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Biomedical subjects

M Esteban

Publications and source records attributed to M Esteban.

At least 55 records · Page 3Linked to original sources
PubMed1999

Enhanced CD8+ T cell response to HIV-1 env by combined immunization with influenza and vaccinia virus recombinants.

With the aim to determine if immunization with two different live recombinant viral vectors could lead to an enhancement of the cellular immune response to HIV-1 antigens, we have characterized the CD8+ T cell response elicited against the V3 loop epitope from HIV-1 env protein in Balb/c mice immunized with either: a recombinant influenza virus (Flu-Env) expressing the V3 loop epitope from HIV-1 strain IIIB, a vaccinia virus recombinant (VV-Env) expressing the complete HIV-1-IIIB env protein, or a combination of both. The CD8+ T cell response, measured by the ELISPOT assay, in animals primed with Flu-Env and boosted with VV-Env was 5 to 6 times higher than in animals inoculated with either Flu-Env or VV-Env alone. Similar results were obtained with recombinant viruses expressing the V3 loop epitope or the complete env protein, respectively, from the MN strain of HIV-1. Our results indicate that the use of two different live vectors for priming and boosting has a synergistic effect on the immune response against HIV-1, and could represent a novel vaccination strategy against AIDS.

Amino Acid Sequence↗
PubMed1999

Identification of functional domains of the interferon-induced enzyme PKR in cells lacking endogenous PKR.

The interferon (IFN)-induced, double-stranded RNA (dsRNA)-activated human protein kinase (PKR) has been shown to exert antiviral and antiproliferative effects. Activation of the enzyme in mammalian cells results in protein synthesis inhibition and cell death by apoptosis. Previous studies on the structure-function relationship of PKR have been based on vectors expressing the enzyme in mammalian cells containing endogenous PKR. As exogenously expressed PKR can form heterodimers with endogenous PKR, the results obtained on the functional characterization of mutant forms of PKR have been taken with caution. To address the natural consequences of heterodimer formation between endogenous and exogenous PKR, we have analyzed the structure-function relationship of PKR ectopically expressed from vaccinia virus (VV) recombinants in cells lacking the endogenous enzyme. We demonstrate that PKR-mediated inhibition of protein synthesis and induction of apoptosis is not dependent on the presence of endogenous PKR. Further, PKR activity is independent of the presence of dsRNA binding motifs (dsRBM). Moreover, single-point mutations of the third basic domain decreased PKR activation. Our findings demonstrate that PKR can be activated in the absence of its N-terminal domain (amino acids 1-232) and that the third basic domain is important for its biologic function.

3T3 Cells↗
PubMed1999

Identification by two-dimensional gel electrophoresis of vaccinia virus and cellular phosphoproteins modified after inducible expression of the dsRNA-activated protein kinase.

The interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) is a serine/threonine kinase that plays an important role in the biology of IFN, exerting antiviral and anticellular actions. These effects have been correlated with phosphorylation of the eukaryotic initiation factor eIF-2alpha and the NF-kappaB inhibitor IkappaB, although it has not been demonstrated that IkappaB is a direct target of PKR in vivo. In view of the various biological effects of PKR, it is likely that other cellular substrates are involved in PKR action. To identify novel substrates of PKR, we have carried out a systematic study of the phosphorylated proteins from cultured cells following PKR activation using high-resolution two-dimensional gel electrophoresis (2D-PAGE). We have used metabolic labeling with [32P]orthophosphate of HeLa cells infected with vaccinia virus (VV) recombinants expressing wild type (wt) or the catalytically inactive mutant form (K296R) of PKR under regulation of the Escherichia coli lacI operator/repressor system. Upon induction of PKR in the presence of isopropyl-beta-D-thiogalactoside (IPTG), the 68-kDA wt enzyme and eIF-2alpha are phosphorylated. These events lead to changes in the phosphorylation state of viral and cellular proteins. A distinct set of VV-induced phosphoproteins remained phophorylated, while the labeling of other viral proteins decreased markedly, probably as a result of a PKR-dependent translational block. Five proteins of unknown origin (68, 26, 20, 19, 15-16 kDA) appeared to be newly phosphorylated after PKR activation. Expression of the catalytically inactive K296R mutant form of PKR did not induce changes in the phosphorylation of VV proteins. Thus, by 2D-PAGE, we identified cellular and VV-induced phosphoproteins modified after PKR activation. Some or all of the phosphoproteins appearing or increasing in amount after PKR activation might not be direct targets of PKR, but rather indirect consequences of PKR activation.

Blotting, Western↗
PubMed1999

Full activation of RNaseL in animal cells requires binding of 2-5A within ankyrin repeats 6 to 9 of this interferon-inducible enzyme.

To define protein domains important for activation of the interferon (IFN)-induced enzyme 2-5A-dependent RNaseL, we have generated vaccinia virus (VV) recombinants able to express in cultured cells truncated forms of this protein and compared their biologic activities with those producing the wild-type enzyme, with and without coexpression of 2-5A synthetase. Our results show that full activation of RNaseL requires binding of 2-5A oligonucleotides within amino acid positions 212-339, corresponding to ankyrin repeats 6 to 9. The protein kinase and ribonuclease domains of RNaseL, amino acids 340-741, are sufficient for a constitutively active enzyme that is unresponsive to excess 2-5A. These results demonstrate in vivo the importance of the ankyrin domains in the biologic function of RNaseL. We suggest that ankyrin repeats act as key modulators of RNaseL activity.

Adenine Nucleotides↗
PubMed1999

Primary motor cortex involvement in Alzheimer disease.

In Alzheimer disease (AD) the involvement of entorhinal cortex, hippocampus, and associative cortical areas is well established. Regarding the involvement of the primary motor cortex the reported data are contradictory. In order to determine whether the primary motor cortex is involved in AD, the brains of 29 autopsy cases were studied, including, 17 cases with severe cortical AD-type changes with definite diagnoses of AD, 7 age-matched cases with discrete to moderate cortical AD-type changes, and 5 control cases without any AD-type cortical changes. Morphometric analysis of the cortical surface occupied by senile plaques (SPs) on beta-amyloid-immunostained sections and quantitative analysis of neurofibrillary tangles (NFTs) on Gallyas-stained sections was performed in 5 different cortical areas including the primary motor cortex. The percentage of cortical surface occupied by SPs was similar in all cortical areas, without significant difference and corresponded to 16.7% in entorhinal cortex, 21.3% in frontal associative, 16% in parietal associative, and 15.8% in primary motor cortex. The number of NFTs in the entorhinal cortex was significantly higher (41 per 0.4 mm2), compared with those in other cortical areas (20.5 in frontal, 17.9 in parietal and 11.5 in the primary motor cortex). Our findings indicate that the primary motor cortex is significantly involved in AD and suggest the appearance of motor dysfunction in late and terminal stages of the disease.

Adult↗
PubMed1999

A human immunodeficiency virus type 1 Env-granulocyte-macrophage colony-stimulating factor fusion protein enhances the cellular immune response to Env in a vaccinia virus-based vaccine.

Vaccinia virus (VV) infection induces protective T- and B-cell responses, making recombinants based on VV good candidates for the development of effective vaccines to other viruses. VV recombinants expressing the human immunodeficiency virus (HIV) envelope protein (Env) have been generated in several laboratories and shown to induce anti-HIV cellular and humoral immune responses in vaccinated humans and in chimpanzees. To increase the immunogenicity of the Env antigen, a VV recombinant was generated that expresses a chimeric antigen consisting of the Env protein fused to an immunostimulatory cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The chimeric protein retained GM-CSF biological activity when expressed by this recombinant virus (VV-GM-gp120) in cells infected in vitro. Infection of BALB/c mice with VV-GM-gp120 triggered a higher HIV-specific cellular immune response, as measured by interferon-gamma production, than that induced by a VV recombinant expressing the native Env protein. Moreover, although anti-gp120 antibody titres were similar in sera from mice inoculated with either of the VV recombinants, immunization with the recombinant expressing the fusion protein elicited antibodies against a broader spectrum of Env epitopes. These results indicate that HIV Env antigen fusion to GM-CSF provides a means to improve the anti-HIV immune response.

AIDS Vaccines↗
PubMed1999

Identification of functional domains in the 14-kilodalton envelope protein (A27L) of vaccinia virus.

The mechanism of entry of vaccinia virus (VV) into cells is still a poorly understood process. A 14-kDa protein (encoded by the A27L gene) in the envelope of intracellular mature virus (IMV) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells. We have previously described the structural organization of the VV 14-kDa protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted Leu zipper-like third alpha helix with an important role in the interaction with a 21-kDa membrane protein (encoded by the A17L gene) thought to anchor the 14-kDa protein to the envelope of IMV (M.-I. Vázquez, G. Rivas, D. Cregut, L. Serrano, and M. Esteban, J. Virol. 72:10126-10137, 1998). To identify the functional domains important for virus entry and release, we have generated VV recombinants containing a copy of the A27L gene regulated by the lacI operator-repressor system of Escherichia coli (VVIndA27L) in the thymidine kinase locus and a mutant form of the A27L gene in the hemagglutinin locus but expressed constitutively under the control of an early-late VV promoter. Cells infected with a VV recombinant that expresses a mutant 14-kDa form lacking the first 29 amino acids at the N terminus failed to form extracellular enveloped virus (EEV). Fusion-from-without assays with purified virus confirmed that the fusion process was mediated by the 14-kDa protein and the fusion domain to be contained within amino acids 29 to 43 of the N-terminal region. Competitive inhibition of the infection process with soluble heparin and synthetic peptides and in vitro experiments with purified mutant proteins identified the heparin binding domain within amino acids 21 to 33, suggesting that this domain is involved in virus-cell binding via heparan sulfate. Thus, the N terminus of the 14-kDa protein contains a heparin binding domain, a fusion domain, and a domain responsible for interacting with proteins or lipids in the Golgi stacks for EEV formation and virus spread.

Amino Acid Sequence↗
PubMed1999

Induction of apoptosis by double-stranded-RNA-dependent protein kinase (PKR) involves the alpha subunit of eukaryotic translation initiation factor 2 and NF-kappaB.

The double-stranded (ds) RNA-dependent protein kinase (PKR) is a key mediator of antiviral effects of interferon (IFN) and an active player in apoptosis induced by different stimuli. The translation initiation factor eIF-2alpha (alpha subunit of eukaryotic translation initiation factor 2) and IkappaBalpha, the inhibitor of the transcription factor NF-kappaB, have been proposed as downstream mediators of PKR effects. To evaluate the involvement of NF-kappaB and eIF-2alpha in the induction of apoptosis by PKR, we have used vaccinia virus (VV) recombinants that inducibly express PKR concomitantly with a dominant negative mutant of eIF-2alpha or a repressor form of IkappaBalpha. We found that while expression of PKR by a VV vector resulted in extensive inhibition of protein synthesis and induction of apoptosis, coexpression of PKR with a dominant negative mutant of eIF-2alpha (Ser-51-->Ala) reversed both the PKR-mediated translational block and PKR-induced apoptosis. Coexpression of PKR with a repressor form of IkappaBalpha (Ser-32, 36-Ala) also leads to the inhibition of apoptosis by abolishing NF-kappaB induction, while translation remains blocked. Treating cells with two different proteasome inhibitors which block IkappaBalpha degradation, prevented PKR-induced apoptosis, supporting results from coexpression studies. Biochemical analysis and transient assays revealed that PKR expression by a VV vector induced NF-kappaB binding and transactivation. In addition, upregulation of Fas mRNA transcription occurred during PKR activation. Our findings provide direct evidence for the involvement of eIF-2alpha and NF-kappaB in the induction of apoptosis by PKR.

3T3 Cells↗
PubMed1998

Vaccinia virus E3L protein is an inhibitor of the interferon (i.f.n.)-induced 2-5A synthetase enzyme.

Induction of apoptosis in mammalian cells by double-stranded (ds) RNA-dependent enzymes, protein kinase (PKR), and 2-5A-synthetase/RNase L (referred to as the 2-5A system) might be a mechanism mediating anticellular and antiviral actions of interferon (i.f.n.). To counteract the effect of i.f.n., animal viruses have acquired genes that block specific i.f.n. pathways. Among poxviruses, vaccinia virus (VV) encodes E3L, a dsRNA-binding protein, which inhibits activation of i.f.n.-induced PKR. It has been proposed that E3L might also block activation of the 2-5A system, but direct proof is lacking. To establish if E3L inhibits the 2-5A system, we have developed a method to assay apoptosis induced by increased production of enzymes in the 2-5A pathway, as well as of their putative modulators. This assay is based on the use of cells derived from homozygous PKR knockout mice (Pkr-/-) infected with a VV mutant lacking E3L (delta E3L) and transiently transfected with a luciferase reporter gene together with plasmid vectors expressing 2-5A-synthetase, RNase L, or E3L, all controlled by the same inducible promoter. We found that expression of 2-5A-synthetase inhibited luciferase activity in a dose-response manner, reaching inhibition values of 80% relative to transfections with control plasmids. Similar results were obtained by transfection with an RNase L vector, although in this case the extent of inhibition was further enhanced upon coexpression of 2-5A-synthetase and RNase L. Inhibition of protein synthesis mediated by the 2-5A system correlated well with induction of apoptosis. Transfection of cells with a plasmid vector expressing E3L together with 2-5A-synthetase completely prevented apoptosis induced by this enzyme. We conclude that VV E3L acts as an inhibitor of the i.f.n.-induced 2-5A-synthetase enzyme.

2',5'-Oligoadenylate Synthetase↗
PubMed1998

Recombinant viruses expressing a human malaria antigen can elicit potentially protective immune CD8+ responses in mice.

Extensive studies on protective immunity to rodent malaria provided the basis for the current experiments in which mice were immunized with recombinant (re) influenza and vaccinia viruses expressing selected sequences of the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum. Mice of different H-2 haplotypes immunized with re influenza viruses expressing the immunodominant B cell epitope of this CS protein produced high titers of antibodies to the parasite. A cytotoxic T lymphocyte epitope of the CS protein of P. falciparum, PF3, recognized by CD8+ T cells of H-2(k) mice, was expressed in a re vaccinia virus (VacPf) and a re influenza virus (FluPf). Immunization of mice with either FluPf or VacPf elicited a modest CS-specific CD8+ T cell response detected by interferon gamma secretion of individual immune cells. Priming of mice with FluPf, followed by a booster with VacPf, resulted in a striking enhancement of this T cell response. The reverse protocol, i.e., priming with VacPf followed by a booster with FluPf, failed to enhance the primary response. VacPf also greatly enhanced the primary response of mice injected with P. falciparum sporozoites or with a lipopeptide containing PF3. A booster with FluPf also amplified the response of lipopeptide- or sporozoite-primed mice but less than a VacPf booster did. Although mice are not susceptible to infection by P. falciparum sporozoites, we demonstrated that administration of two distinct immunogens expressing PF3 elicited activated, extravasating CS-specific T cells that protected against an intracerebral VacPf challenge.

Amino Acid Sequence↗
PubMed1998

Multivariate curve resolution: a possible tool in the detection of intermediate structures in protein folding.

Different multivariate data analysis techniques based on factor analysis and multivariate curve resolution are shown for the study of biochemical evolutionary processes like conformational changes and protein folding. Several simulated CD spectral data sets describing different hypothetical protein folding pathways are analyzed and discussed in relation to the feasibility of factor analysis techniques to detect and resolve the number of components needed to explain the evolution of the CD spectra corresponding to the process (i.e., to detect the presence of intermediate forms). When more than two components (the native and unordered forms) are needed to explain the evolution of the spectra, an iterative multivariate curve resolution procedure based on an alternating least squares algorithm is proposed to estimate the CD spectrum corresponding to the intermediate form.

Circular Dichroism↗
PubMed1998

The vaccinia virus 14-kilodalton (A27L) fusion protein forms a triple coiled-coil structure and interacts with the 21-kilodalton (A17L) virus membrane protein through a C-terminal alpha-helix.

The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.

Amino Acid Sequence↗
PubMed1998

Vaccinia virus 15-kilodalton (A14L) protein is essential for assembly and attachment of viral crescents to virosomes.

Early stages in vaccinia virus (VV) assembly involve the recruitment of cellular membranes from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to virus factories (or virosomes). The key viral factors involved in this process are not yet known. We have previously identified and characterized two viral proteins, of 21 kDa (A17L gene) and 15 kDa (A14L gene), that associate with tubulovesicular elements related to the ERGIC and are localized in viral membranes at all stages of virion assembly. We showed that the 21-kDa protein is not responsible for the recruitment of membranes from the ERGIC to viral factories. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, VVindA14L, in which the expression of the A14L gene is inducibly regulated by the Escherichia coli lacI operator-repressor system. Repression of 15-kDa protein synthesis has a dramatic effect on virus yields and severely impairs plaque formation. Compared to wild-type VV, reduced amounts of 15-kDa protein are produced in VVindA14L-infected cells in the presence of IPTG (isopropyl-beta-D-thiogalactoside), and this correlates with a small-plaque phenotype and reduced VVindA14L yields under these conditions. In the absence of the 15-kDa protein, early and late viral protein syntheses proceed normally; however, proteolytic cleavage of the major core precursors is inhibited. Electron microscopic examination of cells infected with VVindA14L under nonpermissive conditions reveals the presence of numerous membranous elements that look like unfinished or disassembled crescents interspersed between electron-dense masses. These abnormal membrane elements are usually well separated from the surfaces of the dense structures. These findings show that the 15-kDa protein is essential for VV morphogenesis and indicate that this polypeptide is necessary both for the correct assembly of viral crescents and for their stable attachment to the surfaces of viral factories.

HeLa Cells↗
PubMed1997

Activation of the IFN-inducible enzyme RNase L causes apoptosis of animal cells.

The interferon (IFN)-induced enzyme RNase L produced by a recombinant vaccinia virus (VV) causes death of mammalian cells with morphological and biochemical characteristics of apoptosis. Coexpression of 2-5A-synthetase enhances apoptosis induced by RNase L Activation of endogenous RNase L by infection with a VV ts mutant (ts22) or with wild-type virus in the presence of the antipoxvirus drug isatin-beta-thiosemicarbazone, a treatment known to significantly increase the amount of double-stranded RNA late during infection, also causes pronounced apoptosis of infected cells. The effects observed with recombinant virus-derived RNase L or with the endogenous enzyme are specific, since apoptosis also occurs in cells derived from mice lacking the IFN-induced protein kinase (PKR). The apoptosis antagonist Bcl-2 prevents induction of cell death by RNase L activation. Apoptosis of mammalian cells by RNase L activation could be a mechanism mediating anticellular actions of IFN.

2',5'-Oligoadenylate Synthetase↗
PubMed1997

The apoptosis pathway triggered by the interferon-induced protein kinase PKR requires the third basic domain, initiates upstream of Bcl-2, and involves ICE-like proteases.

The interferon-induced double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase which exerts antiviral and anticellular functions. The antiviral effect of PKR is mediated by the phosphorylation of the alpha subunit of the translational initiation factor elF-2 alpha, while it is not known whether the anticellular effect is due to phosphorylation of elF-2 alpha, l kappa B, or other unknown substrates. We have previously shown that activation of PKR during infection of cells with a vaccinia virus recombinant expressing the wild-type kinase resulted in a complete inhibition of viral and cellular protein synthesis and in the induction of apoptosis. Here, we report that expression of the human proto-oncogene bcl-2 blocks PKR-induced apoptosis but not PKR-induced inhibition of translation. In addition, PKR-induced apoptosis resulted in a cleavage of the death substrate poly(ADP-ribose) polymerase (PARP). Moreover, induction of apoptosis by PKR was not observed with a mutant lacking the third basic region (aa 234-272). Taken together, these results suggest that the third basic region of PKR is required for PKR-induced apoptosis, the process is initiated upstream of bcl-2 and involves activation of a cellular protease, CPP32, or its family members that cleave PARP.

Apoptosis↗
PubMed1997

Growth of Escherichia coli in acetate as a sole carbon source is inhibited by ankyrin-like repeats present in the 2',5'-linked oligoadenylate-dependent human RNase L enzyme.

Expression of low levels of the 2',5'-linked oligoadenylate-dependent human RNase L, an enzyme induced by interferons, is highly toxic in Escherichia coli. This protein contains an ankyrin domain responsible for RNase L toxicity. The only known ORF in E. coli containing ankyrin repeats is yjaC in the acetate metabolic cluster. We have investigated if expression of mutant forms of RNase L interfere with metabolism of acetate in E. coli. Our findings demonstrate that E. coli expressing RNase L ankyrin repeats is unable to grow in medium containing acetate as the sole carbon source, while it can grow when expressing other domains of the protein. This defect correlates with a severe decrease in the levels of induction of enzymes in the glyoxylate bypass.

Acetates↗
PubMed1997

Bcl-2 prevents nitric oxide-mediated apoptosis and poly(ADP-ribose) polymerase cleavage.

Toxic effects of nitric oxide (NO) were suggested to be mediated by its metabolite peroxynitrite, a strong oxidizing agent. To determine if antioxidative effects of Bcl-2 protooncogene can prevent NO-mediated apoptosis, we used vaccinia virus recombinants expressing mouse inducible NO-synthase, iNOS, or human bcl-2 genes. Expression of iNOS in HeLa G cells induces apoptosis which can be prevented by co-expression of bcl-2 or by addition of reduced glutathione or N-acetylcysteine. We demonstrate that this NO-induced apoptosis proceeds through the activation of interleukin-1 beta-converting enzyme-like proteases and cleavage of the poly(ADP-ribose) polymerase, an effect which is also prevented by Bcl-2.

Acetylcysteine↗