Estudio del papel funcional de la proteína estructural VP3 en el proceso morfogenético
Author
Clemente Cervera, RobertoEntity
UAM. Departamento de Biología Molecular; CSIC. Centro Nacional de Biotecnología (CNB)Date
2004-11-29Subjects
Proteínas virales-Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-11-2004Abstract
Infectious bursal disease virus (IBDV) is the prototype member of the Avibimavincs
genus of the Birnaviridae family, whose genome is a bipartite double suanded RNA.
IBDV virions are non-enveloped icosahedra (T=13) with a diameter of 65-70 nm. IBDV is
an important avian pathogen, causing a severe immunosuppressive disease and accounting
for important economic losses in the poultry industry world-wide.
The initial aim of the work presented here was double: i) analysis of the structural
VP3 polypeptide during viral morphogenesis; and ii) generation of new tools to further
characterize the IBDV capsid sbucture.
We fiist established the molecular bases that were precluding the assembly of IBDV
vims-like particles (VLPs) after expression of the virus-encoded polyprotein in insect ceiis.
The results of this study showed that the morphogenetic blockade is due to the proteolytic
trimming of the VP3 polypeptide rendering a polypeptide that lacks the 13 C-terminal
amino acid residues. Further analyses allowed us to identify the VP3 oligomenzation
domain. This domain spans residues 224-247, thus overlapping the previously described
VPI-binding domain. The above mentioned features lead to the design of a strategy, based
on the co-expression of the IBDV polyprotein and VP1 polypeptides, that prevents VP3
proteolysis and allows the production of IBDV VLPs in insect cells.
Series of expression vectors designed to produce chimerical VLPs were generated and
tested. A recombinant vaccinia vims, expressing a chimerical protein fonned by the fusion
of the green fluorescent protein (EGFP) to the polyprotein C-terminal end was selected for
further analyses (Poly-EGFP). Poly-EGFP expression in avian cells leads to efficient
assembly of chimerical VLPs (EGFP-VLPs) containing VP3-EGFP fusion polypeptide.
The analysis of EGFP-VLPs by electron cryomicroscopy and image processing allowed us
to generate a 15 A resolution 3D map. The comparison of this map with the 3D
reconstmction of IBDV virions indicated that the icosahedral capsid is built up by VP2,
and VP3 is trapped at the interior volume together with the genome.
Finally, a transducer recombinant baculovirus vector expressing the Poly-EGFP
chimerical gene was generated for analysis in vivo of the IBDV morphogenetic process.
From these analysis, we concluded that IBDV-derived tubular smctures, known as type 1
tubules, are not involved in the morphogenetic process but they constitute aberrant
assemblies. Preliminary results suggest that transducer recombinant baculovirus vector
might be applicable for generation of divalent avian vaccines.
Files in this item
Google Scholar:Clemente Cervera, Roberto
This item appears in the following Collection(s)
Related items
Showing items related by title, author, creator and subject.
-
Estudio de la función y localización de la proteína no estructural VP5 del virus de la bursitis infecciosa
Méndez Hernández, Fernando
2018-07-20 -
Estudio del papel de la vía de señalización de Hedgehog en la determinación del patrón morfogenético del ala de Drosophila
Mullor Sanjosé, José Luís
1999-02-12