Engineered Mutations Change the Structure and Stability of a Virus-Like Particle

Abstract

The single-coat protein (CP) of bacteriophage Qβ self-assembles into T = 3 icosahedral virus-like particles (VLPs), of interest for a wide range of applications. These VLPs are very stable, but identification of the specific molecular determinants of this stability is lacking. To investigate these determinants along with manipulations that confer more capabilities to our VLP material, we manipulated the CP primary structure to test the importance of various putative stabilizing interactions. Optimization of a procedure to incorporate fused CP subunits allowed for good control over the average number of covalent dimers in each VLP. We confirmed that the disulfide linkages are the most important stabilizing elements for the capsid and that acidic conditions significantly enhance the resistance of VLPs to thermal degradation. Interdimer interactions were found to be less important for VLP assembly than intradimer interactions. Finally, a single point mutation in the CP resulted in a population of smaller VLPs in three distinct structural forms.

Monitoring the Disassembly of Virus-like Particles by 19F-NMR

“…Pleasingly, Qβ-CS still formed discrete particles, even without disulfide bonds at C74 and C80 (Figure S2), consistent with prior studies that have identified the dominant contribution of noncovalent interactions between viral subunits. 34 The relative robustness of these Qβ-CS VLPs was probed with 19 F-NMR: in contrast to Qβ-F, Qβ-CS VLPs disassembled directly into monomers under denaturing conditions when treated only with SDS (Figure 2c), consistent with our intended design. The chemical shifts of Qβ-CS VLPs in the absence and presence of SDS were highly similar (−40.63 ppm, FWHH = ∼200 Hz; −40.88 ppm, FWHH = ∼6 Hz, respectively) to those observed for Qβ (Figure 2b).…”

Engineering virus-like particles as vaccine platforms

“…One common technique to improve particle thermostability is the introduction of intersubunit disulfide bonds. For instance, mutations that introduce cysteines at the 5-fold symmetry axes increase the stability of MS2 VLPs [34,35]. Similarly, Hepatitis B core antigen VLPs with enhanced stability were produced by identifying amino acid pairs of interacting dimers with the shortest distances between side chains and substituting cysteines that could form disulfide bonds, creating a disulfide-bond network stabilizing the particle [36].…”

Identification of the Structural Basis of Thermal Lability of a Virus Provides a Rationale for Improved Vaccines

“…In contrast, very few attempts aimed at the rational thermostabilization of FMD virions (Mateo et al, 2008) or capsids (Porta et al, 2013b), other virus particles (Ashcroft et al, 2005;Fiedler et al, 2012) or supramolecular assemblies, have been published so far. This situation severely impairs the establishment of structure-based protein engineering guidelines for developing robust virus particles and protein assemblies for different applications (Mateu, 2011).…”