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(2+) 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 -2.3 x 10(-4) mol ml g(-2) (weak protein-protein attraction) to -2.4 x 10(-3) mol ml g(-2) (strong protein attraction) for metastable and Ca(2+)-triggered self-assembling capsomeres, respectively. An assembly-deficient mutant (VP1CDelta63) 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.

Figure 1.

Measurement of capsomere second virial coefficient during capsid self-assembly by combined SEC and SLS analysis. (a) dRI chromatograms of purified VP1wt capsomeres injected into a Superdex 200 SEC column equilibrated with non-assembly (40 mM Tris (pH 7.2), 200 mM NaCl, 1 mM EDTA, 5 mM DTT, 5% glycerol) or assembly (40 mM Tris (pH 7.2), 200 mM NaCl, 100 µm CaCl₂, 5% glycerol) buffers. Molar mass (M) of the capsomere peak was measured with multi-angle SLS. Thin line, dRI (non-assembly); thick line, dRI (assembly); cross, M (non-assembly); circle, M (assembly). (b) Debye plot generated using 90° SLS and concentration data from several injections of purified capsomeres, each at a different volume (50, 100, 200 or 400 µl), into a column pre-equilibrated with non-assembly or assembly buffer. The plot gradient gives the second virial coefficient (B₂₂), which becomes more negative when the capsomeres are exchanged into the assembly buffer during analysis. See equations (2.1) and (2.2) in §2 for definitions of plot parameters used. Open circle, non-assembly; filled circle, assembly.

Figure 2.

Self-assembly of VP1wt capsomeres triggered by Ca²⁺. (a) dRI chromatograms of purified VP1wt capsomeres injected into a Superdex 200 SEC column equilibrated with Tris buffer (40 mM Tris (pH 7.2), 200 mM NaCl, 5% glycerol) containing 0–200 µM CaCl₂ or with L buffer (L, 40 mM Tris (pH 7.2), 200 mM NaCl, 1 mM EDTA, 5 mM DTT, 5% glycerol). (b) SEC peak fractions (i–v) were collected at the dRI detector outlet, incubated at 4°C for 48 h and analysed with transmission electron microscopy following negative staining. (c) B₂₂ of VP1wt capsomeres as a function of Ca²⁺ or Mg²⁺ concentration in Tris buffer at pH 8.0 or 7.2. Red line, Ca²⁺ pH 8.0; blue line, Ca²⁺ pH 7.2; green line, Mg²⁺ pH 7.2.

Figure 3.

Assembly reaction endproducts analysed with AF4. (a) Endproducts obtained by dialysing purified capsomeres (48 h, 25°C) against L buffer (pH 7.2; dashed line) or assembly buffer (40 mM Tris (pH 7.2), 200 mM NaCl, 5% glycerol, 50 µM CaCl₂; thick line). (b) Endproducts obtained by dialysing purified capsomeres (48 h, 25°C) against 40 mM Tris (pH 7.2 or 8.0), 200 mM NaCl, 5% glycerol, 200 µM CaCl₂. Thick line, UV absorbance (pH 7.2); dashed line, UV absorbance (pH 8.0); open circle, R_h (pH 7.2); inverted triangle, R_h (pH 8.0).

Figure 4.

Self-assembly of VP1wt capsomeres triggered by pH switching. (a) VP1wt B₂₂ in L buffer (40 mM Tris (pH 7.2–8.0) or bis–tris (pH 6.0–6.6), 200 mM NaCl, 5% glycerol, 1 mM EDTA and 5 mM DTT) as pH decreased from 8.0 to 6.0. (b) dRI chromatograms of purified VP1wt capsomeres injected into an SEC column equilibrated with L buffer (40 mM Tris (pH 7.2–8.0) or bis–tris (pH 6.0–6.6), 200 mM NaCl, 1 mM EDTA, 5 mM DTT, 5% glycerol) at pH 6.0–8.0. (c) TEM micrograph of assembly products from incubation (48 h, 4°C) of excluded-volume peak material collected from SEC in L buffer at pH 6.0.

Figure 5.

Dependence of VP1wt B₂₂ as a function of ammonium sulphate. VP1wt B₂₂ decreased in response to increase in (NH₄)₂SO₄ concentration in Tris buffer (40 mM Tris (pH 7.2), 200 mM NaCl, 5% glycerol). Inset shows the assembly products from incubation (48 h, 4°C) of excluded-volume peak materials collected from SEC in Tris buffer with 200 mM (NH₄)₂SO₄.

Figure 6.

The second virial coefficient of an assembly deficient mutant (VP1CΔ63) is invariant across conditions that trigger assembly of VP1wt capsomere. (a) Ribbon diagram of a VP1wt capsomere (blue) depicting the 63 C-terminal residues truncated in the mutant (red) and how the invading arm interacts with a VP1 molecule from a neighbouring capsomere (green). The image is generated with Rasmol (Sayle & Milnerwhite 1995) using PDB code ‘1SID’. (b) dRI chromatograms of purified VP1wt (blue line) and VP1CΔ63 (red line) capsomeres injected into a Superdex 200 column equilibrated with L buffer (L, 40 mM Tris (pH 7.2), 200 mM NaCl, 1 mM EDTA, 5 mM DTT, 5% glycerol). Molar mass (M) of the capsomere peak was measured with multi-angle SLS. Cross, M (VP1wt); circle, M (VP1CΔ63). (c) B₂₂ of VP1CΔ63 as a function of Ca²⁺ concentration in Tris buffer (40 mM Tris (pH 7.2), 200 mM NaCl, 5% glycerol) and (d) as a function of pH in L buffer (40 mM Tris (pH 7.2–8.0) or bis–tris (pH 6.0–6.6), 200 mM NaCl, 5% glycerol, 1 mM EDTA and 5 mM DTT).