C Terminus of Infectious Bursal Disease Virus Major Capsid Protein VP2 Is Involved in Definition of the T Number for Capsid Assembly

J. R. Caston, J. L. Martinez-Torrecuadrada, A. Maraver, E. Lombardo, J. F. Rodriguez, J. I. Casal, J. L. Carrascosa
2001 Journal of Virology  
Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T‫31؍‬ markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up
more » ... VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T‫1؍‬ shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T‫1؍‬ capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis. Infectious bursal disease virus (IBDV) is the prototype member of the Avibirnavirus genus in the Birnaviridae family (44) and an important pathogen of chickens, accounting for important economic losses in the poultry industry worldwide and representing a major hazard for several species of wild birds (27). During the past decade there have been outbreaks of highly virulent strains against which classical vaccines were not protective (61, 77). Improvement in the control of the disease will be obtained only through further understanding of IBDV molecular biology, including viral structure. The IBDV genome is formed by two double-stranded RNA (dsRNA) segments of 3.2 kb (segment A) and 2.8 kb (segment B) (35, 38). Segment A contains two partially overlapping open reading frames (ORFs). The first ORF encodes the nonstructural VP5 protein (17 kDa), whose functional properties are not yet clear, although it is important in virus release and dissemination (49, 60). The second ORF codes for a 110-kDa polyprotein that is autoproteolytically cleaved, yielding three proteins: VPX (ϳ48 kDa), VP3 (32 kDa), and VP4 (28 kDa) (Fig. 1A) . A major proportion of VPX (also designated pVP2) is further proteolytically processed to VP2 (41 kDa) (48, 59). Biochemical analysis of purified capsids from virions revealed that VP2 and VP3 are the major structural proteins in the mature virion (18), while VP4, a serine-lysine protease (7, 43), is involved in the proteolytic maturation of the polyprotein (22). Segment B contains an ORF that encodes VP1 (95 kDa), which is assumed to be the RNA-dependent RNA polymerase, responsible for the reactions of transcription (plus-strand or mRNA synthesis) and replication (minus-strand synthesis) (58, 73). IBDV is a nonenveloped virus that differs from most dsRNA viruses in having a single shell. By cryoelectron microscopy and computer image reconstruction methods Böttcher et al. (8) have shown that the capsid (maximum diameter, 700 Å) is an icosahedron with 780 subunits, clustered as 260 outer trimers, arranged with a triangulation number of Tϭ13. However, the correlation of the structural features of the capsid and the structural proteins of the virus is poorly understood. In capsids with TϾ1, the protein subunits are able to adopt several different conformations depending on their different structural environments in the shell; that is, the bonding properties of subunits are not identical, and several classes of conformers exist (11). High-resolution structural studies have revealed some clues about how a capsid protein is able to know the conformation that it must adopt (see, e.g., references 5, 47, and 69), although the mechanism is still poorly understood (37). These differences are quite subtle and may be controlled by flexible regions in the protein (loops, N ends, and C ends), duplex or single-stranded RNA, metal ions, or some combination of these (36). These factors are referred to as molecular switches. Additionally, capsids with large T numbers can be
doi:10.1128/jvi.75.22.10815-10828.2001 pmid:11602723 pmcid:PMC114663 fatcat:necf7dq2vzha7jpcuoa3pc3bqi