- Volume 67, Issue 11, 1986
Volume 67, Issue 11, 1986
- Review Article
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- Bacterial
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Effect of P22-mediated Receptor Release and of Phage DNA Injection on Cell Viability of Salmonella typhimurium
More LessSummaryInfection with u.v.-inactivated P22 bacteriophage at multiplicities higher than 30 caused a decrease in Salmonella typhimurium viability without cell lysis. Neither the action of the endoglycosidase of the P22 virion on the lipopolysaccharide of S. typhimurium nor the concomitant release of cell wall components was responsible for m.o.i.-dependent cell death. Using both free P22 tails and u.v.-inactivated P22, we have shown that p9 tail protein activity has no effect either on the integrity of host cells or on cell viability. Our results show that cell death is due to the injection of the u.v.-inactivated P22 DNA.
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- Animal
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Nucleotide Sequence of a Cloned Hepatitis B Virus Genome, Subtype ayr: Comparison with Genomes of the Other Three Subtypes
SummaryThe entire nucleotide sequence of genomic DNA was determined for hepatitis B virus (HBV) of subtype ayr, which had been derived from the blood of a Japanese asymptomatic carrier. The genome was 3215 nucleotides long, and differed in DNA sequence by 10% from that of subtypes adw or ayw, but by only 2% from that of subtype adr. Amino acid sequences coded for by the S, C, P and X genes, as well as by the pre-S region, closely resembled those of subtype adr, indicating that the evolution of HBV/ayr from HBV/adr was more recent than the differentiation of the other three subtypes. In the product of the S gene, the mutually exclusive subtypic determinants of the surface antigen, d and y, were associated with variation of amino acid residues at only the 68th and 122nd positions from the N terminus, in contrast to the variation at as many as seven positions for the other set of subtypic determinants, w and r. Sequences representing high local hydrophilicity in the product of the S gene were involved in subtypic variation, although such sequences in the pre-S region were shared by HBV genomes of the various subtypes. In particular, a hydrophilic sequence of 19 amino acid residues, coded for by the pre-S(2) region and implicated in the presumed hepatotropism of HBV, was possessed in common by HBV/adr, HBV/ayr and HBV/ayw, and differed in HBV/adw by only one residue at the 9th position. This amino acid sequence appears to be a promising candidate for a synthetic peptide vaccine.
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Comparative Expression of Hepatitis B Virus Antigens in Several Cell Model Systems
More LessSummaryIn this paper the kinetics of hepatitis B virus (HBV) gene expression were investigated in natural and experimentally transfected cell systems. These systems included four human hepatocellular carcinoma cell lines containing HBV DNA (TONG/PHC, HEp 3B2, PLC/PRF/5 and HA22T/VGH) as well as a mouse and a rat cell line both experimentally transfected with HBV DNA. Comparative results on the kinetics of hepatitis B surface antigen in these cell systems suggested that the S gene in the integrated state is expressed at different levels. No human cell line derived from HBV-associated hepatocellular carcinoma produced hepatitis B e antigen (HBeAg) when medium was concentrated by ultrafiltration. In distinct contrast, the two experimentally transfected cell systems produced e antigen at different levels. When all HBV-containing cell lines were grown as tumours in nude mice, no HBeAg was detected in the serum of these mice inoculated with human hepatocellular carcinoma cell lines, in the tumour homogenates, or in the tumour-derived lines, whereas e antigen was expressed both in vivo and in vitro in the experimentally transfected cell lines. These observations indicate that C gene expression is restricted to transfected cell cultures and this suggests a distinct difference in the mechanisms of HBV gene expression between the two types of in vitro model systems.
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Human Interferon Alpha and Gamma Production by Lymphocytes During the Generation of Influenza Virus-specific Cytotoxic T Lymphocytes
More LessSummaryWe analysed the production of interferons (IFN)-alpha and -gamma during the generation of human influenza-virus specific cytotoxic T lymphocyte (CTL) responses using monoclonal antibodies in a specific radioimmunoassay. The results showed that the peripheral blood mononuclear cells (PBM) of all donors tested produced IFN-gamma and had influenza A virus-specific CTL activity after stimulation. The amount of IFN-gamma produced and the level of CTL activity were significantly correlated. The PBM of some donors also produced IFN-alpha. The level of IFN-gamma produced was low during the first few days and increased subsequently, but IFN-alpha, when it was detected, was produced on day 1. The kinetics of the increase in IFN-gamma correlated with the increase in CTL activity. We also observed an increased percentage of cells bearing interleukin-2 receptors, which may have been a response to the production of IFN-gamma. The T cells active in lysing influenza A virus-infected target cells and in producing IFN-gamma were determined after separating effector cells with monoclonal antibodies. The CTL effector cells were mainly in the T8+ subset, but IFN-gamma-producing cells were found in both T4+ and T8+ subsets. These results suggest that influenza virus-specific T8+ CTL produce IFN-gamma in response to virus, and that T4+ cells which are not CTL effectors also produce IFN-gamma after restimulation with influenza A virus-infected cells.
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Establishment of a Mouse Model for Human Rhinovirus Infection
More LessSummaryWe describe here a mouse model for rhinovirus infection using a variant of human rhinovirus type 2 (HRV2/H) which replicated 50- to 300-fold in the lungs of BALB/c mice. The variant virus differed only marginally from HRV2/H according to various biochemical parameters. Use of a photosensitive inoculum and pretreatment of the animals with actinomycin D were necessary for detection of reproducible and significant levels of virus replication. This mouse model of rhinovirus infection is the first example of human rhinovirus replication in a non-primate mammal, and provides an important link for the development of rhinovirus therapy or prophylaxis.
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Selection and Characterization of an Interferon-responsive Clonal Cell Line of HeLa Cells
More LessSummaryHeLa cells generally do not respond well to interferon (IFN). We have used is-1, an IFN-sensitive mutant of mengovirus, to select a clone of IFN-responsive HeLa cells (F-H12). At moderate levels of human α/β IFN, is-1 yields were fivefold lower in these cells than in similarly protected control cells. In contrast, wild-type mengovirus, vesicular stomatitis virus and a wild-type and thymidine kinase-negative strains of herpes simplex virus type 1 grew equally well in both cell lines. By a cell survival assay, the F-H12 line was up to 100 times more responsive to IFN than the parental line when challenged by is-1. 2′-5′-Oligo(A)-dependent endonuclease activity was the same in both lines. These observations cannot be accounted for by enhanced induction of IFN following infection.
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Characterization of the IE110 Gene of Herpes Simplex Virus Type 1
More LessSummaryWe have determined the DNA sequence of the herpes simplex virus type 1 (HSV-1) gene encoding the immediate early protein IE110, which is involved in transcriptional activation of later virus genes. The locations of the 5′ and 3′ termini of IE110 mRNA, together with the positions of two introns, were identified. Examination of the DNA sequence suggested that translation starts at the first ATG after the 5′ terminus of the mRNA, and that both introns occur in protein-coding sequence. The predicted IE110 polypeptide contains 775 amino acids, and has a molecular weight of 78452. It contains a cysteine-rich region resembling regions found in several proteins which interact functionally with DNA. An antiserum was raised to the predicted C terminal amino acid sequence of the IE110 polypeptide and was shown to immunoprecipitate the native protein from HSV-1-infected cell extracts. The functional importance of regions of the protein was evaluated by construction of frameshift and deletion mutants of a plasmid-borne IE110 gene. The mutants were tested for IE110 function by short-term transfection assays, and the results were correlated with the DNA sequence and RNA mapping studies.
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Transcriptional Organization of Bovine Papillomavirus Type 4
More LessSummarySeven virus-specific RNA transcripts have been identified in tumours induced by bovine papillomavirus type 4 (BPV-4). The RNAs measured 4.2, 3.6, 3.0, 2.8, 1.9, 1.6 and 1.0 kilobases (kb). They were mapped on the viral genome by Northern blot hybridization to subgenomic probes, by cDNA hybridization to viral DNA fragments and by S1 analysis of unlabelled and 3′ and 5′ end-labelled DNA fragments. All the RNA species are transcribed from the same DNA strand, are polyadenylated and with the exception of the 1.0 kb RNA internally spliced. The 3.0, 1.9, 1.6 and 1.0 kb RNAs share the same 3′ polyadenylation site at nucleotide 4009 whereas the 4.2 kb RNA and the 2.8 kb RNA terminate near a polyadenylation site at nucleotide 7187. The 4.2 kb and the 2.8 kb RNAs are transcribed from the late open reading frames and encode the structural polypeptides; the 3.0, 1.9, 1.6 and 1.0 kb RNAs are transcribed from the early open reading frames and are the transcripts involved in viral replication and cellular transformation.
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Characterization of Transforming Viruses Rescued from a Hamster Tumour Cell Line Harbouring the v-src Gene Flanked by Long Terminal Repeats
More LessSummaryThe organization of proviruses derived from infecting transforming viruses rescued from hamster tumour cells was studied. Southern blot analysis indicated that the provirus from the F6 cell line was organized as long terminal repeat (LTR)-src-LTR, and S1 mapping experiments suggested that it was probably derived by reverse transcription of src mRNA followed by integration. In the E6 cell line, the provirus unit was arranged as LTR-Δ gag-src-LTR, indicating a recombination event between the rescued transforming virus and the helper virus. These results suggest that transforming defective viruses containing only the src gene can be rescued from non-permissive mammalian cells.
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The Genome-linked Proteins of Aphthoviruses: Specific Immunoprecipitation of the Three Species Detected on Virus RNA and Identification of Possible Precursors
More LessSummarySynthetic peptides have been made corresponding to the C-terminal portion of each of the three presumptive genome-linked proteins (VPgs) of foot-and-mouth disease virus type A10. Antisera against each of these peptides efficiently precipitated only the homologous VPg, and the reactions were inhibited by prior absorption with homologous, but not heterologous synthetic peptide. The peptide antisera precipitated a number of proteins from infected cell extracts with mol. wt. of 100, 84, 56, 36, 27, 25 and 20, all × 103; all these reactions were inhibited by absorption with homologous peptide, indicating that they were probable precursors of VPg. The relationship between these proteins is at present unclear.
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Production of Monoclonal and Monospecific Antibodies against Non-capsid Proteins of Poliovirus
More LessSummaryNon-capsid poliovirus proteins of the P2 region in extracts of infected cells were solubilized by SDS, separated by PAGE, electroeluted from the gels and used to immunize mice. The sera obtained were rendered monospecific by extensive absorption with proteins from uninfected cell extracts, and the spleen cells of these animals were used to establish antibody-secreting hybridomas. The monoclonal and the monospecific antibodies recognized denatured antigen (proteins 2C, 2BC and P2) in ELISA, immunoblot and immunoprecipitation tests and also combined with the native proteins of the P2 region in immunoprecipitation. In addition, the antibodies could be used successfully for immunofluorescence and electron microscopic immunocytochemistry.
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The Effect of pH on the Early Interaction of West Nile Virus with P388D1 Cells
More LessSummaryThe interaction between the flavivirus West Nile virus (WNV) and cells of the mouse macrophage-like cell line, P388D1, was assayed by transmission electron microscopy, by following the association of [35S]methionine-labelled virus with cells, and by using a radiobinding assay with an 125I-labelled F(ab′)2 fragment of a monoclonal antibody directed against the major viral envelope surface glycoprotein. Using electron microscopy, both fusion and endocytosis were observed at pH 6.4, but at pH 8.0 only endocytosis was observed. When 35S-labelled WNV was bound to the P388D1 cell surface at 0 °C, less virus eluted on warming to 37 °C at mildly acidic than at alkaline or neutral pH values. The monoclonal antibody fragment had an increased affinity for cell surface viral E glycoprotein after prebound WNV was warmed at mildly acidic pH values. It is proposed that the warming of cell-virus mixtures at low pH results in fusion with a consequent reduction in elution of virus and an increase in the recognition of cell surface-expressed viral envelope glycoprotein by labelled antibody.
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Restricted Expression of Measles Virus Proteins in Brains from Cases of Subacute Sclerosing Panencephalitis
More LessSummaryThe presence of five structural proteins of measles virus in brain material obtained at autopsy from four patients with subacute sclerosing panencephalitis (SSPE) was examined by immunofluorescence employing monoclonal antibodies. In addition, the humoral immune response against measles virus antigens in serum and cerebrospinal fluid was analysed by immunoprecipitation in combination with gel electrophoresis, revealing a reduced response mainly to the matrix (M) protein. In none of the brain material were all five structural proteins simultaneously detected. Nucleocapsid protein and phosphoprotein were found in every diseased brain area, whereas haemagglutinin (H) protein was detected in two, fusion (F) protein in three and M protein only in one SSPE case. In two cases, variations in the occurrence of H and F proteins could be observed between regions displaying different degrees of neuropathological changes. No correlation was observed between the humoral immune response and the immunohistological findings. These data support the hypothesis of a restricted synthesis of measles virus proteins, in particular the envelope and M proteins, in SSPE.
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VP7 Serotype-specific Glycoprotein of OSU Porcine Rotavirus: Coding Assignment and Gene Sequence
SummaryWith a reassortant from a cross of human rotavirus DS-1 (serotype 2) and OSU (serotype 5) it was determined that the OSU major neutralization glycoprotein antigen (VP7) was encoded by gene segment 9. A full-sized cloned cDNA copy of the OSU gene 9 was produced and sequenced. Hybridization of such labelled cDNA with the corresponding segment of a reassortant DS-1 × OSU virus confirmed the coding assignment. Comparison of the deduced amino acid sequence of the VP7 of OSU with those previously determined for five other rotavirus strains, representing four distinct serotypes, revealed some hydrophilic regions that exhibited significant homology and other hydrophilic domains with greater amino acid divergence. In one of the latter hydrophilic domains each of the five serotypes had a distinct amino acid substitution at residue 146, suggesting that it may be involved in serotype specificity.
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Antigenic Analysis of Rotavirus Isolates using Monoclonal Antibodies Specific for Human Serotypes 1, 2, 3 and 4, and SA11
More LessSummaryFive neutralizing monoclonal antibodies produced against human rotavirus (HRV) serotypes 1, 2, 3 and 4 and the simian rotavirus (SA11) were used to study 59 rotavirus isolates of human, simian and feline origin previously serotyped using polyclonal antisera. In neutralization tests, 19 of 26 HRV serotype 1 isolates, both strains of HRV serotype 2, 14 of 24 HRV serotype 3 isolates and all of seven serotype 4 isolates were neutralized by the homologous serotype-specific monoclonal antibodies. Use of the panel of monoclonal antibodies revealed antigenic differences between strains within serotypes 1 and 3 and, in the case of the serotype 3 strains, each variant had a unique RNA electropherotype. An enzyme immunoassay (EIA) which utilized the monoclonal antibodies essentially confirmed the neutralization results. Preliminary results show that direct serotyping in faecal extracts by EIA using these monoclonal antibodies is specific but lacks sensitivity.
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Virus-neutralizing Antibodies to Hepatitis B Virus: The Nature of an Immunogenic Epitope on the S Gene Peptide
More LessSummaryUsing a murine monoclonal antibody (RF-HBs-1) which has been shown to be capable of neutralizing both ad and ay subtypes of hepatitis B virus (HBV), we have devised a competitive inhibition assay to measure the presence of virus-neutralizing antibodies in the sera of patients who have recovered from acute type B hepatitis. The majority of patients have this antibody in their serum. We also show that this antibody inhibits the binding of polymerized human serum albumin (pHSA) to the pHSA receptor site of the HBV particle, which has been proposed as an important site for the entry of HBV into liver cells. We have demonstrated that the epitope recognized by this antibody is dependent on the linkage of 24000 and 28000 mol. wt. polypeptides via a disulphide bond. This conformational determinant in the coat of the virus which is part of or near to the pHSA binding site is important in evoking a virus-neutralizing response.
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The Peplomers of Berne Virus
More LessSummaryUsing [3H]glucosamine and [3H]mannose labels, two virus-specific glycosylated polypeptide species with M r values of about 200000 (200K) and in the 75K to 100K range, respectively, were recognized in Berne virus-infected embryonic mule skin cells. In purified virions only the latter glycoprotein occurred. Concanavalin A was bound to the virion as evidenced by reduction in infectivity. Analyses using SDS-PAGE, blotting and glycoprotein identification with concanavalin A and horseradish peroxidase showed coincidence of the virion glycoprotein signals with the maximum infectivity and haemagglutinating activity in an isokinetic sucrose gradient. Polyclonal rabbit immune serum and a neutralizing and haemagglutination-inhibiting monoclonal antibody raised against Berne virus recognized both the 75K to 100K and the ‘200K’ glycoproteins. Using tunicamycin, a concentration-dependent inhibition of infectivity was noted; however, non-infectious particles containing the two major polypeptides (20K and 22K) were released from the cells in small quantities. The glycoproteins were absent from cytoplasmic extracts and a novel polypeptide of about 150K was identified instead. Translation of poly(A)-selected intracellular RNA from infected cells in a rabbit reticulocyte cell-free system also resulted in the appearance of a new high M r polypeptide (about 170K). Using pulse-chase labelling and radioimmunoprecipitation, suggestive evidence for a precursor-product relationship between the intracellular ‘200K’ and the virion glycoproteins has been obtained. These experiments identify the N-glycosylated proteins in the 75K to 100K range as constituents of the peplomeric envelope projection of Berne virus; they probably arise by post-translational processing of a 150K to 170K precursor molecule involving glycosylation and subsequent cleavage.
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The Haemagglutinating Activity of Berne Virus
More LessSummaryBerne virus possesses haemagglutinating activity which is inhibited by antisera that neutralize the infectivity of the virus. In decreasing order, human, rabbit and guinea-pig erythrocytes were agglutinated whereas agglutination was not observed with rat, goose, chicken or horse red blood cells. This pattern is different from that seen with the closely related Breda virus of cattle. Haemagglutinin was found to co-sediment with viral infectivity in sucrose density gradients. Transmission electron microscopy showed that intact virus particles form bridges between adjacent erythrocytes. The viral envelope was seen at a distance from the erythrocyte surface suggesting that the peplomers possess haemagglutinating activity. Haemagglutination was decreased in the presence of fetuin and gangliosides and also by pretreatment of the erythrocytes with periodate, suggesting that the virus binds to glycoproteins and/or glycolipids on the erythrocyte surface.
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Establishment of Herpes Simplex Virus Latency in vitro with Cycloheximide
More LessSummaryHuman embryonic lung cells were infected with herpes simplex virus (HSV), treated with 10 µg/ml or more of cycloheximide for 24 h, incubated at 37 °C, and then shifted to 40.5 °C for various periods of time (0 to 40 days) without cycloheximide treatment. No infectious virus was detected after freezing and thawing of the cultures; however, infectious virus was recovered after temperature shift-down to 37 °C or superinfection with human cytomegalovirus (HCMV). The time course for formation of infectious centres after temperature shift-down was examined with and without HCMV superinfection during incubation at 40.5 °C. Two patterns of latently infected cells were identified: one pattern showed spontaneous reactivation of virus after temperature shift-down, and the second showed reactivation of HSV after superinfection with HCMV. The first pattern showed a rapid decrease in the number of infectious centres with time, whereas the second maintained a steady reactivation rate up to 40 days at 40.5 °C. The same tendency was observed for infectious centre formation at 37 °C with and without HCMV superinfection in the HSV latency system established with (E)-5-(2-bromovinyl)-2′-deoxyuridine and interferon treatment.
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Restoration of Wild-type Pathogenicity to an Attenuated DNA Polymerase Mutant of Herpes Simplex Virus Type 1
More LessSummaryThe drug-resistant variant, RSC-26, which was derived from the herpes simplex virus type 1 wild-type strain SC16, expresses an altered DNA polymerase and has reduced pathogenicity in animal models. To determine whether the attenuation in pathogenicity was due solely to mutation in the polymerase gene, a fragment of the wild-type gene was cloned, transferred into the genome of RSC-26 and recombinants were isolated. Three recombinants examined had similar properties to wild-type virus with respect to their sensitivity to antiviral drugs, DNA polymerase activities and their pathogenicity for mice. These results strongly suggest that expression of the altered polymerase of RSC-26 results in attenuated pathogenicity.
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The Products of Herpes Simplex Virus Type 1 (HSV-1) Immediate Early Genes 1, 2 and 3 Can Activate HSV-1 Gene Expression in trans
More LessSummaryExpression of the early and late genes of herpes simplex virus type 1 (HSV-1) during infection of tissue culture cells requires the prior expression of the immediate early (IE) genes. The requirement for the product of IE gene 3, Vmw175, for the activation of early promoters has been revealed by studies with temperature-sensitive virus mutants. Recent experiments using transfection assays have shown that both Vmw175 and the product of IE gene 1, Vmw110, are involved in the transactivation of a variety of HSV-1 early promoters. This paper describes experiments which compared the activation of two early promoters [those of the glycoprotein gD and thymidine kinase (tk) genes] with that of a member of a later class of genes (the major capsid protein, VP5). Plasmids containing these promoters linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into HeLa cells with plasmids containing one or more HSV-1 IE genes. Promoter activity was estimated by measurement of CAT activity in extracts of transfected cells. The gD and tk promoters were activated by both Vmw175 and Vmw110, and the combination of these two IE gene products resulted in very high levels of activation. Addition of further IE gene products did not result in any significant increase in the activation seen with the combination of Vmw175 and Vmw110. In contrast, the activation of the VP5 promoter brought about by the combination of Vmw175 and Vmw110 was relatively slight, but was increased further when plasmids containing IE gene 2, encoding Vmw63, were included in the transfection. These data suggest that Vmw63, like Vmw175 and Vmw110, is also involved in the activation of transcription from HSV-1 promoters. The effect of Vmw63 may be limited to the activation of a subset of HSV-1 genes.
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Detection of Bovine Herpesvirus Type 1 RNA in Trigeminal Ganglia of Latently Infected Rabbits by in situ Hybridization
More LessSummaryAt times after conjunctival inoculation with bovine herpesvirus type 1 (BHV-1), representing the acute and latent phases of infection, rabbit trigeminal ganglia were examined for the presence of BHV-1 nucleic acids by in situ hybridization using a 3H-labelled BHV-1 DNA probe. During the acute phase of virus infection, both BHV-1 DNA and RNA were detected in ganglionic neurons and occasionally in adjacent satellite cells. However, during the latent phase of infection only viral RNA was detectable in involved neurons. Viral RNA appeared restricted to the nucleus of latently infected cells and was present in varying amounts in individual cells. These results indicate that the BHV-1 genome is transcriptionally active in ganglionic neurons during latent infection.
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Induction of Oligo-2′,5′-adenylate Synthetase in Human Lymphoid Cells Treated with 5-Azacytosine and 5-Iododeoxyuridine
More LessSummaryOligo-2′,5′-adenylate synthetase activity was investigated in several human lymphoblastoid cell lines of B cell origin treated with reagents having effects on gene expression, 5-azacytosine (5AZct), 5-azacytidine (5AZcd) and 5-iodo-2′-deoxyuridine (IUdR). Enzyme activity did not increase in cells treated with 5AZcd, but the other reagents induced significant levels of activity. Interferon (IFN) activity was not detected in culture fluids of cells treated with 5AZcd or 5AZct. On the other hand, treatment of cells with IUdR led to the production of 5 to 10 units/ml IFN by NC-37 and Raji cells, but not by other human B cell lines. Enzyme induction by IUdR occurred in IFN-producing cells, NC-37 and Raji, exposed to anti-IFN-α sera and also in IFN-non-producing human B cells. The effect of 5AZct on enzyme induction was observed only after cultivation of cells for more than 1 month. This compound could not induce enzyme activity in Epstein-Barr virus (EBV)-negative cells, BJAB, but only in the EBV-positive cell lines NC-37 and Raji. In contrast, treatment of cells with IUdR for only 3 days induced the enzyme in all human lymphoblastoid cell lines of B cell origin except P3HR-1, but not in other human cell lines, FL, HeLa and K562. Oligo-2′,5′-adenylate synthetase activity in P3HR-1 cells, which produce EBV particles, was hardly affected by IUdR or IFN treatment.
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Reactivity of Anti-peptide and Anti-poliovirus Type 3 Monoclonal Antibodies with Synthetic Peptides
More LessSummaryMonoclonal antibodies were prepared from mice immunized with an 18-residue synthetic peptide with an amino acid sequence from a major antigenic sequence involved in the neutralization of type 3 poliovirus. Approximately 250 hybridomas secreted antibodies that reacted with the peptide but not the virus, two antibodies reacted with the virus but not the peptide and no antibody reacted with both. Conversely 26 monoclonal antibodies prepared from mice immunized with type 3 poliovirus and known to be directed against the appropriate sequence on the virus, generally failed to react with the peptide. These results might be expected if only a small proportion of the free or coupled peptide molecules adopt molecular conformations which resemble that of the homologous antigenic site in the virus. Antibodies specific for other antigens occasionally reacted well with the synthetic peptides, indicating that antibodies may bind to peptides of inappropriate sequence. The identification of antigenic sites by the use of synthetic peptides therefore requires considerable caution.
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Shedding and Interspecies Type Sero-reactivity of the Envelope Glycopolypeptide gp120 of the Human Immunodeficiency Virus
More LessSummaryTwo glycopolypeptides with molecular weights 160000 and 120000 (gp120) are regularly recognized by human immunodeficiency virus (HIV)-specific antisera in lysates of cells persistently infected with HIV. In the present study, gp120 was characterized as the major envelope glycopolypeptide of HIV. Gp120 was identified as the external viral glycoprotein by radiosequencing and by its presence in purified virus. However gp120 was predominantly shed as a soluble protein into the culture fluid. Furthermore gp120 was precipitated by sera from horses infected with equine infectious anaemia virus (EIAV), but not by sera from uninfected animals. This may indicate conserved epitopes common to the envelopes of HIV and EIAV.
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Antibody-mediated Early Death in vivo after Infection with Yellow Fever Virus
More LessSummaryThe phenomenon known as antibody-dependent enhancement (ADE) has been demonstrated in vitro but its significance in viral pathogenesis is uncertain even though it has been associated with dengue shock syndrome. Here we report for the first time the enhancement of virus virulence in mice using monoclonal antibodies (MAbs) prepared against yellow fever (YF) viruses. Our results show that the average survival time of mice was reduced by up to 33% (i.e. 6.7 to 4.5 days) and that ADE is both antibody dose-dependent and antibody- and virus strain-specific. A total of 12 YF viruses and 11 MAbs were examined and of these only three YF viruses (FNV, Asibi and B11) could be enhanced in vivo by only two MAbs (427 and 126). A particular combination of virus and antibody is required for ADE to take place.
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Complete Sequence of the Major Nucleocapsid Protein Gene of Human Parainfluenza Type 3 Virus: Comparison with Other Negative Strand Viruses
More LessSummaryThe sequence of the major nucleocapsid protein (NP) mRNA and its encoded protein were deduced by sequencing a cDNA clone representing the complete mRNA. The cDNA sequence was confirmed by dideoxynucleotide sequencing of purified viral genomic RNA by primer extension using synthetic oligonucleotides. The NP mRNA contains 1641 nucleotides exclusive of poly(A) and encodes an NP protein of 515 amino acids. Alignment of the human parainfluenza type 3 virus (PF3) NP protein sequence with that of Sendai virus showed that the two proteins shared considerable sequence identity (58.8%). Additional comparisons provided highly significant statistical evidence that the PF3 NP protein sequence is related to those of measles and canine distemper viruses, but there was no evidence of relatedness with the nucleocapsid proteins of respiratory syncytial virus, influenza B virus, or vesicular stomatitis virus.
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Developmental-dependent Replication of Minute Virus of Mice in Differentiated Mouse Testicular Cell Lines
More LessSummaryThe replication of the autonomous parvovirus, minute virus of mice (MVM), requires mitotically active cells and depends on certain factors expressed by cells of particular differentiated phenotype. As an approach to the understanding of these helper functions, we studied the interaction of the fibrotropic [MVM(p)] and the lymphotropic [MVM(i)] strains of MVM with two differentiated cell lines from mouse testicular epithelial origins. The relative support given to viral expression by these cell lines varied extensively. Cells from Sertoli origin (TM4) were permissive to MVM(p) but were mostly restrictive to MVM(i). The other cell line, of Leydig cell origin (TM3), was highly restrictive to both viral strains, but the blocks to their growth in these cells were localized at different stages of their growth cycle, suggesting that the replication of MVM in these cells requires tissue-specific helper functions during at least two stages of viral replication.
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Sequence Reiteration Required for the Efficient Growth of BK Virus
More LessSummaryCompared with wild-type BK virus DNA having tandem triplication of a 68 base pair (bp) element in its transcriptional control region, a mutant viral DNA with a single copy of the 68 bp element induced remarkably delayed virus production in human embryonic kidney (HEK) cells. We molecularly cloned the DNA of progeny viruses using plasmid vector pAT153. Nucleotide sequence analysis of representative clones revealed that all of the altered viral DNAs examined duplicated various segments extending over origin-distal portions of the 68 bp element and their flanking regions. After transfection of HEK cells, most of these rearranged viral DNAs induced viral growth slightly slower than, or at the same rate as, the wild-type viral DNA. Comparison of the structures of these rearranged viral DNAs suggests that reiteration of a 13 bp sequence, which is located in an origin-distal portion of the 68 bp element, is required for the efficient replication of BK virus.
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Nucleotide Sequence of a Portion of the Autographa californica Nuclear Polyhedrosis Virus Genome Containing the EcoRI Site-rich Region (hr5) and an Open Reading Frame just 5′ of the p10 Gene
More LessSummaryThe nucleotide sequence of a 1587 bp region lying within the HindIII-Q fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) DNA has been determined. It begins in the EcoRI-S-EcoRI-X region, continues to the HindIII-P/Q boundary and contains an open reading frame that codes for a polypeptide of 240 amino acids (p26). This open reading frame is also included in the 1100 and 1500 base transcripts previously mapped to this region. The sequence reveals that the 5′ ends of the 1100 and 1500 base transcripts are located 20 bp downstream from the end of a putative TATA box (TAATTAAAT) and 19 bp upstream from the translation start codon (ATG) of the p26 open reading frame. The translation termination codon (TAA) falls in the immediate 5′ flanking region of the major late p10 gene of AcMNPV, 3 bp downstream from the putative TATA box. The probable polyadenylation site for the 1100 base transcript lies 23 bp downstream from the cap site for the 750 and 2500 base transcripts encoding the p10 protein. The 5′ flanking region of the p26 open reading frame contains the EcoRI site-rich region, hr5, whose sequence is included here. The EcoRI site-rich region, hr5, consists of six imperfect tandem repeats of a sequence that includes the EcoRI recognition site. These direct repeats also include many inverted repeats.
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Expression of Potato Virus X Resistance Gene Rx in Potato Leaf Protoplasts
More LessSummaryProtoplasts derived from shoot cultures of potato cv. Cara, which carries immunity gene Rx, supported only limited virus multiplication after inoculation with particles or RNA of isolate DX, a group 3 strain of potato virus X, as compared to similarly inoculated protoplasts of cultivars King Edward and Pentland Ivory which lack Rx. The Cara protoplasts were, however, able to support extensive replication of the resistance-breaking strain HB. This strain-specific resistance did not appear to be mediated by a failure or inhibition of the uncoating mechanism or of virus assembly.
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The Stability of Cowpea Mosaic Virus VPg in Reticulocyte Lysates
More LessSummaryThe ability of the genome-linked protein (VPg) of cowpea mosaic virus (CPMV) to survive incubation in rabbit reticulocyte lysates was investigated. In contrast to the results obtained with picornavirus RNAs, there was no evidence for the specific removal (‘unlinking’) of the VPg from CPMV RNA during incubation. While linked to RNA, CPMV VPg was protected from proteolytic degradation; if the RNA was first digested with nuclease P1, rapid degradation of the VPg occurred. However if as few as 17 nucleotides were left attached to the VPg, stability was retained.
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Effects of Anti-microtubule Agents on the Assembly of Tobacco Mosaic Virus Coat Protein
More LessSummaryAssembly of the coat protein subunits of tobacco mosaic virus (TMV) into 20S aggregates (‘disks’) and long helices was monitored by light scattering and electron microscopy. The anti-microtubule agent methyl benzimidazol-2-yl carbamate, which inhibits TMV multiplication in vivo, did not affect assembly of coat protein in vitro. In contrast, the anti-microtubule agents colchicine and vinblastine inhibited disk formation, but stimulated rod elongation from coat protein subunits in vitro. Both agents also disrupted preformed disks. Vinblastine inhibited virus multiplication in leaf tissue, but colchicine did not.
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