The SV40 capsid is stabilized by a conserved pentapeptide hinge of the major capsid protein VP1

Abstract

The simian virus 40 (SV40) outer shell is composed of 72 pentamers of VP1. The core of the VP1 monomer is a beta-barrel with jelly-roll topology and extending N- and C-terminal arms. A pentapeptide hinge, KNPYP, tethers the C-arm to the VP1 beta-barrel core. The five C-arms that extend from each pentamer insert into the neighbouring pentamers, tying them together through different types of interactions. In the mature virion, this element adopts either of six conformations according to their location in the capsid. We found that the hinge is conserved among 16 members of the Polyomaviridae, attesting to its importance in capsid assembly and/or structure. We have used site-directed mutagenesis to gain an understanding into the structural requirements of this element: Y299 was changed to A, F, and T, and P300 to A and G. The mutants showed reduction in viability to varying degrees. Unexpectedly, assembly was reduced only to a small extent. However, the data showed that the mutants were highly unstable. The largest effect was observed for mutations of P300, indicating a role of the proline in the virion structure. P300G was more unstable than P300A, indicating a requirement for rigidity of the pentapeptide hinge. Y299T and Y299A were more defective in viability than Y299F, highlighting the importance of an aromatic ring at this position. Structural inspection showed that this aromatic ring contacts C-arms of neighbouring pentamers. Computational modelling predicted loss of stability of the Y mutants in concordance with the experimental results. This study provides insights into the structural details of the pentapeptide hinge that are responsible for capsid stability.

Fig. 1

Top: Architecture of the virion shell (reprinted from with permission from Elsevier). (a) Arrangement of the pentavalent (grey) and hexavalent (coloured) pentamers on the T=7d icosahedral lattice. (b) Three distinct types of interactions between pentamers. α monomers (grey) of pentavalent pentamers and monomers, α′ and α″ of hexavalent pentamers (coloured) form a three-helix contact. The hexavalent pentamers interact through two-helix contact of monomers β- β′ and γ-γ. Bottom: The different conformations of the pentapeptide hinge. (c) Superimposition of the pentapeptide hinge of monomers α (grey), α′ (blue), α″ (green), β (red) and β′ (turquoise). Y299 and P300 have distinct orientations in the different monomers. The pentapeptide of γ monomers is not included, as its high B-factor indicates that it is less ordered. (d,e) The two well-ordered conformation groups. Superimposition of monomers β, α′ and β′ shows stabilization by two hydrogen bonds (d), while monomers α and α″ contain only one or no hydrogen bond, respectively (e). Superimpositions were prepared using Swiss-PDB-Viewer.

Fig. 2

Instability of mutant capsids. Virions were harvested by di-detergent and centrifuged in CsCl density gradient. 250 microliter fractions were collected from the top. The refraction index of each fraction was measured and calculated for its density. Fractions were analyzed by western blot with anti-VP1 antibody. The arrow shows the fraction with density of 1.345 g/ml where full SV40 particles are expected.

Fig. 3

Stability analysis of the virions. Stability of mutants was measured as sensitivity to DNase I at 37°C (a) and at 0°C (b). Viral DNA amount was measured by RQ-PCR with primers covering the SV40 Poly A region. Results are presented as percentage of DNA at t=x relative to the DNA amount at the start point and are averages of several experimental repeats, as indicates in parentheses.

Fig. 4

Interactions of Y299. (a). Stacking interactions within the α monomer involving Y299 stacked between P298 and F303. The surface is coloured according to CPK (Grey – carbon, blue – nitrogen, red – oxygen atoms). (b). Y299 covers the hydrophobic patch formed by the three C-terminal α helices. Representation of the hydrophobic interaction interface between VP1 monomer α (grey) α′ (blue) and α″ (green) tying the pentavalent pentamer (grey) with its neighbouring hexavalent pentamers. The three C-terminal α helices are represented as spheres. The monomer core is represented as ribbon. The hinge is shown in white, Y299 in yellow, and P300 in red. (c) Y299 interaction with distal part of the C-arm. The helices of monomers β (red) and β′ (turquoise) form two-fold, tight hydrophobic contacts between hexavalent pentamers. Y299 (yellow spheres) of these monomers interact with M346 and T357 (blue and light-blue spheres) which arrive from distant monomers in other pentamers, contributing to the interpentameric interaction. All Figures were created using CHIMERA.

Fig. 5

Calculated effect of mutations on binding. The predicted ΔΔG values of the mutations Y299F, Y299T, Y299A for each of the five tested chains, α (grey), α′ (blue), α″ (green), β (red) and β′ (turquoise) are shown (in Rosetta Units – an energy decrease of >1–1.5kcal/mol indicates significantly impaired binding). The effect of Y299F is negligible, while truncation of the sidechain to T, and even more to A, results in a significant loss of binding energy.