- Volume 72, Issue 10, 1991
Volume 72, Issue 10, 1991
- Animal
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Transcriptional activity across the Epstein-Barr virus genome in Raji cells during latency and after induction of an abortive lytic cycle
More LessWe have studied the relative rate of transcription across the Epstein-Barr virus genome in the Burkitt’s lymphoma cell line Raji by nuclear run-on analysis during latency and after induction of an abortive lytic cycle with 12-O-tetradecanoylphorbol 13-acetate (TPA) and 5-iodo-2′-deoxyuridine (IUdR). During latency the entire, or almost the entire, viral genome was found to be transcriptionally active to a low or intermediate extent, with some variation in activity along the genome. The fragment with the highest transcriptional activity was EcoRI J, which contains the genes encoding the small nuclear RNAs EBER1 and -2, transcribed predominantly by RNA polymerase III. An intermediate level of transcription was observed between positions 10 and 138 (kb), with areas of slightly higher activity on the large internal repeats and the left duplicated region (DL). The remaining part of the viral genome, between position 138 and the termini, and the termini and position 10 (kb) (with the exception of the EcoRI J fragment), showed very little transcriptional activity, except for the intermediately active regions carrying the righthand oriLyt (DR) and the terminal repeats. Upon induction of the viral genome with TPA and IUdR, the viral genome was transcriptionally active at a rate at least tenfold that seen during latency. Polymerases were not equally distributed along the genome after induction; the highest density was found in regions 48 to 58 kb, 82 to 84 kb, 102 to 104 kb, 118 to 122 kb and 142 to 145 kb of the viral genome. High transcriptional activity correlated with distinct transcription units in some cases, i.e. BamHI HILF1 (DL), BamHI MLF1, BamHI ZLF1/BamHI RLF1 and BamHI X (thymidine kinase), but not in others (BamHI H2). Besides initiation of transcription, other regulatory processes such as stabilization and processing of primary transcripts may also contribute to regulation of virus gene expression. Addition of cycloheximide completely abolished the transcriptional activation of the genome mediated by TPA and IUdR.
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Isolation and sequencing of the Epstein-Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma
The BamHI fragment containing the Epstein-Barr virus (EBV) LMP1 gene was cloned from a genomic library of the nude mouse-propagated Chinese nasopharyngeal carcinoma CAO. The sequence of the LMP1 gene and its promoter and enhancer was determined. The nucleotide sequence of the CAO isolate differed from those of the B95-8 and Raji isolates in the promoter/enhancer region; the amino acid sequence of the protein also differed. Structural differences in the protein were located mainly in the 20 N-terminal residues and the array of repeated amino acids in the C-terminal part of the protein, in which the CAO isolate displays a cluster of seven perfect repeats of 11 amino acids (aa). Three of these repeats have no counterpart in the other virus strains. This, together with two deletions of five and 10 aa in the C-terminal part, yields a protein of 404 aa, compared to 386 aa for B95-8 and Raji. The larger LMP1 protein was detected on immunoblots of tissue samples from the CAO nude mouse tumour, and was also present in EBV-negative B cell lines and immortalized keratinocytes transfected with the cloned gene. A XhoI restriction site in exon 1 of the B95-8 BNLF-1 gene was absent from the CAO EBV isolate, as well as from 36 of 37 Chinese NPC biopsies tested. In contrast, 17 of 19 NPC biopsies of African origin retained this XhoI site.
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Different scrapie-associated fibril proteins (PrP) are encoded by lines of sheep selected for different alleles of the Sip gene
More LessThe incubation period of scrapie in sheep is controlled by the Sip gene which has two alleles (sA and pA). Following experimental challenge with SSBP/1 scrapie, a short incubation period is conferred by the partially dominant sA allele. Restriction fragment length polymorphisms of the scrapie-associated fibril protein (PrP) gene are associated with the Sip alleles. By sequencing the protein coding region of the PrP gene in Cheviot sheep selected for differing Sip genotypes, we have found four PrP protein variants which differ at three positions: amino acid 112 (Ala/Val), amino acid 130 (Arg/His) and amino acid 147 (Arg/Gln). The Val 112 variant can be distinguished at the DNA level by an RspXI restriction site which is not present in the Ala 112 form. Val 112 appears to be linked to a short incubation period of experimentally induced scrapie in the Cheviot sheep and therefore with the Sip sA allele. These results provide new evidence that the PrP protein may be a product of the Sip locus.
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In vivo detection of metabolic changes in a mouse model of scrapie using nuclear magnetic resonance spectroscopy
More LessIn vivo proton nuclear magnetic resonance (NMR) spectroscopy studies of scrapie in a mouse model have shown the appearance of an abnormal peak in the brain early in the incubation period. This abnormal peak was detected weeks before the detection of a protease-resistant form of a membrane protein and vacuolar histopathology in vitro, and several months before clinical signs, and the signal increased in intensity as the disease progressed. In the chronic stage of the disease, a reduction in N-acetyl aspartate levels was observed using in vivo and in vitro proton NMR spectroscopy.
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A reovirus in the brown planthopper, Nilaparvata lugens
More LessA new virus, belonging to the reovirus group, was found in an apparently healthy colony of the brown planthopper, Nilaparvata lugens, and was referred to as the Nilaparvata lugens reovirus (NLRV). The virus was found in the cytoplasm of the insect cells, sometimes associated with tubular structures, which is one of the characteristic features in tissues infected with reoviruses. The virus was purified by carbon tetrachloride clarification, polyethylene glycol precipitation, differential and CsCl equilibrium centrifugations. The virus has double-shelled particles approximately 65 nm in diameter, containing 10 genome segments of dsRNA. The electrophoretic profile of the dsRNA segments differed from those of viruses associated with rice planthoppers and leafhoppers. Seven proteins were detected in a purified preparation of the virus: four were associated with the core particle and three with the outer shell. A virus antigen was detected in individual insects by ELISA. The virus is retained after injection and is vertically transmitted to the offspring.
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A single amino acid change in the E2 glycoprotein of Venezuelan equine encephalitis virus affects replication and dissemination in Aedes aegypti mosquitoes
More LessFour monoclonal antibody-resistant variants (MARVs) of Venezuelan equine encephalitis (VEE) virus were used to study mosquito-virus interactions. In vitro experiments using an Aedes albopictus cell line, C6/36, demonstrated that an amino acid change in the glycoprotein E2h epitope (MARV 1A3B-7) decreased virus growth when compared with the wild-type, Trinidad donkey virus, and its vaccine derivative, TC-83. The MARVs replicated as efficiently as the parent virus when inoculated into Aedes aegypti mosquitoes, but MARV 1A3B-7 was restricted in its ability to infect and disseminate from the midgut following oral infection. These results demonstrate that a single amino acid change in the E2 glycoprotein can affect the ability of VEE virus to replicate and disseminate in Ae. aegypti mosquitoes.
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Replication of lactate dehydrogenase-elevating virus in cells infected with murine leukaemia viruses in vitro
T. Inada and S. YamazakiAlthough the majority of mouse strains infected with lactate dehydrogenase-elevating virus (LDV) do not show any particular symptoms, the virus is able to induce acute poliomyelitis in C58 or AKR mice. Murine leukaemia virus (MuLV) has been detected at a high titre in the spinal cord of affected mice. In this study, we have analysed the possible role of MuLV in the induction of neurological disease by LDV. Immunofluorescent staining, autoradiography and an infectivity assay of virus yield have shown that LDV replicated in continuous mouse and rat cell lines that had been infected with an ecotropic MuLV isolated from C58 mice, but did not replicate in cells not infected with MuLV. No significant differences in infection were observed among the various ecotropic MuLVs employed, except for Friend leukaemia virus which rendered the cells susceptible to LDV least efficiently. The infectivity of the neurovirulent strain, LDV-C, to MuLV-infected cells was 50- to 100-fold greater than that of the avirulent strains (LDV-N, -Nu, -R and -P). The infectivity to macrophages was almost the same for virulent and avirulent strains. Adsorption studies using a radiolabelled virus revealed that LDV-C was adsorbed to MuLV-infected cells more efficiently than the avirulent strain, LDV-N. The difference in infectivity to these cells, therefore, may be due in part to the difference in adsorption rate. This may suggest differences in the interaction of the viral proteins with MuLV-infected cells from those with macrophages at the initiation of virus infection. These results may be relevant to the mechanisms of paralytic disease caused by LDV infection in C58 mice.
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Fine mapping of canine parvovirus B cell epitopes
In this report we describe the topological mapping of neutralizing domains of canine parvovirus (CPV). We obtained 11 CPV-specific monoclonal antibodies (MAbs), six of which are neutralizing. The reactivities were as determined by ELISA and Western blot (immunoblot) analysis. VP2, the most abundant protein of the CPV capsid, seemed to contain all the neutralization sites. Also, an almost full-length genomic clone of CPV was constructed in the bacterial plasmid pUC18 to enable expression of CPV proteins. All the neutralizing MAbs recognized recombinant VP2 when it was expressed as a free protein in Escherichia coli but not when expressed as a fusion protein with glutathione-S-transferase. When two large fragments containing about 85% and 67% of the C terminus of VP2 were expressed, no neutralization sites were detected. When fusion proteins containing the N terminus were expressed, two linear determinants were mapped, one between residues 1 to 10 of VP2, and the other between amino acids 11 and 23. The peptide 11 GQPAVRNERATGS 23, recognized by MAb 3C9, was synthesized chemically and checked for immunogenicity, not being able to induce neutralizing activity. Although the antibody response in rabbits to all the fusion proteins was uniformly high, the anti-CPV response was very variable. Protein from pCPVEx11, which contains a T cell epitope (peptide PKIFINLAKKKKAG) present in the VP1-specific region as well as the B cell epitopes, seemed to be the most effective in inducing virus neutralization.
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Non-major histocompatibility complex-restricted cytotoxicity of bovine coronavirus-infected target cells mediated by bovine intestinal intraepithelial leukocytes
More LessNon-specific cellular mechanisms of defence against intestinal virus infections of cattle were investigated using bovine coronavirus (BCV) as a representative enteric virus. Since BCV infection is limited to the epithelial cells of the intestinal tract, defence mechanisms must be capable of acting at that site to be effective. Therefore, the intraepithelial leukocyte (IEL) population of the intestinal mucosa was chosen for initial study. Treatment of intestinal samples with DTT and EDTA in calcium- and magnesium-free buffers allowed recovery of viable IEL populations appropriate for further functional assessment. Studies of IELs isolated from neonatal calves revealed that non-major histocompatibility complex (MHC)-restricted cytotoxicity of BCV-infected target cells was more prevalent in calves with concurrent virus infection, suggesting in vivo activation of the cytotoxic response. Peripheral blood mononuclear cells from the same calves did not mediate cytotoxicity, emphasizing the difference in function of lymphocytes isolated from different anatomical sites. IELs from normal adult animals rarely showed spontaneous non-MHC-restricted cytotoxicity. However, interleukin-2 (IL-2) was a potent activator of IEL cytotoxicity in vitro, enhancing the killing of BCV-infected target cells after just 18 h of treatment. Incubation of IELs with interferon-γ and tumour necrosis factor (TNF) did not induce cytotoxic activity, but TNF could augment the levels of IL-2-induced cytotoxicity. Although further analysis of the cytotoxic effector cells present in the intestinal epithelium is required, the present study indicates that the IEL population may play a role in enteric antiviral activity.
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Gangliosides as binding sites in SA-11 rotavirus infection of LLC-MK2 cells
More LessThe chemical nature of receptors involved in the attachment of simian rotavirus (SA-11) to a monkey kidney cell line (LLC-MK2) was investigated. Enzymic treatment of cells before virus infection indicated that membrane proteins and phospholipids are not involved in virus attachment, whereas sialic acid and galactose participate in the receptor structure to differing extents. Incubation of SA-11 with bovine brain gangliosides before infection strongly reduced its ability to bind to cell membranes. Similar experiments with individual purified gangliosides from bovine brain showed that virus infection was prevented by preincubation with GM1. Moreover, desialylated cells regained susceptibility to virus infection when coated with whole gangliosides or GM1 immediately after Clostridium perfringens neuraminidase treatment. The binding of SA-11 to whole gangliosides or GM1 was quantified by an ELISA procedure. The results suggest that gangliosides, mainly GM1, are part of the receptor structure for SA-11 of susceptible LLC-MK2 cells.
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Antigenic structure of chimeras of type 1 and type 3 polioviruses involving antigenic sites 2, 3 and 4
Chimeric polioviruses have been made in which regions of the type 1 Sabin strain corresponding to antigenic sites 2, 3 and 4 have been replaced by the corresponding regions of the type 3 Sabin strain. Manipulation of one site or a component of it generally did not affect the reactions of the others, suggesting that they form independent structural features. The extent to which the inserted site expressed the antigenic properties of type 3 could be assessed by reaction with polyclonal or monoclonal antibodies, or by immunogenicity. Site 2 could be expressed on infectious virus and site 3 on heated non-infectious virus (C antigen), but not on the native virion. The results are consistent with the view that sites consisting of a continuous sequence of amino acids may be presented on chimeras, whereas more complex sites, such as site 4 or site 3 of the native virion, are transferred less readily from type 3 to type 1.
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Generation of virus genetic lineages during an outbreak of poliomyelitis
More LessWild poliovirus type 3 isolates collected during the Finnish outbreak (1984 to 1985) in different geographical locations were compared by partial RNA sequencing. The entire 5′ non-coding end and a discontinuous part of the capsid coding region were sequenced from 15 isolates. Combining the present sequence data with previously published data and analysing these by the maximum parsimony method showed that the epidemic strains had diverged in cocirculating lineages. Genetic comparison of strains isolated from a single person often revealed a branched structure in the phylogenetic tree indicating high potential for diversification. The extent of variation generated under immunological pressure during an infection lasting for weeks in one person was high as compared with the observed geographical variation.
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Efficacy of individual measles virus structural proteins in the protection of rats from measles encephalitis
More LessLewis rats were immunized with recombinant vaccinia virus (VV) expressing the nucleocapsid (N), phospho (P), matrix (M), fusion (F), and haemagglutinin (H) proteins of measles virus (MV). Animals developed humoral as well as cell-mediated immune (CMI) responses to the corresponding MV proteins. Rats immunized with recombinants VVN, VVF or VVH survived a MV challenge infection whereas VVP- and VVM-immunized rats were only partially protected. In vivo depletion of CD8+ T lymphocytes did not prevent the protective effect of the N, F or H protein-specific CMI response in rats. VVH and VVF immunization induced neutralizing antibodies, but no such antibodies were detected after VVN immunization. Further investigation of the temporal occurrence of the antiviral antibodies indicated that the observed protection provided by VVN and VVF immunization depends on CD4+ N- or F-specific T cells in the absence of neutralizing antibodies and CD8+ T cells. A role for neutralizing antibodies induced by VVH cannot be ruled out.
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Defective synthesis of envelope proteins by temperature-sensitive mutants representing complementation groups B and D of respiratory syncytial virus
More LessThe phenotypes of two complementing temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus indicate that the mutational lesions involve the attachment (G) and matrix (M) proteins of the viral envelope. Synthesis of the G protein was affected in cells infected with mutant tsA2 (complementation group B); the p50 precursor of the G protein was synthesized normally, but further maturation to the fully glycosylated form was defective at 39 °C. A non-ts alteration in the efficiency of cleavage of the F0 precursor to the F1 and F2 subunits of the fusion protein was also observed in tsA2-infected cells, which is consistent with the aberrant non-syncytial plaque morphology induced by tsA2 in certain cells. In cells infected with mutant tsN1 (complementation group D) the M protein disappeared from the soluble cytoplasmic fraction soon after synthesis at 39 °C and had a slightly decreased electrophoretic mobility. The M protein of non-ts revertants was stable at 39 °C, which links the defect in M protein stability with the tsN1 phenotype. However, the aberrant mobility phenotype remained, suggesting pseudoreversion. These results assign two of the eight complementation groups of ts mutants of RS virus.
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Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study
An ultrastructural study was performed on rabbit epithelial RK-13 cells and CD4+ human T lymphocyte lines infected with various recombinant vaccinia viruses (RVVs) expressing genes of human immunodeficiency virus (HIV): the mature p17 or p24 gag domain alone, the entire or truncated gag gene, the reverse transcriptase domain, or the gag-pol genes with a frameshift mutation. Cells infected with RVVs that produced the gag polyprotein with a predicted M r of more than 48K showed budding and release of HIV-like particles into the extracellular space. These particles were not observed in cells expressing a truncated gag gene (p17 and p24 regions). Mature HIV-like particles were observed extracellularly when the entire gag gene and the protease region of the pol gene were expressed. In contrast, in cells infected with RVVs that contained the gag-pol gene with a frameshift mutation, neither recognizable budding structures nor extracellular HIV-like particles could be detected. These results suggest that the gag gene, particularly its 3′ terminus, is necessary for the assembly of HIV particles. In addition, the protease region of the pol gene seems to be required for morphological maturation of HIV particles, but complete proteolytic cleavage of the gag protein may prevent bud formation.
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Neutralizing activity of anti-peptide antibodies against the principal neutralization domain of human immunodeficiency virus type 1
More LessMonoclonal antibodies (MAbs) raised against a 15-mer peptide representing the centre of the principal neutralization domain of human immunodeficiency virus type 1 (strain BH10) showed wide variations in neutralizing activity against the homologous strain. The nature of this difference in neutralizing activity was studied by measuring antibody concentration, their affinity for peptide and specificity, by reaction with peptides which differed in the extent of sequence overlap, length and the presence of single amino acid replacements. All MAbs bound to approximately the same region in the principal neutralization domain, within the sequence RIQRGPGRAFV. The peptides with which each antibody was able to react differed by only a few amino acids. The neutralizing activity of each MAb preparation was related to its afinity and concentration; the affinity is related in part to the fine structure of the epitope recognized. MAbs with high affinity for the peptide tended to react only with peptides in which amino acid replacements did not affect the β-turn potential of the peptide, whereas the reactivity of MAbs with low affinity was relatively insensitive to amino acid replacements affecting the β-turn potential.
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Construction and functional characterization of mutants of the bovine leukaemia virus trans-activator protein p34tax
More LessThe p34tax protein [p38tax, p34, p38(XBL), XBL-I] of bovine leukaemia virus (BLV) activates transcription from the BLV long terminal repeat (LTR) promoter. To analyse the functional properties of this protein, in frame insertions and internal deletions were systematically introduced in a plasmid-encoded copy of the p34tax gene. The abilities of wild-type and mutant genes to activate gene expression from the LTR promoter linked to the chloramphenicol acetyltransferase gene and to inhibit trans-activation by the wild-type protein were studied. The trans-activating activity of 14 of the 18 mutants tested was completely abolished, but four mutants each containing a lesion in the internal portion of the polypeptide retained activity. Taken together, these results suggest the presence of an internal region of the polypeptide where structural integrity is less strictly required for the functional activity of this protein. Among the mutants incompetent in the transactivation assay, only two with mutations in the N-terminal region of the polypeptide inhibited trans-activation by the wild-type protein in a dose-dependent manner. These results facilitate understanding of the physiological function of the tax protein family.
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Enzootic nasal tumour of goats: demonstration of a type D-realated retrovirus in nasal fluids and tumours
More LessNasal exudate and tumour tissue from goats with enzootic nasal tumours were shown to contain a reverse transcriptase activity associated with a particle of buoyant density typical of retroviruses. The same particle contained a 25000 M r protein that cross-reacted with the p27 of Mason-Pfizer monkey virus (MPMV) and with p25 of sheep pulmonary adenomatosis retrovirus. It also contained a low M r protein related to p10–12 of MPMV.
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Spontaneously productive C-type retrovirus infection of fish cell lines
More LessThe spontaneous production and release of morphologically typical, 85 to 90 nm diameter C-type retrovirus particles from four cell lines derived from three species of warmwater fish have been identified. Virus pellets from cell culture supernatants showed high levels of Mn2+-dependent reverse transcriptase activity at 24 °C. Peak enzyme activity was associated with a 1.16 g/ml sucrose gradient fraction. All four isolates induced a cytolytic infection of a bluegill fry cell line within 6 to 10 days.
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Influence of different ionic and pH environments on structural alterations of poliovirus and their possible relation to virus uncoating
More LessPoliovirus eclipse products were totally precipitated from infected HeLa cells after different times of infection by using TCA, suggesting that cellular enzymic digestion of parental proteins was not involved in virus uncoating. In an investigation of poliovirus thermal stability in vitro, progressive degradation of native virus into 80S empty capsids occurred upon incubation at 37 °C in a buffer of low ionic strength containing 20 mm-Tris-HCl pH 7.5, whereas in Eagle’s medium or in the presence of L cells degradation was very slow. Degradation was faster at alkaline than at acid pH. Furthermore, liberation of the viral RNA was prevented and 135S particles were produced upon treatment of virus at 37 °C in 20-mm-Tris-HCl pH 7.5 containing 2 mm-CaCl2. Although the poliovirus receptor is able to induce conformational alterations of the capsid, low ion concentration could contribute to virus uncoating as well.
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Genes 1 and 2 of pneumonia virus of mice encode proteins which have little homology with the 1C and 1B proteins of human respiratory syncytial virus
More LessGenes 1 and 2 of pneumonia virus of mice (PVM) consist of 410 and 571 nucleotides and encode proteins of 113 and 156 amino acids respectively. The proteins show no extensive (gene 1 analogous to 1C) or low (gene 2 analogous to 1B) homology to their presumed counterparts in human respiratory syncytial virus (HRSV). The strongest homology is between regions of approximately 35 amino acids located near the carboxy termini of the gene 2 product and the 1B protein with 29% identity, although a lower level of homology can be detected throughout much of these proteins (18% identity overall). These observations contrast with the conservation of 1C and 1B proteins between subgroups of HRSV and with the conservation of nucleocapsid proteins between HRSV and PVM.
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Transcriptional analyses of baculovirus polyhedrin and foreign gene expression relative to baculovirus p10 mRNA levels
More LessComparisons have been made between the p10 and polyhedrin mRNA levels recovered from Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV). In molar terms and from 18 h post-infection (p.i.), the polyhedrin mRNA species increased to levels one and a half times to twice as high as the p10 levels. The influence of the polyhedrin leader sequence on the expression of a foreign gene under the control of the polyhedrin promoter was investigated using a series of four recombinant baculoviruses expressing the lymphocytic choriomeningitis virus nucleocapsid (N) protein gene. The different recombinants varied in the length and composition of the upstream polyhedrin mRNA leader sequence. The recombinant containing the full-length polyhedrin leader sequence gave levels of N mRNA comparable to those of AcNPV polyhedrin mRNA. These levels were either equal to (12 h p.i.) or higher (18 to 42 h p.i.) than the p10 levels at corresponding times. Three other recombinants, with different lengths of leader sequence, accumulated significantly lower quantities of N mRNA in comparison to the p10 mRNA levels. However the mRNA levels for the three recombinants were similar (20 to 50% of the p10 level) and did not correspond to their N protein expression levels. By comparing the mRNA and protein levels, it is concluded that the sequence between -8 to +1 of the AcNPV polyhedrin translation-initiating ATG has an important function for mRNA transcription (or accumulation), while the sequences between -32 to -8 affect the overall translation efficiencies.
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Partial nucleotide sequence analysis of a French hepatitis C virus: implications for HCV genetic variability in the E2/NS1 protein
More LessTo contribute to the study of the genetic variability of hepatitis C virus (HCV) we have determined the nucleotide sequence of the E2/NS1 and NS3/NS4 regions of a French isolate using the polymerase chain reaction. Comparison of these nucleotide sequences with those available for American and Japanese isolates showed a significant genetic variability: 5 to 33% and 2 to 30% at the nucleic acid and amino acid levels, respectively. The genetic variability is higher in the E2/NS1 (13 to 33% and 12 to 30% at the nucleic acid and amino acid levels, respectively) than in the NS3/NS4 (5 to 21% and 2 to 7%) regions. The sequence of the French isolate is more closely related to that of the American HCV prototype than to the Japanese HCV isolates. This study confirms the extent of HCV genetic variability.
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Characterization of the VPg-dsRNA linkage of infectious pancreatic necrosis virus
More LessBy the use of strong denaturing agents, a genome-linked protein (VPg)-RNA complex was purified from infectious pancreatic necrosis virus. Ribonuclease treatment of 125I-labelled VPg-RNA released a 90K polypeptide identical to the minor structural polypeptide VP1 (the putative RNA polymerase), as determined by peptide mapping. The polypeptide is linked to the RNA by a serine-5′ GMP phosphodiester bond. The results identify birnaviruses as the only dsRNA viruses with a VPg, the size of which is the largest of the VPgs of RNA viruses.
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Presence and integration of human papillomavirus type 6 in a tonsillar carcinoma
Human papillomavirus type 6 subtype a (HPV-6a0 was detected in a human invasive tonsillar carcinoma. Southern blot hybridization analysis showed the presence of additional bands when using non-cutting and single-cut restriction enzymes. Molecular cloning yielded two recombinant clones of 8.0 and 1.4 kb in size. The first represents the complete HPV-6a genome. Sequence analysis of the second clone showed a 0.6 kb DNA sequence corresponding to the L2 region of HPV-6a, whereas the rest belongs to cellular sequences. These data show the presence of a usually low risk HPV type in an invasive carcinoma, at an unusual infection site, with viral DNA integrated into the host genome. These findings add evidence in support of the hypothesis of a relationship between HPV infection and at least some ororespiratory cancers.
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Sequencing data on the long control region of human papillomavirus type 16
More LessA one base change (adenine at position 7861) in the long control region (LCR) of the prototype sequence of human papillomavirus type 16 was reported. With this correction, a new E2-binding site was revealed, 111 bp and 143 bp upstream from the TATA box and P97, respectively, and 105 bp downstream of the keratinocyte-dependent enhancer. An A to C mutation at position 41 was found in a patient with invasive carcinoma. This mutation falls within an E2-binding site.
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Comparison of ELISA and Western blotting for human papillomavirus type 16 E7 antibody determination
A total of 140 sera originating from healthy women and women with either cervical intraepithelial neoplasia or cervical cancer were tested for the presence of IgG antibody against E7 of human papillomavirus type 16 (HPV-16) by ELISA using a synthetic icosapeptide, denoted 16/E7-2, representing amino acids 11 to 30, and by Western blotting (WB) using a genetically engineered HPV-16 E7 fusion protein. Eighteen sera were found positive in either one or the other test. Positive reactions were more frequently detected in cervical carcinoma patients (12 of 34, 35.2%) than in the other individuals (six of 106, 5.7%). Ten children’s (1 to 3 years of age) sera reacted in neither ELISA nor WB with HPV-16 E7. A high degree of concordance between the two tests was found suggesting that both tests detect the same or similar activity. To locate the reacting epitopes in the E7 protein, absorption tests were performed with peptides corresponding to various sections of the protein. Based on the results obtained, sera possessing antibody to HPV-16 E7 could be differentiated into those reactive with only the 16/E7-2 peptide and those reactive with other HPV-16 E7 epitopes.
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Evolution of herpesvirus thymidine kinases from cellular deoxycytidine kinase
More LessThe thymidine kinases encoded by herpesviruses of higher vertebrates form a distinct group and are unrelated to the thymidine kinases (TKs) of other organisms. Their evolutionary source has not been identified, but our analysis has revealed a clear relationship with a sequence of human deoxycytidine kinase (dCK) published recently. We report the sequence of the putative TK of channel catfish virus, a herpesvirus of a lower vertebrate, and show that it is also related to dCK. We propose, therefore, that the TKs of herpesviruses of higher and lower vertebrates have evolved, either independently or successively, from a cellular dCK.
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The nucleotide sequence and genome structure of the geminivirus miscanthus streak virus
A tandem dimer of miscanthus streak virus (MiSV) DNA was inserted into the T-DNA of the binary plasmid vector pBIN19 and agroinoculated into several monocotyledonous plants (monocots) using Agrobacterium tumefaciens or A. rhizogenes. Disease symptoms and geminate particles were produced in maize and Panicum milaceum plants, and MiSV-specific double-stranded and single-stranded DNAs were found in these plants. The nucleotide sequence of the infectious MiSV clone, consisting of 2672 nucleotides, was determined. Four open reading frames (ORFs) for proteins of M r greater than 10K were identified, two (V0 and V2) in the virus (+) sense and two (C1 and C2) in the complementary (-) sense, although C2 did not have an ATG start codon. Unlike other geminiviruses infecting monocots, complementary-sense ORFs did not overlap. Potential splicing donor and acceptor sites were identified in the sequence of the border region between the C terminus of ORF C1 and the N terminus of ORF C2. Amino acid sequences predicted from three (V2, C1 and C2) of these ORFs showed significant homology with the corresponding ORFs of other geminiviruses infecting monocots. A fifth ORF (V1), which showed some homology with ORF V1 of other monocot-infecting geminiviruses despite having a coding capacity for a product of M r 8.8K, was found just upstream of ORF V2 as observed in those geminiviruses. ORF V0 showed no significant homology with ORFs present in any other geminiviruses. A mutation of V0 indicated that the C-terminal 30% of this ORF was not necessary for infection in maize, but that sequences around the mutated LspI site might have some regulatory role.
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Nucleotide sequence and gene organization of the 3′-terminal region of chrysanthemum virus B genomic RNA
K. Levay and S. ZavrievDNA clones complementary to the 3′-terminal 3426 nucleotides of the genomic RNA of the carlavirus chrysanthemum virus B (CVB) have been sequenced. The sequence contains six open reading frames (ORFs) which encode putative proteins (in the 5′ → 3′ direction) of M r 25749 (ORF2), M r 11435 (ORF3), M r 6984 (ORF4), the triple gene block proteins, a protein M r 34638 (ORF5); the coat protein, and a protein of M r 12609 (ORF6). The latter protein is basic and contains a putative zinc finger motif. The 5′-proximal ORF1 encodes a product with substantial homology to the C-terminal portions of the putative RNA replicases of two other carlaviruses, potato viruses M and S. The analysis of the minus-sense sequence shows one ORF which encodes a polypeptide of M r 16817. The sequenced portion of the CVB genomic RNA contains three short internal non-coding regions, two of which are typical for carlaviruses (those between ORFs 1 and 2, and between ORFs 4 and 5), and one (between ORFs 5 and 6) is unusual. There is significant similarity in the amino acid sequences of the CVB RNA-encoded proteins and the corresponding proteins of other carlaviruses.
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The sequence between nucleotides 161 and 512 of cowpea mosaic virus M RNA is able to support internal initiation of translation in vitro
More LessCowpea mosaic virus M RNA is translated in vitro as well as in vivo into two C-coterminal polyproteins of M r 105K and 95K. Initiation of translation of the 95K protein gene occurs at an AUG codon at position 512 of M RNA, 351 nucleotides downstream of the initiation codon of the 105K protein gene at position 161. By employing an in vitro transcription and translation system it was determined that this 351 nucleotide sequence has the capacity to direct ribosomes to initiate translation at a downstream start codon. This effect is independent of the position of this sequence in an mRNA. Furthermore, evidence has been obtained that scanning ribosomes can bypass the AUG at position 161. Thus, both leaky scanning and internal entry are mechanisms for the initiation of translation of the 95K protein gene.
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Nucleotide sequence analysis and genomic organization of the NY-RPV isolate of barley yellow dwarf virus
More LesscDNA clones representing the ssRNA genome of the NY-RPV isolate of barley yellow dwarf luteovirus (BYDV) were sequenced and 5600 nucleotides of the genome were determined. The deduced genome organization has limited similarity to that of another BYDV isolate, Vic-PAV, but is identical to that of beet western yellows (BWYV) and potato leafroll (PLRV) luteoviruses. NY-RPV has six major positive-sense open reading frames (ORFs) and, by comparison with RNA-dependent RNA polymerase and nucleic acid helicase consensus sequence motifs, it is postulated that NY-RPV ORF2 and ORF3 encode the viral replicase, which is expressed by a translational frame-shift mechanism. The region of the NY-RPV genome containing the 22K coat protein ORF, the apparently associated internal apparent VPg ORF and the ORF immediately 3′-proximal (ORF6) to the coat protein ORF are organized as reported for other luteoviruses. Evidence is presented showing that ORF6 is expressed by readthrough of the coat protein gene termination codon, and that this protein is associated with the intact virus as a 65K protein. Although NY-RPV infects graminaceous rather than dicotyledonous plants, the taxonomic relationships between BYDV isolates and other luteoviruses deduced from the genome organization and sequence data strongly suggest that NY-RPV is distinct from the PAV-like isolates of BYDV and is more closely related to BWYV and PLRV.
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Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA1
More LessThe nucleotide sequence of the genomic RNAI, 7342 nucleotides (nt) of grapevine fanleaf virus strain F13 (GFLV-F13) has been determined from cDNA clones. The complete sequence contained only one long open reading frame (ORF) of 6852 nucleotides extending from nucleotide 243 to 7101. The putative polyprotein encoded by this ORF is 2284 amino acids in length with an M r of 253K. The location of genome-linked protein and comparison of the primary structure of the 253K polyprotein to that of other closely related viral proteins of the picornavirus-like family allows the proposal of a scheme for the genetic organization of GFLV-F13 RNA1. The primary structure of the polyprotein includes a putative RNA-dependent RNA polymerase of 92K and a cysteine protease of 25K. This protease shares not only major structural homologies, particularly in the substrate-binding pocket, with the trypsin-like serine proteases of other picorna-like viruses, but also their specificity in terms of cleavage. The large region of M r 133K upstream of the VPg was found to contain at least two domains, one of which could be easily aligned with the NTP-binding sequence pattern and another which may have the characteristics of a protease cofactor. Thus, the 253K protein possesses the same general genetic organization as the corresponding protein of other picorna-like viruses.
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Non-replicating deletion mutants of brome mosaic virus RNA-2 interfere with viral replication
More LessNaturally occurring defective interfering RNAs (DI-RNAs) and satellite RNAs greatly reduce the accumulation of their helper virus in vivo, but often modulate symptom expression in an unpredictable manner. Deletion mutants Nc/S, Na/M and Sa/Nc + M/S, derived from brome mosaic virus (BMV) RNA-2, failed to replicate when co-inoculated with BMV RNAs-1 and -2 to barley protoplasts. However, the inoculum RNA corresponding to these deletion mutants was extremely stable and could have been mistaken for plus-strand progeny had minus-strand progeny analysis been omitted. These results accentuate the need for such tests in evaluating the ability of mutant viral sequences to replicate. One of the mutants, Nc/S, effectively interfered with the accumulation of BMV RNAs-1 and -2 in barley protoplasts. This non-replicating interfering RNA was termed NRI RNA-2 Nc/S. When present with RNAs-1 and -2 at low inoculum amounts (1 µg), NRI RNA-2 Nc/S reduced replication of RNA-2, the parental RNA, by 63% and preferentially interfered with minus-strand RNA accumulation. At higher levels (4 µg), it completely displaced replication of both RNAs-1 and -2. Mutations eliminating translation of a truncated p2a protein from NRI RNA-2 Nc/S did not alleviate the interference effect, demonstrating that a defective replicase protein was not responsible for the decreased accumulation of genomic RNA. At an NRI RNA:genomic RNA inoculum molar ratio of 1:1, NRI RNA-2 Nc/S reduced the accumulation of all helper virus RNAs by 55%. Since this reduction was seen for both wild-type RNA-3 and ASGP RNA-3, a deletion mutant of RNA-3 that lacks the subgenomic promoter necessary for coat protein expression, it was evident that the effective interference mediated by NRI RNA-2 Nc/S was not mitigated by encapsidation. The ability of the NRI RNAs to mimic satellite DI RNAs in depressing helper virus replication suggests that their expression in transgenic plants may provide a new and widely applicable approach for inducing resistance to viral infection.
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Generation of envelope and defective interfering RNA mutants of tomato spotted wilt virus by mechanical passage
During a series of mechanical transfers of tomato spotted wilt virus, two distinct types of mutants were generated. Firstly, a morphologically defective isolate was obtained which had lost the ability to produce the membrane glycoproteins and, as a consequence, was not able to form enveloped particles. Analysis of the genomic RNAs of this isolate suggested that this defect was caused by either point mutations or very small deletions in the medium genomic RNA segment. Secondly, isolates were obtained which had accumulated truncated forms of the large (L) RNA segment. These shortened L RNA molecules most likely represented defective interfering RNAs, since they replicated more rapidly than full-length L RNA and their appearance was often associated with symptom attenuation. Defective L RNAs of different sizes were generated after repeated transfers, and hybridization analysis using L RNA-specific cDNA probes showed that the internal regions deleted varied in length. The presence of defective L RNAs in nucleocapsid fractions as well as in enveloped virus particles indicates that all defective molecules retained the sequences required for replication, encapsidation by nucleocapsid proteins and packaging of the nucleocapsid into virus particles.
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Reciprocal phenotype alterations between two satellite RNAs of cucumber mosaic virus
More LessCucumber mosaic virus Y satellite RNA (Y-satRNA) induces distinctive yellow mosaic symptoms on tobacco, whereas S19 satellite RNA (S19-satRNA) causes an attenuated green mosaic on tobacco, although they show considerable sequence identity. Biological assays of infectious chimeric satellite RNA molecules synthesized from cDNA clones of Y-satRNA and S19-satRNA using common restriction sites showed that the determinant for the induction of yellow mosaic symptoms lies in the BstXI-NheI fragment, in which 14 nucleotide differences are found between the two satellite RNAs. To define more precisely the yellow mosaic determinant(s) in this fragment, several site-directed mutants of Y-satRNA were created. The replacement of AUU, at nucleotides 191 to 193 in Y-satRNA, with GC, which mimicks the S19-satRNA sequence at the corresponding site, abolished the ability of Y-satRNA to elicit a yellow mosaic. Conversely, a mutant RNA molecule derived from S19-satRNA in which GC at nucleotides 192 and 193 was changed to AUU induced the yellow mosaic symptoms. Thus, the phenotypes of two satellite RNAs on tobacco can be altered reciprocally by changing the sequences in this limited region.
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Coat protein gene sequences of two cucumber mosaic virus strains reveal a single amino acid change correlating with chlorosis induction
More LessThe coat protein genes of two chlorosis-inducing strains of cucumber mosaic virus (CMV) were compared by nucleotide sequence analysis. The predicted amino acid sequences of the encoded coat proteins were compared with those of two other chlorosis-inducing and four mosaic-inducing CMV strains. Overall, the sequences were highly conserved, with more than 95% amino acid sequence identity between any two strains. However, a proline is present at amino acid 129 of all the mosaic-inducing strains, whereas that position is occupied by either a serine or a leucine in the coat proteins of all the chlorosis-inducing strains. The correlation of chlorosis induction and a substitution for proline with leucine or serine at amino acid 129 suggests that this residue is the determinant of chlorosis induction.
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The 5′-terminal sequence of potato leafroll virus RNA: evidence of recombination between virus and host RNA
More LessThe discrepancy between published sequences of the 5′ non-coding regions of RNA of a Scottish (S) and that of Dutch (D), Australian and Canadian isolates of potato leafroll virus (PLRV) was investigated. Reverse transcription followed by amplification by polymerase chain reaction showed that RNA from three distinct Scottish isolates of PLRV contained molecules with 5′ ends like that of the original Scottish isolate. However, determination of the 5′-terminal sequences of RNA in two of these preparations showed that most RNA molecules had 5′ termini like those of the Dutch and other non-Scottish sequences. Northern blot analysis confirmed that only a small fraction of PLRV RNA contained sequences homologous to the 5′-terminal 119 nucleotides of the PLRV-S sequence. Most PLRV-S RNA molecules therefore have termini like that reported for PLRV-D. The 5′-terminal 119 nucleotides of the minor species of PLRV-SRNA were very similar (109/119 nucleotides were identical) in sequence to an exon of tobacco chloroplast DNA open reading frame 196. The results therefore suggest that recombination has occurred between virus RNA and host RNA.
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Nucleotide sequence of the 3′ non-coding region and N gene of the S RNA of a serologically distinct tospovirus
M. D. Law, J. Speck and J. W. MoyerA tomato spotted wilt-like virus (TSWV-I) is a distinct member of the Tospovirus genus of the Bunyaviridae and is distinguished from the typical TSWV by having a serologically distinct nucleoprotein (N). A cDNA clone extending from the 3′ terminus of the viral RNA through the entire N open reading frame (ORF) was sequenced. The TSWV-IN ORF is capable of encoding a polypeptide of 262 amino acids with a predicted M r of 28.8K. In vitro transcription and translation of the clone produced a protein which comigrated with TSWV-I N and was immunoprecipitated by TSWV-I antibodies. Hybridization analysis of lithium chloride-precipitated RNA from healthy and TSWV-I-infected tissue detected a virus-specific 1.2 kb subgenomic RNA. The TSWV-I S RNA terminal consensus sequence (8 nucleotides) was identical to that of TSWV; the remaining TSWV-I untranslated region showed only 51% identity with that of TSWV. Comparison of the TSWV-I and TSWV N proteins showed 67% identity at the amino acid level. The degree of similarity in the terminal sequence, untranslated region and N ORF is similar to that expected between distinct serogroups within certain genera of the Bunyaviridae.
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Comparison of viral nucleic acid intermediates at early and late stages of cauliflower mosaic virus infection suggests a feedback regulatory mechanism
More LessAn important phase of the multiplication cycle of the pararetrovirus cauliflower mosaic virus (CaMV) is transcription of the viral minichromosome in the nucleus. Leaves of infected turnip plants at the vein clearing stage were found to contain a relatively low level of minichromosome DNA, and abundant viral transcripts and characteristic reverse transcription products. In contrast, at the much later stage of severe leaf chlorosis, an elevated level of minichromosome DNA but less RNA, especially the 35S RNA reverse transcription template, was observed. Changes in the composition of virus nucleic acid intermediates were also seen in roots and stems early, compared with late, in infection. A possible feedback mechanism controlling the level of viral minichromosome DNA and its importance in regulation of the CaMV multiplication cycle are discussed in the light of these observations.
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Volumes and issues
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Volume 105 (2024)
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