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1.
PBMC from healthy adult individuals seropositive for measles virus (MV) were tested for their capacity to proliferate to UV-inactivated MV (UV-MV) or to autologous MV-infected EBV-transformed B cell lines (EBV-BC). MV-specific T cell responses were observed in 11 of 15 donors tested (stimulation index greater than 2), when optimal doses of UV-MV were used in proliferative assays. T cell clones were generated from PBMC of three donors responding to MV, by using either UV-MV or MV-infected autologous EBV-BC as APC. Stimulation with UV-MV generated exclusively CD3+ CD4+ CD8- MV-specific T cells, whereas after stimulation of PBMC with MV-infected EBV-BC, both CD3+ CD4+ CD8- and CD3+ CD4- CD8+ MV-specific T cell clones were obtained. Of 19 CD4+ T cell clones tested so far, 7 clones reacted specifically with purified fusion protein and 1 with purified hemagglutinin protein. Seven clones proliferated in response to the internal proteins of MV. Three clones reacted to whole virus but not to one of the purified proteins, whereas one clone seemed to recognize more than one polypeptide. Some of the T cell clones, generated from in vitro stimulation of PBMC with UV-MV, failed to recognize MV Ag when MV-infected EBV-BC were used as APC instead of UV-MV and PBMC. CD3+ CD4+ CD8- T cell clones recognized MV in association with HLA class II Ag (HLA-DQ or -DR), and most of them displayed CTL activity to autologous MV-infected EBV-BC. All CD4+ HLA class II-restricted CTL clones thus far tested were capable of assisting B lymphocytes for the production of MV-specific antibody. The CD4- CD8+ T cell clone MARO 1 recognized MV in association with HLA class I molecules and displayed cytotoxic activity toward MV-infected EBV-BC.  相似文献   

2.
Nonstructural proteins encoded by measles virus (MV) include the V protein which is translated from an edited P mRNA. V protein is not associated with intracellular or released viral particles and has recently been found to be dispensable for MV propagation in cell culture (H. Schneider, K. Kaelin, and M. A. Billeter, Virology 227:314–322, 1997). Using recombinant MVs (strain Edmonston [ED]) genetically engineered to overexpress V protein (ED-V+) or to be deficient for V protein (ED-V−), we found that in the absence of V both MV-specific proteins and RNAs accumulated to levels higher than those in the parental MV molecular clone (ED-tag), whereas MV-specific gene expression was strongly attenuated in human U-87 glioblastomas cells after infection with ED-V+. The titers of virus released from these cells 48 h after infection with either V mutant virus were lower than those from cells infected with ED-tag. Similarly, significantly reduced titers of infectious virus were reisolated from lung tissue of cotton rats (Sigmodon hispidus) after intranasal infection with both editing mutants compared to titers isolated from ED-tag-infected animals. In cell culture, expression of V protein led to a redistribution of MV N protein in doubly transfected Cos-7 cells, indicating that these proteins form heterologous complexes. This interaction was further confirmed by using a two-hybrid approach with both proteins expressed as Gal4 or VP16 fusion products. Moreover, V protein efficiently competed complexes formed between MV N and P proteins. These findings indicate that V protein acts to balance accumulation of viral gene products in cell culture, and this may be dependent on its interaction with MV N protein. Furthermore, expression of V protein may contribute to viral pathogenicity in vivo.  相似文献   

3.
4.
Measles virus (MV)-specific murine helper T cell clones (Thy-1.2+, CD4+, CD8-) were generated from mice immunized with MV-infected mouse brain homogenate by limiting dilution and in vitro stimulation of spleen cells with UV-inactivated MV Ag. The protein specificity of 7 out of 37 stable T cell clones, which displayed MHC-restricted MV Ag recognition, could be assessed by using purified MV proteins. Two fusion (F) protein-specific, two hemagglutinin-specific, and three nucleoprotein- or matrix protein-specific clones were shown to be established. The F protein-specific T cell clones together with a panel of previously generated F protein-specific T cell clones were characterized for their fine specificity by using beta-galactosidase fusion products, which contained different parts of the F protein. It was shown that at least two epitopes on the major part of the F protein (amino acid 2-513) can be recognized by mouse T cells. Functional characterization of three T cell clones showed that they were able to assist MV-specific B cells and bystander B cells for antibody production. Furthermore, they were shown to produce the lymphokines IL-2 and IFN-gamma. It was also shown that these T cell clones induced a MV-specific delayed type hypersensitivity response. These observations suggest that all of the T cell clones characterized belong to the TH1 helper subset.  相似文献   

5.
Measles remains a significant childhood disease, and is associated with a transient immune suppression. Paradoxically, measles virus (MV) infection also induces robust MV-specific immune responses. Current hypotheses for the mechanism underlying measles immune suppression focus on functional impairment of lymphocytes or antigen-presenting cells, caused by infection with or exposure to MV. We have generated stable recombinant MVs that express enhanced green fluorescent protein, and remain virulent in non-human primates. By performing a comprehensive study of virological, immunological, hematological and histopathological observations made in animals euthanized at different time points after MV infection, we developed a model explaining measles immune suppression which fits with the “measles paradox”. Here we show that MV preferentially infects CD45RA memory T-lymphocytes and follicular B-lymphocytes, resulting in high infection levels in these populations. After the peak of viremia MV-infected lymphocytes were cleared within days, followed by immune activation and lymph node enlargement. During this period tuberculin-specific T-lymphocyte responses disappeared, whilst strong MV-specific T-lymphocyte responses emerged. Histopathological analysis of lymphoid tissues showed lymphocyte depletion in the B- and T-cell areas in the absence of apoptotic cells, paralleled by infiltration of T-lymphocytes into B-cell follicles and reappearance of proliferating cells. Our findings indicate an immune-mediated clearance of MV-infected CD45RA memory T-lymphocytes and follicular B-lymphocytes, which causes temporary immunological amnesia. The rapid oligoclonal expansion of MV-specific lymphocytes and bystander cells masks this depletion, explaining the short duration of measles lymphopenia yet long duration of immune suppression.  相似文献   

6.
The complete nucleotide sequence of the phosphoprotein (P) gene of the Yamagata-1 strain of a defective subacute sclerosing panencephalitis (SSPE) virus was determined. Comparison with the P gene of the Edmonston strain of measles virus (MV) revealed 44 differences of which 23 nucleotides substitutions were identical with those revealed between other SSPE viruses and MV (Cattaneo et al. (1989) Virology 173, 415-425). The consensus sequence of the G insertion site was completely conserved, whereas mRNAs with one or three non-templated G residue insertions were found in addition to the mRNA of the exact genome copy. As a result of the frameshift downstream of the site of G insertion, the cysteine-rich V protein was predicted from the one G-inserted mRNA besides the P and C proteins predicted from the genome-copied mRNA.  相似文献   

7.
Measles virus (MV) vaccine effectively protects seronegative individuals against infection. However, inhibition of vaccine-induced seroconversion by maternal antibodies after vaccination remains a problem, as it leaves infants susceptible to MV infection. In cotton rats, passive transfer of MV-specific IgG mimics maternal antibodies and inhibits vaccine-induced seroconversion. Here, we report that immunization in the presence of passively transferred IgG inhibits the secretion of neutralizing antibodies but not the generation of MV-specific B cells. This finding suggested that MV-specific B cells require an additional stimulus to mature into antibody-secreting plasma cells. In order to provide such a stimulus, we generated a recombinant Newcastle disease virus (NDV) expressing the MV hemagglutinin (NDV-H). In contrast to MV, NDV-H induced high levels of type I interferon in plasmacytoid dendritic cells and in lung tissue. In cotton rats immunized with NDV-H, neutralizing antibodies were also generated in the presence of passively transferred antibodies. In the latter case, however, the level and kinetics of antibody generation were reduced. In vitro, alpha interferon stimulated the activation of MV-specific B cells from MV-immune spleen cells. NDV infection (which induces alpha interferon) had the same effect, and stimulation could be abrogated by antibodies neutralizing alpha interferon, but not interleukin 6 (IL-6). In vivo, coapplication of UV-inactivated MV with NDV led to increased MV-specific antibody production in the presence and absence of passively transferred antibodies. These data indicate that MV-specific B cells are being generated after immunization in the presence of maternal antibodies and that the provision of alpha interferon as an additional signal leads to antibody secretion.  相似文献   

8.
The presence of increased IgG in the brains of humans with infectious and inflammatory CNS diseases of unknown etiology such as multiple sclerosis may be a clue to the cause of disease. For example, the intrathecally synthesized oligoclonal bands (OGBs) in diseases such as subacute sclerosing panencephalitis (SSPE) or cryptococcal meningitis have been shown to represent Ab directed against the causative agents, measles virus (MV) or Cryptococcus neoformans, respectively. Using SSPE as a model system, we have developed a PCR-based strategy to analyze the repertoire of IgG V region sequences expressed in SSPE brain. We observed abnormal expression of germline V segments, overrepresentation of particular sequences that correspond to the oligoclonal bands, and substantial somatic mutation of most clones from the germline, which, taken together, constitute features of Ag-driven selection in the IgG response. Using the most abundant or most highly mutated gamma H chain and kappa or lambda L chain sequences in various combinations, we constructed functional Abs in IgG mammalian expression vectors. Three Abs specifically stained MV-infected cells. One Ab also stained cells transfected with the MV nucleoprotein, and a second Ab stained cells transfected with the MV-fusion protein. This technique demonstrates that functional Abs produced from putative disease-relevant IgG sequences can be used to recognize their corresponding Ags.  相似文献   

9.
10.
In the present study we describe a live vaccine against measles virus (MV) infection on the basis of attenuated Salmonella typhimurium aroA secreting MV antigens via the Escherichia coli alpha-hemolysin secretion system. Two well-characterized MV epitopes, a B-cell epitope of the MV fusion protein (amino acids 404-414) and a T-cell epitope of the MV nucleocapsid protein (amino acids 79-99) were fused as single or repeating units to the C-terminal secretion signal of the E. coli hemolysin and expressed in secreted form by the attenuated S. typhimurium aroA SL7207. Immunization of MV-susceptible C3H mice revealed that S. typhimurium SL7207 secreting these antigens provoked a humoral and a cellular MV-specific immune response, respectively. Mice vaccinated orally with a combination of both recombinant S. typhimurium strains showed partial protection against a lethal MV encephalitis after intracerebral challenge with a rodent-adapted, neurotropic MV strain.  相似文献   

11.
The nucleotide sequence of the P gene of human parainfluenza virus type 1 (PIV1) was determined from cloned cDNA copies of the mRNA. By analogy with the gene organization of Sendai virus, two open reading frames in the mRNA sense of the gene were identified as coding sequences for the P protein (568 amino acids with an estimated molecular weight of 64,655) and the C protein (204 amino acids with an estimated molecular weight of 24,108). Comparison of the deduced amino acid sequences of the P and C proteins of PIV1 with those of Sendai virus showed a high degree of homology. However, a sequence for the cysteine-rich V protein, which was considered a common feature of other paramyxoviruses, was interrupted by the presence of multiple stop codons. The sequence analysis of three P-gene-specific cDNA clones generated from genomic RNA by polymerase chain reaction and one additional clone generated from mRNA confirmed that the coding sequence for the cysteine-rich region is silent in the PIV1 gene and thus is not translated into protein. Two potential editing sites with the consensus sequence 3'UUYUCCC were found in the PIV1 P gene at positions 564 to 570 and 1430 to 1436. However, examination of the PIV1 mRNA population by a primer extension method indicated that neither of these sites is utilized. These results indicate that the PIV1 P gene has a coding strategy different from those of other paramyxovirus P genes.  相似文献   

12.
Recently, it has been observed that the infection of human target cells with certain measles virus (MV) strains leads to the downregulation of the major MV receptor CD46. Here we report that CD46 downregulation can be rapidly induced in uninfected cells after surface contact with MV particles or MV-infected cells. Receptor modulation is detectable after 30 min of cocultivation of uninfected cells with MV-infected cells and is complete after 2 to 4 h, a time after which newly synthesized MV hemagglutinin (MV-H) cannot be detected in freshly infected target cells. This contact-mediated receptor modulation is also induced by recombinant MV-H expressed by vaccinia virus and is inhibitable with antibodies against CD46 and MV-H. By titrating the effect with MV Edmonston strain-infected cells, a significant contact-mediated CD46 modulation was detectable up to a ratio of 1 infected to 64 uninfected cells. As a result of CD46 downregulation, an increased susceptibility of uninfected cells for complement-mediated lysis was observed. This phenomenon, however, is MV strain dependent, as observed for the downregulation of CD46 after MV infection. These data suggest that in acute measles or following measles vaccination, uninfected cells might also be destroyed by complement after contacting an MV-infected cell.  相似文献   

13.
14.
Measles virus (MV) C protein is a small and basic non-structural protein, but its function is not well understood. We have found that a FLAG-tagged wild-type MV C protein expressed from cDNA was accumulated exclusively in the nucleus. To analyze the amino acid sequence important for the nuclear localization of C protein, a plasmid expressing C protein fused to the enhanced green fluorescent protein (EGFP) was generated. Mutation analysis revealed that (41)PPARKRRQ(48), belonging to the classical nuclear localization signal was important for nuclear localization. Analysis of the amino acid sequence of C protein revealed that it has a nuclear export signal (NES)-like sequence, (76)LEKAMTTLKL(85). Addition of the putative NES to the EGFP resulted in the translocation of EGFP to the cytoplasm. The Rev(1.4)-EGFP nuclear export assay showed that this putative NES has a CRM1-dependent NES activity. C-EGFP accumulated in HeLa nuclei could be translocated to NIH3T3 nuclei in heterokaryon assays. In MV-infected cells, C-EGFP was accumulated in the nuclei in early phase but in the cytoplasm in late phase. These results indicate that the putative NES is functional and that C protein has the ability to shuttle between the nucleus and the cytoplasm.  相似文献   

15.
A procedure for selective full length cDNA cloning of specific RNA species.   总被引:7,自引:0,他引:7  
A method allowing routine establishment of full length and functionally competent cDNA clones of particular mRNAs from small preparations of polyadenylated RNA is described. Pairs of synthetic primers are used for first and second strand synthesis. They include sequences complementary to the 3' terminal regions of the mRNAs and of the full length first cDNA strands, respectively and bear a few additional nucleotides at their 5' ends. After synthesis of both cDNA strands in one tube, they are precisely trimmed back with T4 DNA polymerase in presence of only two nucleoside triphosphates, to yield sticky ends fitting into a vector plasmid cleaved with two restriction endonucleases. The procedure was first applied to the simultaneous cloning of all five major measles virus (MV) mRNA species from a persistently infected cell line. Two thirds of all clones contained full length MV-specific cDNAs. Screening of less than 200 clones was sufficient to obtain several independent clones corresponding to each mRNA, except for gene F which was represented only once.  相似文献   

16.
Komune N  Ichinohe T  Ito M  Yanagi Y 《Journal of virology》2011,85(24):13019-13026
Inflammasomes are cytosolic protein complexes that stimulate the activation of caspase-1, which in turn induces the secretion of the inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. Recent studies have indicated that the inflammasome known as the NOD-like-receptor-family, pyrin domain-containing 3 (NLRP3) inflammasome recognizes several RNA viruses, including the influenza and encephalomyocarditis viruses, whereas the retinoic acid-inducible gene I (RIG-I) inflammasome may detect vesicular stomatitis virus. We demonstrate that measles virus (MV) infection induces caspase-1-dependent IL-1β secretion in the human macrophage-like cell line THP-1. Gene knockdown experiments indicated that IL-1β secretion in MV-infected THP-1 cells was mediated by the NLRP3 inflammasome but not the RIG-I inflammasome. MV produces the nonstructural V protein, which has been shown to antagonize host innate immune responses. The recombinant MV lacking the V protein induced more IL-1β than the parental virus. THP-1 cells stably expressing the V protein suppressed NLRP3 inflammasome-mediated IL-1β secretion. Furthermore, coimmunoprecipitation assays revealed that the V protein interacts with NLRP3 through its carboxyl-terminal domain. NLRP3 was located in cytoplasmic granular structures in THP-1 cells stably expressing the V protein, but upon inflammasome activation, NLRP3 was redistributed to the perinuclear region, where it colocalized with the V protein. These results indicate that the V protein of MV suppresses NLRP3 inflammasome-mediated IL-1β secretion by directly or indirectly interacting with NLRP3.  相似文献   

17.
A computer program combining of hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus (MV) fusion protein was used to select four possible epitopes. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using the homobifunctional cross-linker bis-sulfosuccinimidyl suberate. Immune stimulating complexes were prepared with the peptides conjugated to the purified protein derivative carrier using a dialysis method. All antisera raised in rabbits against the peptide conjugates had a high titer to the homologous peptides and reacted well with denatured MV as tested by plate ELISA. None of the sera had neutralizing antibody. Human sera positive for MV antibody reacted strongly with the synthesized peptides indicating that the selected locations function as partial antigenic sites. Antisera against peptide conjugates reacted weakly in immunofluorescence and none of these antisera reacted with purified MV proteins in Western blot. The results obtained in this study indicated that although the computer program could not predict epitopes important for the neutralization of the MV, the predicted epitopes are useful for detecting antibodies against MV.  相似文献   

18.
The fusion (F) glycoproteins of measles virus strains Edmonston (MV-Edm) and wtF (MV-wtF) confer distinct cytopathic effects and strengths of hemagglutinin (H) interaction on a recombinant MV-Edm virus. They differ in just two amino acids, V94 and V101 in F-Edm versus M94 and F101 in F-wtF, both of which lie in the relatively uncharacterized F(2) domain. By comparing the sequence of MV F with those of the parainfluenza virus SV5 and Newcastle disease virus (NDV) F proteins, the structures of which are known, we show that MV F(2) also possesses a potential heptad repeat (HR) C domain. In NDV, the N-terminal half of HR-C interacts with HR-A in F(1) while the C-terminal half is induced to kink outward by a central proline residue. We found that this proline is part of an LXP motif conserved in all three viruses. Folding and transport of MV F require this motif to be intact and also require covalent interaction of cysteine residues that probably support the potential HR-A-HR-C interaction. Amino acids 94 and 101, both located in "d" positions of the HR-C helical wheel, lie in the potentially outwardly kinked region. We demonstrate that their effect on MV fusogenicity and glycoprotein interaction is mediated solely by amino acid 94. Substitutions at position 94 with polar or charged amino acids are tolerated poorly or not at all, while changes to smaller and more hydrophilic amino acids are tolerated in both transiently expressed F protein and recombinant virus. MV F V94A and MV F V94G viruses induce extensive syncytium formation and are relatively, or almost completely, resistant to a known inhibitor of MV glycoprotein-induced fusion. We propose that the conformational changes in MV F protein required to expose the fusion peptide involve the C-terminal half of the HR-C helix, specifically amino acid 94.  相似文献   

19.
The mRNA of a putative small hydrophobic protein (SH) of mumps virus was identified in mumps virus-infected Vero cells, and its complete nucleotide sequence was determined by sequencing the genomic RNA and cDNA clones and partial sequencing of mRNA. The SH mRNA is 310 nucleotides long excluding the poly(A) and contains a single open reading frame encoding a protein of 57 amino acids with a calculated molecular weight of 6,719. The predicted protein is highly hydrophobic and contains a stretch of 25 hydrophobic amino acids near the amino terminus which could act as a membrane anchor region. There is no homology between the putative SH protein of mumps virus and the SH protein of simian virus 5, even though the SH genes are located in the same locus in the corresponding genome. One interesting observation is that the hydrophobic domain of simian virus 5 SH protein is at the carboxyl terminus, whereas that of mumps virus putative SH protein is near the amino terminus.  相似文献   

20.
Editing of P-gene mRNA of Newcastle disease virus (NDV) enables the formation of two additional proteins (V and W) by inserting one or two nontemplated G residues at a conserved editing site (5'-AAAAAGGG). The V protein of NDV plays an important role in virus replication and is also a virulence factor presumably due to its ability to counteract the antiviral effects of interferon. A recombinant virus possessing a nucleotide substitution within the A-stretch (5'-AAgAAGGG) produced 20-fold-less V protein and, in consequence, was impaired in replication capacity and completely attenuated in pathogenicity for chicken embryos. However, in a total of seven serial passages, restoration of replication and pathogenic capacity in 9- to 11-day-old chicken embryos was noticed. Determining the sequence around the editing site of the virus at passage 7 revealed a C-to-U mutation at the second nucleotide immediately upstream of the 5'-A(5) stretch (5'-GuUAAgAAGGG). The V mRNA increased from an undetectable level at passage 5 to ca. 1 and 5% at passages 6 and 7, respectively. In addition, similar defects in another mutant possessing a different substitution mutation (5'-AAAcAGGG) were restored in an identical manner within a total of seven serial passages. Introduction of the above C-to-U mutation into the parent virus (5'-GuUAAAAAGGG) altered the frequency of P, V, and W mRNAs from 68, 28, and 4% to 15, 44, and 41%, respectively, demonstrating that the U at this position is a key determinant in modulating P-gene mRNA editing. The results indicate that this second-site mutation is required to compensate for the drop in edited mRNAs and consequently to restore the replication capacity, as well as the pathogenic potential, of editing-defective NDV recombinants.  相似文献   

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