Diversity in Qβ Virus-like Particle Cage Assembly via Coat Protein Monomers and AYGG-linked Dimers

The single-stranded RNA (ssRNA) bacteriophage Qβ is a virion comprised of coat proteins (CPs) that can assemble to form virus-like particle (VLP) cage structures by the generation of pentamers and hexamers. Previously, Qβ VLPs have been shown to form geometrically distinct cages with varying numbers of CPs. In particular, capsid symmetries that have been seen include T4, T3, D3 (small prolate), D5 (oblate), and D5-elongated (D5e, or prolate) [1]. The monomeric asymmetric CP subunit contains 132 amino acids with a notable α-helical structure connecting two β-sheets. When two monomeric units associate, the α-helices come together to provide spatial stabilization. Here, we present the discovery of three additional cage assemblies formed from CP monomers that have not been described before. Specifically, high resolution cryoEM maps of C3, T (tetrahedral), and C2 geometries have been resolved. Previously, we demonstrated that CP-dimer fusion constructs for a related PP7 bacteriophage promote formation of larger assemblies [2]. We set to test whether a similar CP-fusion will have the effect on structures formed by Qβ. Here, the same AYGG linker was used to covalently connect two CP monomers to generate a CP dimer (Fig. 1). Single-particle analysis of the cages formed by these dimers revealed a completely different structural profile of capsid formation. The dimeric CP Qβ VLP structures revealed T, D3 (pseudo D6 barrel), and D5 (pseudo-icosahedral) symmetries. At the same time, all other monomeric cage symmetries were no longer present. We used cryoSPARC processing suite to analyze cryoEM data [3]. Exhaustive 2D and 3D classification were performed on the selected particles to generate subsets of particles with different numbers of CPs and symmetries. In short, the particles were first picked using A generic blob picker, extracted from micrographs, and analyzed by 2D classification to remove “junk classes.” Ab-initio refinement with multiple classes, followed by homogenous refinement, was run for the particle lists to resolve low-resolution maps and to obtain initial insight about the variety of structural assemblies. 3D classification of the initial models provided cleaner subsets of particles and were used for homogenous refinement with symmetries applied. The cryoEM map resolutions and percent abundances for all geometries are represented in Table 1 (monomeric Qβ VLPs) and Table 2 (dimeric Qβ CP-fusion VLPs). An overview of all Qβ VLP cages overlayed with the respective maps appear in Figure 2 and 3. Structure of Qβ CP-dimer fusion with two monomeric CPs connected by an AYGG linker. Map resolutions, particle totals, and percentage of different Qβ VLP cages formed by monomeric CPs. Map resolutions, particle totals, and percentage of different Qβ VLP cages formed by dimeric CP-fusion CPs. CryoEM maps of Qβ VLPs assembled from monomeric CPs. (A) T4 symmetry; (B) T3 symmetry; (C) D3 symmetry; (D) D5 symmetry; (E) D5e symmetry; (F) C3 symmetry; (G) T symmetry; (H) C2 symmetry. CryoEM maps of Qβ VLPs assembled from dimeric CP-fusions. (A) T symmetry; (B) D3 symmetry; (C) D5 symmetry. Structural overlay of (A) hexamers and (B) pentamers from four Qβ VLPs (D3, C3, T, C2) assembled from monomeric CPs showing highly flexible nature of hexamers and the more rigid conformations of pentamers. It has been shown previously that pentamers in Qβ VLP cages are rigid, while the hexamers are more flexible and can bend to accommodate different geometries of the cages [1]. Here, we analyzed the D3-barrel and the newly discovered T, C2, and C3 monomeric CP assemblies, revealing that the hexamers do exhibit a range of flexibilities amongst the cage geometries (Fig. 4A). Consistent with the previous reports, pentamers remain primarily static, with slight variations in plasticity for these capsids (Fig 4B). While the structures of the Qβ VLP assemblies assists in the understanding of cage formation, one key question remains – why does the presence of a flexible linker alter the assembly profile for these cages? We speculate that the ability of a dimer to dissociate could play a role in cage assemblies. If the assembly is happening co-translationally, then the CP-fusion dimer being twice the size of the monomer may hinder the CPs assembly of some geometries, while promoting formation of others. Interestingly, the only overlapping assembly between CP and CP-dimer constructs is tetrahedral, which is apparently not affected by the CP-fusion.