Biomedical subjects

José L Carrascosa

Publications and source records attributed to José L Carrascosa.

At least 19 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

All three chaperonin genes in the archaeon Haloferax volcanii are individually dispensable.

The Hsp60 or chaperonin class of molecular chaperones is divided into two phylogenetic groups: group I, found in bacteria, mitochondria and chloroplasts, and group II, found in eukaryotic cytosol and archaea. Group I chaperonins are generally essential in bacteria, although when multiple copies are found one or more of these are dispensable. Eukaryotes contain eight genes for group II chaperonins, all of which are essential, and it has been shown that these proteins assemble into double-ring complexes with eightfold symmetry where all proteins occupy specific positions in the ring. In archaea, there are one, two or three genes for the group II chaperonins, but whether they are essential for growth is unknown. Here we describe a detailed genetic, structural and biochemical analysis of these proteins in the halophilic archaeon, Haloferax volcanii. This organism contains three genes for group II chaperonins, and we show that all are individually dispensable but at least one must be present for growth. Two of the three possible double mutants can be constructed, but only one of the three genes is capable of fully complementing the stress-dependent phenotypes that these double mutants show. The chaperonin complexes are made up of hetero-oligomers with eightfold symmetry, and the properties of the different combinations of subunits derived from the mutants are distinct. We conclude that, although they are more homologous to eukaryotic than prokaryotic chaperonins, archaeal chaperonins have some redundancy of function.

Archaeal Proteins↗

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

Structure of the connector of bacteriophage T7 at 8A resolution: structural homologies of a basic component of a DNA translocating machinery.

The three-dimensional structure of the bacteriophage T7 head-to-tail connector has been obtained at 8A resolution using cryo-electron microscopy and single-particle analysis from purified recombinant connectors. The general morphology of the T7 connector is that of a 12-folded toroidal homopolymer with a channel that runs along the longitudinal axis of the particle. The structure of the T7 connector reveals many structural similarities with the connectors from other bacteriophages. Docking of the atomic structure of the varphi29 connector into the three-dimensional reconstruction of T7 connector reveals that the narrow, distal region of the two oligomers are almost identical. This region of the varphi29 connector has been suggested to be involved in DNA translocation, and is composed of an alpha-beta-alpha-beta-beta-alpha motif. A search for alpha-helices in the same region of the T7 three-dimensional map has located three alpha-helices in approximately the same position as those of the varphi29 connector. A comparison of the predicted secondary structure of several bacteriophage connectors, including among others T7, varphi29, P22 and SPP1, reveals that, despite the lack of sequence homology, they seem to contain the same alpha-beta-alpha-beta-beta-alpha motif as that present in the varphi29 connector. These results allow us to suggest a common architecture related to a basic component of the DNA translocating machinery for several viruses.

Bacteriophage T7↗

PubMed2005

Cryo-electron tomography of vaccinia virus.

The combination of cryo-microscopy and electron tomographic reconstruction has allowed us to determine the structure of one of the more complex viruses, intracellular mature vaccinia virus, at a resolution of 4-6 nm. The tomographic reconstruction allows us to dissect the different structural components of the viral particle, avoiding projection artifacts derived from previous microscopic observations. A surface-rendering representation revealed brick-shaped viral particles with slightly rounded edges and dimensions of approximately 360 x 270 x 250 nm. The outer layer was consistent with a lipid membrane (5-6 nm thick), below which usually two lateral bodies were found, built up by a heterogeneous material without apparent ordering or repetitive features. The internal core presented an inner cavity with electron dense coils of presumptive DNA-protein complexes, together with areas of very low density. The core was surrounded by two layers comprising an overall thickness of approximately 18-19 nm; the inner layer was consistent with a lipid membrane. The outer layer was discontinuous, formed by a periodic palisade built by the side interaction of T-shaped protein spikes that were anchored in the lower membrane and were arranged into small hexagonal crystallites. It was possible to detect a few pore-like structures that communicated the inner side of the core with the region outside the layer built by the T-shaped spike palisade.

Cryoelectron Microscopy↗

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

Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation.

To attain its native conformation, the cytoskeletal protein tubulin needs the concourse of several molecular chaperones, among others the cytosolic chaperonin CCT. It has been previously described that denatured tubulin interacts with CCT in a quasi-folded conformation using several loops located throughout its sequence. These loops are also involved in microtubule formation and are absent in its prokaryote homologue FtsZ, which in vitro folds by itself and does not interact with CCT. Several FtsZ/tubulin chimeric proteins were generated by inserting consecutively one, two or three of the CCT-binding domains of tubulin into the corresponding sequence of FtsZ from Methanococccus jannaschii. The insertion of any of the CCT-binding loops generates in the FtsZ/tubulin chimeras the ability to interact with CCT. The accumulation of CCT-binding loops induces in the FtsZ/tubulin chimeras unfolding and refolding properties that are more similar to tubulin than to its prokaryote counterpart. Finally, the insertion of some of these loops generates in the FtsZ/tubulin chimeras more complex polymeric structures than those found for FtsZ. These results reinforce the notion that CCT has coevolved with tubulin to deal with the folding problems encountered by the eukaryotic protein with the appearance of the new sequences involved in microtubule formation.

Archaeal Proteins↗

PubMed2004

Structure of the complex between the cytosolic chaperonin CCT and phosducin-like protein.

The three-dimensional structure of the complex formed between the cytosolic chaperonin CCT (chaperonin containing TCP-1) and phosducin (Pdc)-like protein (PhLP), a regulator of CCT activity, has been solved by cryoelectron microscopy. Binding of PhLP to CCT occurs through only one of the chaperonin rings, and the protein does not occupy the central folding cavity but rather sits above it through interactions with two regions on opposite sides of the ring. This causes the apical domains of the CCT subunits to close in, thus excluding access to the folding cavity. The atomic model of PhLP generated from several atomic structures of the homologous Pdc fits very well with the mass of the complex attributable to PhLP and predicts the involvement of several sequences of PhLP in CCT binding. Binding experiments performed with PhLP/Pdc chimeric proteins, taking advantage of the fact that Pdc does not interact with CCT, confirm that both the N- and C-terminal domains of PhLP are involved in CCT binding and that several regions suggested by the docking experiment are indeed critical in the interaction with the cytosolic chaperonin.

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↗

PubMed2003

3D structure of the influenza virus polymerase complex: localization of subunit domains.

The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent boundaries among subunits. The position of specific regions of the PB1, PB2, and PA subunits was determined by 3D reconstruction of either RNP-mAb complexes or tagged RNPs. This structural model is available for the polymerase of a negative-stranded RNA virus and provides a general delineation of the complex and its interaction with the template-associated nucleoprotein monomers in the RNP.

Image Processing, Computer-Assisted↗

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

Structural maturation of rubella virus in the Golgi complex.

Rubella virus is a small enveloped virus that assembles in association with Golgi membranes. Freeze-substitution electron microscopy of rubella virus-infected cells revealed a previously unrecognized virion polymorphism inside the Golgi stacks: homogeneously dense particles without a defined core coexisting with less dense, mature virions that contained assembled cores. The homogeneous particles appear to be a precursor form during the virion morphogenesis process as the forms with mature morphology were the only ones detected inside secretory vesicles and on the exterior of cells. In mature virions potential remnants of C protein membrane insertion were visualized as dense strips connecting the envelope with the internal core. In infected cells Golgi stacks were frequently seen close to cytopathic vacuoles, structures identified as the sites for viral RNA replication, along with the rough endoplasmic reticulum and mitochondria. These associations could facilitate the transfer of viral genomes from the cytopathic vacuoles to the areas of rubella assembly in Golgi membranes.

Animals↗

PubMed2003

Differences in virus-induced cell morphology and in virus maturation between MVA and other strains (WR, Ankara, and NYCBH) of vaccinia virus in infected human cells.

Live recombinants based on attenuated modified vaccinia virus Ankara (MVA) are potential vaccine candidates against a broad spectrum of diseases and tumors. To better understand the efficacy of MVA as a human vaccine, we analyzed by confocal and electron microscopy approaches MVA-induced morphological changes and morphogenetic stages during infection of human HeLa cells in comparison to other strains of vaccinia virus (VV): the wild-type Western Reserve (WR), Ankara, and the New York City Board of Health (NYCBH) strains. Confocal microscopy studies revealed that MVA infection alters the cytoskeleton producing elongated cells (bipolar), which do not form the characteristic actin tails. Few virions are detected in the projections connecting neighboring cells. In contrast, cells infected with the WR, Ankara, and NYCBH strains exhibit a stellated (multipolar) or rounded morphology with actin tails. A detailed transmission electron microscopy analysis of HeLa cells infected with MVA showed important differences in fine ultrastructure and amounts of the viral intermediates compared to cells infected with the other VV strains. In HeLa cells infected with MVA, the most abundant viral forms are intracellular immature virus, with few intermediates reaching the intracellular mature virus (IMV) form, at various stages of maturation, which exhibit a more rounded shape than IMVs from cells infected with the other VV strains. The "IMVs" from MVA-infected cells have an abnormal internal structure ("atypical" viruses) with potential alterations in the core-envelope interactions and are unable to significantly acquire the additional double envelope to render intracellular envelope virus. The presence of potential cell-associated envelope virus is very scarce. Our findings revealed that MVA in human cells promotes characteristic morphological changes to the cells and is able to reach the IMV stage, but these virions were not structurally normal and the subsequent steps in the morphogenetic pathway are blocked.

Animals↗

PubMed2003

Polymorphism and structural maturation of bunyamwera virus in Golgi and post-Golgi compartments.

The Golgi apparatus is the assembly site for a number of complex enveloped viruses. Using high-preservation methods for electron microscopy, we have detected two previously unknown maturation steps in the morphogenesis of Bunyamwera virus in BHK-21 cells. The first maturation takes place inside the Golgi stack, where annular immature particles transform into dense, compact structures. Megalomicin, a drug that disrupts the trans side of the Golgi complex, reversibly blocks transformation, showing that a functional trans-Golgi is needed for maturation. The second structural change seems to take place during the egress of viral particles from cells, when a coat of round-shaped spikes becomes evident. A fourth viral assembly was detected in infected cells: rigid tubular structures assemble in the Golgi region early in infection and frequently connect with mitochondria. In Vero cells, the virus induces an early and spectacular fragmentation of intracellular membranes while productive infection progresses. Assembly occurs in fragmented Golgi stacks and generates tubular structures, as well as the three spherical viral forms. These results, together with our previous studies with nonrelated viruses, show that the Golgi complex contains key factors for the structural transformation of a number of enveloped viruses that assemble intracellularly.

Animals↗

PubMed2002

Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT.

The biogenesis of the cytoskeletal proteins actin and tubulin involves interaction of nascent chains of each of the two proteins with the oligomeric protein prefoldin (PFD) and their subsequent transfer to the cytosolic chaperonin CCT (chaperonin containing TCP-1). Here we show by electron microscopy that eukaryotic PFD, which has a similar structure to its archaeal counterpart, interacts with unfolded actin along the tips of its projecting arms. In its PFD-bound state, actin seems to acquire a conformation similar to that adopted when it is bound to CCT. Three-dimensional reconstruction of the CCT:PFD complex based on cryoelectron microscopy reveals that PFD binds to each of the CCT rings in a unique conformation through two specific CCT subunits that are placed in a 1,4 arrangement. This defines the phasing of the CCT rings and suggests a handoff mechanism for PFD.

Actins↗

PubMed2002

Structure and function of a protein folding machine: the eukaryotic cytosolic chaperonin CCT.

Chaperonins are large oligomers made up of two superimposed rings, each enclosing a cavity used for the folding of other proteins. Among the chaperonins, the eukaryotic cytosolic chaperonin CCT is the most complex, not only with regard to its subunit composition but also with respect to its function, still not well understood. Unlike the more well studied eubacterial chaperonin GroEL, which binds any protein that presents stretches of hydrophobic residues, CCT recognises in its substrates specific binding determinants and interacts with them through particular combinations of CCT subunits. Folding then occurs after the conformational changes induced in the chaperonin upon nucleotide binding have occurred, through a mechanism that, although still poorly defined, clearly differs from the one established for GroEL. Although CCT seems to be mainly involved in the folding of actin and tubulin, other substrates involved in various cellular roles are beginning to be characterised, including many WD40-repeat, 7-blade propeller proteins.

Actins↗

PubMed2002

Importance of the N-terminal region of the phage GA-1 single-stranded DNA-binding protein for its self-interaction ability and functionality.

The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages phi 29 and Nf. In this work, the self-interaction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo cross-linking of bacterial cultures infected with phage GA-1. GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of phi 29 and Nf. Three deletion mutant proteins have been characterized, Delta N19, Delta N26, and Delta N33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB. Mutant protein Delta N19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins Delta N26 and Delta N33 no longer stimulate viral DNA replication or display helix-destabilizing activity. Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein. Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein.

Amino Acid Sequence↗