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An infectious center viral plaque assay has been utilized to quantitate activated T suppressor (Ts) cells. This assay is based on two observations. Namely, resting T cells do not serve as good replicative hosts for many viruses, including vesicular stomatitis virus (VSV), and that Ts cells can be enriched by their ability to bind to antigen-coated dishes. Our data show that Ts cells specific for either the TNP hapten or for dextran will replicate VSV upon antigenic and/or mitogenic activation, whereas resting Ts and hapten-specific B cells are less efficient in this process. This system will now allow the direct quantitation of Ts cells and their activation properties. 相似文献
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Temperature-Sensitive Mutants of Complementation Group E of Vesicular Stomatitis Virus New Jersey Serotype Possess Altered NS Polypeptides
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In vesicular stomatitis virus New Jersey serotype polyacrylamide gel electrophoresis was unable to distinguish the polypeptides of the temperature-sensitive (ts) mutants of complementation groups A, B, C, and F from those of the wild-type virus. However, the NS polypeptide of the representative mutant of group E, ts E1, had a significantly greater electrophoretic mobility than that of the wild-type virus NS polypeptide. The electrophoretic mobilities of the NS polypeptides of the three mutants of complementation group E varied, being greatest in the case of ts E1, slightly less for ts E2, and only a little greater than that of wild-type virus NS polypeptide in the case of ts E3. Since the NS polypeptides of the revertant clones ts E1/R1 and ts E3/R1 have mobilities identical to that of wild-type NS polypeptide, the observed altered mobilities of the group E mutants are almost certainly the direct result of the ts mutations in the E locus. The electrophoretic mobilities of the intracellular NS polypeptides of the group E mutants were indistinguishable from those of their virion NS polypeptides. The electrophoretic mobilities of the NS polypeptides of the group E mutants synthesized in vitro using mRNA synthesized in vitro by TNP were identical to those of the NS polypeptides of their purified virions. The NS polypeptides of all three mutants were labeled with (32)P(i) to approximately the same extent as wild-type virus NS polypeptide, indicating that gross differences in phosphorylation of this polypeptide are unlikely to account for the altered mobilities. We propose a model in which the NS polypeptide consists of at least three loops held in this configuration by hydrophobic or ionic forces or both and stabilized by phosphodiester bridges. If a mutation affects one of the amino acids to which the phosphate is covalently linked, the phosphodiester bridge cannot be formed, and, as a result, in the presence of sodium dodecyl sulfate the affected loop opens and thus the NS polypeptide migrates further into the gel. Such a configuration may also explain the multifunctional nature of the NS polypeptide. 相似文献
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Zhang XM Ellis S Sriratana A Mitchell CA Rowe T 《The Journal of biological chemistry》2004,279(41):43027-43034
Rab/Ypt GTPases play key roles in the regulation of vesicular trafficking. They perform most of their functions in a GTP-bound form by interacting with specific downstream effectors. The exocyst is a complex of eight polypeptides involved in constitutive secretion and functions as an effector for multiple Ras-related small GTPases, including the Rab protein Sec4p in yeast. In this study, we have examined the localization and function of the Sec15 exocyst subunit in mammalian cells. Overexpressed Sec15 associated with clusters of tubular/vesicular elements that were concentrated in the perinuclear region. The tubular/vesicular clusters were dispersed throughout the cytoplasm upon treatment with the microtubule-depolymerizing agent nocodazole and were accessible to endocytosed transferrin, but not exocytic cargo (vesicular stomatitis virus glycoprotein). Consistent with these observations, Sec15 colocalized selectively with the recycling endosome marker Rab11 and exhibited a GTP-dependent interaction with the Rab11 GTPase, but not with Rab4, Rab6, or Rab7. These findings provide the first evidence that the exocyst functions as a Rab effector complex in mammalian cells. 相似文献
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Sequence Determination of the (+) Leader RNA Regions of the Vesicular Stomatitis Virus Chandipura, Cocal, and Piry Serotype Genomes 总被引:10,自引:5,他引:5
Using 3'-end-labeled genome probes, cells infected with vesicular stomatitis virus Chandipura, Cocal, and Piry serotypes were shown to contain (+) leader RNAs of approximately 50 nucleotides in length. The nucleotide sequence of the leader RNA regions of these genomes was determined and compared with the previously reported sequences of both the (+) and (-) leader RNA regions of other vesicular stomatitis virus serotypes. Regions of strong conservation of nucleotide sequence among the various vesicular stomatitis virus serotypes suggest those nucleotides thought to be involved in control functions during vesicular stomatitis virus replication. 相似文献
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Comparisons of nucleotide sequences in the genomes of the New Jersey and Indiana serotypes of vesicular stomatitis virus. 总被引:7,自引:5,他引:2
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Nucleotide sequences of around 200 residues were determined adjacent to the 3' terminus of the genome RNA of vesicular stomatitis virus, New Jersey serotype, and adjacent to the 3'-terminal polyadenylic acid tract of the N protein mRNA of the same virus. These sequences were compared with the corresponding sequences previously determined for the Indiana serotype of vesicular stomatitis virus. The sequences obtained for the two strains were readily aligned, showing 70.8% homology overall. Examination of the sequences allowed identification of the translation initiation and termination codons for the N mRNA of each serotype. The deduced N-terminal and C-terminal amino acid sequences of the two N polypeptides were each similar, and most of the differences between them consisted of substitution by a clearly homologous amino acid. It was proposed that these nucleotide sequences, within limits imposed by their functions, comprise reasonably representative measures of the extent of sequence homology between the genomes of the two serotypes, and that this is higher than previously estimated, but with little exact homology over extended regions. 相似文献
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Vesicular stomatitis virus contains a single structural glycoprotein whose carbohydrate sequences are probably specified by the host cell. The glycopeptides derived by Pronase digestion of the glycoprotein of vesicular stomatitis virus grown in HeLa cells have an average molecular weight of 1,800. There are multiple oligosaccharide chains on the vesicular stomatitis virus glycoprotein with protein-carbohydrate linkages that are cleaved only by strong alkali under reducing conditions, suggesting that they contain asparagine and N-acetylglucosamine. The oligosaccharide moieties, in addition, appear to be heterogeneous in sequence on the basis of their mobilities during electrophoresis and their sensitivities to cleavage by an endoglycosidase. The carbohydrate-peptide linkage region of the major class of oligosaccharides of the vesicular stomatitis virus glycoprotein has the proposed sequence: (see article). 相似文献
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Comparative Membrane Microviscosity of Fish and Mammalian Rhabdoviruses Studied by Fluorescence Depolarization
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Norman F. Moore Yechezkel Barenholz Philip E. McAllister Robert R. Wagner 《Journal of virology》1976,19(1):275-278
The microviscosity of the hydrophobic region of the membrane of infectious hematopoietic necrosis virus was determined using fluorescence depolarization analysis of the probe 1,6-diphenyl-1,3,5-hexatriene and was found to be much lower at 37 C than that of another rhabdovirus, vesicular stomatitis virus. However, the microviscosity of this fish virus at 18 C, the temperature at which it was grown, corresponded to the microviscosity of vesicular stomatitis virus at 37 C. Data obtained with the fish virus host cell (chinook salmon embryo cells) grown at 18 C suggest that its membranes have a lower microviscosity than either L-929 or BHK-21 cells (the vesicular stomatitis virus host cells) grown at 37 C. 相似文献
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Mx gene control of interferon action: different kinetics of the antiviral state against influenza virus and vesicular stomatitis virus. 总被引:3,自引:2,他引:1
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The allele Mx regulates the extent to which interferon alpha/beta inhibits the growth of influenza viruses in mouse cells such as peritoneal macrophages. The time course of induction of the antiviral state against an influenza A virus is comparable in macrophages with and without Mx and is similar to that found with vesicular stomatitis virus. In contrast, the decay of the antiviral state against influenza virus is markedly slower in Mx-positive cells and slower than that against vesicular stomatitis virus observed in either Mx-positive or Mx-negative cells. Thus, after removal of interferon alpha/beta, Mx-positive cells remain protected against influenza virus at times when they have lost protection against vesicular stomatitis virus. These results suggest that interferon alpha/beta treatment activates different antiviral mechanisms, each acting against distinct groups of viruses and each independently controlled by host genes. 相似文献
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CER cells infected with vesicular stomatitis virus showed a morphology similar to that observed after cytochalasin B treatment. Temperature-sensitive mutants affected in envelope protein maturation did not induce those morphological changes at a nonpermissive temperature. In addition, the cytoskeleton was not implicated in vesicular stomatitis virus reproduction. 相似文献
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Enhanced Mutability Associated with a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus 总被引:5,自引:4,他引:1
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C. R. Pringle V. Devine M. Wilkie C. M. Preston A. Dolan D. J. McGeoch 《Journal of virology》1981,39(2):377-389
Temperature-sensitive (ts) mutant tsD1 of vesicular stomatitis virus, New Jersey serotype, is the sole representative of complementation group D. Clones derived from this mutant exhibited three different phenotypes with respect to electrophoretic mobility of the G and N polypeptides of the virion in sodium dodecyl sulfate-polyacrylamide gel. Analysis of non-ts pseudorevertants showed that none of the three phenotypes was associated with the temperature sensitivity of mutant tsD1. Additional phenotypes, some also involving the NS polypeptide, appeared during sequential cloning, indicating that mutations were generated at high frequency during replication of tsD1. Furthermore, mutations altering the electrophoretic mobility of the G, N, NS, and M polypeptides were induced in heterologous viruses multiplying in the same cells as tsD1. These heterologous viruses included another complementing ts mutant of vesicular stomatitis virus New Jersey and ts mutants of vesicular stomatitis virus Indiana and Chandipura virus. Complete or incomplete virions of tsD1 appeared to be equally efficient inducers of mutations in heterologous viruses. Analysis of the progeny of a mixed infection of two complementing ts mutants of vesicular stomatitis virus New Jersey with electrophoretically distinguishable G, N, NS, and M proteins yielded no recombinants and excluded recombination as a factor in the generation of the electrophoretic mobility variants. In vitro translation of total cytoplasmic RNA from BHK cells indicated that post-translational processing was not responsible for the aberrant electrophoretic mobility of the N, NS, and M protein mutants. Aberrant glycosylation could account for three of four G protein mutants, however. Some clones of tsD1 had an N polypeptide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel than did the wild type, suggesting that the polypeptide might be shorter by about 10 amino acids. Determination of the nucleotide sequence to about 200 residues from each terminus of the N gene of one of these clones, a revertant, and the wild-type parent revealed no changes compatible with synthesis of a shorter polypeptide by premature termination or late initiation of translation. The sequence data indicated, however, that the N-protein mutant and its revertant differed from the parental wild type in two of the 399 nucleotides determined. These sequencing results and the phenomenon of enhanced mutability associated with mutant tsD1 reveal that rapid and extensive evolution of the viral genome can occur during the course of normal cytolytic infection of cultured cells. 相似文献
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Injection of mice with antibody to mouse interferon alpha/beta decreases the level of 2''-5'' oligoadenylate synthetase in peritoneal macrophages. 总被引:10,自引:6,他引:4
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Injection of conventional or axenic weanling mice with potent sheep or goat antibody to mouse interferon alpha/beta resulted in a decrease in the basal level of 2-5A synthetase in resting peritoneal macrophages and rendered these cells permissive for vesicular stomatitis virus. There was a good inverse correlation between the level of 2-5A synthetase in peritoneal macrophages and the permissivity of these cells for vesicular stomatitis virus. The peritoneal macrophages of 1- and 2-week-old mice had low levels of 2-5A synthetase and were permissive for vesicular stomatitis virus, whereas at 3 weeks (and after) there was a marked increase in the level of 2-5A synthetase in peritoneal macrophages, and these cells were no longer permissive for vesicular stomatitis virus. We suggest that low levels of interferon alpha or beta or both are produced in normal mice, and that this interferon contributes to host defense by inducing and maintaining an antiviral state in some cells. 相似文献
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The guanine-nucleotide exchange factor Vav is a crucial regulator of B cell receptor activation and B cell responses to nonrepetitive antigens. 总被引:1,自引:0,他引:1
M F Bachmann L Nitschke C Krawczyk K Tedford P S Ohashi K D Fischer J M Penninger 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(1):137-142
The proto-oncogene product Vav is required for receptor clustering, proliferation, and differentiation of T cells, and Vav was identified as a substrate in the TCR and B cell receptor signaling pathway. The role of Vav in B cell responses to Ag challenge in vivo is not known. In this study, we show that Vav regulates B cell proliferation following in vitro activation of Ag receptors, but Vav has no apparent role in CD40-, IL-4-, or LPS-induced B cell activation. Increased degrees of Ag receptor cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav-/- B cells. In vivo, vav-/- mice mounted protective antiviral IgM and IgG responses to infections with vesicular stomatitis virus and recombinant vaccinia virus expressing the vesicular stomatitis virus glycoprotein, which harbor repetitive surface epitopes that directly cross-link the Ag receptor and activate B cells in the absence of T cell help. vav-/- B cells also responded normally to the polyvalent, repetitive hapten Ag trinitrophenyl (TNP)-Ficoll that effectively cross-links B cell receptors. However, vav-/- mice failed to mount immune responses to the nonrepetitive, T cell-dependent hapten Ag (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)-OVA. These results provide the first genetic evidence on the role of the guanine exchange factor Vav in immune responses to viral infections and antigenic challenge in vivo, and suggest that Vav adjusts the threshold for Ag receptor-mediated B cell activation depending on the nature of the Ag. 相似文献
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Specificity of initiation factor preparations from poliovirus-infected cells. 总被引:4,自引:3,他引:1
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Crude initiation factor preparations from poliovirus-infected cells stimulated the translation of poliovirus RNA in vitro, but were inactive for the translation of host cell or vesicular stomatitis virus mRNA's. In contrast, similar preparations from either uninfected or vesicular stomatitis virus-infected cells supported the initiation of translation of host cell mRNA's and both viral mRNA's. These results reflect a specific alteration of some components(s) of the initiation factor preparation from poliovirus-infected cells which is consistent with the ability of the virus to inhibit the translation of host cell and vesicular stomatitis virus-directed protein synthesis. 相似文献