- Volume 71, Issue 8, 1990
Volume 71, Issue 8, 1990
- Bacterial
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Corynephage Cog, a Virulent Bacteriophage of Corynebacterium Glutamicum, and its Relationship to 𝜙GA1, an Inducible Phage Particle from Brevibacterium Flavum
More LessThe host range of the virulent bacteriophage Cog among several strains of amino acid-producing corynebacteria is limited to Corynebacterium glutamicum LP-6. Cog is a typical corynephage of the Siphoviridae family (B1 morphotype) with an isometric head of 52 nm and a medium length, striated tail of 190 nm. It has a linear genome of 39·7 kb with cohesive ends and 53% G+C; a restriction map is presented, including regions of DNA homology (by hybridization) with the inducible phage particle 𝜙GA1 Brevibacterium flavum.
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Characterization of Filamentous Bacteriophage 𝜙Lf from Xanthomonas Campestris pv. Campestris
More LessA filamentous phage, øLf, which specifically infects Xanthomonas campestris pv. campestris was isolated. The phage particle measured 1000 (±200) × 8 nm. It formed turbid plaques of about 1 mm in diameter. During multiplication, the progeny virions extruded into the medium without retarding host cell growth. Stocks were stable for 6 months at 4 °C and survived treatment at 80 °C for 10 min. Treatment with chloroform, ethanol or acetone completely destroyed infectivity; ethyl ether and methanol inactivated 98 to 99% of the phage. SDS-polyacrylamide gel electrophoresis showed a major coat protein band of approximate Mr 4000 whereas an immunoprecipitation test detected the existence of two coat protein species. The phage genome was shown to be a single-stranded DNA molecule. A physical map was constructed and the DNA size was calculated to be 5·9 kb. Cells treated with Tris-HCl containing CaCl2 and polyethylene glycol 6000 were transfected by replicative form DNA at a frequency of 3·4 × 103 p.f.u./ µg.
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Comparative Study of Virion Structure, Protein Composition and Genomic DNA of Three Ascovirus Isolates
More LessThe virions of three ascoviruses isolated from the noctuids Heliothis zea, Spodoptera frugiperda and Trichoplusia ni were compared with respect to their size and structure, protein composition and the size and relatedness of their DNAs. The virions of the isolates from H. zea (HAV) and T. ni (TAV) were allantoid in shape (400×130 nm), enveloped and contained an inner particle which appeared to have an internal lipid bilayer surrounding the DNA core. The virions of the S. frugiperda isolate (SAV) were similar in structure and size, but were bacilliform in shape, and after formation, were often occluded in vesiculate occlusion bodies. In preparations of purified virions of each isolate, at least 12 polypeptides were detected that ranged in size from 10K to 200K and contained a major species of about 50K. The genome of SAV was about 140 kbp in size, whereas those of TAV and HAV were approximately 180 kbp. Analysis of DNA fragment patterns of the three isolates generated with BamHI, HindIII or XhoI, as well as DNA-DNA dot blot and Southern blot hybridization studies, demonstrated that HAV and TAV were closely related but not identical. The DNA from SAV, however, did not hybridize with the DNA from either of the other isolates. Thus the ascovirus isolates from T. ni and H. zea are considered variants of the same virus, whereas the isolate from S. frugiperda is a separate member of the ascovirus group.
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Cloning and Sequencing the Messenger RNA of the N Gene of Viral Haemorrhagic Septicaemia Virus
More LessThe mRNA transcribed from the N gene of viral haemorrhagic septicaemia virus (VHSV) of salmonids has been cloned in Escherichia coli and expressed. Fusion proteins were recognized by monoclonal antibody directed against the N protein from the viral particle. A 1212 bp long open reading frame (ORF) coding for 404 amino acids with a calculated Mr of 44590 was deduced from the nucleotide sequence. The ORF was preceded by a 93 bp segment including in position 42 the AACAC pentanucleotide which is presumed to be the start signal for transcription by analogy with other rhabdoviral mRNAs. The upstream 41 bp region could correspond to the covalently linked positive polarity leader RNA as also found on the N mRNA from infectious haematopoietic necrosis virus (IHNV). This may be a characteristic of fish lyssaviruses. The AAACC sequence, which is part of the leader, was not found. Amino acids 44 to 359 from IHNV and 45 to 360 from VHSV are 45·3% homologous. A strong homology which could reflect functional importance was also found for potential phosphorylation sites and hydrophobic peaks despite the fact that the two viruses evolved on different continents.
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Tumour Necrosis Factor Enhances induction by β-Interferon of a Ubiquitin Cross-reactive Protein
More LessTumour necrosis factor α (TNF-α) elicited an antiviral response in some cell lines (MG-63 and HEp-2) but not in others (MDBK). Cell lines that generated an antiviral response to TNF-α also showed induction of a 15K protein which shared sequence homology with ubiquitin and reacted with an antibody to ubiquitin. This ubiquitin cross-reactive protein (UCRP) had been demonstrated previously to be induced by interferon. The TNF-α induction of UCRP occurred at the level of transcription. TNF-α induction of both the antiviral state and the 15K protein was blocked by either monoclonal or polyclonal anti-β-interferon (IFN-β) antibody. However no measurable increase in the mRNA specific for IFN-β was detected after TNF-α treatment. Nonetheless, in supernatants from cell cultures, the presence of an antiviral activity inhibitable by anti-IFN-β antibody indicates that these cells are making IFN-β already. We conclude that the TNF-α induction of antiviral activity and UCRP in cells is dependent upon the presence of constitutive low levels of IFN-β in the responding cells. Furthermore TNF functions to enhance the existing IFN-β activity.
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The Antigenic Structure of a Human Influenza A (H1N1) Virus Isolate Grown Exclusively in MDCK Cells
More LessA panel of monoclonal antibodies has been produced against a 1983 human influenza A (H1N1) virus that has been isolated and grown exclusively in MDCK cells. Several of these antibodies were used to select variants from MDCK-derived virus in cells and their epitopes were then located by determining the HA1 amino acid sequence. The operational antigenic map of the haemagglutinin indicates the presence of two distinct immunodominant antigenic regions which correspond but are not identical to antigenic sites Sa and Sb of the A (H1N1) virus A/PR/8/34. Also during this study, we characterized a unique group of antibodies for which variants require two distinct and specific HA1 amino acid substitutions to escape neutralization.
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Human Monoclonal Antibodies Specific for the Rabies Virus Glycoprotein and N Protein
More LessHuman monoclonal antibodies to rabies virus were established by Epstein-Barr virus infection of peripheral blood lymphocytes collected from a rabies- vaccinated donor, and fusion with a heteromyeloma line. Two human monoclonal antibodies, HUM1 and HUM2, both IgG2, reacted with the envelope glycoprotein of the rabies virus. The antibody HUM1 neutralized rabies virus (lyssavirus serotype 1) and Mokola virus (lyssavirus serotype 3), but did not neutralize European bat lyssavirus, suggesting that some common antigenicity exists between the glycoproteins of serotypes 1 and 3. In addition, this antibody neutralized a series of viruses resistant to neutralization by antibodies recognizing, in a murine system, anti genic sites I, II and III; however, it failed to neutralize viruses altered at site VI, indicating that human monoclonal antibody HUM1 is directed against antigenic site VI. The other human anti-glycoprotein antibody, HUM2, neutralized the European bat lyssavirus in addition to serotypes 1 and 3, but none of the resistant variant viruses altered at the sites mentioned above. A third human monoclonal antibody, HUM3 (IgM), was reactive with the internal nucleoprotein of the rabies virus. This antibody contained a murine light chain corresponding to the cytoplasmic murine chain not secreted in the heteromyeloma line. The potential use of monoclonal antibodies in post-exposure treatment of rabies is discussed.
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Protection of mice against yellow fever virus encephalitis by immunization with a vaccinia virus recombinant encoding the yellow fever virus non-structural proteins, NS1, NS2a and NS2b
More LessRecent evidence of a protective immune response to the flavivirus non-structural protein, NS1, has suggested its incorporation into possible recombinant vaccines. The region of the 17D yellow fever virus (YFV) genome encoding the C terminus of envelope glycoprotein and extending to the N terminus of non-structural protein NS3 (NSl-NS2a-NS2b; nucleotides 2030 to 4940) was expressed in vaccinia virus and physical and immunogenic properties of the NS1 moiety were studied. Recombinant NS1 protein, and native YFV NS1, was detected at the surface of infected cells by immunofluorescence and by immune cytolysis after treatment with anti-NSl antibody and complement. NS1 was also detected in the extracellular medium as a secreted form. Recombinant NS1 was immunoprecipi- tated as a single protein of approximately the same size as native 17D YFV NS1. Unboiled, both recombinant and native NS1 formed polymers of high M r. Immunization of mice with this recombinant vaccinia virus stimulated production of non-neutralizing, complement-fixing cytolytic antibody and conferred partial protection against lethal intracerebral inoculation of mice with live 17D YFV.
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Synthesis of Foot-and-mouth Disease Virus Capsid Proteins in Insect Cells Using Baculovirus Expression vectors
More LessFoot-and-mouth disease virus (FMDV) cDNA cassettes containing sequences encoding the capsid precursor PI-2A with and without those encoding the proteases L and 3C were introduced into Autographa californica nuclear polyhedrosis virus (AcMNPV) expression vectors. Procapsid proteins 1AB, 1C and ID were produced in cells infected with recombinant baculoviruses, when L and 3C were present in the constructs, indicating that these FMDV proteases were active in insect cells. Unlike µl processing in poliovirus, which has been shown to be catalysed mainly by the 3CD gene product, the 3C protease of FMDV was able to process P1 independently of 3D. Cytotoxicity of the L protease for insect cells prevented the use of the optimized transfer vector, pAcRP23, for inserting L- containing cassettes into AcMNPV. By contrast, viable AcMNPV-FMDV recombinants could be made without restriction on choice of the transfer vector when the L gene was either not expressed or inactivated by an inframe deletion. In the latter case, normal cleavage at the L-Pl junction no longer occurred in cis, and a new processing event, probably catalysed by 3C, was observed within the C-terminal region of the residual L protein. Analysis of baculovirus-expressed products in sucrose gradients showed that a fraction of the capsid proteins is present in an aggregated form, migrating at 70S and possibly resembling FMDV empty capsid particles.
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The Expression of the Adenovirus 12 Early Region 1B 19K Protein Using a Recombinant Simian Virus 40 System
Using a simian virus 40/adenovirus 12 (Adl2)-recom binant virus, the Ad 12 early region IB (E1B) 19K protein has been produced at high levels after infection of Cos 1 cells. Expression of the 19K polypeptide reaches a maximum at about 48 h post-infection, declining at later times as host cell death occurs. Using two-dimensional isoelectric focusing/SDS-polyacryl- amide gel electrophoresis, we have shown that the Ad 12 E1B protein is a major species following infection of Cos 1 cells with recombinant virus. Two forms of 19K polypeptide can be distinguished following isoelectric focusing. Using subcellular fractionation of infected cells, it was found that a similar distribution of 19K protein occurred after recombinant virus and Ad 12 infection, with the polypeptide being most abundant in nuclear and membranous fractions. Similarly, as in Adl2-infected cells, a certain proportion of the protein is located on the outside surface of the cell after recombinant viral infection. Immuno- histochemical studies suggest that, at early times postinfection, the E1B 19K protein is located in the nuclear membrane, the Golgi apparatus and the endoplasmic reticulum. At later times, it can be seen to have spread to the cytoplasm as well as to the other organelles. These results are discussed in relation to the known functions of the 19K E1B protein.
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The Complete Nucleotide Sequence of Bovine Polyomavirus
More LessThe complete sequence of the genome of bovine polyomavirus (BPyV), formerly known as the CK isolate of the stump-tailed macaque virus, is presented. The genomic organization of BPyV is similar to that of the non-rodent polyomaviruses. With a genome size of 4697 bp, BPyV has the smallest polyomavirus genome known so far. When compared to simian virus 40 (SV40), the shortness of the BPyV genome is due mainly to differences in the coding capacity of the BPyV early region. The first exon of the proposed large T antigen encodes only 35 amino acids; also, a coding region corresponding to the C-terminal 64 amino acids of the SV40 large T antigen is absent in BPyV. It is proposed that the nucleotide sequence encompassing the small t antigen coding sequence contains an intron sequence of 71 nucleotides. Together the two exon sequences encode a 124 amino acid protein. We conclude that this may be the first example of a polyomavirus that has a small t antigen which is translated from two exon sequences. The enhancer region of BPyV does not show homology to the SV40 enhancer sequences. An agnogene is present with a coding capacity of 118 amino acid residues. The highest degree of homology to SV40 and PyV is present in the VP1 molecule.
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Molecular Cloning and Sequencing of an Australian Isolate of Proviral Bovine Leukaemia Virus DNA: Comparison with other Isolates
The molecular cloning and characterization of an EcoRI fragment, 8·26 kb in size, of an Australian isolate of bovine leukaemia virus (pBLV-Al) is described. This fragment includes most of the proviral genome as well as 340 bp of flanking bovine DNA sequence at the 5′ end. Approximately 790 bp, including the 3′ long terminal repeat, was missing from this clone. At the level of restriction enzyme mapping, this isolate could be distinguished from American, Belgian and Japanese isolates. DNA sequencing of the entire clone demonstrated some variation at the amino acid level between pBLV-Al and the Japanese and Belgian isolates, particularly in the gag gene. In that gene there were 59 amino acid changes compared to the Japanese isolate and 24 compared to the Belgian isolate. The greater number in the case of the Japanese isolate was due to both single nucleotide changes and frameshift in a single region of the gene. This study also demonstrates that there are large tracts of amino acid sequence, particularly within the env andpol genes, that are highly conserved in different isolates. Some of these conserved sequences exist in regions containing epitopes important in virus infectivity.
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Identification of Two Genes in the Unique Short Region of Pseudorabies Virus; Comparison with Herpes Simplex Virus and Varicella-zoster Virus
More LessWe have determined the nucleotide sequence of two genes in the unique short region of the genome of pseudorabies virus (PRV). Near the internal repeat, upstream of the gene encoding glycoprotein gX, we identified an open reading frame (ORF) encoding a protein of 390 amino acids. We designated this gene PK because the predicted protein contains most of the conserved motifs of a eukaryotic protein kinase. The protein shares amino acid homology with the protein kinases encoded by gene US3 of herpes simplex virus type 1 (HSV-1) and gene 66 of varicella-zoster virus. Near the terminal repeat, downstream of a gene encoding an 11K protein, we identified an ORF encoding a protein of 256 amino acids. We designated this gene 28K, the Mr of the predicted protein. Part of the amino acid sequence of 28K is homologous to the predicted US2 protein of HSV-1. Northern blot analysis revealed a 2·7 kb mRNA encoding the putative protein kinase and a 1·2 kb mRNA encoding the 28K protein in PRV-infected cells. The 5′ ends of the mRNAs were mapped by primer extension. Two transcriptional start sites were identified for the PK mRNA: a minor start site immediately upstream of the ORF and a major start site (>95% of the mRNA) within the ORF, 64 nucleotides upstream of an internal ATG codon. A single transcriptional start site was identified for the 28K mRNA immediately upstream of the ORF. Immunoblot analysis with anti-peptide sera revealed that, in cells infected with PRV, the PK gene was translated into two proteins with Mr s of 53K and 4IK, and the 28K gene into a single protein with an M r of 28K.
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The Protein Kinase Encoded in the Short Unique Region of Pseudorabies Virus: Description of the Gene and Identification of its Product in Virions and in Infected Cells
More LessWe have cloned and determined the nucleotide sequence of a gene, pk, that lies immediately upstream from the gene encoding glycoprotein X in the short unique region of the alphaherpesvirus, pseudorabies virus (PRV). The gene has the potential to encode a protein of 334 amino acids, and is related to gene US3 of herpes simplex virus type 1 (HSV-1), which has been shown to encode a protein kinase. The predicted amino acid sequence encoded by the PRV pk gene is homologous to the corresponding sequence encoded by the HSV-1 US3 gene in the C-terminal catalytic domain, but diverges markedly in the N-terminal domain. As with HSV-1, the mRNA for the pk gene appears to be 3′ coterminal with that for the glycoprotein downstream. An antiserum was raised against a protein generated from the fusion of part of the PRV pk catalytic domain with Escherichia coli β-galactosidase. This specifically reacted with a previously described physically homogeneous protein kinase, PRV-PK, isolated from hamster fibroblasts lytically infected with PRV. Although the majority of the PRV-PK is found in the cytoplasm, some was also detected in purified PRV virions by using the same antibody; a similar distribution was found for the HSV-1 protein kinase, using an antiserum raised against the corresponding HSV-1 fusion protein. When presented with heat- inactivated virions, purified PRV-PK (in common with certain cellular protein kinases also present in the virion) was able to phosphorylate in vitro the major virion phosphoprotein phosphorylated in vivo.
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Virus Neutralizing Activity Induced by Synthetic Peptides of Glycoprotein D of Herpes Simplex Virus Type 1, selected by their Reactivity with Hyperimmune Sera from Mice
Mice were immunized with synthetic peptides covering the first 56 amino acids of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) and a fusion protein, produced in Escherichia coli, containing the first 55 amino acid residues of gD. It was found that mice immunized with peptides composed of amino acid residues 1 to 13, 18 to 30, 22 to 38 and 38 to 56 of gD were not significantly protected against a lethal challenge with HSV-1. Immunization with peptide 9–21 and the gD fusion protein resulted in significant protection. Antisera, from mice immunized with HSV-1, were investigated for reactivity with a series of 57 overlapping gD peptides covering the entire amino acid sequence, except for the membrane-spanning region. All antisera reacted with peptides 9–21, 10–24, 151–165, 216–232, 282–301 and with peptide 340–354 located in the anchoring region of gD, and 15 other peptides were recognized by at least one antiserum. Twelve peptides (10–24, 151–165, 216–232, 244–267, 260–274, 270–284, 260–284, 282–301, 300–314, 340–354, 348–362 and 355–369) reacted most frequently with the hyperimmune sera from mice and were selected for further study. These were conjugated to bovine serum albumin and used to immunize rabbits. Only antisera against peptide 10–24, which covers the same epitope as peptide 9–21, neutralized HSV-1 in vitro.
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A Prominent Serine-rich Region in Vmw175, the Major Transcriptional Regulator Protein of Herpes Simplex Virus Type 1, is not Essential for Virus Growth in Tissue Culture
More LessHerpes simplex virus type 1 (HSV-1) encodes five immediate early (IE) genes of which at least three are involved in the transcriptional regulation of later classes of viral genes. Perhaps the most important of these regulatory proteins is Vmw175, a nuclear phosphoprotein of 1298 predicted amino acid residues. In the absence of functional Vmw175 the virus fails to activate early or late genes or to repress IE gene expression. All viruses of the sub-family alphaherpesvirinae encode polypeptides that are closely related to Vmw175. Mutational studies have shown that regions of homology within this family of gene regulators are generally of functional importance. One of the most striking conserved stretches of amino acid sequence is a run of serine residues followed by a highly acidic region in the amino-terminal fifth of the polypeptide. We have constructed an HSV-1 virus which lacks this serine-rich run within Vmw175. Surprisingly, the virus was viable in tissue culture cells and expressed apparently normal amounts of viral polypeptides. In plaque assays it was very slightly temperature-sensitive and, depending on the state of the host cells, could generate plaques with a syncytial morphology. The mutant protein was able to bind to DNA in a manner indistinguishable from that of the wild-type polypeptide. We conclude that despite its conservation in all of the alphaherpesvirinae so far sequenced, the serine-rich homology is not important for virus growth in tissue culture.
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High Level Expression in 293 Cells of the Herpes Simplex Virus Type 2 Ribonucleotide Reductase Subunit 2 Using an Adenovirus Vector
More LessThe herpes simplex viruses (HSV-1 and HSV-2) encode a ribonucleotide reductase consisting of two non-identical subunits (RR1 and RR2) which associate to form the active holoenzyme. To facilitate the purification and subsequent biochemical characterization of this enzyme, we have cloned the small subunit 2 of the HSV-2 ribonucleotide reductase (RR2 HSV-2) in a helper-independent adenovirus type 5 vector under the control of the adenovirus type 2 major late promoter. After infection of 293 cells with the recombinant virus, the amount of RR2 HSV-2 protein produced was eightfold higher than in HSV-2-infected cells. The specific activities of the RR2 HSV-2 recombinant subunit and the RR2 HSV-2 protein in HSV-2-infected cells were determined by their mixing with saturating amounts of isolated RR1 HSV-1 subunit. By comparison of the relative amount of each RR2 HSV-2 subunit with its specific activity, we calculated that the recombinant protein intrinsic activity was similar to that of the protein produced in HSV-2-infected cells. These results demonstrated that the adenovirus expression vector is a good system to produce an active RR2 HSV-2 subunit in fairly high amounts.
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The Nucleotide Sequence of an Equine Herpesvirus 4 Gene Homologue of the Herpes Simplex Virus 1 Glycoprotein H Gene
More LessThe equine herpesvirus 4 (EHV-4) gene glycoprotein H (gH) gene homologue was localized by virtue of the conserved genomic position of this gene throughout members of the herpesvirus family. The gene maps immediately downstream of the thymidine kinase gene at approximately 0.49 to 0.51 map units within genomic fragment BamHI C. The EHV-4 gH primary translation product is predicted to be a polypeptide of M r 94100, 855 amino acids long, which possesses features characteristic of a membrane glycoprotein, namely an N-terminal signal sequence, a large hydrophilic domain containing 11 putative N-linked glycosylation sites, a C-terminal transmembrane domain, and a charged cytoplasmic tail. Comparison to other herpesvirus glycoproteins revealed identities of 85%, 26% and 32% with the gH counterparts of the alphaherpesviruses EHV-1, herpes simplex virus 1 and varicella-zoster virus, respectively, and of 17% and 18% with those of human cytomegalovirus, herpesvirus saimiri and Epstein-Barr virus. The EHV-4 gH exhibits features previously reported to be conserved throughout the gH polypeptides of herpesviruses of all three subgroups. A region of direct repeat elements and a possible origin of DNA replication are located immediately downstream of the gH gene.
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The Nucleotide Sequence of the Equine Herpesvirus 4 Thymidine Kinase Gene
More LessWe have determined the genomic location and nucleotide sequence of the equine herpesvirus 4 thymidine kinase (TK) gene. The gene is positioned at approximately 0·48 map units within the long unique component of the genome and is flanked by genes encoding a herpes simplex virus 1 (HSV-1) UL24 homologue and glycoprotein H. The predicted protein is composed of 352 amino acids, has an M r of 38 800 and exhibits 36 % identity to the predicted TK of HSV-1.
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Synthesis and Processing of a gp28/32 Membrane Glycoprotein Induced by Marek's Disease Virus Serotype 2
More LessThe post-translational events leading from the precursor to the processed forms of a glycoprotein with an M t of 28K to 32K (gp28/32) of Marek’s disease virus (MDV) serotype 2 were examined with pulse-chase experiments and treatment with tunicamycin and monensin. Cell-free translation of infected cell mRNA followed by immunoprecipitation analysis suggested that a polypeptide with a size of 22K is the initial precursor. Experiments with endo-β-N-acetylglucosa- minidase H and endo-β-N-acetylglucosaminidase F indicated that gp28/32 contains mostly N-linked oligosaccharides of the complex type. These studies showed that 22K, the initial product, is then processed through intermediates to the 28K to 32K form.
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Flow Cytometric Analysis of Epstein-Barr Virus (EBV) Latent Membrane Protein Expression in EBV-infected Raji cells
More LessIn cultures of the latently Epstein-Barr virus (EBV)- infected Burkitt’s lymphoma cell line Raji, the detectable amount of the EBV-encoded latent membrane protein (LMP) is transiently increased after addition of fresh nutrient medium containing foetal calf serum. In the current study, the relative amount of LMP and DNA in Raji cells was determined by biparametric flow cytometry analysis at different times after the addition of fresh medium with 10% foetal calf serum to a dense Raji culture. A transient increase in the proportion of LMP-positive cells was observed during the lag phase of the culture. Subsequently, a subpopulation of cells, which had been arrested in the G0 or G1 phase, simultaneously started to progress through the cell cycle. Neither the amount of LMP in the cells, nor the enhanced expression of LMP, was restricted to a certain phase of the cell cycle. Further analysis revealed that the number of LMP-positive cells proceeding simultaneously from the G, to the S phase of the cell cycle is about the same as the total number of cells changing phases. These results suggest that LMP expression might be one step in the pathway leading to growth activation of resting cells in cultures of the immortalized Raji cell line.
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Papillomavirus Trans-activator Protein E2 Activates Expression from the Promoter for the Ribonucleotide Reductase Large Subunit from Herpes Simplex Virus Type 2
More LessActivation of the herpes simplex virus type 2 (HSV-2) large subunit of the ribonucleotide reductase (ICP10) gene by papillomavirus DNA encoding the E2 or E7 proteins was studied directly by immunofluorescence or by chloramphenicol acetyltransferase (CAT) analysis with hybrid ICP10 or IE175 and 38K promoter constructions. Cotransfection with bovine papillomavirus type 1 or human papillomavirus type 16 (HPV-16) E2 DNA enhanced CAT expression from constructions in which CAT is regulated by the ICP10 but not by other HSV promoters. Expression was not enhanced by cotransfection with HPV-16 E7 DNA. Sequence analysis of the ICP10 promoter identified a consensus E2-binding motif. Activation was significantly reduced by site-directed mutagenesis of the consensus motif.
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Human Papillomavirus Type 16 DNA Expresses a Replication Modulation Factor in cos-1 Cells
More LessPlasmid DNA including the simian virus 40 (SV40) origin of replication undergoes uncontrolled, runaway replication in cos-1 cells owing to the intracellular production of SV40 large T antigen. Covalent linkage of such plasmids with human papillomavirus type 16 (HPV-16) DNA was found to prevent runaway replication. Replication control was found to be dependent on the presence of HPV-16 DNA sequences including the El open reading frame and part of the non-coding region.
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A New Transcription Element in the JC Virus Enhancer
More LessThe enhancer of JC virus restricts its gene expression to the brain. In a previous study, we demonstrated an enhancer element, nuclear factor I (NFI) motif, in the middle of the enhancer. Here, we demonstrate that the NFI motif is a tissue-specific element. We further present a new tissue-specific element, SacI motif, just upstream from the NFI motif. These motifs showed transcriptional enhancement both in vitro and in vivo and acted upon a heterologous adenovirus major late promoter. DNase I footprint analyses demonstrated that the SacI motif bound to a brain nuclear factor, and that its binding region overlapped with the NFI motif. Gel shift experiments with the SacI motif revealed that the populations of SacI motif factors in the brain and HeLa cell extracts were different. Together with our previous findings about tissue-specific NFI-like factor(s), cooperation of NFI motif and SacI motif factors may be required for the strong brain specificity of the viral gene expression.
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Production of Infectious Swine Vesicular Disease Virus from Cloned cDNA in Mammalian Cells
More LessFull-length cDNA clones of the swine vesicular disease virus (SVDV) were constructed from subgenomic cDNA clones in the expression vector pSVL (pSVLSOO). The direct transfection of mammalian cells with plasmid pSVLSOO results in the production of infectious virus. The recovered virus was neutralized completely by anti-SVDV guinea-pig serum, but did show a difference in plaque morphology from the parental virus.
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Biophysical Studies on the Morphology of Baculovirus-expressed Bluetongue Virus Tubules
More LessBluetongue virus tubules were purified from Spodop- tera frugiperda cells infected with a recombinant baculovirus containing the NS 1 gene from bluetongue virus serotype 10, and expressed under control of the Autographa californica nuclear polyhedrosis virus polyhedrin promoter. These tubules were subjected to a variety of chemical and physical treatments and the resulting effects on tubule morphology were examined by electron microscopy. A number of morphological similarities were noted between bluetongue virus tubules and cellular microtubules despite a lack of homology between the component proteins at the primary sequence level. A possible multistranded helical configuration is proposed for the tubule structure.
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Low pH-induced Cell Fusion in Flavivirus-infected Aedes Albopictus Cell Cultures
More LessCell-to-cell fusion of Aedes albopictus (mosquito) cells infected with dengue and St Louis encephalitis (SLE) flaviviruses was induced by exposure to low pH. The parameters of this low pH-induced fusion were examined. Syncytium formation was maximal in cultures 36 to 48 h post-infection and occurred when cultures were maintained at the acid pH for 15 min at 35 °C. The optimal pH range for fusion was 5·0 to 6·5 for dengue virus-infected cells and 5·0 to 5·5 for SLE virus- infected cells. Syncytia were not observed in vertebrate cells (Vero and BHK) under these conditions despite similar virus yields. Fusion was shown to be ATP- dependent and could be prevented by the addition of either polyclonal antiviral antibodies or monoclonal antibody to the envelope glycoprotein. The lysosomotropic amine ammonium chloride inhibited the replication of SLE virus in both mosquito and vertebrate cells, consistent with the idea that low pH-induced fusion is necessary for virus entry into both types.
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Molecular Cloning and Physical Mapping of the Genome of Bombyx Mori Nuclear Polyhedrosis Virus
More LessA restriction fragment library which covered the entire genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) was constructed using plasmid vectors. By analysis of cloned and viral DNA by double digestion with endonucleases and by hybridization techniques, a complete physical map of BmNPV was constructed for BamHI, Eco RI, Hin dIII, Kpn I, PstI and SmaI. Five regions of repeated sequences containing EcoRI sites were also found and mapped.
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Serotyping and Strain Identification of Maize Streak Virus Isolates
More LessFour strains of maize streak virus, namely the Panicum maximum, Digitaria setigera and sugarcane strains, have been identified from 19 isolates by ELISA using polyclonal antisera cross-absorbed with particles of the maize strain. The results suggest there is an epitope of the maize strain which is not dependent on the capsid being intact and which is common to all the members of the group; other strain-specific epitopes are probably conformation-dependent. A specific epitope (probably internal) occurs on the coat protein of a maize strain isolate, D(R)D, grown in D. velutina, that is also present on the coat protein of the D. setigera (previously reported as D. sanguinalis) strain. Specific internal epitopes also occur in the coat proteins of sugarcane and P. maximum strains. The use of indirect ELISA was necessary for accurate serotyping. The serological reactivities of particles of all the members of each type were identical irrespective of the host from which they were extracted. Sap extracts proved to be more reliable sources of antigen than virus preparations, which could vary in their serological reactivity. Nevertheless, serological differentiation index values determined in tests using either type of antigen proved reliable and consistent.
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Susceptibility of Brassica Species to Cauliflower Mosaic Virus Infection is Related to a Specific Stage in the Virus Multiplication Cycle
More LessThe relative susceptibilities and symptom responses of different Brassica species to infection by cauliflower mosaic virus (CaMV) have been compared and related to molecular events of the virus multiplication cycle. Variants of B. rapa (genome descriptor aa) were highly susceptible to infection by CaMV strain Cabb B-JI and contained relatively large amounts of virus; B. oleracea (cc) variants showed low susceptibility and contained small amounts of virus. B. nigra (bb) and allotetraploid species, B. juncea (aabb), B. napus (aacc) and B. carinata (bbcc), showed moderate responses to CaMV. CaMV unencapsidated DNA forms were isolated from different Brassica plants and examined by two-dimensional gel electrophoresis and blot hybridization. Viral RNA was estimated by dot blot analysis. These analyses showed differences in accumulation of key viral replication cycle intermediates within the broad range of host plants studied. The most susceptible species contained relatively small amounts of super-coiled (SC) DNA, a component of the CaMV minichromosome, but abundant viral transcripts and reverse transcription replication products. Tolerant plant hosts contained high levels of SC DNA but low levels of viral transcripts and reverse transcription DNA products. Allotetraploids contained SC DNA, RNA transcripts and replication product levels which were generally intermediate between those of their respective progenitor species. Evidence is presented that accumulation of CaMV SC DNA in the less susceptible host species is probably not due to autonomous DNA replication or tissue-specific expression. We conclude that a major component of the susceptibility of Brassica plants (and probably all CaMV host species) to CaMV infection is the level of viral minichromosome expression, influenced directly by the host genotype.
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Analysis of the in Vitro Cleavage Products of the Tomato Black Ring Virus RNA-1-encoded 250K Polyprotein
More LessTomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of M r 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (PI90, PI20, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. PI90 is an intermediate cleavage product which is further cleaved to form P60 and PI20. PI20, which contains the region that has been assigned to the virus protease and the virus polymerase, was not further cleaved in vitro.
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Sequence Analysis of Cymbidium Ringspot Virus Satellite and Defective Interfering RNAs
L. Rubino, J. Burgyan, F. Grieco and M. RussoThe nucleotide sequences of cloned cDNA copies of satellite and defective interfering (DI) RNAs of cymbidium ringspot virus were determined. DI RNA is 499 nucleotides long and is composed of six stretches of sequence derived from CyRSV genomic RNA. Four of these stretches share common 5′ sequences and the 5′ and 3′ ends are identical to those of genome RNA. Satellite RNA is 621 nucleotides long. In some regions the sequence is very similar (60% to 100% identical) to genomic RNA. A consensus sequence is proposed to be involved in the replication of genomic, DI and satellite RNAs.
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Synthesis of Infectious RNA from Full-length Cloned cDNA to RNA of Cymbidium Ringspot Tombusvirus
More LessA full-length DNA copy of cymbidium ringspot virus RNA was cloned downstream of a phage T7 promoter. In vitro transcripts had no extra nucleotides at the 3′ terminus, and a 5′ end likely to be precisely as in genomic RNA. Transcripts were infective when inoculated into test plants. Northern blots from inoculated plants revealed the presence of genomic and subgenomic RNAs, but not of satellite RNA. Virus particles isolated from infected plants had the same outward aspect and size as those of the wild-type virus and were decorated by an antiserum to CyRSV in immune electron microscopy tests.
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Nucleotide Sequence of Segment S9 of the Genome of Rice Gall Dwarf Virus
DNA complementary to the ninth largest (S9) of the 12 genome segments of rice gall dwarf virus (RGDV) was cloned and its sequence was determined. It is 1202 nucleotides in length and contains one open reading frame which extends for 969 nucleotides from nucleotide 26. It encodes a polypeptide of 323 amino acids with an M r of 35560. The dinucleotide sequence at the 5′ end and the trinucleotide sequence at the 3′ end of the plus strand, 5′ GG—GAU 3′, which are present in the RNA of both wound tumour virus (WTV) and rice dwarf virus (RDV), were also found in RGDV genome segment S9. The nucleotide sequences in the non-coding region at the 5′ terminus and in the 15 nucleotides at the 3′ terminus, which form an imperfect inverted repeat of 10 bp together with the 5′ terminus, are approximately 70% homologous with those of the WTV genome segment S9, but only 30% and 50% homologous with the respective termini of RDV S9.
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Expression of Resistance to Barley Stripe Mosaic Virus in Barley and Oat Protoplasts
More LessMesophyll protoplasts from both susceptible and resistant hosts were inoculated with RNA purified from barley stripe mosaic virus (BSMV) strains CV52 and CV42 using the polyethylene glycol (PEG) method. Protoplasts derived from the susceptible Hordeum vulgare L. cv. Black Hulless were susceptible to both BSMV strains, as indicated by fluorescein isothiocyanate staining and ELISA. More than 80% of protoplasts derived from an oat cultivar resistant to CV52, but not to CV42, were readily infected by either CV52 or CV42. Protoplasts from 10 barley lines resistant to CV42 remained resistant to CV42, although a limited number of protoplasts were infected. Functional resistance in the cultured barley protoplasts, but not in those from oat plants, suggests that resistance in these barley lines may be the result of restriction of replication, whereas resistance in oat plants is more likely due to restriction of cell-to-cell movement.
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Nucleotide Sequence of the Coat Protein Gene of Pea Seed-borne Mosaic Potyvirus
More LessThe nucleotide sequence of a 1355 bp cDNA representing the 3′-terminal sequences of pea seed-borne mosaic virus (PSbMV) was determined. This sequence contained a single long open reading frame (ORF) of 1189 bp ending with a single TAA termination codon. Downstream from the ORF was an untranslatable region of 189 bp followed by eight bp of polyadenylate. The probable location of the PSbMV coat protein codons within the long ORF was determined by comparing the inferred amino acid sequence with other potyviral coat protein sequences and by examining the sequence for a potyviral polyprotein cleavage cassette sequence. Direct chemical sequencing of the PSbMV coat protein revealed it to be blocked at its amino terminus. A partial amino acid sequence representing the N terminus of the protease-resistant core of the coat protein was determined, however. Alignment of the PSbMV coat protein sequence and the sequences of seven other potyviral coat proteins revealed significant homology, ranging from 53.7% for potato virus Y strain D to 43.2% for tobacco vein mottling virus.
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Isolation of a Subterranean Clover Mottle Virus-like Satellite RNA from Lucerne Infected with Lucerne Transient Streak Virus
More LessA circular, viroid-like satellite RNA (sat RNA) was detected in lucerne transient streak virus (LTSV), from lucerne in South Australia. It was larger than the previously reported sat RNA of LTSV, being similar in size and sequence homology to the 388 nucleotide sat RNA previously shown to be encapsidated by subterranean clover mottle virus (SCMoV) isolated from subterranean clover in Western Australia. This indicates that under field conditions, very similar sat RNAs can be associated with two distantly related sobemoviruses, LTSV and SCMoV. The natural hosts of these viruses are lucerne and subterranean clover, respectively.
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Nucleotide Sequence of the 3′-Terminal Region of Helenium Virus S RNA
More LessThe sequence of 1390 nucleotides of the 3′-terminal region of Helenium virus S RNA was determined from cloned cDNA. This portion of the viral RNA contains two major open reading frames (ORFs) encoding proteins of Mr 32·9K and 12·6K. The 32·9K protein, which is similar in size to the capsid protein of the virus, shares marked homology with the coat proteins of potexviruses and two other carlaviruses. The ORF, corresponding to Mr 12·6K, nearest to the 3′-terminal poly(A) tail shows extensive similarity with the corresponding proteins of potato virus S and potato virus M. We propose that this protein may be involved in nucleic acid binding within infected cells.
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