- Volume 70, Issue 5, 1989
Volume 70, Issue 5, 1989
- Animal
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Anatomical Basis of Thogoto Virus Infection in BHK Cell Culture and in the Ixodid Tick Vector, Rhipicephalus appendiculatus
More LessSummaryInfection by Thogoto (THO) virus, a tick-borne virus related to the orthomyxo-viruses, has been compared in vertebrate cell culture and in Rhipicephalus appendiculatus ticks using infectivity titrations, immunofluorescence, and immune electron microscopy with colloidal gold markers to detect cell surface and intracellular antigens. Morphogenesis of THO virus in cell culture was similar to that of influenza virus, with polymorphic virus particles budding at the plasma membrane. In the tick, THO viral infection caused no obvious pathology; virions or budding profiles were not observed in electron micrographs, although replication, trans-stadial persistence and transmission to a susceptible host occur. THO virus was not detected in the salivary glands of trans-stadially infected ticks until about 7 days after the commencement of feeding on a host. The synganglion (brain) appears to be the major organ involved in trans-stadial persistence of the virus; viral antigens were detected in the neural cortex (cell bodies) but not in nerve fibres and axons. The detection of THO viral antigen in basement membranes and connective tissue, but its absence from nerve fibres, suggests that dissemination occurs via the haemolymph rather than a neural route.
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A Novel Replicative Form DNA of Aleutian Disease Virus: the Covalently Closed Linear DNA of the Parvoviruses
More LessSummaryThe analysis of replicative form (RF) DNA of Aleutian disease virus (ADV) by alkaline gel electrophoresis revealed that all RF DNA species segregate into DNA single strands which represent integral multiples of a genome equivalent. This demonstrates that as with other autonomous parvoviruses, the virion and complementary DNA strands are frequently linked by hairpin structures and that also, nicks are present at subterminal sites. Approximately 50 % of the 5′-terminal hairpins contain a subterminal nick whereas no nick is detectable in the 3′-terminal hairpin. This finding together with the presence of nicks in the 3′ palindrome sequence of the dimer RF DNA (D RF DNA) bridge fragment is the first experimental proof for the so far hypothetical substrate specificity of a nickase. A novel DNA structure was identified in the monomer (M) RF DNA population. This molecule, designated ‘monomer covalently closed linear RF DNA’ (Mccl RF DNA), consists of a continuous, self-complementary, circular polynucleotide chain of twice the genome length. It was directly visualized by electron microscopy that denatured ADV M RF DNA is a single-stranded circular molecule of twice the genome length with covalently closed terminal hairpins on either end. Alkaline gradient centrifugations, enzymic assays and electrophoretic techniques confirmed the proposed structure. Moreover, evidence was obtained that the D RF DNA species contains an analogous Dccl RF DNA. It is suggested that the newly described Mccl RF DNA form is an important intermediate common to the DNA replication of all autonomously replicating parvoviruses.
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The Temperature Sensitivity of the Sabin Type 3 Vaccine Strain of Poliovirus: Molecular and Structural Effects of a Mutation in the Capsid Protein VP3
SummaryThe growth of the Sabin strain of type 3 poliovirus is reduced at high temperatures compared to that of its virulent precursor strain Leon. Recombinant viruses have been generated from infectious cDNA clones and demonstrate that the temperature-sensitive (ts) phenotype is mainly attributable to a difference in residue 91 of the virion protein VP3. Examination of non-ts mutants derived in vitro or in vivo reveals the existence of second site mutations some of which are clearly able to suppress the ts phenotype. The location of residue 91 of VP3, and of a number of candidate suppressor mutations, in the atomic structure of the virion suggests that the ts phenotype may result in destabilization of the particle and that the suppressors may function by stabilizing specific interfaces. It is not yet clear whether the ts phenotype is expressed at the level of the particle or in the form of defects in assembly or uncoating of the virion, or all three.
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Junin Virus Monoclonal Antibodies: Characterization and Cross-reactivity with Other Arenaviruses
SummaryTwenty-one monoclonal antibodies reactive with Junin virus structural proteins were produced and characterized. Using radioimmunoprecipitation and Western blot assays, 13 were found to react with the nucleoprotein, seven with the surface glyco-protein and one failed to react, but showed a fluorescent antibody staining pattern consistent with other glycoprotein-specific antibodies. In radioimmunoprecipitation assays, glycoprotein-specific monoclonal antibodies reacted not only with the 35K structural glycoprotein, but also with what is presumed to be the glycoprotein precursor. Four of seven glycoprotein-specific antibodies neutralized Junin virus to high titres. Cross-reactivity with other arenaviruses was found to be restricted to nucleoprotein-specific monoclonal antibodies and occurred only with New World arenaviruses. Cross-reactivity also shows the Junin virus to be most closely related to Machupo and Tacaribe viruses.
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Identification of L2 Open Reading Frame Gene Products of Bovine Papillomavirus Type 1 Using Monoclonal Antibodies
More LessSummaryFour hybridoma cell lines producing monoclonal antibodies (MAbs) to bovine papillomavirus type 1 (BPV-1) L2 open reading frame (ORF) gene products have been established from mice immunized with a BPV-1 L2-β-galactosidase fusion protein. Hybridomas were selected and cloned (from over 700 hybridomas) on the basis of specific reactivity of supernatant fluids with BPV-1 L2 epitopes on disrupted BPV-1 particles and L2-β-galactosidase fusion proteins by ELISA and Western blotting, and with acetone-fixed frozen sections of BPV-1-induced fibropapillomas by immunofluorescence. These MAbs were not reactive with intact BPV-1 particles or BPV-1 L1-β-galactosidase fusion proteins by ELISA or with β-galactosidase by ELISA and Western blotting. The four MAbs detected viral structural proteins of M r 76K, 68K and possibly 55K in purified BPV-1 preparations by Western blotting. Two of the four MAbs were cross-reactive with BPV-2-induced fibropapillomas. These findings suggest that (i) the BPV-1 L2 ORF encodes the minor capsid protein(s), (ii) the gene products of the BPV-1 L2 ORF have M r values of 76K, 68K and possibly 55K, (iii) minor capsid epitopes are internal to the BPV-1 particle, and (iv) MAbs reactive with genetically engineered truncated BPV-1 L2 ORF gene products can distinguish between BPV-1 and BPV-2 productive infections.
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Preliminary Characterization of the Alcelaphine Herpesvirus 1 Genome
More LessSummaryAlcelaphine herpesvirus type 1 (AHV-1) is a causative agent of the fatal lymphoproliferative disease malignant catarrhal fever in deer and cattle. The genomes of the attenuated WC11 isolate and the virulent C500 isolate have been studied. The genome of WC 11 comprises a region of unique DNA of approximately 130 kbp, which has a G + C content of 50%, and approximately 30 kbp of additional tandem direct repeat sequences with a G + C content of 72 %. WC11 possesses a major repeat sequence of 950 bp interspersed with a small number of related sequences of different length; these sequences are probably terminal in location. DNA from the C500 isolate has a similar restriction profile to that of WC11 in the unique region, but only one repeat sequence of 1050 bp is present. We propose, on the basis of biological and structural properties, that AHV-1 be included within the γ 2 group of herpes viruses of which herpesvirus ateles is the prototype.
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Alpha-, Beta- and Gammaherpesviruses Encode a Putative Phosphotransferase
More LessSummaryWe have sequenced a gene in human cytomegalovirus and a homologous gene in human herpesvirus 6 which could specify a product related to protein kinases. This gene appears to be generic in the herpesvirus family as homologues were found in three other human herpesviruses. The five sequences were aligned and found to be quite divergent. Some of the differences occur at amino acid positions which are functionally important and highly conserved in known protein kinases. Hence these genes may represent a significant departure from known protein kinases in terms of structure and/or function.
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Identification of the gB Homologues of Equine Herpesvirus Types 1 and 4 as Disulphide-linked Heterodimers and Their Characterization Using Monoclonal Antibodies
SummaryEquine herpesvirus types 1 and 4 (EHV-1 and EHV-4) labelled with [14C]glucosamine were purified from infected cell culture medium and profiles of their structural proteins were obtained that enabled identification of the major glycoproteins. Nine glycosylated polypeptides were identified for each virus. Preparations of the purified viruses each contained a glycoprotein which was linked by disulphide bonds, as determined by diagonal gel electrophoresis under reducing/non-reducing conditions. High M r forms of this glycoprotein were detected for EHV-1 when the sample was not heated. The EHV-1 protein consisted of three polypeptides of M r 108K, 76K and 58K and the EHV-4 protein consisted of three polypeptides of M r 112K, 74K and 61K. Western blotting and immunoprecipitation with monoclonal antibodies confirmed that the EHV-1 gB homologue migrates with an apparent M r of 108K (140K under non-reducing conditions) but is cleaved to give glycoproteins of 76K and 58K which are held together by disulphide bonds. The EHV-4 gB homologue consists of a 112K glycoprotein which is cleaved to give glycoproteins of 74K and 61K which are also linked by disulphide bonds.
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A Hamster Model of Equine Herpesvirus Type 1 (EHV-1) Infection; Passive Protection by Monoclonal Antibodies to EHV-1 Glycoproteins 13, 14 and 17/18
More LessSummaryFollowing intraperitoneal or intranasal inoculation of the Syrian hamster with equine herpesvirus type 1 (EHV-1), strain Kentucky D, virus replicated in the liver and lungs reaching a peak at 4 days post-infection (p.i.). By day 6 p.i. virus titres in these organs had reduced and the spleen contained virus-specific cytotoxic cells. This cytotoxicity was mediated by T cells since treatment of effector cells with a monoclonal antibody to hamster T lymphocytes inhibited the effect. An antiviral humoral immune response was present by day 4 when antibodies capable of lysing EHV-1-infected target cells in the presence of complement were detected. The transfer of serum from recovered hamsters into naive recipients 24 h before challenge prevented virus infection, whereas serum transferred 24 h after challenge reduced the titre of virus recovered from target organs. Inoculation of hamsters with monoclonal antibodies directed to glycoproteins 13, 14 and 17/18 of a subtype 1 virus (Army 183) before virus challenge protected hamsters. This hamster model will prove useful for studying the immune response to EHV-1 and evaluating the immunogenicity of individual virus components.
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Construction and Characterization of Herpes Simplex Virus Type 1 Mutants with Defined Lesions in Immediate Early Gene 1
More LessSummaryTranscription from the early and late classes of the herpes simplex virus type 1 (HSV-1) promoters requires prior immediate early (IE) gene expression. Although the product of IE gene 1, Vmw110, is not absolutely essential for virus growth in tissue culture, transfection experiments have demonstrated that Vmw110 can activate gene expression both by itself and in a synergistic manner with the product of IE gene 3, Vmw175. This paper describes the construction of 10 mutant HSV-1 viruses with deletion and insertion mutations in Vmw110. The mutant viruses were then studied in single-step growth curve experiments, by assaying for plaques in a variety of cell types and by analysis of viral polypeptide synthesis during productive infection at high and low multiplicities. The results show that mutations in Vmw110 reduce the efficiency of plaque formation by HSV-1; the extent of this reduction depends on cell type and the position of the mutation in the polypeptide. In particular, a potential zinc finger domain is crucial for Vmw110 function. The patterns and amounts of viral polypeptide synthesis during high multiplicity infections with mutant and wild-type viruses were similar in all cell types. At low multiplicity, mutations in Vmw110 reduced viral gene expression in the least permissive cell type. The data suggest that the role of Vmw110 during virus infection in tissue culture is at a very early stage of low multiplicity infections; its inactivity leads to the failure to express viral genes so that the virus does not enter the lytic cycle.
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Epstein-Barr Virus DNA Sequences in Precursor Monocyte–Macrophage Cell Lines Established from the Bone Marrow of Children with Maturation Defects of Haematopoiesis
More LessSummaryEpstein-Barr virus (EBV) DNA sequences were detected in four established monoblast or early monocytic cell lines (CM-S, ROV-S, CV-S and AD-S) obtained from bone marrow of children suffering from maturation defects of haematopoiesis. EBV is present in these cells in a latent state. The viral DNA in these cell lines was analysed by Southern blot hybridization, using a set of cloned EBV DNA fragments from the EBV strain B95-8 as probes. A common spectrum of highly related but distinguishable EBV DNA restriction enzyme sequences was found, suggesting some genomic diversity. Propagation of the cells in long-term culture revealed a gradual decrease of EBV copies per cell in all lines with some minor changes in the restriction pattern of the EBV DNA. These findings demonstrate that human precursor monocyte cells may be susceptible to infection by EBV.
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The Type-specific Epitopes of the Epstein-Barr Virus Nuclear Antigen 2 Are Near the Carboxy Terminus of the Protein
More LessSummaryThe Epstein-Barr virus nuclear antigen 2 (EBNA 2) shows serotype variation and two serologically distinct groups of viruses have been identified. These correspond to the two hybridization groups of viruses (A and B) that are distinguished by a highly substituted nucleic acid sequence in the middle of the open reading frame of the EBNA 2 gene. An epitope survey of the EBNA 2-coding region was carried out using a new prokaryotic expression vector tailored to express DNA fragments from the M13 sequencing libraries of the B95-8 (type A) and Jijoye (type B) prototype virus strains. Short overlapping stretches of EBNA 2 sequence were expressed as fusion proteins and used in Western blotting with human sera that contained serotype-specific antibodies. The type A-specific epitope was located between residues 378 and 435 of the B95-8 EBNA 2 polypeptide and the type B-specific epitope mapped between residues 390 and 454, at the carboxy terminus of the Jijoye polypeptide chain. All of the type-specific anti-EBNA 2 sera tested reacted with fusion proteins containing one or other of these epitopes. Despite the direct correlation between the hybridization and serological phenotypes, the type-specific epitopes appear to lie in the relatively conserved carboxy-terminal region of EBNA 2. There was no indication that the residues of the non-homologous region contributed to the formation of antibody-combining sites.
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Enhanced Expression of the Epstein–Barr Virus Latent Membrane Protein by a Recombinant Vaccinia Virus
More LessSummaryThe complete coding sequence of the Epstein–Barr virus strain B95-8 latent membrane protein (LMP) was cloned using a Raji cell cDNA library and genomic B95-8 DNA. The clone was characterized by sequencing and then used to make a recombinant vaccinia virus. This virus (VLMP) was shown to express a relatively high level of LMP in an authentic fashion. Antisera raised in rabbits against VLMP were shown to react with B95-8 LMP as well as cross-reacting with a 50K cellular protein.
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Pseudorabies Virus Displays Variable Numbers of a Repeat Unit Adjacent to the 3′ End of the Glycoprotein gII Gene
More LessSummaryThe coding region of the glycoprotein complex gII of pseudorabies virus (PRV) is located in the unique long part of the genome on SalI subfragments 1A and 1G of BamHI fragment 1 (map units 0·105 to 0·130). Fragment 1G which includes the 3′ end of the gII gene displays a size heterogeneity among different PRV strains and also within plaque isolates of a given strain. To reveal the cause of this heterogeneity and whether it might affect the gII-coding region we sequenced different 1G fragments of the PRV strains Ka, Phylaxia and Dessau, and determined the 3′ end of the gII mRNA by SI analysis. These data show that the size heterogeneity is caused by the presence of a variable number of tandemly repeated DNA sequence downstream but adjacent to the coding region of the glycoprotein gII gene. The 3′ end of the gII mRNA was mapped about 24 bp upstream of the first repeat unit. A 15 bp sequence 5′ GGGACGGAGGGGAGA 3′ is repeated from three to over 50 times in different 1G fragments. It is the only repeat unit present in strain Ka, whereas the Phylaxia and Dessau strains show additional modifications.
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Identification and Preliminary Use of Recombinant Lambda gt11 Fusion Proteins in Human Cytomegalovirus Diagnosis
More LessSummaryWe have isolated reactive clones from a λgt11 expression library of human cytomegalovirus (HCMV) DNA using HCMV-positive human sera. Among the recombinant clones obtained, one carried a fragment encoding a portion of p52, the major non-structural DNA-binding protein of 52K (p52) and another carried a part of the gene coding for p150, the major structural phosphoprotein. These two fusion proteins were examined by immunoblot analysis to test their ability to bind specific antibodies in human sera. The results showed that high titres of antibody to the DNA-binding protein are present in sera of patients undergoing acute HCMV infection, whereas high titres of antibodies to the structural phosphoprotein are widespread in the healthy HCMV-seropositive population. The use of these fusion proteins as antigens for differential screening of serum as a way of detecting recent HCMV infection is discussed.
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The Effect of Cyclosporin on Major Histocompatibility Complex-linked Resistance to Murine Cytomegalovirus
More LessSummaryThe ability of mice to survive infection with murine cytomegalovirus (MCMV) is known to be influenced by genes of the major histocompatibility complex (MHC). One hypothesis to account for this association is that MHC-linked resistance to MCMV is an ‘immune response’ gene effect, caused by differences in the strength of the MHC-restricted T cell response of mouse strains with different MHC haplotypes. Therefore, removal of T cell responses in mouse strains differing only at the MHC should render them equally susceptible to the virus infection. To test this hypothesis, the immunosuppressive drug cyclosporin (CsA) was used to reduce T cell responses in inbred congenic mouse strains carrying either a resistant or susceptible MHC haplotype. CsA reduced the delayed-type hypersensitivity (DTH) response to MCMV in both resistant and susceptible mouse strains to background levels, equivalent to control uninfected mice. CsA treatment had little effect on the susceptibility of C57BL/10 and B10. BR mice to the virus and the differences in susceptibility between these strains remained. In contrast, CsA increased the susceptibility of the genetically susceptible BALB/c mice (H-2d) by 100-fold and increased the susceptibility of resistant BALB.K mice (H-2k) by 15-fold. Thus the H-2-determined difference in susceptibility between these strains was increased after CsA treatment. The results obtained with congenic strains show that MHC-linked resistance patterns to MCMV are not eliminated by CsA and suggest therefore that T cells are not responsible for this phenomenon. Interestingly, the mean time to death was delayed for CsA-treated BALB/c mice compared with untreated mice given equivalent virus doses. In addition, although CsA prevented DTH responses in both genetically susceptible A/J (H-2a) and resistant CBA (H-2k) mice, CsA treatment markedly increased the susceptibility of A/J mice (32-fold) but had little effect on the susceptibility of CBA mice to the virus.
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Immortalization of Primary Human Epithelial Cells by Cloned Cervical Carcinoma DNA Containing Human Papillomavirus Type 16 E6/E7 Open Reading Frames
More LessSummaryPrimary human epithelial cells were transfected with a subgenomic fragment of DNA cloned from a cervical carcinoma, containing the putative transforming genes E6 and E7 from the human papillomavirus type 16 prototype. Several immortalized cell lines were generated, all retaining tumour DNA and expressing the viral genes, suggesting that either one or both of these genes is sufficient for the immortalization of primary human genital keratinocytes.
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Effect of Interferon on the Accumulation of RNA Transcripts of Genes Coding for Cellular and Viral Proteins
More LessSummaryUsing plasmids containing sequences complementary to the genes that code for oligo-2′,5′-adenylate synthetase, actin and H4 histone, we have shown that although interferon does not affect the accumulation of RNA transcripts of the actin and H4 histone genes, it activates the accumulation of RNA transcripts of the oligo-2′,5′-adenylate synthetase gene. However, interferon inhibits the accumulation of RNA transcripts of simian virus 40 (SV40) genes in SV40-infected cells.
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Studies on the Control Region of the p10 Gene of the Autographa californica Nuclear Polyhedrosis Virus
More LessSummary5′ deletion mutants of the Autographa californica nuclear polyhedrosis virus very late p 10 gene promoter have been prepared and subjected to a transient expression assay in infected Spodoptera frugiperda cells. The control plasmid contained the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the p 10 promoter, which was included in a 230 bp sequence upstream from the p 10 translation initiation codon. The control plasmid also contained a segment of the hr5 enhancer downstream from the CAT gene. Promoter activity was unaffected by 5′ deletion to position –77, which lies about 11 bp upstream from the p10 cap site. However, deletion of 12 more bp completely eliminated p10 promoter activity. Thus, the 5′ border of the p10 promoter lies downstream from position –77, and the region between positions –77 and –65 contains an element that is important to promoter activity. This is the region that is conserved near the cap sites of late baculovirus genes. Our studies also show that transient expression of CAT under the control of the p10 promoter and hr5 enhancer is higher when transfection occurs prior to infection by virus.
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Nucleotide Sequence Analysis of the Small (S) RNA Segment of Bunyamwera Virus, the Prototype of the Family Bunyaviridae
More LessSummaryThe nucleotide sequence of the small (S) RNA segment of the Bunyamwera virus genome has been determined. The S RNA is 961 bases in length and, in common with other bunyaviruses, encodes two proteins, N and NSs, in overlapping reading frames. A six-way alignment of the amino acid sequences of the N and NSs proteins of viruses representing three serogroups within the Bunyavirus genus indicates regions which are strongly conserved, and provides targets for future analysis of protein function.
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Effect of Anti-haemagglutinin-esterase Glycoprotein Monoclonal Antibodies on the Receptor-destroying Activity of Influenza C Virus
More LessSummaryFive monoclonal antibodies (J14, J9, Q5, K16, S16), directed to three distinct antigenic sites (A-1, A-2, B-1) on the haemagglutinin–esterase glycoprotein of influenza C virus, were analysed for their ability to inhibit the receptor-destroying enzyme (RDE) activity of the virus, utilizing various assay systems. The ability of influenza C virus to destroy the receptors on chicken erythrocytes was inhibited efficiently by the antibodies to site A-1 (J14, J9, Q5) but not by those to site A-2 (K16) and site B-1 (S16). Of the three antibodies to site A-1, J14 showed the highest inhibitory activity. Antibodies to sites A-1 and A-2 inhibited the ability of RDE to inactivate the haemagglutination inhibition activity of rat serum inhibitors, but the highest activity was observed again with J14. Thus the RDE site of influenza C virus may be located closest to the epitope recognized by J14. The removal of O-acetyl groups from either 9-O-acetyl-N-acetylneuraminic acid or p-nitrophenylacetate, caused by the viral RDE, was not prevented at all by any of the monoclonal antibodies tested. Furthermore, none of several polyclonal antiviral sera prepared in different animal species was able to block the hydrolysis of these small substrates, raising the possibility that the catalytic site of influenza C viral RDE is antigenically silent.
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- Plant
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Nucleotide Sequence of Potato Leafroll Luteovirus RNA
More LessSummaryA sequence of 5987 nucleotides is reported for the RNA of potato leafroll luteovirus (PLRV). The sequence contains six large open reading frames, and non-coding regions of 174 nucleotides at the 5′ end, 141 nucleotides at the 3′ end and 197 nucleotides between two large blocks of coding sequences. The 5′ coding region encodes two polypeptides of 28000 (28K) and 70K which overlap in different reading frames and circumstantial evidence suggests that the third open reading frame in the 5′ block is translated by frameshift readthrough near the end of the 70K polypeptide to give a 118K polypeptide. The C-terminal part of the 118K protein contains the consensus sequence for RNA-dependent RNA polymerases. In vitro translation of PLRV RNA resulted in the synthesis mainly of 28K and 70K polypeptides and the largest product made was about 125K; these sizes are similar to those predicted for the translation products of the 5′ block of coding sequence. The 3′ block of coding sequence codes for three polypeptides: a 23K coat protein, a 17K polypeptide which is encoded in a different frame, and a 53K polypeptide which immediately follows the coat protein coding sequence, and is in the same reading frame. Circumstantial evidence suggests that the 53K polypeptide is translated by readthrough of the amber termination codon of the coat protein gene. The amino acid sequences encoded by the 3′ block of coding sequence show many similarities with analogous polypeptides translated from the nucleotide sequences of RNA of barley yellow dwarf virus, PAV strain (BYDV) and, in particular, beet western yellows virus (BWYV). The 118K polypeptide has some similarities with the putative polymerase of southern bean mosaic virus and much more extensive similarities with the corresponding BWYV polypeptide but almost none with that of BYDV. In contrast, the amino acid sequence of the 28K polypeptide is not like that of proteins of the other luteoviruses or of viruses in other groups. The nucleotide sequences reported will appear in the EMBL, GenBank and DDBJ databases under the accession number X14600.
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Organization and Interviral Homologies of the 3′-terminal Portion of Potato Virus S RNA
More LessSummaryThe sequence of 3553 nucleotides corresponding to the 3′-terminal region of potato virus S (PVS) has been determined from cloned cDNA. The sequence obtained contains six open reading frames (ORFs) encoding proteins of M r 10734, M r 32515, M r 7222, M r 11802, M r 25092 and at least M r 41052. The sequence of the 33K ORF has been confirmed to be that of the viral coat protein gene. The nucleotide sequence of this ORF was obtained from plasmids which were isolated by colony hybridization with a specific monoclonal antibody to PVS, and the expression of coat protein fusion products was verified by Western blots of bacterial cell lystates. The deduced amino acid sequence of a 70 amino acid portion from the central region of the PVS coat protein was 59% identical to the analogous region of potato virus X. In addition, the 7K, 12K and 25K ORFs displayed significant sequence homology with the similarly sized ORFs from a number of potexviruses. The partial 41K ORF product was homologous with the C-terminal portion of the viral replicase proteins of potato virus X and white clover mosaic virus.
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Nucleotide Sequences of the Coat Protein Genes and Flanking Regions of Cucumber Mosaic Virus Strains C and WL RNA 3
More LessSummarySeveral strains of cucumber mosaic virus (CMV) have been classified, and nucleic acid hybridization data indicate that these strains differ widely in nucleotide sequence. We have constructed cDNA clones of the coat protein coding regions of CMV strains C and WL, and have compared the nucleotide sequences of the RNA 3 intergenic region, coat protein gene, and 3′ untranslated region with published CMV sequences from the same regions of the Q, D and Y strains. These comparisons show that the C and WL strains belong to different CMV subgroups, and that the subgroups are more closely related in sequence than suggested by previous nucleic acid hybridization studies.
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Characterization of a Satellite RNA Associated with Pea Enation Mosaic Virus
More LessSummaryThe bipartite genome of pea enation mosaic virus (PEMV) is often accompanied by a non-essential third RNA (M r 0·3 × 106) of unknown origin and function. Although the Wisconsin strains of PEMV originally lacked this RNA, we have monitored the appearance of a putative replicative form of this species in PEMV-infected tissue. In later generations encapsidated single-stranded RNA 3 appeared. We have used a 750 bp clone generated against the ds replicative form of RNA 3 to probe viral and host-derived nucleic acids to establish the relationship of this RNA to PEMV infection. Northern blot analysis showed that RNA 3 is distinct from viral genomic RNA and from host RNA. Similarly, Southern blot analysis showed that RNA 3 is distinct from the host genome. Infectivity analysis of fractionated viral RNAs coupled with Northern blot analysis confirmed that RNA 3 is both non-essential for PEMV infection, and non-infectious when inoculated on its own. RNA 3 does not influence symptom expression, aphid transmission or particle morphology. We conclude that RNA 3 of PEMV is a satellite RNA.
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Distribution of Cylindrical Inclusion, Amorphous Inclusion and Capsid Proteins of Watermelon Mosaic Virus 2 in Systemically Infected Pumpkin Leaves
More LessSummaryMonoclonal antibodies (MAbs) were prepared against the cylindrical inclusion protein (CIP), amorphous inclusion protein (AIP) and capsid protein (CP) of watermelon mosaic virus 2 (WMV2). Using the MAbs, CIP, AIP and CP were detected, roughly quantitatively, in WMV2-infected pumpkin leaves showing various symptoms by using electroblot-ELISA. From symptomless leaves and most dark green areas in the mosaic pattern no protein or small amounts of the three proteins were detected, but from most yellow areas in the mosaic almost equal amounts of each protein were detected in abundance. Leaves showing mild vein-yellowing and vein-clearing (respectively, the first and second leaves of the plants tested) contained AIP and CP in large amounts, but little CIP. On the other hand, expanding leaves contained CIP and AIP in large quantities, butCP in traces only. Therefore the distributions of CIP, AIP and CP in pumpkin plants were very uneven, but correlated with symptoms. In addition, the ratio of the concentrations of CIP, AIP and CP varied from tissue to tissue.
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Use of Monoclonal Antibodies in the Purification of an Inhibitor of Virus Replication by Affinity Chromatography
More LessSummaryMouse monoclonal antibodies (MAbs) were prepared to an inhibitor of virus replication (IVR), released from protoplasts or leaf tissue of hypersensitive tobacco plants infected with tobacco mosaic virus. The MAbs were highly specific for IVR and reduced its antiviral activity. Using these MAbs in affinity chromatography enabled the recovery of purified IVR. SDS-PAGE of the immunoaffinity-purified IVR gave a single M r 23K band. Immunoblots of IVR from extracts of protoplast or leaf tissue also revealed a single M r 23K band which suggests that protoplast and tissue IVR are closely related.
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Nucleotide Sequence of Rice Dwarf Virus Genome Segment 9
SummaryThe complete nucleotide sequence of the phytoreovirus rice dwarf virus (RDV) genome segment 9 is presented. It consisted of 1305 nucleotides and had an open reading frame that codes for a putative polypeptide of 351 amino acids. M r of the protein was calculated to be 38 598. The terminal nucleotides 5′ GGUAAA--GAU 3′ were the same as those of RDV genome segment 10. The fourth nucleotide from the 3′end of an expected conserved sequence was a C rather than the U found in the previously sequenced genome segment 10. A structure similar to the segment-specific inverted repeat of wound tumour virus was also found in the terminal region of segment 9.
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- Fungal
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Conservative Transcription of Helminthosporium victoriae 190S Virus Double-stranded RNA in vitro
More LessSummaryIn in vitro reactions, the Helminthosporium victoriae 190S virus-associated RNA polymerase catalysed the synthesis and release of full-length ssRNA transcripts of genomic dsRNA. The transcriptase activity, which was dependent on virus concentration, required all four nucleoside triphosphates and magnesium ions. In reaction mixtures containing [3H]UTP, 99·0% to 99·5% of the incorporated label was in ssRNA. Hybridization analysis and in vitro translation of the reaction products showed that transcription was asymmetric and that the product of transcription was the message strand. In rabbit reticulocyte lysates, the in vitro transcript directed the synthesis of the capsid polypeptide p88. In transcription reactions containing [3H]UTP, no incorporated label was detected in genomic dsRNA during the time it took for the ssRNA transcript to reach full length. These results support the idea that transcription of dsRNA of this virus, like that of dsRNA of other members of the family Totiviridae, is conservative.
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Volume 54 (1981)
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Volume 53 (1981)
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Volume 52 (1981)
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Volume 51 (1980)
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Volume 50 (1980)
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Volume 49 (1980)
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Volume 48 (1980)
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Volume 47 (1980)
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Volume 46 (1980)
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Volume 45 (1979)
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Volume 44 (1979)
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Volume 43 (1979)
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Volume 42 (1979)
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Volume 41 (1978)
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Volume 40 (1978)
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Volume 39 (1978)
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Volume 38 (1978)
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Volume 37 (1977)
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Volume 36 (1977)
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Volume 35 (1977)
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Volume 34 (1977)
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Volume 33 (1976)
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Volume 32 (1976)
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Volume 31 (1976)
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Volume 30 (1976)
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Volume 29 (1975)
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Volume 28 (1975)
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Volume 27 (1975)
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Volume 26 (1975)
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Volume 25 (1974)
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Volume 24 (1974)
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Volume 23 (1974)
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Volume 22 (1974)
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Volume 21 (1973)
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Volume 20 (1973)
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Volume 19 (1973)
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Volume 18 (1973)
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Volume 17 (1972)
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Volume 16 (1972)
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Volume 15 (1972)
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Volume 14 (1972)
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Volume 13 (1971)
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Volume 12 (1971)
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Volume 11 (1971)
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Volume 10 (1971)
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Volume 9 (1970)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)