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Biomedical subjects

E Domingo

Publications and source records attributed to E Domingo.

At least 19 recordsLinked to original sources
PubMed1999

Multiple molecular pathways for fitness recovery of an RNA virus debilitated by operation of Muller's ratchet.

Repeated bottleneck passages of RNA viruses result in fitness losses due to accumulation of deleterious mutations. We have analysed the molecular events underlying fitness recovery of a highly debilitated foot- and-mouth disease virus (FMDV) clone, upon serial passage in BHK-21 cells. The debilitated clone included an unusual, internal polyadenylate extension preceding the second functional AUG initiation codon, and a number of additional mutations scattered throughout the genome. Comparison of entire genomic nucleotide sequences in the course of passaging documented that loss of the internal polyadenylate was the first event in the process of fitness recovery. Further increases of fitness were associated with very few true reversions and with the accumulation of additional mutations affecting non-coding and coding regions. Remarkably, four biological subclones of the same debilitated FMDV clone gained fitness through three separate molecular pathways regarding correction of the internal polyadenylate: (i) a true reversion to yield the wild-type sequence at the second functional AUG; (ii) a shortening of the internal polyadenylate tract; or (iii) a deletion of 69 residues spanning the site of the polyadenylate extension. The results document that an RNA virus can find multiple pathways to reach alternative high fitness peaks on the fitness landscape.

Adaptation, Physiological↗
PubMed1999

Exponential fitness gains of RNA virus populations are limited by bottleneck effects.

Fitness is a parameter that quantitatively measures adaptation of a virus to a given environment. We have previously reported exponential fitness gains of large populations of vesicular stomatitis virus replicating in a constant environment (I. S. Novella et al., Proc. Natl. Acad. Sci. USA 92:5841-5844, 1995). In this paper, we report that during long-term passage of such large viral populations, fitness values reached a high-fitness plateau during which stochastic fitness variations were observed. This effect appears likely to be due to bottleneck effects on very high fitness populations.

Adaptation, Biological↗
PubMed1998

Quasispecies and the implications for virus persistence and escape.

BACKGROUND: In the 1970s Manfred Eigen and colleagues proposed a new model of molecular evolution to explain adaptability and rapid evolution of simple replicons, as those that probably populated the earth at the onset of life. This model of evolution placed emphasis on mutant generation, to the point of invalidating the concept of wild-type genomes as a defined sequence of nucleotides. In striking similarity with the proposals for such early replicons, present-day RNA viruses consist of complex distributions of nonidentical but closely related genomes termed quasispecies. OBJECTIVES: To discuss indeterminations inherent to a quasispecies structure and to the analytical procedures to define it, biological implications of quasispecies, and the need to take into account this type of population structure, in order to design effective strategies to prevent and control diseases caused by highly variable viruses. RESULTS: Quasispecies have many biological implications, extending from viral pathogenesis to the emergence of new pathogens, rapid antigenic variation, and alterations in cell tropism, virulence, host range and viral gene expression. CONCLUSIONS: Diseases caused by highly variable RNA viruses prove very difficult to control and vaccine development against such viruses are largely unsuccessful. It is important to understand quasispecies composition and dynamics, as quasispecies are an important step in the natural history of RNA viruses.

Animals↗
PubMed1998

Population dynamics in the evolution of RNA viruses.

RNA virus quasispecies are subjected to processes of positive Darwinian selection, to a very active and continuous negative selection and to random genetic drift. The course of RNA virus evolution is often unpredictable, and recent results suggest that even highly conserved motifs, once regarded as essential for infectivity, may be rendered dispensable by singular evolutionary events. An immediate consequence of the quasispecies genetic organization of RNA viruses is a surprising ability to gain fitness once a minimal replication ability is established in a biological environment. The unique features of RNA genetics should not be underestimated since they are at the basis of the emergence of new viral diseases and of the current difficulties to control many diseases associated variable viruses.

Animals↗
PubMed1998

Mutational analysis of discontinuous epitopes of foot-and-mouth disease virus using an unprocessed capsid protomer precursor.

An unprocessed capsid precursor (P1) of foot-and-mouth disease virus (FMDV) has been expressed in mammalian cells to study discontinuous epitopes involved in viral neutralization. Amino acid replacements found in virus-escape mutants were engineered in the P1 precursor by site-directed mutagenesis of the plasmid. In all cases the replacements abolished recognition of unprocessed P1 by the relevant monoclonal antibodies (MAbs), paralleling the effects of the corresponding substitutions in neutralization of infectious FMDV. Five capsid surface residues within the same discontinuous antigenic area that were never found replaced in escape mutants were also engineered in P1. None of the substitutions affected antibody recognition, suggesting that these residues were not directly involved in the interaction with the antibodies tested. The results validate site-directed mutagenesis of constructs encoding capsid precursors as an approach to probe the structure of viral discontinuous epitopes not amenable to analysis with synthetic peptides.

Amino Acid Sequence↗
PubMed1998

LTR and tat variability of HIV-1 isolates from patients with divergent rates of disease progression.

The genetic heterogeneity and transcription activity of the human immunodeficiency virus type 1 (HIV-1) LTR region and tat gene have been examined. Comparison involved the relevant genomic regions of viruses isolated from twenty long-term survivors and from ten typical progressors. No significant differences were observed in mutation frequencies among the two groups, although there was a significant higher proportion of synonymous substitutions in the tat gene of viruses from typical progressors. Four LTR sequences showed an insertion of 20-31 residues at the junction between the LTR Nef-coding and the LTR noncoding region. Neither these insertions nor other genetic changes found in these sequences affected the LTR transcription function, as measured in transient expression assays using transfection of both established cell lines and peripheral blood lymphocytes with plasmid DNA. The results did not allow the association of structural or functional alterations in LTR or tat with a degree of disease progression. The results reinforce the concepts of complexity of HIV-1 evolution in infected individuals, and the multifactorial nature of progression to AIDS.

Adult↗
PubMed1998

Noninvasive mechanical ventilation in a patient with respiratory failure after hematopoietic progenitor transplantation.

Respiratory failure requiring orotracheal intubation (OTI) and mechanical ventilation (MV) is almost always a fatal complication in patients who undergo hematopoietic progenitor transplantation (HPT). We present the case of a woman who suffered respiratory failure with bilateral infiltrates on a chest X-ray taken on day +14 following autologous bone marrow transplantation. We managed the patient satisfactorily with noninvasive ventilation, avoiding OTI. We believe that patients with non-progressive pulmonary lesions and without multiple system organ failure, may be correctly managed with noninvasive positive-pressure ventilation (NPPV).

Aged↗
PubMed1998

Fasting and postprandial ischemic threshold in patients with unstable angina with and without postprandial angina at rest.

BACKGROUND: Postprandial angina develops within minutes after a meal in patients with unstable angina, but the clinical characteristics of these patients and why it develops in only some of those with advanced coronary artery disease remain largely unknown. A severely reduced coronary reserve associated with postprandial increases in heart rate could be a contributory mechanism. METHODS: The clinical and angiographic characteristics of 277 patients with unstable angina with (23) or without (254) postprandial angina were analyzed. The coronary reserve was also analyzed by measuring the ischemic threshold by atrial pacing in a fasting state in all patients and 15 minutes after a 900-calorie meal in 54. RESULTS: Patients with postprandial angina were older, more likely to be women, and had a higher incidence of hypertension and three-vessel disease than those without (p < 0.005) and had a lower fasting ischemic threshold (131.8 [SD 13.0] vs 147.5 [SD 23.4] beats/min, p < 0.0001). However, 67 of the 79 patients with the lowest fasting thresholds (< or =130 beats/min) (84.8%) had no postprandial angina. Moreover, among patients with and without postprandial angina who were matched for age, sex, and extent of coronary disease, the ischemic threshold was also lower in those with postprandial angina (p < 0.005) and there were no differences in left ventricular end-diastolic pressure or volume. Postprandial pacing was positive in 37 patients but postprandial ischemic threshold was comparable to fasting threshold (132 [SD 14] vs 132 [SD 16] beats/min). Moreover, in the 10 patients who experienced in-hospital postprandial angina, heart rate during postprandial angina was similar to nonpostprandial angina (93.1 [SD 14.7] vs 90.3 [SD 17.6]) and lower than the fasting ischemic threshold (132.0 [SD 10.8] beats/min, p < 0.0001). CONCLUSIONS: Thus postprandial angina tends to occur among elderly and hypertensive patients with advanced coronary disease and severely reduced ischemic threshold. The fact that the postprandial ischemic threshold was clearly higher than the heart rate attained during postprandial angina suggest that factors others than increases in heart rate account for postprandial angina. Furthermore, the lack of a decline in the postprandial ischemic threshold suggests that, in the absence of postprandial angina, there is not a consistent postprandial change in coronary tone or that the increases in myocardial oxygen demands due to increased myocardial contractility-wall tension do not seem to play a major role in postprandial ischemia.

Adult↗
PubMed1998

An RNA virus can adapt to the multiplicity of infection.

RNA viruses evolve as complex distributions of mutants termed viral quasispecies. For this reason it is relevant to explore those environmental parameters that favour the selective advantage of some viral subpopulations over others. In the present study we provide direct evidence that the relative fitness of two competing viral subpopulations may depend on the multiplicity of infection (m.o.i.). Two closely related subpopulations of foot-and-mouth disease virus (FMDV) of serotype C, which differed in their history of cytolytic passages in BHK-21 cells, were subjected to growth-competition experiments in BHK-21 cells. One of the populations, termed S, was found to have a selective advantage over the other population, termed L, only when the competition passages were carried out at low m.o.i. In contrast, both populations, L and S, coexisted during serial passages carried out at high m.o.i. No differences between S and L were detected in assays of inhibition of infectivity by synthetic peptides, in cell binding-competition experiments, or in virulence for BHK-21 cells. However, FMDV S displayed increased heparin binding compared with L, and L higher virulence for Chinese hamster ovary (CHO) cells than S. These results with FMDV suggest that small differences in the interaction of the virus with the host cell may contribute to an m.o.i.-dependent selective advantage of one viral subpopulation over a closely related subpopulation. Therefore, different viral mutants from quasispecies replicating in vivo may be selected depending on the number of variant viruses relative to the number of susceptible cells.

Adaptation, Biological↗
PubMed1998

A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation.

The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C1, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.

Amino Acid Sequence↗
PubMed1998

Rapid selection in modified BHK-21 cells of a foot-and-mouth disease virus variant showing alterations in cell tropism.

With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the cells and the resident virus; the virulence of the virus for the parental BHK-21 cells is gradually increased, and the cells become partially resistant to FMDV. Here we report that variants of FMDV C3Arg/85 were selected in a single infection of partially resistant BHK-21 cells (termed BHK-Rb cells). Indirect immunofluorescence showed that the BHK-Rb cell population was heterogeneous with regard to susceptibility to C3Arg/85 infection. Infection of BHK-Rb cells with C3Arg/85 resulted in an early phase of partial cytopathology which was followed at 6 to 10 days postinfection by the shedding of mutant FMDVs, termed C3-Rb. The selected C3-Rb variants showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect wild-type Chinese hamster ovary (CHO) cells. A comparison of the genomic sequences of the parental and modified viruses revealed only two amino acid differences, located at the surface of the particle, at the fivefold axis of the viral capsid (Asp-9-->Ala in VP3 and either Gly-110-->Arg or His-108-->Arg in VP1). The same phenotypic and genotypic modifications occurred in a highly reproducible manner; they were seen in a number of independent infections of BHK-Rb cells with viral preparation C3Arg/85 or with clones derived from it. Neither amino acid substitutions in other structural or nonstructural proteins nor nucleotide substitutions in regulatory regions were found. These results prove that infection of partially permissive cells can promote the rapid selection of virus variants that show alterations in cell tropism and are highly virulent for the same cells.

Amino Acid Substitution↗
PubMed1998

Multiple virulence determinants of foot-and-mouth disease virus in cell culture.

Hypervirulent variants of foot-and-mouth disease virus (FMDV) of serotype C arise upon serial cytolytic or persistent infections in cell culture. A specific mutation in the internal ribosome entry site of persistent FMDV was previously associated with enhanced translation initiation activity that could contribute to the hypervirulent phenotype for BHK-21 cells. Here we report that several hypervirulent FMDV variants arising upon serial cytolytic passage show an invariant internal ribosome entry site but have a number of mutations affecting structural and nonstructural viral proteins. The construction of chimeric type O-type C infectious transcripts has allowed the mapping of a major determinant of hypervirulence to the viral capsid. Tissue culture-adapted FMDV displayed enhanced affinity for heparin, but binding to cell surface heparan sulfate moieties was not required for expression of the hypervirulent phenotype in Chinese hamster ovary (CHO) cells. Virulence was identical or even higher for glycosaminoglycan-deficient CHO cells than for wild-type CHO cells. FMDV variants with decreased affinity for heparin were selected from a high-binding parental population and analyzed. Substitutions associated with decreased heparin binding were located at positions 173 of capsid protein VP3 and 144 of capsid protein VP1. These substitutions had a moderate effect on virulence for BHK-21 cells but completely abrogated infection of CHO cells. The comparative results with several FMDV isolates show that (i) increased affinity for heparin and alterations in cell tropism may be mediated by a number of independent sites on the viral capsid and (ii) the same capsid modifications may have different effects on different cell types.

Amino Acid Substitution↗
PubMed1997

Characterization of the reverse transcriptase of a human immunodeficiency virus type 1 group O isolate.

The catalytic properties and sensitivity to different inhibitors have been determined for the reverse transcriptase (RT) of group O human immunodeficiency virus type 1 (HIV-1). The RT-coding region was cloned from a new HIV-1 group O isolate from Spain, expressed in Escherichia coli, and purified by affinity chromatography. This new RT showed 79% amino acid sequence identity with the corresponding enzyme of group M subtype B strain BH10. The two enzymes showed very similar kinetics of RNA-dependent DNA polymerization using homopolymeric template-primers and RNase H specific activity. Inhibitor sensitivity to ddTTP and 3'-azido-2',3'-dideoxythymidine triphosphate (AZTTP) was also similar for both enzymes. However, the two enzymes differed dramatically in their sensitivity to several inhibitors. While the RT of the BH10 isolate was sensitive to nevirapine and loviride (IC50 ranged from 0.16 to 8.2 microM, depending on the substrates used), the enzyme of the Spanish HIV-1 group O isolate showed high-level resistance to those compounds (IC50 > 200 microM). The amino acid sequence of the RT of group O HIV-1 contains three amino acids (Cys-181, Glu-179, and Gly-98), which are found in group M subtype B strains resistant to nonnucleoside RT inhibitors. The recombinant group O HIV-1 RT should be useful for studies aimed at discovering and designing drugs directed toward group O isolates of HIV-1.

Adult↗
PubMed1997

Evolution subverting essentiality: dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus.

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet-which is also a widespread motif involved in cell-to-cell adhesion-can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature.

Aphthovirus↗
PubMed1997

Structure of the complex of an Fab fragment of a neutralizing antibody with foot-and-mouth disease virus: positioning of a highly mobile antigenic loop.

Data from cryo-electron microscopy and X-ray crystallography have been combined to study the interactions of foot-and-mouth disease virus serotype C (FMDV-C) with a strongly neutralizing monoclonal antibody (mAb) SD6. The mAb SD6 binds to the long flexible GH-loop of viral protein 1 (VP1) which also binds to an integrin receptor. The structure of the virus-Fab complex was determined to 30 A resolution using cryo-electron microscopy and image analysis. The known structure of FMDV-C, and of the SD6 Fab co-crystallized with a synthetic peptide corresponding to the GH-loop of VP1, were fitted to the cryo-electron microscope density map. The SD6 Fab is seen to project almost radially from the viral surface in an orientation which is only compatible with monovalent binding of the mAb. Even taking into account the mAb hinge and elbow flexibility, it is not possible to model bivalent binding without severely distorting the Fabs. The bound GH-loop is essentially in what has previously been termed the 'up' position in the best fit Fab orientation. The SD6 Fab interacts almost exclusively with the GH-loop of VP1, making very few other contacts with the viral capsid. The position and orientation of the SD6 Fab bound to FMDV-C is in accord with previous immunogenic data.

Antibodies, Monoclonal↗
PubMed1997

Mispair extension fidelity of human immunodeficiency virus type 1 reverse transcriptases with amino acid substitutions affecting Tyr115.

The role of Tyr115 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) in the mispair extension fidelity of DNA dependent DNA synthesis was analysed by using a series of 15 mutant enzymes with substitutions at Tyr115. Their kinetic parameters for elongation using homopolymeric RNA-DNA and heteropolymeric DNA-DNA complexes showed major effects of the amino acid substitutions on the Km value for dNTP. Enzymes with large hydrophobic residues at position 115 displayed lower Km values than enzymes with small and charged amino acids at this position. The influence of all these amino acid replacements in mispair extension fidelity assays was analyzed using three different mismatches (A:C, A:G and A:A) at the 3'-terminal position of the primer DNA. For the A:C mispair, a 2. 6-33.4-fold increase in mispair extension efficiency (fext) was observed as compared with the wild-type enzyme. Unexpectedly, all the mutants tested as well as the wild-type RT were very efficient in extending the A:G and A:A transversion mispairs. This effect was due to the template-primer sequence context and not to the buffer conditions of the assay. The data support a role of Tyr115 in accommodating the complementary nucleotide into the nascent DNA while polymerization takes place.

DNA Repair↗