- Volume 75, Issue 4, 1994
Volume 75, Issue 4, 1994
- Articles
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- Bacterial
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Structural proteins and DNA characteristics of 14 Listeria typing bacteriophages
More LessThe major structural proteins of 13 temperate and one virulent Listeria typing bacteriophages were analysed and compared using isoelectric focusing in immobilized pH gradients (IPG), ultrathin-layer two-dimensional electrophoresis, amino acid analysis and N-terminal amino acid sequences of selected proteins. Isoelectric points for major capsid and tail proteins of the 12 members of the siphoviridae family included in this study ranged from 4·70 to 5·92, whereas one of the two myoviridae investigated (B054) showed structural proteins in the 6·1 to 6·3 range. In comparison to protein profiles from one-dimensional SDS gels, the IPG technique gave better resolution and improved discrimination of phage proteins. Combination of this technique and SDS gel electrophoresis made it possible to correlate M r and isoelectric points of major structural proteins. Tail polypeptides of all siphoviridae are smaller and, with one exception, more acidic than their corresponding capsid counterparts. We also determined the amino acid composition of capsid and tail proteins. When compared with an average protein, they were found to be fairly rich in acidic and short-chain hydrophobic amino acids, as well as in lysine. In addition, the N-terminal amino acid sequences of major capsid and tail proteins of four representative listeria- phages were compared. The base composition of listeriaphage DNAs was between 37 % and 39 % G + C, reflecting that of their bacterial hosts. Each phage had a distinct restriction endonuclease pattern, and genome sizes ranged from 35 to 116 kb. DNA-DNA hybridization permitted the identification of five DNA homology groups. The two myoviruses studied (A511 and B054) showed no DNA homology to other phages, confirming their unique nature. The 12 siphoviruses were classified into three DNA homology groups with little cross-homology. Furthermore, phage A006 was found to share little DNA homology with the other investigated members of species 2671. Therefore, a new species (A006) is proposed. With respect to phage classification and taxonomy, a good correlation between the various approaches was observed, mostly corresponding to particle morphology.
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- Animal
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Characterization of the herpes simplex virus type 1 strain 17+ neurovirulence gene RL1 and its expression in a bacterial system
More LessThe DNA sequence of herpes simplex virus type 1 (HSV-1) strain 17+ in the region coding for the polypeptide ICP34.5 predicts a protein of 248 amino acids with a proposed Mr of26158. The entire RL1 open reading frame was cloned into the expression vector pET8c to enable over-expression of ICP34.5 in Escherichia coli. The expressed protein was partially purified and used as an immunogen to produce a polyclonal antiserum in rabbits. Construction of an ICP34.5 null mutant (1771), demonstrated that the predicted open reading frame for ICP34.5 in strain 17+ is correct and confirmed that HSV-1 strain 17+ ICP34.5 specifically determines neurovirulence. The specificity of the antiserum directed against the E. coli -expressed ICP34.5 was defined by Western blotting of wild-type and RL1- negative infected cell extracts.
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Localization of a functional site on herpes simplex virus type 1 glycoprotein C involved in binding to cell surface heparan sulphate
The amino acid residues critical for interaction between herpes simplex virus type 1 (HSV-1) glycoprotein C (gC- 1) and cell surface heparan sulphate (HS) were localized to two separate regions within antigenic site II of this glycoprotein. These amino acids were Arg-143, Arg-145, Arg-147 and Thr-150 in one region and Gly-247 in the other. This conclusion is based on the following observations, (i) Monoclonal antibodies defining gC-1 antigenic site II, and not those reactive with antigenic site I, inhibited HSV-1-induced haemagglutination and virus binding to susceptible cells, (ii) A number of HSV- 1 mar mutants, altered at these critical residues, were impaired in attachment to cells, (iii) Synthetic peptides, corresponding to these two regions inhibited virus attachment to cells and infectivity. In addition these peptides were found to agglutinate red blood cells. This agglutination was inhibited by soluble HS, and was prevented by the pretreatment of red blood cells with heparitinase suggesting that cell surface HS was a site of peptide binding. The same was observed with the polycationic substances neomycin and poly-l-lysine. In conclusion, we propose that the regions of gC-1 represented by the HS-binding peptides may form a functional site of a polycationic nature, active in attachment to the polyanionic glycosaminoglycan chain of cell surface HS.
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Binding and repression of the latency-associated promoter of herpes simplex virus by the immediate early 175K protein
More LessWe demonstrate that the immediate early 175K protein (IE175K) of herpes simplex virus type 1 binds to the cap site of the latency-associated promoter (LAP) in an unusual manner. The complex formed on the LAP cap site was significantly larger than that formed on the IE175K cap site and the requirements for binding were qualitatively distinct with respect to both the primary sequence determinants at the site, and the regions of IE175K protein required for binding compared to those for the IE175K cap site. Although purified IE175K was sufficient for this larger complex formed on the LAP cap site, the DNA-binding domain was unable to bind efficiently. This contrasted strikingly with the IE175K cap site where, using precisely analogous probes, the DNA-binding domain exhibited a strong interaction. Surprisingly, from dissociation kinetics we show that binding of the intact protein to the LAP cap site is considerably more stable than the binding of IE175K to its own cap site (half-lives of the complexes 15 min and < 1 min respectively), and this was reflected in more efficient repression of LAP-driven expression than IE175K promoter-driven expression by IE175K. Moreover, primary sequence requirements for IE175K binding to the LAP cap site region differed from previously identified IE175K recognition sequences in that in addition to a partially conserved consensus sequence, neighbouring bases were necessary for binding. Although the LAP cap site exhibits a pseudopalindromic arrangement of core consensus sites, we show that this is not the basis for the higher order, more stable binding to this region. Together these results indicate that IE175K forms an unusual complex at the LAP cap site, broadening the range of previously defined sequences recognized by IE175K.
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Precipitation of the Epstein—Barr virus protein EBNA 2 by an EBNA 3c-specific monoclonal antibody
More LessTwo monoclonal antibodies, E3cD8 and E3cA10, were generated to the EBNA 3 c nuclear protein from the B95.8 isolate of Epstein-Barr virus (EBV). Both antibodies efficiently precipitate EBNA 3c from B95.8- transformed lymphoblastoid cell lines, and E3cA10 also detects EBNA 3c on Western blots. Whereas E3cD8 reacts with all 11 Type-1 isolates of EBV tested, and E3cA10 reacts with 14 of 17 Type-1 isolates, neither antibody detects the EBNA 3 c protein encoded by Type-2 isolates. E3cD8 recognizes a peptide sequence (PA/pPQAPYQGY) in a repeat region of the B95.8 EBNA 3c coding sequence which is not present in the prototype Type-2 AG876 sequence. The E3cA10 antibody epitope has been mapped to the minimal five amino acid B95.8 peptide, WAPSV, which has an alanine to valine substitution in the AG876 virus isolate. This substitution was also found in three Type-1 EBV isolates that expressed EBNA 3c proteins not detected by E3cA10. In immunoprecipitation studies E3cA10 additionally coprecipitated the EBNA 2 protein from Type-1 isolates of EBV. The possibility of a direct interaction between EBNA 2 and EBNA 3 c was ruled out by the demonstration that the antibody precipitated EBNA 2 from the Raji cell line which carries a virus with a deleted EBNA 3c gene. Since the WAPSV epitope identified in EBNA 3c is not present in EBNA 2, and no EBNA 2 linear peptide reactivity was detected in ELISA, it seems likely that E3cA10 recognizes a conformational epitope on EBNA 2. However, from the present data we cannot exclude the possibility that the antibody reacts with a cellular protein that physically associates with EBNA 2.
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Tumour necrosis factor-α production during cytomegalovirus infection in immunosuppressed rats
The production of tumour necrosis factor (TNF)-α, interleukin (IL)-l and IL-6, all proinflammatory cytokines, was investigated in radiation-immunosuppressed rats infected with rat cytomegalovirus (RCMV). At day 7 post-infection, when the animals showed disease signs, high TNF-α levels were detected in the serum and in homogenates of various organ tissues. In contrast, IL-1 and IL-6 levels were not significantly elevated. Moreover, replication of RCMV induced TNF-α expression in different types of cells grown in vitro. When frozen tissue sections were examined by immunohistology, TNF-α-producing cells were found in areas with extensive pathology in the lungs, spleen and liver. Both lymphocytes and RCMV-infected cells were identified as the sources of TNF-α. Its abundance in RCMV-infected rats suggests an important role for TNF-α in CMV pathogenesis.
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Characterization of the 55K adenovirus type 5 E1B product and related proteins
More LessIn addition to major proteins of 19K and 55K (176 and 496 residues, 176R and 496R, respectively), early region 1B (E1B) of human adenovirus type 5 (Ad5) is predicted to encode at least three other polypeptides of 156R, 93R and 84R that share 79 amino-terminal residues with 496R. We have used a series of specific antipeptide sera to identify and partially characterize these proteins. 84R was produced in large amounts, 156R somewhat less, and 93R at very low levels. Synthesis of 176R, 496R, as well as the E2A 72K DNA-binding protein commenced shortly after that of E1A proteins in Ad5-infected KB cells. Production of 156R, 93R and 84R began somewhat later, but prior to the synthesis of the late structural protein IX and hexon. 156R, which is composed of the 79 amino-terminal and 77 carboxy-terminal amino acids of 496R, migrated on SDS-PAGE as two species which appeared to differ by their degree of phosphorylation. 156R and 496R yielded identical tryptic phosphopeptides that contained both phosphoserine and phosphothreonine, and one of these was immunoprecipitated by a serum specific for the carboxy terminus. These results suggested that Ser-490 and/or Ser-491 as well as Thr-495 are major sites of phosphorylation in these proteins.
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Cytotoxic T lymphocytes raised against Japanese encephalitis virus: effector cell phenotype, target specificity and in vitro virus clearance
More LessSeveral H-2 defined cell lines were examined for their ability to support infection and replication of Japanese encephalitis virus (JEV) before their use in in vitro and in vivo stimulation protocols for generating cytotoxic T lymphocytes (CTLs) against JEV. Among 11 different cell lines tested, two H-2d macrophage tumour lines (P388D1, RAW 264.7), an H-2d hybridoma (Sp2/0), an H-2KkDd neuroblastoma (Neuro 2a), and H-2k fibroblast cell line (L929) were found to support JEV infection and replication. These cell lines were used to generate anti-JEV CTLs by using in vivo immunization followed by in vitro stimulation of BALB/c mice. We observed that not only syngeneic and allogeneic infected cells but also JEV-infected xenogeneic cells could prime BALB/c mice for the generation of JEV-specific CTLs upon subsequent in vitro stimulation of splenocytes with JEV- infected syngeneic cells. Although infected xenogeneic cells were used for immunization, the anti-JEV effectors that were generated lysed infected syngeneic targets but not JEV-infected xenogeneic or allogeneic target cells in a 5 h 51Cr release assay. These anti-JEV effectors recognized syngeneic target cells infected with West Nile virus to a lesser extent and were shown to be Lyt-2.2+ T cells. The results of unlabelled cold target competition studies suggested alterations in the cell surface expression of viral antigenic determinants recognized by these CTLs. We further demonstrate that the JEV- specific CTLs generated could virtually block the release of infectious virus particles from infected P388D1 and Neuro 2a cells in vitro.
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The nucleoprotein of Marburg virus is phosphorylated
More LessThe nucleoprotein (NP) of Marburg virus (MBG), a filovirus, is encoded by the gene closest to the 3′ end of the non-segmented negative-strand RNA genome. Sequence comparison has indicated that NP is the functional equivalent to the nucleoproteins of paramyxoviruses and rhabdoviruses. Expression of recombinant NP in two eukaryotic systems using vaccinia virus and baculovirus (vectors pSCll and pAcYMBl, respectively) and analysis of MBG-specific proteins have demonstrated that the NP of MBG is phosphorylated. The NP appeared in two forms differing in Mr by about 2K (94K and 92K respectively). Dephosphorylation clearly demonstrated that the 94K form is phosphorylated whereas the 92K form is unphosphorylated. In virion particles NP was exclusively present in the phosphorylated form. These findings suggest that only the phosphorylated NP can form nucleocapsid complexes and interact with the genomic RNA.
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Reversal of the measles virus-mediated increase of phosphorylating activity in persistently infected mouse neuroblastoma cells by anti-measles virus antibodies
To investigate the effect of persistent measles virus infection on signal transduction in cells of neuronal origin, the mouse neuroblastoma cell line NS20Y/MS, which is persistently infected with measles virus, was used. The results demonstrate an approximate 50% increase in total phosphorylation and a similar increase in protein kinase C (PKC) activity. Western blot analysis with anti-total PKC or anti-PKC-α antibodies revealed a significant increase in the level of an 80K immuno-reactive PKC in NS20Y/MS cells. Following incubation of NS20Y/MS cells with polyclonal anti-measles virus antibodies, which down-regulate the level of measles virus proteins, total and PKC-mediated phosphorylation returned to the basal level of uninfected cells. This effect was reversible and removal of the antibodies resulted in restoration of the high level of total and PKC-mediated phosphorylation. The release of infectious measles virus was strongly inhibited by incubation of NS20Y/MS cells with the PKC inhibitor, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H-7). These results demonstrate that measles virus induces elevation in cellular phosphorylation which is essential for measles virus production.
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Identification and characterization of the ecdysteroid UDP-glucosyltransferase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus
More LessWe have located, cloned, sequenced and characterized the ecdysteroid UDP-glucosyltransferase gene (egt) gene from the baculovirus Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.(LdMNPV), which is specific for the gypsy moth (L. dispar). The egt gene from the related baculovirus Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) disrupts the hormonal balance of the host larva by galactosylating ecdysone, which prevents moulting. The location of the LdMNPV egt gene, determined by hybridization analysis using a cloned coding segment of the AcMNPV egt gene, was mapped to between 79·1 and 80·2 map units on the viral genome. This region contains an open reading frame of 1464 nucleotides capable of encoding a 55K polypeptide. This predicted protein exhibits a 42% amino acid identity with the AcMNPV egt polypeptide. Transcripts of the egt gene were analysed by Northern blot and primer extension. The egt gene is transcribed from approximately 12 to 48 h, and maximally at about 16 h post-infection. Transcription occurred in the presence of aphidicolin, a viral DNA synthesis inhibitor, but not in the presence of cycloheximide, a protein synthesis inhibitor. Therefore the LdMNPV egt gene is classified as a delayed early gene. The egt gene is transcribed in a clockwise direction with respect to the circular map, and transcription initiates at a single site. Comparisons between the two baculoviral egt proteins and mammalian UDP-glucuronosyltransferases reveal areas which are conserved between the mammalian and baculoviral genes, as well as areas that are only conserved in the viral egt proteins. The LdMNPV protein sequence appears to include a signal peptide, which would allow the protein to be secreted into the haemolymph.
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Analysis of the human immunodeficiency virus type 1 envelope protein interaction with the CD4 host cell receptor
More LessA secreted form of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp160s), expressed in HeLa cells from a vaccinia virus recombinant was analysed by velocity-gradient centrifugation and chemical cross-linking. We showed that gp160s existed predominantly as a dimer, but higher forms corresponding to trimers and tetramers were also found. Soluble CD4 (sCD4) and native CD4 expressed by recombinant vaccinia viruses were analysed by sucrose- gradient sedimentation alone or after complexing with gp160s. The sCD4 sedimented in sucrose gradients as a monomer, whereas after solubilization the native CD4 was in a dimeric state. Both forms of CD4 were able to form complexes when incubated with gp160s. In the case of the sCD4, the M r corresponded to a (sCD4)2- (gp160s)2 complex, whereas with CD4 the complexes were of a greater order of magnitude. HIV gpl20 was secreted into the medium in a monomeric state. With sCD4 it gave a one-to-one complex, whereas with the native CD4 high M r complexes were formed. The importance of the oligomeric state of the virus- and cell-receptor proteins are discussed regarding their avidities.
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Phagocytosis of Mycobacterium tuberculosis modulates human immunodeficiency virus replication in human monocytic cells
More LessMacrophage activation resulting from phagocytosis has the potential to modulate human immunodeficiency virus (HIV) replication. We have determined the effects of phagocytosis of particulate stimuli on transcription and release of HIV. Using THP-1 and Mono Mac 6 human monocytic cell lines transfected with HIV long terminal repeat sequence chloramphenicol acetytransferase (LTR CAT) constructs we have demonstrated that phagocytosis of Mycobacterium tuberculosis enhanced HIV-1 and -2 LTR CAT expression. However phagocytosis of zymosan or inert latex beads had little or no effect on CAT expression. Enhancement of HIV LTR CAT expression was dependent upon intact NF-κB binding sites and was independent of tumour necrosis factor alpha secretion. M. tuberculosis strains of different degrees of virulence induced similar levels of enhanced CAT expression. In contrast, phagocytosis of M. tuberculosis by HIV-1-infected THP-1 cells reduced supernatant reverse transcriptase (RT) activity without suppression of p24 antigen release. Phagocytosis of zymosan granules or latex particles did not alter released RT activity. However, phagocytosis of either M. tuberculosis, zymosan granules or latex particles by HIV-1-infected human peripheral blood monocyte-derived macrophages reduced supernatant RT activity. These data indicate that phagocytosis of M. tuberculosis may enhance HIV transcription in monocytic cells although it may reduce release of intact HIV.
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Correlation between high level gp160 expression and reduced CD4 biosynthesis in clonal derivatives of human immunodeficiency virus type 1-infected U-937 cells
More LessWe have compared cytoplasmic CD4 mRNA accumulation, CD4 biosynthesis and steady-state levels of both CD4 protein and mRNA in a variety of clonal derivatives of U-937 cells, chronically infected with human immunodeficiency virus type 1IIIB (HIV-1), that express various cellular and viral phenotypes. These phenotypes included defective processing of either gpl60 or Gag-Pol, viruses with severely limited host-range, and inability to generate viral products. All clones, with the exception of the one that failed to generate viral mRNA and proteins, did not express cell surface CD4. Furthermore, each of these clones had steady-state levels of CD4 mRNA which were either equivalent to or higher than those of the parental U-937 cell line. Patterns of cytoplasmic CD4 mRNA levels resembled those of total RNA, suggesting that CD4 mRNA transport from the nucleus to the cytoplasm was unaffected by HIV-1 infection. Profiles of steady-state levels of the CD4 protein resembled those of CD4 mRNA in the UHC clones, but CD4 biosynthesis was reduced in all clones with the exception of that which failed to express viral products. This report is the first demonstration that steady-state CD4 biosynthesis is reduced in HIV-l-infected cells. In general, there was a good correlation between high levels of expression of gpl60 and reduced CD4 biosynthesis. These results suggest that HIV-1 env gene products may contribute to the observed reduction in levels of CD4 biosynthesis.
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Genomic variation of human immunodeficiency virus type 1 (HIV-1): molecular analyses of HIV-1 in sequential blood samples and various organs obtained at autopsy
More LessLength polymorphisms and partial nucleotide sequences were determined for the hypervariable regions, V1 to V5, of the human immunodeficiency virus type 1 (HIV-1) env gene obtained from proviral DNA of sequential peripheral blood samples, from viral RNA in plasma, and from proviral DNA obtained from different organs of individuals at autopsy. The lengths of several env regions of HIV-1 proviral DNA differed markedly when obtained from different organs of an individual. Nucleotide sequences of the hypervariable V3 region of HIV-1 obtained from different organs of one patient demonstrated distinct viral variants. Most proviral DNA sequences found in organs were also present in viral RNA obtained from plasma. The majority of HIV-1 V3 variants present in the lymph tissue could be found in the plasma viral population obtained at autopsy and in the sequential blood samples obtained before death, but were absent from the cardiac blood provirus population obtained at autopsy. However, sequence variants found in the brain proviral DNA were not detected in either plasma or the sequential blood samples. Sequence differences were observed at the apex of the V3 loop between HIV-1 variants present in sequential blood samples and in blood lymphocytes and nervous tissue, lymph tissue and plasma obtained post-mortem. The potential effect of lymph tissue on the long-term persistence of different viral variants is discussed. Virus obtained from the two sequential blood samples produced syncytia in primary cultures and was easily transmitted to the continuous JM cell line. Consensus (majority) V3 loop sequences determined for the adapted viruses demonstrated that some, but not all, sequences were represented within the in vivo viral population.
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Molecular cloning and characterization of a murine AIDS virus-related endogenous transcript expressed in C57BL/6 mice
The murine AIDS (MAIDS) virus has a unique sequence in the gag p12 region, which could be responsible for MAIDS development. RNA preparations from the spleens of normal uninfected C57BL/6 mice contain a transcript hybridizing with this sequence. Levels of the transcript in the kidney of C57BL/6 mice were higher than in the spleen, liver or thymus. Although BALB/c, NFS, DBA/2 and SL murine strains also contained genomic sequences hybridizing with the MAIDS virus- specific probe, no transcript hybridizing with the probe was detected in these strains of mice. The cDNAs carrying the transcript expressed in C57BL/6 mice were molecularly cloned. The complete nucleotide sequence of the clone indicates that the transcript is one of the endogenous murine leukaemia virus-related sequences containing large deletions from the R and U5 regions of the 5′ long terminal repeat (LTR) to gagpl5, from the C- terminal region of pol p40 (integrase) to the N-terminal region of env pl5E, and many short deletions in the 3′ LTR U3 region. The nucleotide sequence in the gag pi 2 region of the transcript was closely similar to that of the MAIDS virus, but the amino acid sequence was less similar because of frameshifting, even when translated. As the MAIDS virus was isolated from C57BL/6 mice with radiation-induced leukaemia, this transcript may be the progenitor of the MAIDS virus. To determine whether the gag p12 region of the transcript contains a functional sequence, a recombinant virus was generated by replacing the gag p12 region of a replication- competent BM5eco virus with that of the endogenous transcript. The recombinant virus was replication- competent, and the p12 region of the transcript retained the functional sequence present in the BM5eco virus.
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Monoclonal antibodies to immunodominant and neutralizing domains of the envelope surface protein of feline immunodeficiency virus
Hybridomas secreting monoclonal antibodies (MAbs) specific for the surface protein (SU) of feline immunodeficiency virus were generated. Four MAbs were obtained which could be assigned to two groups based on their neutralization and competition behaviour. Using SU protein fragments expressed in Escherichia coli the antigenic site recognized by one of the MAbs (2H11) could be mapped to the c terminus. The neutralizing MAb 1E1 did not bind to any of the SU protein fragments and was directed to a conformational epitope. Binding of the MAb 1E1 to native SU protein could be blocked with a rabbit serum raised against the SU3 fragment (amino acids 361 to 445). These data indicate that at least part of the epitope is located on this SU3 domain. In competition experiments most sera of naturally infected cats were able to inhibit binding of the MAbs. This shows the conserved and immunodominant nature of the epitopes involved.
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Expression of functional protease and subviral particles by vaccinia virus containing equine infectious anaemia virus gag and 5′ pol genes
Cells infected with vaccinia viruses expressing the equine infectious anaemia virus (EIAV) gag gene (VGag) or gag plus the 5′ pol encoding protease (VGag/PR) were evaluated with monoclonal antibody to a p26 capsid protein linear epitope (QEISKFLTD). Both recombinant viruses expressed Gag precursor protein (55K) whereas only VGag/PR expressed a detectable Gag-Pol fusion protein (82K) with a functional protease, shown by subviral particles containing processed p26. Horses inoculated with VGag/PR produced antibodies reactive with EIAV Gag proteins.
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Expression of bovine herpesvirus 1 glycoprotein gIII by a recombinant baculovirus in insect cells
K. Okazaki, E. Honda and Y. KonoBovine herpesvirus 1 (BHV-1) glycoprotein gill functions both as a major virus attachment protein and haemagglutinating protein. Here we constructed recombinant baculovirus incorporating the BHV-1 gill coding sequence to characterize the expression, function and immunogenicity of the glycoprotein in insect cells. The recombinant gill had an Mr of 72K and seemed to form homodimers. The gill was expressed on the surface of insect cells and a rosette formation assay demonstrated haemadsorbing activity of the glycoprotein. Antigenic authenticity of the recombinant gill was confirmed by a panel of monoclonal antibodies specific for the glycoprotein produced in mammalian cells. Antisera raised to recombinant gill neutralized the infectivity of BHV-1. These data suggest that recombinant gill produced in insect cells may be a useful immunogen in a BHV-1 vaccine.
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Transcriptional analysis and genome expression of chicken anaemia virus
More LessStrand-specific riboprobes representative of either strand of the chicken anaemia virus (CAV) replicative form (RF) DNA indicated that only one strand of the RF was transcribed to produce a major 2·0 kb transcript and that the encapsidated DNA strand was of negative sense. Primer extension analysis located a single transcriptional start site at nucleotide position 360 of the CAV sequence. Amplification, cloning and sequencing of the 3′ end of the major transcript revealed the polyadenylation site at nucleotide position 21. Northern blot analysis using a series of genomic probes indicated that the 2·0 kb transcript was devoid of splicing and identified a non-transcribed region of the genome. This non-transcribed region was shown to possess promoter activity, enhancing the expression of the human growth hormone reporter gene in a transient gene expression assay. These observations suggest a simple strategy of genome expression involving a functional polycistronic message.
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Sequence variation in the capsid protein genes of human papillomavirus type 16
More LessWe have cloned and sequenced the L1 and L2 genes from human papillomavirus type 16 (HPV16) DNA- containing cervical cytology samples collected from the U.K. and Trinidad. Samples containing high copy numbers of HPV16 DNA were selected as being likely to contain fully functional virus DNA molecules in an episomal state, rather than in an integrated and possibly altered state. In comparison with the previously published sequence of HPVI6 isolated from an invasive cancer a variety of differences were detected in both L1 and L2. The pattern of changes appears to be different in samples from the two geographic regions. One of the differences (resulting in D at position 202 of the LI protein) reported recently to be functionally important for virus particle assembly was found to occur in all the samples examined. Variations in LI found within known immunoreactive regions or hydrophobic domains should be taken into account in design of prophylactic vaccines for HPV16 based on virus-like particles. All variations within L2 protein were found in hydrophilic domains in the carboxy-terminal half of L2. These positions were highly variable among other types of papillomavirus and are located outside the known L2 immunoreactive region.
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Effect of frameshift mutation in the pre-C region of hepatitis B virus on the X and C genes
More LessWe have previously cloned a mutant hepatitis B virus (HBV) genome which had one thymidine addition in the pre-C region resulting in a frameshift mutation in the pre-C region and fusion of the X and C genes. We constructed plasmids containing serially deleted and/or back-mutated (authentic) pre-C regions to study the effect of the frameshift mutation. COS cells transfected with plasmids containing the frameshifted pre-C region produced a 21K C protein (P21c) but not a 22K partially processed pre-C protein (P22). On the other hand, COS cells transfected with plasmids containing the back-mutated pre-C region produced P22. This result was also observed in HepG2-K8 cells producing the mutant HBV particles. Therefore, the pre-C region of HBV is likely to be non-essential for virus replication. COS cells transfected with the plasmid containing a fused X-C open reading frame (ORF) produced a 40K X-C fusion protein. This X-C fusion protein exerted transcriptional trans-activation. These results suggest that the mutant HBV has a C gene with a defective pre-C region and a fused X-C ORF, and hence cannot synthesize 16K HBeAg (P16e).
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Determination of the 5′ end of the lactate dehydrogenase-elevating virus genome by two independent approaches
More LessWe have determined the 5′ end of the lactate dehydrogenase-elevating virus (LDV) genome (strain LDV-P) using two independent approaches. In one approach, methylmercuric hydroxide-denatured genomic RNA was reverse-transcribed using as primer an oligonucleotide complementary to the 5′ end of open reading frame (ORF) la. The first-strand cDNA was ligated with T4 RNA ligase to an oligonucleotide of which the 3′ end was blocked. The ligated product was amplified by PCR, cloned and sequenced. In the second approach, untreated or decapped genomic RNA was ligated between the 3′ and 5′ ends, reverse-transcribed across the ligation junction and the product was amplified by PCR, cloned and sequenced. Both approaches yielded the same results, indicating that the 5′ leader of LDV-P is 156 nucleotides long, inclusive of the 5′ UAUAACC 3′ sequence involved in the linkage of the 5′ leader to the bodies of the seven subgenomic mRNAs of LDV. The 5′ leader of LDV is about 50 nucleotides shorter than those of the related viruses, equine arteritis virus and Lelystad virus, but at least twice as long as the leaders of the coronaviruses. The finding that untreated LDV RNA was ligated 5′ to 3′ end as efficiently as RNA treated with decapping enzyme suggests that genomic LDV RNA may not possess a 5′ cap but terminates with 5′ phosphoryl-A.
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Hepatitis C virus variants from Nepal with novel genotypes and their classification into the third major group
Five isolates of hepatitis C virus (HCV) RNA from patients with chronic liver disease in Nepal were not classifiable into the known genotypes I/1a, Il/lb, III/2a, IV/2b or V/3a using PCR with type-specific primers deduced from the HCV core gene. Their nucleotide sequences were determined for the 5′ -terminal 1·5 ldlobases and 3′ -terminal 1·2 kilobases, covering 30% of the entire genome, and compared with each other and with reported sequences of HCV isolates of various genotypes. They were more similar to a reported HCV isolate (NZL1) of genotype V/3a (in 81·6 to 84T % of their nucleotides and 85·7 to 88·7% of the deduced amino acid sequence) compared with the genotypes I/1a to IV/2b (in 69·3 to 74·7% and 72·3 to 77·4%, respectively). Hence they were considered to be variants of the third major group (group 3). The five HCV isolates shared 81·3 to 85·2% of nucleotide sequence and 85·4 to 89·3 % of deduced amino acid sequence. Thus they were substantially different from each other. One of them was classified as genotype VI/3b due to an 88·2 % similarity in nucleotide sequence to that of the reported HCV isolates of this genotype, whereas the remaining four were classified into provisional genotypes 3c, 3d, 3e and 3f. These HCV variants have evolved and remained in Nepal, and have not been observed in the other areas of the world.
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Common distribution of antigenic determinants and complementation activity on matrix proteins of two vesicular stomatitis virus serotypes
More LessTo compare the antigenic and functional domains of the matrix (M) proteins of vesicular stomatitis virus (VSV) serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ), deletion mutants and chimeras were cloned in pBSM13 and expressed as in-frame lacZ fusion proteins in Escherichia coli. Non-cross-reactive monoclonal antibodies directed to the two antigenically distinct M proteins were tested by Western blot analysis to map three epitopes of VSV-Ind M protein and four epitopes of VSV-NJ M protein. Epitope 1 of the VSV-Ind M protein and epitope II of the VSV-NJ M protein both mapped to the highly basic N-terminal 34 amino acids of each homotypic M protein. Epitopes 2 and 3 of the VSV-Ind M protein and epitopes III and IV of the VSV-NJ M protein mapped to a region spanning amino acids 35 to 74. Epitope I of the VSV-NJ M protein mapped to a region between amino acid 75 and the C terminus. The similarity in location of the serotypically unique antigenic determinants of the two M proteins suggested that they may have a common functional domain. This hypothesis was substantiated by the finding that the two M proteins and various chimeras expressed in CV-1 cells by a recombinant vaccinia virus system were able to rescue M gene temperature-sensitive mutants of both VSV serotypes.
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Role of virion M2 protein in influenza virus uncoating: specific reduction in the rate of membrane fusion between virus and liposomes by amantadine
More LessThe anti-influenza virus drug amantadine was shown to reduce the rate of fusion of liposomes with influenza A viruses whose replication is inhibited by this drug. The fusion with amantadine-resistant viruses was unaffected. Experiments with reassortant and mutant viruses showed that this effect was linked to the M2 protein and not to the haemagglutinin of the virus. The proton ionophore monensin, on the other hand, substantially increased the rate of fusion of the viruses tested. These results indicate that the kinetics of virus-liposome fusion can be modulated by the virus M2 protein, the target of amantadine action, and it is postulated that the M2 ion channel functions by transporting protons into the virion interior and facilitating virus uncoating.
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Nucleotide sequence of the carlavirus associated with blueberry scorch and similar diseases
We have synthesized and mapped a library of cDNA clones representing the RNA genome of a strain of blueberry scorch carlavirus (BBScY) associated with a disease known locally, in New Jersey, U.S.A., as Sheep Pen Hill disease. The nucleotide sequence of that strain was determined to be 8514 residues, excluding the poly(A) tail. In addition, cDNA clones representing the 3′ terminus of another strain of the virus from the same field were synthesized, mapped and sequenced. The overall identity between sequences of these two strains was approximately 90% spanning the 1634 residue overlap, confirming their identity as distinct strains and not simply different isolates of a single strain. Finally, the coat protein gene of a distinct strain of the virus, isolated from plants with blueberry scorch disease in the Puyallup Valley in Washington State, U.S.A., was cloned from total cDNA by PCR. Sequence analysis revealed that the strain from Washington was more divergent from the two New Jersey strains than they were from each other. Comparisons of these sequences with other carlavirus sequences indicated that BBScV is more closely related to lily symptomless virus and potato virus S than to potato virus M, Helenium virus S, carnation latent virus or poplar mosaic virus. BBScV and potato virus M shared approximately 54 % nucleotide sequence identity overall.
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Simultaneous regulation of tomato golden mosaic virus coat protein and AL1 gene expression: expression of the AL4 gene may contribute to suppression of the AL1 gene
More LessThe tomato golden mosaic virus (TGMV) coat protein and AL1 genes are located in opposite directions on either side of an intergenic region. To enable the effects of the AL1, AL2 and AL3 gene products on expression of the coat protein and AL1 genes to be studied simultaneously, a plasmid was constructed, containing the intergenic region linked on one side to a 5′ -terminal portion of the AL1 gene fused to a β -glucuronidase (GUS) reporter gene (to replace most of the AL1 gene) and on the other side to a neomycin phosphotransferase (NEO) reporter gene (to replace the coat protein gene). This GUS-NEO plasmid was mixed with plant expression plasmids containing the AL1, AL2 or AL3 coding regions, the DNA was transformed into Nicotiana benthamiana protoplasts and GUS activities and NEO protein levels were measured. Control transformations were carried out with the GUS-NEO plasmid mixed with the AL1, AL2 or AL3 plasmids in which mutations were introduced to prevent translation of the open reading frames (ORFs). The results showed that transactivation of the coat protein gene by the AL2 gene product and suppression of the AL1 gene by the expression of AL1 DNA (both reported previously) can occur simultaneously. It was also shown that expression of AL4, a small ORF contained within AL1 DNA but in a different reading frame, as well as expression of ORF AL1, can cause significant suppression of AL1 gene expression. Neither the AL1 nor the AL3 gene products affected the expression of the coat protein gene.
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A stable 463 nucleotide variant of citrus exocortis viroid produced by terminal repeats
More LessAn unusual variant of citrus exocortis viroid (CEV) was detected when an inoculum source from Gynura auran- tiaca D.C. was used to infect a hybrid tomato (Lycopersicon esculentum Mill, × L. peruvianum). The 92 nucleotide larger variant, CEV D-92, which displayed the characteristic circular and linear viroid structural forms, contained two repeated sequences spanning the V and T2 domains. A dramatic moderation of symptom expression in Gynura accompanied the incorporation of these repeated sequences. A comparison of the sequence and structure of CEV D-92 with coconut cadang-cadang viroid revealed similarities in the regions generating the naturally occurring terminal repeats suggesting a possible preferred site for RNA recombination between viroids.
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