Abstract
The simian virus 40 (SV40) outer shell is composed of 72 pentamers of VP1. The core of the VP1 monomer is a β-barrel with jelly-roll topology and extending N- and C-terminal arms. A pentapeptide hinge, KNPYP, tethers the C-arm to the VP1 β-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.
| Original language | English |
|---|---|
| Pages (from-to) | 1382-1391 |
| Number of pages | 10 |
| Journal | Journal of Molecular Biology |
| Volume | 386 |
| Issue number | 5 |
| DOIs | |
| State | Published - 13 Mar 2009 |
Bibliographical note
Funding Information:Prof. Amos Oppenheim contributed ideas and know-how through helpful discussions and guided and assisted with the recombineering procedure. We thank Dr. Gali Prag for helpful discussions. Seth Salpeter assisted in the construction and preparation of the pUC-SV40 and P300 mutants. Profs. Steve Harrison and Thilo Stehle kindly gave us permission to use a figure from their publication. We thank an anonymous reviewer for most helpful comments. This work was supported in part by U.S. Public Health Service grant CA100479 and by the US–Israel Binational Science Foundation grant 2005050. Molecular graphics images were produced using the University of California, San Francisco (UCSF) Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the UCSF (supported by National Institutes of Health P41 RR-01081).
Keywords
- SV40
- computational binding prediction
- major capsid protein VP1
- protein structure-function
- site-directed mutagenesis