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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.
Mol Cell Proteomics 2006 May
PMID:Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection. 1643 54

Aims-To evaluate factors which ameliorate false positive artefacts with direct in situ PCR using labelled dNTPs; to investigate the use of labelled primers to overcome this artefact whilst maintaining sensitivity.Methods-Sections of measles (RNA virus) infected Vero cells with cytoplasmic signal or cytomegalovirus (DNA virus) infected fibroblasts with nuclear signal were collected. In situ PCR (or in situ RT-PCR) was carried out by methods permitting evaporation. Reagents or conditions which may control false positive artefacts using labelled dNTPs were investigated systematically. Labelled primers were tested to overcome artefacts, with adjuncts which improve sensitivity.Results-No reagent nor condition investigated was able to control the artefact with labelled dNTPs. Excessive digestion and incomplete DNAse treatments exacerbated the artefact, whereas novobiocin decreased both specific signal and artefact. However, the artefact was controlled by labelled primers, albeit with relatively low sensitivity. Sensitivity using labelled primers could be increased using alcohol fixation, albumin or Perfectmatch.Conclusions-A repair process is implicated for the artefact using labelled dNTPs. Excessive digestion or DNAse treatment may exacerbate DNA damage by disrupting histones or the DNA, respectively. Labelled primers control this artefact, albeit with reduced sensitivity, which may be improved by precipitation fixatives (alcohol) and reagents which enhance specific reaction.
Clin Mol Pathol 1996 Dec
PMID:Direct in situ nucleic acid amplification: control of artefact and use of labelled primers. 1669 1

Signaling lymphocyte activation molecule (SLAM; also known as CD150) is a newly identified cellular receptor for measles virus (MV). The interaction between MV Haemagglutin (MVH) and SLAM is an initial step for MV entry. We have identified several novel SLAM binding sites at residues S429, T436 and H437 of MVH protein and MVH mutants in these residues dramatically decrease the ability to interaction with the cell surface SLAM and fail to coprecipitation with SLAM in vivo as well as malfunction in syncytium formation. At the same time, K58, S59 and H61 of SLAM was also identified to be critical for MVH and SLAM binding. Further, these residues may be useful targets for the development of measles therapy.
J Biochem Mol Biol 2006 Jul 31
PMID:Identification of amino acid residues involved in the interaction between measles virus Haemagglutin (MVH) and its human cell receptor (signaling lymphocyte activation molecule, SLAM). 1688 84

Pancreatic cancer has a poor prognosis with an annual mortality rate close to the annual incidence rate. We evaluated whether the expression of measles virus fusogenic membrane glycoproteins (FMG) H and F will enhance chemotherapy. Using Chou-Talalay analysis, we showed in vitro in pancreatic cancer cells that the expression of FMG often synergistically enhances clinically relevant chemotherapy. Furthermore, cell fusion in combination with chemotherapy resulted in strongly enhanced Annexin V binding, an early marker for apoptosis, when compared with single treatment. We showed in an i.p. and s.c. pancreatic xenograft model that the administration of a replication-defective adenoviral vector Ad.H/F encoding tumor-restricted FMG in combination with gemcitabine significantly enhanced treatment outcome when compared with treatment with each compound individually. To improve tumor transduction efficiency, the Ad.H/F vector was also transcomplemented with an oncolytic replication-restricted adenovirus (Ad.COX*MK), resulting in significantly improved treatment efficacy. We assessed treatment efficacy by survival analysis or measuring growth, respectively. In the i.p. model, on day 120, three of eight animals treated with this novel triple therapy consisting of Ad.H/F, gemcitabine, and Ad.COX*MK were alive and tumor free. Treatment with Ad.H/F and Ad.COX*MK resulted in one long-term survivor. In all other treatment groups, there were no long-term survivors. The significantly improved therapeutic outcome of animals receiving the triple therapy was attributed to multiple factors, including most likely improved FMG expression throughout the tumor and enhanced sensitivity of the tumor cells to gemcitabine by adenoviral gene products but also FMG expression. Qualitatively similar results were obtained in a s.c. pancreatic xenograft model.
Mol Cancer Ther 2006 Aug
PMID:Enhanced killing of pancreatic cancer cells by expression of fusogenic membrane glycoproteins in combination with chemotherapy. 1692 22

Attenuated measles viruses (MVs) propagate selectively in human tumor cells, and phase I clinical trials are currently underway to test their oncolytic activity. A major theoretical impediment to systemic MV application is the presence of pre-existing antiviral immunity. We hypothesized that autologous MV-infected cells might be a more reliable vehicle than cell-free virions to deliver the infection to tumor cells in subjects with neutralizing titers of anti-measles antibodies. Our in vitro studies, using a dual-color fluorescent model, demonstrated efficient cell-to-cell transfer of infection via heterofusion. In contrast to infection by naked virions, heterofusion between infected cell carriers and tumor cells was more resistant to antibody neutralization. Infected monocytic, endothelial, or stimulated peripheral blood cells could deliver oncolytic MV to tumor lesions in vivo, after intravenous (i.v.) or intraperitoneal (i.p.) administration. Single or repeated i.p. injections of monocytic carriers significantly improved survival of animals bearing human ovarian cancer xenografts. Systemic or i.p. injection of MV-infected cells successfully transferred infection by heterofusion to Raji lymphomas or hepatocellular carcinoma tumors in the presence of neutralizing antibodies. These results suggest a novel strategy for systemic delivery of oncolytic virotherapy in cancer patients that can "bypass" the pre-existing humoral immunity against MV.
Mol Ther 2007 Jan
PMID:Infected cell carriers: a new strategy for systemic delivery of oncolytic measles viruses in cancer virotherapy. 1716 82

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.
Mol Cell 2007 Jan 12
PMID:Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus. 1721 73

The innate antiviral responses of tumor cells are often impaired but may still be sufficient to impede the intratumoral spread of an oncolytic virus. Here, we establish that the oncolytic measles virus (MV-eGFP) induces interferon (IFN) production in human myeloma and ovarian cancer cells. In addition, MV gene expression and virus progeny production were inhibited by IFN treatment of these tumor cells. The P gene of wild-type measles virus encodes P/V/C proteins known to antagonize IFN induction and/or response. We therefore engineered MV-eGFP for IFN evasion and more efficient intratumoral spread by arming it with the P gene from wild-type IC-B strain MV, thus generating MV-eGFP-Pwt. The chimeric virus exhibited reduced IFN sensitivity and diminished capacity to induce IFN in BJAB lymphoma, ARH-77 myeloma cells, and activated peripheral blood mononuclear cells. Interestingly, unlike the wild-type MV, MV-eGFP-Pwt was unable to shut down IFN induction completely. In immunocompromised mice bearing human myeloma xenografts, intravenously administered MV-eGFP-Pwt showed significantly enhanced oncolytic potency compared to MV-eGFP. These results indicate that oncolytic viruses are subject to control by the innate immune defenses of human tumor cells and may therefore be more effective if their natural ability to combat innate immunity is maintained.
Mol Ther 2007 Mar
PMID:Engineering oncolytic measles virus to circumvent the intracellular innate immune response. 1724 55

A retargeted measles virus strain MV-GFP-H(AA)-scEGFR was generated by engineering the MV-NSe Edmonston vaccine strain to incorporate both CD46 (Y481A) and signaling lymphocyte activation molecule (SLAM) (R533A) ablating mutations in the hemagglutinin protein in combination with the display of a single-chain antibody against epidermal growth factor receptor (EGFR) at the C terminus of hemagglutinin. The unmodified MV-GFP virus was used as a positive control. Specificity of the EGFR retargeted virus was demonstrated in non-permissive Chinese hamster ovary (CHO) cells stably transfected to express either the natural receptors CD46 or SLAM or the target receptors EGFR and EGFRvIII. In vitro, the retargeted virus had potent antitumor activity against EGFR- or EGFRvIII-overexpressing primary glioblastoma multi-forme (GBM) cell lines that was comparable to the activity of the unmodified MV-GFP virus. Intratumoral administration of MV-GFP-H(AA)-scEGFRvIII in orthotopic GBM12 xenografts resulted in tumor regression, as demonstrated by bioluminescence imaging and significant prolongation of survival, that was comparable to the effect of the unmodified strain. In contrast to MV-GFP, central nervous system administration of the targeted MV-GFP-H(AA)-scEGFR virus in measles replication-permissive Ifnar(ko) CD46 transgenic mice resulted in no neurotoxicity. In conclusion, EGFR-retargeted measles virus strains have comparable therapeutic efficacy to the unmodified virus in glioma cells overexpressing EGFR or EGFRvIII in vivo and in vitro, and improved therapeutic index, a finding with potential translational implications in glioma virotherapy.
Mol Ther 2007 Apr
PMID:Epidermal growth factor receptor (EGFR)-retargeted measles virus strains effectively target EGFR- or EGFRvIII expressing gliomas. 1729 4

An immunocompetent model is required to test therapeutic regimens for clinical trials with the oncolytic measles virus (MV). Toward developing this model, a retargeted MV that enters murine colon adenocarcinoma cells forming tumors in syngeneic C57BL/6 mice was generated. Since MV infection tends to be less efficient in murine than in human cells, the targeted virus was also armed with the prodrug convertase, purine nucleoside phosphorylase (PNP), and named MV-PNP-antiCEA. We have shown before that in cultured cells, infection with this virus activated the prodrug, 6-methylpurine-2'-deoxyriboside (MeP-dR), causing extensive cytotoxicity. When injected intratumorally (IT), MV-PNP-antiCEA inhibited subcutaneous tumor growth marginally, but subsequent administration of the prodrug enhanced the oncolytic effect. Systemic delivery of MV-PNP-antiCEA alone had no substantial oncolytic effects, but in combination with the prodrug it was therapeutic, revealing synergistic effects between virus and prodrug. Immunosuppression with cyclophosphamide (CPA) retarded the appearance of MV neutralizing antibodies and enhanced oncolytic efficacy: survival was 100%, with 9 out of 10 animals going into complete remission. This immunocompetent murine model facilitates the testing of therapeutic regimens for clinical trials.
Mol Ther 2007 Nov
PMID:An immunocompetent murine model for oncolysis with an armed and targeted measles virus. 1771 31

Measles virus is a highly pathogenic virus that infects roughly 20 million people per year. We report here the crystal structure of the measles virus hemagglutinin, the surface glycoprotein responsible for the binding of measles virus to its host cell receptors. Although the protein lacks neuraminidase activity, its structure resembles a 'dead' neuraminidase fold, presenting spatially distinct receptor-binding sites for its receptors CD46 and SLAM.
Nat Struct Mol Biol 2007 Dec
PMID:Structure of the measles virus hemagglutinin. 1802 16


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