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E Domingo

Publications and source records attributed to E Domingo.

At least 19 recordsLinked to original sources
PubMed2000

Role of a dipeptide insertion between codons 69 and 70 of HIV-1 reverse transcriptase in the mechanism of AZT resistance.

The 3'-azido-3'-deoxythymidine (AZT)-resistant pheno type of a heavily mutated human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) carrying a dipeptide (Ser-Ser) insertion between codons 69 and 70 as well as other mutations related to resistance to RT inhibitors has been studied. Recombinant virus carrying this variant RT (termed SS RT) showed reduced susceptibility to all nucleoside RT inhibitors in clinical use, particularly to AZT. In the presence of ATP, recombinant SS RT had an increased ability to remove the 3'-terminal nucleotide from AZT- terminated primers and extend the unblocked primer, compared with wild-type HIV-1 RT (BH10 isolate). Insertion of two serines in the sequence context of BH10 RT did not affect the ATP-dependent phosphorolytic activity of the enzyme, and had no influence in resistance to RT inhibitors. However, SS RT mutants lacking the dipeptide insertion or bearing a four-serine insertion showed reduced ATP-dependent phosphorolytic activity that correlated with increased AZT sensitivity, as determined using a recombinant virus assay. Therefore, the insertion appears to be critical to enhance AZT resistance in the sequence context of multidrug-resistant HIV-1 RT.

Amino Acid Sequence↗
PubMed2000

Viruses at the edge of adaptation.

How vulnerable is the line that separates adaptation from extinction? Viruses, in particular RNA viruses, are well known for their high rates of genetic variation and their potential to adapt to environmental modifications (Drake and Holland, 1999; Domingo et al., 2000). Yet, fitness variations-both increases and decreases-can be spectacularly rapid, and the simple genetic stratagem of forcing virus multiplication to go through repeated genetic bottlenecks can induce fitness losses, at times near viral extinction. New information has been recently obtained on the two sides of the survival line: the edge of adaptation and the edge of extinction.

Adaptation, Physiological↗
PubMed2000

Dynamics of dominance of a dipeptide insertion in reverse transcriptase of HIV-1 from patients subjected to prolonged therapy.

A small proportion (0.8%) of individuals of a cohort of HIV-1 infected patients subjected to prolonged therapy with nucleoside analogues included a recently recognised dipeptide insertion in their RT (Ser-Ser or Ser-Gly between RT codons 69 and 70). To study the dynamics of dominance of genomes with this genetic change, sequential HIV-1 isolates from two patients were analyzed with regard to consensus sequences and complexity of mutant spectra. The two patients displayed completely different, complex evolutionary patterns leading to temporary dominance of dipeptide insertions. In one patient, a virus very closely related to an ancestor virus from the same patient overtook the population at late times, displacing genomes encoding a Ser-Ser insertion. In another patient the sequential dominance of genomes with Ser-Ser insertion-->no insertion-->Ser-Gly insertion was observed. These three types of genomes coexisted in the mutant spectrum of one HIV-1 isolate. Complexity was also reflected in the shape of phylogenetic trees derived with genomes from the mutant spectrum at each time point. The results suggest that HIV-1 genomes encoding a dipeptide insertion between RT codons 69 and 70 do not show a clear selective advantage over other genomes lacking the insertion. Such an absence of a clear selective advantage will favor that such genomes encoding this RT insertion become dominant only in a transient fashion, and following disparate kinetics in different patients.

Adult↗
PubMed2000

A multiply substituted G-H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: a role for water molecules.

The crystal structure of a 15 amino acid synthetic peptide, corresponding to the sequence of the major antigenic site A (G-H loop of VP1) from a multiple variant of foot-and-mouth disease virus (FMDV), has been determined at 2.3 A resolution. The variant peptide includes four amino acid substitutions in the loop relative to the previously studied peptide representing FMDV C-S8c1 and corresponds to the loop of a natural FMDV isolate of subtype C(1). The peptide was complexed with the Fab fragment of the neutralizing monoclonal antibody 4C4. The peptide adopts a compact fold with a nearly cyclic conformation and a disposition of the receptor-recognition motif Arg-Gly-Asp that is closely related to the previously determined structure for the viral loop, as part of the virion, and for unsubstituted synthetic peptide antigen bound to neutralizing antibodies. New structural findings include the observation that well-defined solvent molecules appear to play a major role in stabilizing the conformation of the peptide and its interactions with the antibody. Structural results are supported by molecular-dynamic simulations. The multiply substituted peptide developed compensatory mechanisms to bind the antibody with a conformation very similar to that of its unsubstituted counterpart. One water molecule, which for steric reasons could not occupy the same position in the unsubstituted antigen, establishes hydrogen bonds with three peptide amino acids. The constancy of the structure of an antigenic domain despite multiple amino acid substitutions has implications for vaccine design.

Amino Acid Substitution↗
PubMed2000

Response of foot-and-mouth disease virus to increased mutagenesis: influence of viral load and fitness in loss of infectivity.

Passage of foot-and-mouth disease virus (FMDV) in cell culture in the presence of the mutagenic base analog 5-fluorouracil or 5-azacytidine resulted in decreases of infectivity and occasional extinction of the virus. Low viral loads and low viral fitness enhanced the frequency of extinction events; this finding was shown with a number of closely related FMDV clones and populations differing by up to 10(6)-fold in relative fitness in infections involving either single or multiple passages in the absence or presence of the chemical mutagens. The mutagenic treatments resulted in increases of 2- to 6.4-fold in mutation frequency and up to 3-fold in mutant spectrum complexity. The largest increase observed corresponded to the 3D (polymerase)-coding region, which is highly conserved in nonmutagenized FMDV populations. As a result, nucleotide sequence heterogeneity for the 3D-coding region became very similar to that for the variable VP1-coding region in FMDVs multiply passaged in the presence of chemical mutagens. The results suggest that strategies to combine reductions of viral load and viral fitness could be effectively associated with extinction mutagenesis as a potential new antiviral strategy.

Amino Acid Substitution↗
PubMed2000

Unusual distribution of mutations associated with serial bottleneck passages of human immunodeficiency virus type 1.

Repeated bottleneck passages result in fitness losses of RNA viruses. In the case of human immunodeficiency virus type 1 (HIV-1), decreases in fitness after a limited number of plaque-to-plaque transfers in MT-4 cells were very drastic. Here we report an analysis of entire genomic nucleotide sequences of four HIV-1 clones derived from the same HIV-1 isolate and their low-fitness progeny following 7 to 15 plaque-to-plaque passages. Clones accumulated 4 to 28 mutations per genome, with dominance of A --> G and G --> A transitions (57% of all mutations) and 49% nonsynonymous replacements. One clone-but not three sibling clones-showed an overabundance of G --> A transitions, evidencing the highly stochastic nature of some types of mutational bias. The distribution of mutations along the genome was very unusual in that mutation frequencies in gag were threefold higher than in env. Particularly striking was the complete absence of replacements in the V3 loop of gp120, confirmed with partial nucleotide sequences of additional HIV-1 clones subjected to repeated bottleneck passages. The analyses revealed several amino acid replacements that have not been previously recorded among natural HIV-1 isolates and illustrate how evolution of an RNA virus genome, with regard to constant and variable regions, can be profoundly modified by alterations in population dynamics.

Base Sequence↗
PubMed2000

Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in aphthovirus receptor usage.

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143-->Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin alpha(v)beta(3). In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.

Amino Acid Sequence↗
PubMed2000

Memory in viral quasispecies.

Biological adaptive systems share some common features: variation among their constituent elements and continuity of core information. Some of them, such as the immune system, are endowed with memory of past events. In this study we provide direct evidence that evolving viral quasispecies possess a molecular memory in the form of minority components that populate their mutant spectra. The experiments have involved foot-and-mouth disease virus populations with known evolutionary histories. The composition and behavior of the viral population in response to a selective constraint were influenced by past evolutionary history in a way that could not be predicted from examination of consensus nucleotide sequences of the viral populations. The molecular memory of the viral quasispecies influenced both the nature and the intensity of the response of the virus to a selective constraint.

Animals↗
PubMed2000

[Diagnostic accuracy of tomographic myocardial imaging for evaluation of aortocoronary graft patency].

INTRODUCTION AND OBJECTIVES: Conventional ergometry has limitations in the evaluation of coronary artery bypass graft patency. The aim of the present study was to determine the efficacy of exercise single photon emission tomography with 99mTc-compounds for the diagnosis of coronary artery bypass grafts disease. PATIENTS AND METHODS: The state of sixty-seven coronary artery bypass grafts (31 with mammary artery, 36 with safein vein; 35 to left descending anterior artery, 15 to circumflex and 17 to right coronary artery) were analyzed retrospectively in 38 patients (mean age 63+/-8.7; 35 men). The time that elapsed between the coronary surgery and the exercise tomography was 9,7 years. In 16 cases, exercise tomography was performed with 99mTc-MIBI and in the 22 remaining with 99mTc-tetrofosmin. In 6 cases, dipiridamol was administred simultaneously during an insufficient exercise test. RESULTS: Sensitivity (73.1%), specificity (93%), positive predictive value (86.3%), negative predictive value (84.4%), global value (85%), positive likelihood ratio (10.4) and negative likelihood ratio (0.29) of exercise tomography were significantly (p<0.01) better than those obtained with the exercise test alone (53.8%, 43.6%, 38.9%, 58. 6%, 47.7%, 0.95 and 1.06, respectively). CONCLUSIONS: Exercise tomography with 99mTc-compounds is a highly effective for the detection of coronary artery bypass grafts disease.

Coronary Artery Bypass↗
PubMed2000

Uveal melanoma model with metastasis in rabbits: effects of different doses of cyclosporine A.

OBJECTIVE: To compare the toxicity and efficacy of different doses of cyclosporine A (CsA) in a rabbit model of uveal melanoma. METHODS: We used four experimental groups: control, no CsA; group 1, 15 to 10 mg/kg/day; group 2, 15 mg/kg/day; and group 3, 20 mg/kg/day. The MKT-BR cell line was implanted in the choroid. All animals underwent ophthalmoscopic evaluation; the animals were weighed and blood levels of CsA, blood urea nitrogen (BUN), creatinine and alanine aminotransferase (ALT) were measured weekly. Necropsies and histologic study were performed to detect intraocular tumors and metastasis. RESULTS: A difference in survival rates was found between groups 2 and 3 (p = 0.0042). Differences were observed in the mean BUN and creatinine levels (p < 0.001 and p < 0.003, respectively) between groups but not in the ALT. Intraocular tumors were detected ophthalmoscopically in 50%, 65%, and 70% of the animals in groups 1, 2, and 3 respectively, and histologically in 70%, 90%, and 100% of the same groups. Lung metastases were found in 26.8% of animals with intraocular tumors. Differences were observed in mean CsA blood levels between animals with and without histologically demonstrated uveal tumors (p = 0.001) but not in animals with or without metastasis. CONCLUSIONS: Different doses of CsA affect survival, tumor development and renal toxicity. Metastatic disease is independent of CsA dose and the subsequent CsA blood levels. A blood level of CsA ranging from 500 to 1000 ng/ml and doses of 10 to 15 mg/kg/day may effectively develop this model of uveal melanoma.

Alanine Transaminase↗
PubMed1999

Mutational analysis of Phe160 within the "palm" subdomain of human immunodeficiency virus type 1 reverse transcriptase.

The highly conserved Phe160 residue is located in the "palm" subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), and makes contact with Tyr115, a residue which is involved in deoxynucleoside triphosphate (dNTP) binding and fidelity of DNA synthesis. Five mutant RTs having Tyr, Trp, Ile, Ala or Gln instead of Phe160 were obtained by site-directed mutagenesis. F160Y and F160W retained substantial DNA polymerase activity, whereas the catalytic efficiency of nucleotide incorporation of mutants F160I, F160A and F160Q was less than 10 % that of the wild-type RT, using poly(rA).oligo(dT)20 as the template-primer. The low catalytic efficiency of mutants F160I, F160A and F160Q was due to their lower affinity for the dNTP substrate. F160Y displayed similar kinetic parameters as the wild-type RT in nucleotide insertion assays carried out with heteropolymeric DNA/DNA template-primers. However, nucleotide affinity was two- to sixfold reduced in the case of mutant F160W. Fidelity assays revealed similar misinsertion and mispair extension ratios for the three enzymes, although F160W showed a slightly higher accuracy of DNA synthesis, particularly in the presence of high concentrations of dNTP. When introduced in an infectious proviral clone, mutations F160I, F160A and F160Q rendered non-viable virus. The importance of Phe160 for polymerase function and viral replication could be mediated by its interaction with Tyr115, as suggested by the analysis of the available crystal structures of HIV-1 RT.

Amino Acid Substitution↗
PubMed1999

Red queen dynamics, competition and critical points in a model of RNA virus quasispecies.

RNA viruses offer a unique opportunity for the study of evolution at the molecular level. Recent experiments involving clonal populations of RNA viruses have shown that competition among virus strains of approximately equal relative fitness can result in the eventual competitive exclusion of one of the species. As competition proceeds in time, both the winners and the losers exhibited absolute gains in fitness, consistent with the "Red Queen" hypothesis of evolution. Further experiments involving closely related evolving quasispecies revealed a highly predictable nonlinear behavior suggesting a deterministic component in the underlying quasispecies dynamics. This is apparently in contradiction with the standard view of RNA virus evolution as a highly unpredictable process. In this paper we present a simple model which allows previous hypothesis to be tested and provides an interpretation for the observed experimental results.

Cloning, Molecular↗
PubMed1999

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells.

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.

Adaptation, Physiological↗
PubMed1999

Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralising antibody: structure and neutralisation.

The interaction of foot-and-mouth disease virus (FMDV) serotype C (clone C-S8c1) with a strongly neutralising monoclonal antibody (MAb) 4C4 has been studied by combining data from cryoelectron microscopy and x-ray crystallography. The MAb 4C4 binds to the exposed flexible GH-loop of viral protein 1 (VP1), which appears to retain its flexibility, allowing movement of the bound Fab. This is in striking contrast to MAb SD6, which binds to the same GH-loop of VP1 but exhibits no movement of the bound Fab when observed under identical conditions. However, MAbs 4C4 and SD6 have very similar neutralisation characteristics. The known atomic structure of FMDV C-S8c1 and that of the 4C4 Fab cocrystallised with a synthetic peptide corresponding to the GH-loop of VP1 were fitted to the cryoelectron microscope density map. The best fit of the 4C4 Fab is compatible only with monovalent binding of the MAb in agreement with the neutralisation data on 4C4 MAbs, Fab2s, and Fabs. The position of the bound GH-loop is related to other known positions of this loop by a hinge rotation about the base of the loop. The 4C4 Fab appears to interact almost exclusively with the G-H loop of VP1, making no other contacts with the viral capsid.

Animals↗
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

Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus.

The function of a loop exposed on the aphthovirus capsid (the G-H loop of protein VP1) has been explored by combining genetic and structural studies with viral mutants. The loop displays a dual function of receptor recognition and interaction with neutralizing antibodies. Remarkably, some amino acid residues play a critical role in both such disparate functions. Therefore residues subjected to antibody pressure for variation may nevertheless maintain a role in receptor recognition for which invariance is a requirement. Evolution of FMDV in cell culture may relax the requirements at this site and allow further increase of antigenic diversification. Essential residues at one stage of virus evolution may become dispensable at another not very distant point in the evolutionary landscape. Implications for FMDV evolution and vaccine design are discussed.

Animals↗