Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1

In this study, we have analyzed the morphogenesis of the birnavirus infectious pancreatic necrosis virus throughout the infective cycle in CHSE-214 cells by using a native agarose electrophoresis system. Two types of viral particles (designated A and B) were identified, isolated, and characterized both molecularly and biologically. Together, our results are consistent with a model of morphogenesis in which the genomic doublestranded RNA is immediately assembled, after synthesis, into a large (66-nm diameter) and uninfectious particle A, where the capsid is composed of both mature and immature viral polypeptides. Upon maturation, particles A yield particles B through the proteolytic cleavage of most of the remaining viral precursors within the capsid, the compaction of the particle (60-nm diameter), and the acquisition of infectivity. These studies will provide the foundation for further analyses of birnavirus particle assembly and RNA replication.

Section: Discussionmentioning

confidence: 88%

Genome Assembly and Particle Maturation of the Birnavirus Infectious Pancreatic Necrosis Virus

Villanueva

Galaz

Valdés

et al. 2004

“…Primary bursa cells were isolated from 14-day-old SPF embryos and were maintained in Eagle's modified minimal essential medium (EMEM) supplemented with 15% FCS, 0.125% lactoalbumin hydrolysate (Oxoid), 1,000 units of penicillin/ml (Yamanouchi), and 1 mg of streptomycin (Radiumfarma)/ml. Polyclonal rabbit antiserum against VP1 was produced by injecting rabbits with purified recombinant VP1 (30). A polyclonal rabbit serum against VP3 and VP4 of IBDV was produced as follows.…”

Section: Methodsmentioning

confidence: 99%

Rescue of Very Virulent and Mosaic Infectious Bursal Disease Virus from Cloned cDNA: VP2 Is Not the Sole Determinant of the Very Virulent Phenotype

Boot¹,

Huurne²,

Hoekman³

et al. 2000

Self Cite

114

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A-or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.

“…The cleavage sites for the proteolytic processing of the polyprotein and pVP2 have been characterized (9,36), allowing faithful expression of the capsid polypeptides. The virus RdRp, VP1, interacts with VP3, forming a complex that facilitates VP1 encapsidation (23,38). The VP3 domain responsible for this interaction is located at the 16 C-terminal residues of the protein (25).…”

mentioning

confidence: 99%

The Oligomerization Domain of VP3, the Scaffolding Protein of Infectious Bursal Disease Virus, Plays a Critical Role in Capsid Assembly

Maraver¹,

Oña²,

Abaitua³

et al. 2003

Infectious bursal disease virus (IBDV) capsids are formed by a single protein layer containing three polypeptides, pVP2, VP2, and VP3. Here, we show that the VP3 protein synthesized in insect cells, either after expression of the complete polyprotein or from a VP3 gene construct, is proteolytically degraded, leading to the accumulation of product lacking the 13 C-terminal residues. This finding led to identification of the VP3 oligomerization domain within a 24-amino-acid stretch near the C-terminal end of the polypeptide, partially overlapping the VP1 binding domain. Inactivation of the VP3 oligomerization domain, by either proteolysis or deletion of the polyprotein gene, abolishes viruslike particle formation. Formation of VP3-VP1 complexes in cells infected with a dual recombinant baculovirus simultaneously expressing the polyprotein and VP1 prevented VP3 proteolysis and led to efficient virus-like particle formation in insect cells.