Selective involvement of proteasomes and cysteine proteases in MHC class I antigen presentation.

CTL recognize peptides derived from protein Ags bound to MHC-class I molecules. Proteasomes probably participate in the generation of these peptide epitopes. We investigated the role of proteasomes in the presentation of endogenously synthesized short viral proteins. To this end, we employed proteasome and cysteine protease inhibitors and two closely related recombinant vaccinia viruses that code for 17- and 19-amino acid-long products encompassing murine CMV 9pp89 epitope. Presentation of both minigene products required processing to shorter peptides and was independent of ubiquitination. Proteasomes were necessary for processing the 17-mer product, and cysteine proteases were not required. In contrast, the 19-mer product could be processed in parallel either by proteasomes or by cysteine proteases independently. These results highlight the diversity of alternative processing pathways even for short peptidic Ags, provide evidence for the involvement of cysteine proteases in MHC class I presentation, and show that cleavage by cysteine proteases is governed by sequences flanking the epitope.S

Caspases in virus-infected cells contribute to recognition by CD8+ T lymphocytes

CD8(+) cytotoxic T lymphocytes recognize infected cells in which MHC class I molecules present pathogen-derived peptides that have been processed mainly by proteasomes. Many infections induce a set of proteases, the caspases involved in apoptosis or inflammation. In this study, we report that processing and presentation of a short vaccinia virus-encoded Ag can take place also by a nonproteasomal pathway, which was blocked in infected cells with chemical inhibitors of caspases. By cleaving at noncanonical sites, at least two caspases generated antigenic peptides recognized by T lymphocytes. The sites and the peptidic products were partially overlapping but different to those used and produced by proteasomes in vitro. Antigenic natural peptides produced in infected cells by either pathway were quantitatively and qualitatively similar. Finally, coexpression of the natural vaccinia virus protein B13, which is an inhibitor of caspases and apoptosis, impaired Ag presentation by the caspase pathway in infected cells. These data support the hypothesis that numerous cellular proteolytic systems, including those induced during infection, such as caspases involved in apoptosis or in inflammation, contribute to the repertoire of presented peptides, thereby facilitating immunosurveillance.This work was supported by grants provided by the European Union, Ministerio de Ciencia e Innovación, Comunidad de Madrid, and Instituto de Salud Carlos III (to M.D.V.) and Programa Ramón y Cajal, Comunidad de Madrid, and Instituto de Salud Carlos III (to D.L.). G.L.S. is a Wellcome Principal Research Fellow.S

Protective immunization against murine cytomegalovirus infection using adenoviruses and poxviruses expressing hepatitis B virus chimeras

Recombinant adenoviruses, poxviruses, and plasmid DNA vaccines encoding different hepatitis B virus (HBV)/murine cytomegalovirus (MCMV) protein chimeras were used to immunize mice. Processing of the chimeras resulted in presentation of a protective Ld/CD8+ T-cell epitope of the immediate early 1 protein pp89 (IE1 pp89) of MCMV to the immune system. Different levels of immunogenicity were observed depending on: (i) the type of viral vector used, (ii) whether the antigens were included in the cellular secretion pathway, and (iii) the location of the protective epitope within the chimeric protein. An adenovirus expressing a secretory HBV core protein with the MCMV epitope in its C-terminus induced the highest immune response. When the most immunogenic adenovirus and vaccinia virus were used in a heterologous prime-boost immunization protocol, even higher levels of epitope-specific T cells were obtained. Furthermore, responses were protective against a challenge with MCMV, inducing up to a 96% reduction of viral load in immunized animals, as determined by a sensitive real-time PCR assay. Together, these results confirmed previous observations of the efficient use of adenoviral and poxviral vectors in prime-boost protocols for immunization against diseases whose resolution depends on cellular immunity, as well as the aptness of correctly designed chimeric carrier proteins to facilitate this goal. [Int Microbiol 2007; 10(4):261-269

Cytomegalovirus prevents antigen presentation by blocking the transport of peptide-loaded major histocompatibility complex class I molecules into the medial-Golgi compartment

Selective expression of murine cytomegalovirus (MCMV) immediate-early (IE) genes leads to the presentation by the major histocompatibility complex (MHC) class I molecule L a of a peptide derived from MCMV IE protein pp89 (Reddehase, M. J., J. B. Rothbard, and U. H. Koszinowski. 1989. Nature (Lond.). 337:651). Characterization of endogenous antigenic peptides identified the pp89 peptide as the nonapeptide msYPHFMFFNLt76 (del Val, M., H.-J. Schlicht, T. Ruppert, M. J. Reddehase, and U. H. Koszinowski. 1991. Cell. 66:1145). Subsequent expression of MCMV early genes prevents presentation of pp89 (del Val, M., K. Mfinch, M. J. Reddehase, and U. H. Koszinowski. 1989. Cell. 58:305). We report on the mechanism by which MCMV early genes interfere with antigen presentation. Expression of the IE promoter-driven bacterial gene lacZ by recombinant MCMV subjected antigen presentation of B-galactosidase to the same control and excluded antigen specificity. The La-dependent presence of naturally processed antigenic peptides also in nonpresenting cells located the inhibitory function subsequent to the step of antigen processing. The finding that during the E phase of MCMV gene expression the MHC class I heavy chain glycosylation remained in an Endo H-sensitive form suggested a block within the endoplasmic reticulum/c/s-Golgi compartment. The failure to present antigenic peptides was explained by a general retention of nascent assembled trimolecular MHC class I complexes. Accordingly, at later stages of infection a significant decrease of surface MHC class I expression was seen, whereas other membrane glycoproteins remained unaffected. Thus, MCMV E genes endow this virus with an effective immune evasion potential. These results also indicate that the formation of the trimolecular complex of MHC dass I heavy chain, ~2-microglobulin, and the finally trimmed peptide is completed before entering the medial-Golgi compartment

Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein

Processing of endogenously synthesized proteins generates short peptides that are presented by MHC class I molecules to CD8 T lymphocytes. Here it is documented that not only the sequence of the presented peptide but also the residues by which it is flanked in the protein determine the efficiency of processing and presentation. This became evident when a viral sequence of proven antigenicity was inserted at different positions into an unrelated carrier protein. Not different peptides, but different amounts of the antigenic insert itself were retrieved by isolation of naturally processed peptides from cells expressing the different chimeric proteins. Low yield of antigenic peptide from an unfavorable integration site could be overcome by flanking the insert with oligo-alanine to space it from disruptive neighboring sequences. Notably, the degree of protection against lethal virus disease related directly to the amount of naturally processed antigenic peptide

An endogenous HIV envelope-derived peptide without the terminal NH3+ group anchor is physiologically presented by major histocompatibility complex class I molecules

Cytotoxic T lymphocytes (CTL) recognize viral peptidic antigens presented by major histocompatibility complex (MHC) class I molecules on the surface of infected cells. The CTL response is critical in clearance and prevention of HIV infection. Yet, there are no descriptions of physiological peptides derived from the viral envelope protein. In the few reports on endogenous MHC class I viral peptidic ligands from HIV internal proteins, definitive positive identification by mass spectrometry is lacking. The HIV-1 envelope glycoprotein gp160 induces a strong specific CTL response restricted by several human and murine MHC class I molecules, including H-2Dd. Previous analyses showed that this response can be optimally mimicked with the synthetic decameric peptide 318RGPGRAFVTI327. We aim to identify the endogenous natural peptides mediating the response to this epitope. Our data indicate the presence of, at least, two peptidic species of different length and sharing the same antigenic core, which are associated with the Dd presenting molecule in infected cells. One species is at least, probably, the optimal decapeptide. The second species, identified by mass spectrometry for the first time in HIV, is, unexpectedly, a nonamer, which lacks the correctly positioned N-terminal group to bind to Dd. And yet, it is present in similar amounts and, notably, is equally antigenic. Thus, the physiological set of HIV-derived MHC class I ligands is richer and different than expected from studies with synthetic peptides. This may help raise the plasticity and thus the effectiveness of the immune response against the viral infection. These data have implications for HIV vaccine development.This work was supported by grants from the European Union, Ministerio de Educación y Ciencia, Comisión Interministerial de Ciencia y Tecnología, Comunidad de Madrid, Instituto de Salud Carlos III, and Red Temática de Investigación Cooperativa en SIDA del Fondo de Investigaciones Sanitarias. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.S

A long N-terminal-extended nested set of abundant and antigenic major histocompatibility complex class I natural ligands from HIV envelope protein

Viral antigens complexed with major histocompatibility complex (MHC) class I molecules are recognized by cytotoxic T lymphocytes on infected cells. Assays with synthetic peptides identify optimal MHC class I ligands often used for vaccines. However, when natural peptides are analyzed, more complex mixtures including long peptides bulging in the middle of the binding site or with carboxyl extensions are found, reflecting lack of exposure to carboxypeptidases in the antigen processing pathway. In contrast, precursor peptides are exposed to extensive cytosolic aminopeptidase activity, and fewer than 1% survive, only to be further trimmed in the endoplasmic reticulum. We show here a striking example of a nested set of at least three highly antigenic and similarly abundant natural MHC class I ligands, 15, 10, and 9 amino acids in length, derived from a single human immunodeficiency virus gp160 epitope. Antigen processing, thus, gives rise to a rich pool of possible ligands from which MHC class I molecules can choose. The natural peptide set includes a 15-residue-long peptide with unprecedented 6 N-terminal residues that most likely extend out of the MHC class I binding groove. This 15-mer is the longest natural peptide known recognized by cytotoxic T lymphocytes and is surprisingly protected from aminopeptidase trimming in living cells.This work was supported by grants from European Union, Ministerio de Educación y Ciencia, Comunidad de Madrid, Instituto de Salud Carlos III, Red Temática de Investigación Cooperativa en Sindrome de Inmunodeficiencia Adquirida (SIDA) del Fondo de Investigaciones Sanitarias (to M. D. V.), Comunidad de Madrid, Instituto de Salud Carlos III, Fundación para la Investigación y la Prevención del Sindrome de Inmunodeficiencia Adquirida en España (to D. L.), and by European Commission Grant QLK2-CT-2001-01167 (to P. M. V. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.S

H-2Ld class I molecule protects an HIV N-extended epitope from in vitro trimming by endoplasmic reticulum aminopeptidase associated with antigen processing.

In the classical MHC class I Ag presentation pathway, antigenic peptides derived from viral proteins by multiple proteolytic cleavages are transported to the endoplasmic reticulum lumen and are then exposed to ami-nopeptidase activity. In the current study, a long MHC class I natural ligand recognized by cytotoxic T lymphocytes was used to study the kinetics of degradation by aminopeptidase. The in vitro data indicate that this N-extended peptide is efficiently trimmed to a 9-mer, unless its binding to the MHC molecules protects the full-length peptide.We thank Dr. A. K. Stout of the NIH Tetramer Facility for providing the peptide/MHC complex reagent. This work was suppor ted by grants provi ded by Programa Ramón y Cajal, and Fundación FIPSE to D. L.; by grants provided by Comunidad de Madrid and Ministerio de Educación y Ciencia to M. D. V.; and by a joint grant provided by Instituto de Salud Carlos III to D. L., and M. D. V.S

Presentation of CMV immediate-early antigen to cytolytic T lymphocytes is selectively prevented by viral genes expressed in the early phase

The regulation of antigen processing and presentation to MHC class I-restricted cytolytic T lymphocytes was studied in cells infected with murine cytomegalovirus. Recognition by cytolytic T lymphocytes of the phosphoprotein pp89, the immunodominant viral antigen expressed in the immediate-early phase of infection, was selectively prevented during the subsequent expression of viral early genes. The surface expression of MHC class I glycoproteins and their capacity to present externally added pp89-derived antigenic peptides were not affected. Because recognition of several other antigens occurred during the early phase, a general failure in processing and presentation was excluded. Since neither rate of synthesis, amount, stability, nor nuclear transport of pp89 was modified, the failure in recognition indicates a selective interference with pp89 antigen processing and presentation

Espacios míticos: historias verdaderas, historias literarias

314 p.Las páginas de este libro buscan hurgar en las costuras abiertas del mito, seguir el hilo de sus metamorfosis, contemplarlo como sucesión dinámica y conflictiva de espacios míticos. Esa es la razón por la que el lector podrá encontrar aquí artículos atentos a la pre-historia y a la preliteratura del mito, a sus manifestaciones en el mundo clásico grecolatino, en el arte barroco o en el cine actual, en el santoral cristiano, en las culturas precolombinas o en las literaturas europeas y americanas