- Volume 73, Issue 2, 1992
Volume 73, Issue 2, 1992
- Animal
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The effect of cicloxolone sodium on the replication in cultured cells of adenovirus type 5, reovirus type 3, poliovirus type 1, two bunyaviruses and Semliki Forest virus
More LessThe effect of cicloxolone sodium (CCX) on the replication of typical representatives of different virus families [adenovirus type 5 (Ad-5), reovirus type 3 (Reo-3), Bunyamwera and Germiston viruses, poliovirus type 1 (Polio-1) and Semliki Forest virus (SFV)] in tissue culture was investigated. The Golgi apparatus inhibitor monensin (Mon) and CCX were shown to have analogous effects on some aspects of virus replication. Although the Mon-like effect of CCX played no role in the antiviral activity against Ad-5, Reo-3 or Polio-1, it could entirely account for the antiviral activity against the Bunyamwera and Germiston viruses, for which inhibition of glycoprotein processing was responsible for the antiviral activity. In the case of SFV, the Mon-like activity of CCX caused cytoplasmic assembly of fully infectious SFV within vacuoles and thus impaired virus release without altering total infectious virus yield. Fewer Ad-5 and Reo-3 progeny were produced in the presence of the drug. CCX had a dose-dependent biphasic effect on the particle:p.f.u. ratio of the Reo-3 yield. At low CCX concentration (<50 µm) the virus yield contained poor quality, non-infectious virus, but at higher CCX concentration (⩾ 100 µm) low quality virus could no longer be successfully assembled. We conclude that the antiviral effect can be manifested in three ways: (i) by a reduction in the virus particle yield produced; (ii) by a loss of quality (relative infectivity); (iii) by a virucidal effect of the drug. We have previously defined three CCX sensitivity classes. Mechanisms (i), (ii) and (iii) operate against viruses belonging to class CCXs-1 [herpes simplex virus (HSV) type 1, HSV-2 and vesicular stomatitis virus], but essentially only (i) and (ii) affect Reo-3 (CCXs-2), whereas (i) and possibly (iii) affect Ad-5 (CCXs-2). In the case of SFV (CCXs-3) none of these mechanisms operate, but relocation of assembled virus is found.
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The 24K protein of Borna disease virus
Based on partial amino acid sequences obtained from tryptic peptides of the purified 24K antigen of Borna disease virus (BDV), we identified and sequenced four independent cDNA clones established from BDV-infected MDCK cells. Each of the clones encodes a polypeptide of 201 residues (M r 22461) that differs considerably from an amino acid sequence published recently. In vitro transcription/translation of both the wild-type and a 5′ truncated clone lacking the first ATG codon yielded a peptide that comigrates on electrophoresis with a polypeptide immunoprecipitated from BDV-infected cells. The deduced amino acid sequence contains a putative signal for nuclear targeting.
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Human immunodeficiency virus type 1 envelope glycoprotein gp120-mediated killing of human haematopoietic progenitors (CD34+ cells)
More LessThe effects of human immunodeficiency virus type 1 (HIV-1) and recombinant envelope glycoprotein gp120 on the in vitro growth of enriched human haematopoietic progenitors (CD34+ cells) have been investigated. A 2 h exposure to HIV-1 resulted in a progressive and significant reduction of viable CD34+ cell number in liquid cultures and of granulocyte-macrophage, erythroid and megakaryocytic progenitors in semisolid cultures. In virus-treated CD34+ cells, no signs of active virus replication were observed and the possibility of latent infection was excluded by quantitative polymerase chain reaction. Recombinant HIV-1 envelope glycoprotein gp120 added to CD34+ cell cultures displayed a dose-dependent inhibitory activity on CD34+ cell viability. Neutralizing antibody against gp120 was able to block completely the inhibitory activity on CD34+ cells of either HIV-1 or recombinant gp120. These results demonstrate that HIV-1 envelope glycoprotein gp120 has a direct cytotoxic effect on CD34+ cells.
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Human papillomavirus type 6a DNA in the lung carcinoma of a patient with recurrent laryngeal papillomatosis is characterized by a partial duplication
More LessTranscriptionally active human papillomavirus type 6a (HPV-6a) DNA was detected in a lung carcinoma of a patient with recurrent laryngeal papillomatosis. The carcinoma contained episomal HPV-6a genomes that had a duplication of the upstream regulatory region, the late region and a portion of the early region. HPV-6a genomes found in benign laryngeal papillomas from the same patient did not contain this duplication. A role for the mutant molecules in the pathogenesis of the malignancy is suggested.
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Epitope mapping of the human papillomavirus type 16 E4 protein by means of synthetic peptides
More LessEight overlapping icosapeptides covering the entire sequence of the E4 protein of human papillomavirus type 16 (HPV-16), were prepared and tested for their reactivity with human sera in IgG-specific ELISA. The strongest reactivity of sera from HPV-16 DNA-positive invasive cervical carcinoma (INCA) patients was detected with the peptide denoted 16/E4-6, covering amino acids 51 to 70. Subsequently nearly 200 sera were tested for the presence of the 16/E4-6-specific antibody. Reactivity was more frequent in cervical intraepithelial neoplasia patients and INCA patients than in matched control subjects. Sera from INCA patients were also tested for antibody reactive with peptide 16/E7-2 covering the major type-specific reactive region of the HPV-16 E7 protein. Only four of 13 sera possessing the 16/E4-6-specific antibody were reactive with the 16/E7-2 peptide.
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Repression of human cytomegalovirus major immediate early gene expression in a monocytic cell line
More LessWe have previously shown that a major site of persistence of human cytomegalovirus (HCMV) in healthy carriers is in peripheral blood monocytes. However, monocytes are difficult to infect in vitro with HCMV, and HCMV gene expression cannot be reproducibly detected in peripheral blood cells of healthy carriers. Here we show that the monocytic cell line THP1 is non-permissive for HCMV infection due to a block in expression of the HCMV major immediate early (IE) promoter. This repression is correlated with the presence of a differentiation-specific cellular factor which binds to the imperfect dyad symmetry and the 21 bp enhancer repeats of the major IE promoter regulatory region and which has characteristics of MBF1, a factor which we have previously defined in HCMV non-permissive, undifferentiated teratocarcinoma cells. Both differentiation of THP1 cells into macrophages, which results in a decrease in this factor, or deletion of the factor's binding sites from the IE promoter/enhancer lifts this repression and permits expression from the major IE promoter.
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Nuclear expression of the lower matrix protein of human cytomegalovirus in peripheral blood leukocytes of immunocompromised viraemic patients
More LessPeripheral blood leukocytes (PBL), namely polymorphonuclear leukocytes (PMNL), are the major carrier of human cytomegalovirus (HCMV) in the blood of immunocompromised patients with HCMV viraemia. By using monoclonal antibodies (MAbs) directed against different early and late viral proteins, we showed that the protein accumulating in PBL, originally reported to be an immediate early (IE) gene product, is the 65K lower matrix early structural protein (ep65). This protein is detectable by immunofluorescence before IE proteins during early stages of the replication cycle of HCMV in permissive human embryonic lung fibroblast cells. However, the appearance of ep65 in the nucleus within 1 h post-infection in the presence of cycloheximide indicates that it represents uptake from the virus inoculum rather than newly synthetized protein. The ep65 MAbs staining PBL did not react with Vero cells infected with a recombinant vaccinia virus encoding the major IE gene (IE1) product, whereas MAbs reactive with the 72K major IE protein stained only faintly a small number of infected PBL. A group of four ep65 MAbs was tested in competitive binding assays to show that ep65 possesses at least three distinct epitopes. These were recognized by all four MAbs in AD169-infected Vero cell cultures when fixed with formaldehyde, whereas only one MAb recognizing a distinct epitope was reactive with methanol-acetone (MA)-fixed AD169-infected Vero cells. In formalin-fixed PBL the number of infected cells stained by the four ep65 MAbs was about twofold that found using MA-fixed cells. Using fluorescence-activated cells orter-purified leukocyte subpopulations from viraemic patients with different levels of viraemia, the ratio of ep65-positive to ep65-negative cells was found to be 1:100 to 1:100000 for PMNL, and only 1:10000 to 1:100000 for mononuclear cells.
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Analysis of human herpesvirus 6 glycoproteins recognized by monoclonal antibody OHV1
More LessA virus-specific glycoprotein (gp) from human herpesvirus 6 (HHV-6) was studied using the anti-HHV-6 monoclonal antibody OHV1. Immunoprecipitation with extracts from infected cells revealed that the antibody recognized four glycosylated proteins (gps) with M rs of 106K, 102K, 65K and 63K under reducing conditions. However, only two gps, of 106K (gp106) and 102K, were detected under non-reducing conditions. Pulse-chase experiments revealed that gp65 and gp63 were cleavage products of gp106 and gp102. When infected cells were treated with tunicamycin, none of these gps was detected. With endo-β-N-acetylglucosaminidase H (endo H) and endo-β-N-acetylglucosaminidase F (endo F) treatment, gp106 and gp102 disappeared. Moreover, gp65 and gp63 were not affected by endo H treatment but were sensitive to endo F treatment. These data suggest that sugar residues of gp106 and gp102 are high-mannose type N-linked oligosaccharides, whereas those of gp65 and gp63 are complex type N-linked oligosaccharides.
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Protective immunization against Epstein-Barr virus-induced disease in cottontop tamarins using the virus envelope glycoprotein gp340 produced from a bovine papillomavirus expression vector
More LessInoculation with Epstein-Barr virus (EBV) induces malignant lymphomas in the cottontop tamarin (Saguinus oedipus oedipus). This provides an experimental animal model for assessing the efficacy of candidate EBV vaccines which are intended to reduce the incidence of human tumours associated with EBV infection. Previous work has shown that experimental vaccines based on the major virus envelope glycoprotein gp340 prepared from the membranes of EBV-infected cells are effective in protecting cottontop tamarins against EBV-induced disease. However, not all purified gp340 preparations induce protective immunity against EBV lymphoma in the tamarin. In this work, cottontop tamarins were immunized with recombinant gp340, produced using a bovine papillomavirus (BPV) expression vector, and a threonyl muramyl dipeptide adjuvant formulation. Although the recombinant-derived gp340 lacked the membrane anchor sequence of authentic gp340 and was expressed in mouse cells, it was immunogenic and induced virus-neutralizing antibodies. Healthy vaccinated tamarins were protected against EBV-induced disease. The demonstration that a recombinant gp340 product is able to elicit protective immunity in the cottontop tamarin is a significant step in the development of an EBV vaccine because previously it had not been clear whether a recombinant product would have the exact tertiary structure, including the necessary carbohydrate components, to induce protective immunity. A recombinant gp340 vaccine offers various advantages over production of the authentic molecule by laborious biochemical separation, including lower cost and the absence of potentially oncogenic EBV DNA. Therefore, recombinant gp340 produced using the BPV expression vector is suitable for development as a candidate EBV vaccine for a human Phase I trial and beyond.
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One-step typing of Epstein-Barr virus by polymerase chain reaction: predominance of type 1 virus in Japan
More LessThe prevalence of two types of Epstein-Barr virus (EBV) in Japan was studied by using the polymerase chain reaction (PCR). The U2 region encoding EBV nuclear antigen 2 (EBNA-2) was chosen as the target of amplification. Consensus primers were synthesized from sequences common to the two types but encompassing a large stretch of deletion in the sequence of type 1 EBV. The primers were capable of amplifying both types at the same time but allowed differentiation of each type by the size of the amplification products. Thus we could carry out detection and typing of EBV in a one-step PCR. EBV was detected in mouth washings of 21 (23%) of 91 seropositive healthy adults. Twenty samples (22%) contained type 1 and only one (1%) type 2. Seventy-nine patients suffering from various types of tonsillitis were also studied. EBV was detected in mouth washings of 37 patients (47%). Thirty-four (43%) contained type 1 and three (4%) type 2. Double infection was not seen in either healthy donors or patients. These results indicate that type 1 EBV is highly dominant and the type 2 variant is quite rare in Japan.
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Prevalence of the A and B types of Epstein-Barr virus DNA in nasopharyngeal carcinoma biopsies from Southern China
More LessEpstein-Barr virus (EBV) exists in the human population in two genetic forms, usually referred to as type A and type B. Although many earlier studies had indicated that the A type was generally predominant, there were suggestions that the B type may exhibit a preferential tropism for nasopharyngeal epithelial cells. This study examines the prevalence of the two forms of EBV DNA present in nasopharyngeal carcinoma biopsies obtained from the high incidence area of Southern China. The results obtained by Southern blot or polymerase chain reaction analyses show that in this patient group the A type of EBV is predominant.
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Identification and characterization of the virion-induced host shutoff product of herpes simplex virus gene UL41
More LessThe virion-induced host shutoff product of the herpes simplex virus UL41 gene is required for shutoff of host translation and degradation of cellular mRNAs. We employed a rabbit antipeptide antiserum to identify a 58K UL41-related phosphoprotein in infected cells. We also provide evidence that this protein is a component of the virus particle, consistent with its role in virion-induced shutoff.
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Cooperative binding of the red clover necrotic mosaic virus movement protein to single-stranded nucleic acids
More LessThe movement protein of red clover necrotic mosaic dianthovirus was produced in Escherichia coli using an expression vector. Gel retardation analysis and u.v. cross-linking studies showed that the movement protein bound cooperatively to ssRNA and ssDNA, but not to dsDNA. Binding competition experiments established that the movement protein bound to ssRNA and ssDNA with similar affinities and that the binding was not sequence-specific in the experimental conditions employed. A truncated movement protein lacking the C-terminal 88 amino acids was also shown to bind to ssRNA.
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The topography of the surface of potato virus X: tritium planigraphy and immunological analysis
Thermally activated tritium atoms were used to probe the surface topography of the coat protein of potato virus X (PVX) potexvirus. The accessibility profile of amino acid residues in the polypeptide chain was determined from data on the intramolecular distribution of tritium label in the PVX coat protein. Tryptic peptides T1 and T2, as well as parts of peptides T3 and T5, from the PVX particles were all located in the N-terminal region of the PVX coat protein and were accessible to tritium labelling, whereas the C-terminal region of the coat protein was practically inaccessible to it. Indirect ELISA and immunoblotting with two PVX-specific monoclonal antibodies confirmed that the N terminus of the coat protein (residues 1 to 56) was exposed on the virus surface, and furthermore that this region forms a highly immunogenic virus-specific antigenic region. The data obtained support the spatial model of PVX, in which the N-terminal amino acids of the coat protein are exposed at the particle surface, and the C-terminal region is buried in the particle. The spatial organization of the PVX coat proteins differs from the model proposed for other filamentous plant viruses such as potyviruses and tobamoviruses where both the N and C termini of the coat protein are located at the particles’ surface.
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A number of subgenomic DNAs are produced following agroinoculation of plants with beet curly top virus
More LessIn addition to ss and ds genomic DNA, agroinoculation of Nicotiana benthamiana plants with the Logan strain of the geminivirus beet curly top virus (BCTV) consistently resulted in de novo production of subgenomic DNAs on initial passage. Single-stranded and dsDNA forms representing at least seven size classes (0.8 to 1.8 kb) of subgenomic DNA were observed in total DNA extracts from inoculated plants. Extracts from infected sugar beet and tomato contained variable but usually smaller amounts of subgenomic DNAs, suggesting that their production may be influenced by the host species. Restriction endonuclease mapping and partial nucleotide sequencing of three independent clones of a 1.5 kb size class indicated that this subgenomic DNA is produced from the standard viral genome by two separate deletion events. One deletion of 941 bp includes portions of the leftward open reading frames (ORFs) L1, L2 and L3, while the other deletion of 579 bp encompasses portions of the intergenic region and the rightward ORFs R1, R2 and R3. The data indicate that the 1.5 kb BCTV subgenomic DNA is a defective DNA that has retained ciselements essential for replication.
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Production of the tobacco mosaic virus (TMV) transport protein in transgenic plants is essential but insufficient for complementing foreign virus transport: a need for the full-length TMV genome or some other TMV-encoded product
More LessWe have reported previously that tobamoviruses enable the transport of red clover mottle comovirus (RCMV) in tobacco plants normally resistant to RCMV. Here we show that RCMV transport does not take place in transgenic tobacco plants (line To-4) producing the 30K transport protein of tobacco mosaic virus (TMV), whereas the transport of the TMV Ls1 mutant, the cell-to-cell movement of which is temperature sensitive, is complemented in these plants. However, RCMV transport is observed when these transgenic plants are infected with both RCMV and TMV Ls1 at the non-permissive temperature (33 °C). It is suggested that (i) the hypothetical modification of transgenic plant plasmodesmata by the TMV 30K transport protein can specifically mediate the cell-to-cell movement of the homologous virus (TMV), but is insufficient to mediate RCMV transport; (ii) the presence of the full-length TMV genome or a certain TMV-encoded product(s) besides the 30K protein is essential for complementation of the RCMV transport function. The possibility that line To-4 might provide enough 30K protein to complement TMV Ls1 but not RCMV cannot be ruled out. During double infection the mutant 30K protein may, in concert with the wild-type 30K protein, provide the transport function for RCMV.
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The nucleotide sequence and genome organization of strawberry mild yellow edge-associated potexvirus
More LessThe nucleotide sequence (5966 nucleotides) of cDNA clones of strawberry mild yellow edge-associated potexvirus was determined. The genome contains six open reading frames (ORFs) encoding putative proteins with M rs of 149423, 25344, 11576, 8079, 25714 and 11216. In the first three putative proteins and the coat protein considerable similarity was found to comparable polypeptides of the potexviruses potato virus X, clover yellow mosaic virus, narcissus mosaic virus, papaya mosaic virus, white clover mosaic virus and lily virus X.
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A 1.5 kb sequence homology in 3′-terminal regions of RNA-1 and RNA-2 of a birch isolate of cherry leaf roll nepovirus is also present, in part, in a rhubarb isolate
More LessA series of cDNA clones has been made from the birch isolate of cherry leaf roll nepovirus. Restriction enzyme analysis and sequencing showed that at the 3′ end, RNA-1 and RNA-2 are identical for 1.5 kb. Also a 0.7 kb 3′ end homology exists between the birch and rhubarb isolate. These sequences do not seem to code for any proteins; however, the sequence conservation points to a role in virus replication.
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Nucleotide sequence analysis of the genomes of the MAV-PS1 and P-PAV isolates of barley yellow dwarf virus
More LessThe MAV-PS1 and P-PAV isolates of barley yellow dwarf virus (BYDV) are serologically related, but not identical. Both are transmitted by the aphid Macrosiphum avenae, but P-PAV is also transmitted by Rhopalosiphum padi. To evaluate the basis for these and other differences, overlapping clones from cDNA libraries representing the genome of each isolate were characterized by restriction enzyme digestion and by hybridization, and subsequently sequenced. Each genome has six positive strand open reading frames (ORFs) which are similar to those identified from a BYDV isolate from Australia (Vic-PAV). The greatest diversity between MAV-PS1 and P-PAV sequences was found in ORFs located in the 3′ half of the respective genomes, in particular ORFs 5 and 6, suggesting that these regions of the genome may be involved in the properties that differentiate MAV-PS1 and P-PAV. Sequence comparisons between P-PAV and Vic-PAV showed a high degree of identity in that all ORFs showed > 90% amino acid similarity, except ORF6 which had only 69% similarity.
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Comparison of the strategies of expression of five tymovirus RNAs by in vitro translation studies
More LessTotal nucleotide sequencing of the RNA genome of various tymoviruses has demonstrated that the overall genome organization of these viruses is identical. Furthermore, the strategies of expression of the turnip yellow mosaic virus (TYMV) genome have been established by in vitro translation studies; these include the synthesis of a subgenomic RNA, the utilization of overlapping open reading frames (ORFs) and maturation of a polyprotein. In the experiments described here, the strategies of expression of other tymovirus (eggplant mosaic virus, ononis yellow mosaic virus, belladonna mottle virus and physalis mottle virus) genomes have been compared to those used by the TYMV genome, in particular to determine whether these tymoviruses also resort to the expression of overlapping ORFs and maturation of a polyprotein.
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Volumes and issues
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Volume 105 (2024)
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Volume 73 (1992 - 2024)
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Volume 104 (2023)
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Volume 103 (2022)
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Volume 102 (2021)
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Volume 101 (2020)
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Volume 100 (2019)
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