Virus assembly occurs following a pH- or Ca²⁺-triggered switch in the thermodynamic attraction between structural protein capsomeres

Abstract

Viral self-assembly is of tremendous virological and biomedical importance. Although theoretical and crystallographic considerations suggest that controlled conformational change is a fundamental regulatory mechanism in viral assembly, direct proof that switching alters the thermodynamic attraction of self-assembling components has not been provided. Using the VP1 protein of polyomavirus, we report a new method to quantitatively measure molecular interactions under conditions of rapid protein self-assembly. We show, for the first time, that triggering virus capsid assembly through biologically relevant changes in Ca 2þ concentration, or pH, is associated with a dramatic increase in the strength of protein molecular attraction as quantified by the second virial coefficient (B 22 ). B 22 decreases from 22.3 Â 10 24 mol ml g 22(weak protein -protein attraction) to 22.4 Â 10 23 mol ml g 22 (strong protein attraction) for metastable and Ca 2þ -triggered self-assembling capsomeres, respectively. An assemblydeficient mutant (VP1CD63) is conversely characterized by weak protein -protein repulsion independently of chemical change sufficient to cause VP1 assembly. Concomitant switching of both VP1 assembly and thermodynamic attraction was also achieved by in vitro changes in ammonium sulphate concentration, consistent with protein salting-out behaviour. The methods and findings reported here provide new insight into viral assembly, potentially facilitating the development of new antivirals and vaccines, and will open the way to a more fundamental physico-chemical description of complex protein self-assembly systems.

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

“…Assembly Buffer 1 in this chapter were sufficient to maintain the value of B 22 at lower than -20 x 10 -4 mol ml g -2 . This value is considerably lower than -8 x 10 -4 mol ml g -2 , the limit of B 22 below which protein-protein interactions are classified as a strong attraction (Chuan et al 2010, Curtis et al 1998, Curtis et al 2002a, George and Wilson 1994 (Chuan et al 2010). This value is higher than the limit of B 22 for a strong attraction, which suggests that the pH was not sufficient to promote a strong attraction.…”

“…(i) the presence of Ca 2+ ions, (ii) increased ionic strength, and (iii) the absence of reduction agents , Chuan et al 2010, Salunke et al 1989). The formed VLPs are then analysed using asymmetric flow-field flow fractionation (AF4) coupled with multi-angle light scattering (MALS), and transmission electron microscopy (TEM) ).…”

“…was at approximately 12.5 ml (Chuan et al 2010). In SEC, proteins are separated according to their hydrodynamic radius.…”

“…Highly soluble GST-tagged protein is then purified using GST-affinity chromatography . Following tag removal, capsomeres are separated from aggregates and cleaved GST-tag using size-exclusion chromatography (SEC).Purified capsomeres spontaneously self-assemble in vitro under the following conditions:(i) the presence of Ca 2+ ions, (ii) increased ionic strength, and (iii) the absence of reduction agents , Chuan et al 2010, Salunke et al 1989). The formed VLPs are then analysed using asymmetric flow-field flow fractionation (AF4) coupled with multi-angle light scattering (MALS), and transmission electron microscopy (TEM) ).…”

Murine polyomavirus VLPs as a platform for cytotoxic T cell epitope-based influenza vaccine candidates

“…The purified capsomeres may then be directed to self-assemble under defined conditions into VLPs and either platform (capsomeres and VLPs) may be used to develop vaccine designs (Chuan et al 2010). Multiple matrix 2 ectodomain (M2e) epitopes have been inserted to the VP1 protein to improve immunogenicity, and adjuvanted capsomeres presenting the M2e epitopes at the external loop of the protein were able to stimulate humoral immunity in mice .…”