Biomedical subjects

José R Castón

Publications and source records attributed to José R Castón.

10 recordsLinked to original sources

PubMed2006

Three-dimensional structure and stoichiometry of Helmintosporium victoriae190S totivirus.

Most double-stranded RNA viruses have a characteristic capsid consisting of 60 asymmetric coat protein dimers in a so-called T = 2 organization, a feature probably related to their unique life cycle. These capsids organize the replicative complex(es) that is actively involved in genome transcription and replication. Available structural data indicate that their RNA-dependent RNA polymerase (RDRP) is packaged as an integral capsid component, either as a replicative complex at the pentameric vertex (as in reovirus capsids) or as a fusion protein with the coat protein (as in some totivirus). In contrast with members of the family Reoviridae, there are two well-established capsid arrangements for dsRNA fungal viruses, exemplified by the totiviruses L-A and UmV and the chrysovirus PcV. Whereas L-A and UmV have a canonical T = 2 capsid, the PcV capsid is based on a T = 1 lattice composed of 60 capsid proteins. We used cryo-electron microscopy combined with three-dimensional reconstruction techniques and hydrodynamic analysis to determine the structure at 13.8 A resolution of Helminthosporium victoriae 190S virus (Hv190SV), a totivirus isolated from a filamentous fungus. The Hv190SV capsid has a smooth surface and is based on a T = 2 lattice with 60 equivalent dimers. Unlike the RDRP of some other totiviruses, which are expressed as a capsid protein-RDRP fusion protein, the Hv190SV RDRP is incorporated into the capsid as a separate, nonfused protein, free or non-covalently associated to the capsid interior.

Capsid↗

PubMed2006

The 2.6-Angstrom structure of infectious bursal disease virus-derived T=1 particles reveals new stabilizing elements of the virus capsid.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 A resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.

Capsid↗

PubMed2005

Maturation of phage T7 involves structural modification of both shell and inner core components.

The double-stranded DNA bacteriophages are good model systems to understand basic biological processes such as the macromolecular interactions that take place during the virus assembly and maturation, or the behavior of molecular motors that function during the DNA packaging process. Using cryoelectron microscopy and single-particle methodology, we have determined the structures of two phage T7 assemblies produced during its morphogenetic process, the DNA-free prohead and the mature virion. The first structure reveals a complex assembly in the interior of the capsid, which involves the scaffolding, and the core complex, which plays an important role in DNA packaging and is located in one of the phage vertices. The reconstruction of the mature virion reveals important changes in the shell, now much larger and thinner, the disappearance of the scaffolding structure, and important rearrangements of the core complex, which now protrudes the shell and interacts with the tail. Some of these changes must originate by the pressure exerted by the DNA in the interior of the head.

Bacteriophage T7↗

PubMed2005

Preliminary analysis of two and three dimensional crystals of vault ribonucleoprotein particles.

Vaults are large ribonucleoprotein particles found in a wide variety of eukaryotes. When imaged by electron-microscopy vaults present a strikingly conserved barrel-shaped structure with an invaginated waist and two protruding caps. In this work, we present two dimensional (2D) and three dimensional (3D) crystals of naturally produced vaults in murine and monkey cells, respectively. The 2D-crystals presented a hexagonal packing with the lattice parameter defined by the diameter of the vault barrel. Fourier transforms from images of the negatively stained 2D-crystals showed spots till about 45 A resolution. The 3D-crystals reached about 0.15 x 0.15 x 0.02 mm3 in size and presented a flat triangular morphology with well-developed faces. The preliminary characterization of these 3D-crystals, which diffract very weakly to approximately 10 A resolution, suggests a trigonal packing with the R32 space group symmetry. The 3D-crystals appear to be formed by adding layers of vaults, which retain the hexagonal organization seen in the 2D-crystals, with relative shifts that maximize the interdigitation of particles in adjacent layers. Accurate crystal symmetry in the 2D- and 3D-crystals requires neighbor particles interacting according to a 6-fold and a 3-fold dihedral symmetry, respectively. Compatibility with the reported 8-fold symmetry would imply multiples of 24-fold rotational symmetry, in agreement with the recently proposed 48-fold dihedral symmetry for reconstituted recombinant vaults.

Animals↗

PubMed2005

Structural polymorphism of the major capsid protein of a double-stranded RNA virus: an amphipathic alpha helix as a molecular switch.

The infectious bursal disease virus T=13 viral particle is composed of two major proteins, VP2 and VP3. Here, we show that the molecular basis of the conformational flexibility of the major capsid protein precursor, pVP2, is an amphipatic alpha helix formed by the sequence GFKDIIRAIR. VP2 containing this alpha helix is able to assemble into the T=13 capsid only when expressed as a chimeric protein with an N-terminal His tag. An amphiphilic alpha helix, which acts as a conformational switch, is thus responsible for the inherent structural polymorphism of VP2. The His tag mimics the VP3 C-terminal region closely and acts as a molecular triggering factor. Using cryo-electron microscopy difference imaging, both polypeptide elements were detected on the capsid inner surface. We propose that electrostatic interactions between these two morphogenic elements are transmitted to VP2 to acquire the competent conformations for capsid assembly.

Amino Acid Sequence↗

PubMed2004

The coat protein of Rabbit hemorrhagic disease virus contains a molecular switch at the N-terminal region facing the inner surface of the capsid.

To function adequately, many if not all proteins involved in macromolecular assemblies show conformational polymorphism as an intrinsic feature. This general strategy has been described for many essential cellular processes. Here we describe this structural polymorphism in a viral protein, the coat protein of Rabbit hemorrhagic disease virus (RHDV), which is required during virus capsid assembly. By combining genetic, structure modeling, and cryo-electron microscopy and image processing analysis, we have established the mechanism that allows RHDV coat protein to switch among quasi-equivalent conformational states to achieve the appropriate curvature for the formation of a closed shell. The RHDV capsid structure is based on a T = 3 lattice, containing 180 copies of identical subunits, similar to those of other caliciviruses. The quasi-equivalent interactions between the coat proteins are achieved by the N-terminal region of a subset of subunits, which faces the inner surface of the capsid shell. Mutant coat protein lacking this N-terminal sequence assembles into T = 1 capsids. Our results suggest that the polymorphism of the RHDV T = 3 capsid might bear resemblance to that of plant virus T = 3 capsids.

Amino Acid Sequence↗

PubMed2004

The C-terminal domain of the pVP2 precursor is essential for the interaction between VP2 and VP3, the capsid polypeptides of infectious bursal disease virus.

The interaction between the infectious bursal disease virus (IBDV) capsid proteins VP2 and VP3 has been analyzed in vivo using baculovirus expression vectors. Data presented here demonstrate that the 71-amino acid C-terminal-specific domain of pVP2, the VP2 precursor, is essential for the establishment of the VP2-VP3 interaction. Additionally, we show that coexpression of the pVP2 and VP3 polypeptides from independent genes results in the assembly of virus-like particles (VLPs). This observation demonstrates that these two polypeptides contain the minimal information required for capsid assembly, and that this process does not require the presence of the precursor polyprotein.

Baculoviridae↗

PubMed2003

Three-dimensional structure of penicillium chrysogenum virus: a double-stranded RNA virus with a genuine T=1 capsid.

Although double-stranded (ds) RNA viruses are a rather diverse group, they share general architectural principles and numerous functional features. All dsRNA viruses, from the mammalian reoviruses to the bacteriophage phi6, including fungal viruses, share a specialized capsid involved in transcription and replication of the dsRNA genome, and release of the viral plus strand RNA. This ubiquitous capsid consists of 120 protein subunits in a so-called T=2 organization. The stringent requirements of dsRNA metabolism may explain the similarities observed in capsid architecture among a broad spectrum of dsRNA viruses. We have used cryo-electron microscopy combined with three-dimensional reconstruction techniques and complementary biophysical techniques, to determine the structure at 26A resolution of the Penicillium chrysogenum virus (PcV) capsid. In contrast to all previous studies of dsRNA viruses, PcV capsid is an authentic T=1 capsid with 60 equivalent protein subunits. This T=1 capsid is built with the largest structural protein (110 kDa). Structural comparison between viral particles and capsids devoid of RNA show changes along the inner surface of the capsid, mostly located around the icosahedral 5 and 3-fold axis. Considering that there may be numerous interactions between the inner surface of the protein shell and the underlying RNA, the genome could have an important role in the conformation of the structural subunits. The empty capsid structure suggests a mechanism for transcript release from actively transcribing particles. Furthermore, sequence analysis of the PcV coat protein revealed that both halves of the protein share numerous regions of similar amino acid residues. These results open new perspectives when considering the structural organization of dsRNA virus capsids.

Amino Acid Sequence↗

PubMed2003

Structure-dependent efficacy of infectious bursal disease virus (IBDV) recombinant vaccines.

The immunogenicity and protective capability of several baculovirus-expressed infectious bursal disease virus (IBDV)-derived assemblies as VP2 capsids, VPX tubules and polyprotein (PP)-derived mixed structures, were tested. Four-week-old chickens were immunised subcutaneously with one dose of each particulate antigen. VP2 icosahedral capsids induced the highest neutralising response, followed by PP-derived structures and then VPX tubules. All vaccinated animals were protected when challenged with a very virulent IBDV (vvIBDV) isolate, however the degree of protection is directly correlated with the levels of neutralising antibodies. VP2 capsids elicited stronger protective immunity than tubular structures and 3 micrograms of them were sufficient to confer a total protection comparable to that induced by an inactivated vaccine. Therefore, VP2 capsids represent a suitable candidate recombinant vaccine instead of virus-like particles (VLPs) for IBDV infections. Our results also provide clear evidence that the recombinant IBDV-derived antigens are structure-dependent in order to be efficient as vaccine components.

Animals↗

PubMed2003

Structure-dependent efficacy of infectious bursal disease virus (IBDV) recombinant vaccines.

The immunogenicity and protective capability of several baculovirus-expressed infectious bursal disease virus (IBDV)-derived assemblies as VP2 capsids, VPX tubules and polyprotein (PP)-derived mixed structures, were tested. Four-week-old chickens were immunised subcutaneously with one dose of each particulate antigen. VP2 icosahedral capsids induced the highest neutralising response, followed by PP-derived structures and then VPX tubules. All vaccinated animals were protected when challenged with a very virulent IBDV (vvIBDV) isolate, however the degree of protection is directly correlated with the levels of neutralising antibodies. VP2 capsids elicited stronger protective immunity than tubular structures and 3& mgr;g of them were sufficient to confer a total protection comparable to that induced by an inactivated vaccine. Therefore, VP2 capsids represent a suitable candidate recombinant vaccine instead of virus-like particles (VLPs) for IBDV infections. Our results also provide clear evidence that the recombinant IBDV-derived antigens are structure-dependent in order to be efficient as vaccine components.

Animals↗