domain (PBD) of TP

a, Shaded surface representation of TP viewed from inside, showing the two methylase domains (purple), GTase domain (blue), and CPV’s unique PBD (orange). The PBD is absent from other dsRNA viruses and consists of residues 1–79 of TP. b, Ribbon model of the CPV polyhedrin trimer, which is the building block of the crystalline polyhedra. c, View of one entire turret, showing the orientation and position of one TP (coloured) in the turret. d, Stereo view of a cryo-electron microscopy density map superimposed with the Cα model of GTase domain (green) and PBD (blue), which are connected together through a linker. e, Blown-up view of the model of PBD superimposed with its cryo-electron microscopy density.