Autoproteolytic Activity Derived from the Infectious Bursal Disease Virus Capsid Protein

doi:10.1074/jbc.M808942200

Autoproteolytic Activity Derived from the Infectious Bursal Disease Virus Capsid Protein^*

Departments of ^{^‡}Molecular and Cellular Biology and ^{^¶}Structure of Macromolecules, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas (CSIC), Cantoblanco, 28049 Madrid, Spain and ^{^§}Institut de Biologia Molecular de Barcelona/CSIC, Parc Científic de Barcelona, Josep Samitier 1-5, 08028-Barcelona, Spain

3 To whom correspondence should be addressed: Dpto. de Estructura de Macromoléculas, Centro Nacional de Biotecnología/CSIC, C/Darwin no. 3, Cantoblanco, 28049-Madrid, Spain. Tel.: 34-91-5854971; Fax: 34-91-5854506; E-mail: jrcaston{at}cnb.csic.es.

Abstract

Viral capsids are envisioned as vehicles to deliver the viral genome to the host cell. They are nonetheless dynamic protective shells, as they participate in numerous processes of the virus cycle such as assembly, genome packaging, binding to receptors, and uncoating among others. In so doing, they undergo large scale conformational changes. Capsid proteins with essential enzymatic activities are being described more frequently. Here we show that the precursor (pVP2) of the capsid protein VP2 of the infectious bursal disease virus (IBDV), an avian double-stranded RNA virus, has autoproteolytic activity. The pVP2 C-terminal region is first processed by the viral protease VP4. VP2 Asp-431, lying in a flexible loop preceding the C-terminal most α-helix, is responsible for the endopeptidase activity that cleaves the Ala-441—Phe-442 bond to generate the mature VP2 polypeptide. The D431N substitution abrogates the endopeptidase activity without introducing a significant conformational change, as deduced from the three-dimensional structure of the mutant protein at 3.1 Å resolution. Combinations of VP2 polypeptides containing mutations affecting either the cleavage or the catalytic site revealed that pVP2 proteolytic processing is the result of a monomolecular cis-cleavage reaction. The D431N mutation does not affect the assembly of the VP2 trimers that constitute the capsid building block. Although VP2 D431N trimers are capable of assembling both pentamers and hexamers, expression of a polyprotein gene harboring the D431N mutation does not result in the assembly of IBDV virus-like particles. Reverse genetics analyses demonstrate that pVP2 self-processing is essential for the assembly of an infectious IBDV progeny.

Footnotes

↵⁴ The abbreviations used are: dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; IBDV, infectious bursal disease virus; IPNV, infectious pancreatic necrosis virus; CP, capsid protein; rBV, recombinant baculovirus; rVV, recombinant vaccinia virus; SVP, subviral particle; VLP, virus-like particle; DMEM, Dulbecco's modified Eagle's medium; pi, postinfection.
The atomic coordinates and structure factors (code 3FBM) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
↵^* This work was supported by Spanish Dirección General de Investigación (MEC) Grants BIO2006-09407, BFU2005-02376, and BFU2005-06487. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2.
↵¹ Supported by “Residencia de Estudiantes” and Gobierno de Aragón and by an Formación de Personal Universitario fellowship from the Spanish Ministry of Education (MEC).
↵² Recipient of an I3P fellowship from Consejo Superior de Investigaciones Científicas.
- Received November 25, 2008.
- Revision received December 19, 2008.
The American Society for Biochemistry and Molecular Biology, Inc.