- Volume 70, Issue 8, 1989
Volume 70, Issue 8, 1989
- Animal
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β 2 Microglobulin Binds to the Tegument of Cytomegalovirus: an Immunogold Study
More LessSUMMARYPrevious reports have provided evidence for the ability of human cytomegalovirus (HCMV) to bind the host protein β 2 microglobulin (β 2m) from body fluids or culture medium, and thus enhance infectivity of the virus, both by evasion of immune neutralization and the capacity to employ the bound β 2m for attachment to the host cell. Immunocytochemical techniques and negative stain electron microscopy were used to identify the ultrastructural components of HCMV involved in its interaction with β 2m. Probes comprising colloidal gold coupled to β 2m were seen to bind not to the envelope as previously suspected, but to material closely surrounding the nucleocapsids. It is postulated that the tegument proteins of HCMV, via their capacity to bind β 2m, play an important role in the preservation of infectivity of disrupted virions by enabling unenveloped capsids to bind to cells and gain entry by a pathway other than that normally taken by intact virions.
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Immunosuppression of the Antibody Response to Respiratory Syncytial Virus (RSV) by Pre-existing Serum Antibodies: Partial Prevention by Topical Infection of the Respiratory Tract with Vaccinia Virus-RSV Recombinants
SUMMARYImmunization strategies to prevent respiratory syncytial virus (RSV) disease will involve immunization of infants less than 2 months of age who possess maternally derived RSV antibodies. Vaccinia-RSV recombinant viruses are useful tools for defining parameters important in immunization against RSV and also are being considered as live virus vaccines for use in humans. Previous studies demonstrated that passively acquired RSV antibodies can suppress the immune response and the protective efficacy of vaccinia-RSV recombinants administered by the intradermal route. The present study demonstrates that the suppressive effects of passively acquired antibody on immunity induced by intradermally administered vaccinia-RSV recombinants in cotton rats can be partially overcome by administration of the recombinants by the intranasal route.
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Matrix Protein of Cell-associated Subacute Sclerosing Panencephalitis Viruses
More LessSUMMARYThe nucleotide sequence has been determined for the matrix (M) protein gene of three strains, Niigata-1, ZH and Biken, of cell-associated subacute sclerosing panencephalitis (SSPE) virus. The M proteins of the Niigata-1 and ZH strains were found to terminate prematurely as a result of nonsense mutations at nucleotide positions 68 and 96 respectively. On the other hand it was predicted that the Biken strain would express M protein with 22 amino acid differences and eight additional amino acids at its C terminus in comparison to the M protein of the Edmonston strain of measles virus. Radiolabelling of cells carrying the Biken strain showed the production of an M protein with considerably altered immunoreactivity and a marked reduction in intracellular stability. Either premature termination or rapid degradation of the M protein may underlie the defectiveness of these three strains of SSPE virus.
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Monoclonal Antibodies to Feline Calicivirus
More LessSUMMARYWe have prepared monoclonal antibodies to the capsid protein of feline calicivirus (FCV). These antibodies are directed against two close but distinct epitopes, only one of which is involved in virus neutralization. We have used these antibodies and immune cat serum in immunoprecipitation and Western blotting experiments and have identified novel proteins in FCV-infected cells which we term P78, P41, P35 and P29. The number and sizes of FCV proteins now known resemble those made by other caliciviruses.
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The Design and Use of Specific Genetic Probes to Identify Closely Related Bunyaviruses and to Determine the Genotype of Their Recombinants
More LessSUMMARYViruses that are very closely related to each other at the genetic and gene product level can prove difficult to distinguish, although they may differ in phenotype (for example in their viruslence or vector preferences). A chimeric genetic probe has been developed and tested to distinguish the S RNAs of two closely related bunyaviruses, snowshoe hare and La Crosse viruses. The technique is applicable to other RNA species of these two bunyaviruses.
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Carboxy-terminal Analysis of Nine Proteins Specified by the Flavivirus Kunjin: Evidence that Only the Intracellular Core Protein Is Truncated
More LessSUMMARYNine proteins specified by Kunjin virus were labelled with [3H]lysine and digested with carboxypeptidase B which specifically cleaves carboxy-terminal Lys or Arg. The theoretical amount of [3H]lysine was released from the non-structural (ns) proteins NS2A, NS2B, NS3 and NS4B, which have a common carboxy terminus Lys-Arg deduced from cleavage sites established in the viral polyprotein by previous N-terminal amino acid analyses. This is a flavivirus consensus site, always followed by Gly, Ala or Ser. These results indicate that no truncations had occurred despite anomalous electrophoretic migrations of NS2A, NS2B and NS4B observed in some gel systems. No [3H]lysine was released from NS4A or NS5 which terminate in Ala and Leu, respectively, thus establishing that NS5 (observed M r 98 000, theoretical M r 103 600) was not cleaved post-translationally at any internal Lys-Arg site. Unexpectedly, [3H]lysine residues were apparently released from P14(C), the intracellular equivalent of virion C protein which terminates in Ala (adjacent to the established N-terminus of prM). However, a putative internal cleavage site (Lys-Arg↓Gly) exists 18 residues upstream from the carboxy terminus of C, prior to the transmembrane spanning domain. Apparent internal cleavage in the cytosol at this site to produce P 14(C) would expose [3H]lysine residues to carboxypeptidase B, and account for the previously observed differences in size and composition between C and P14(C).
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The Morphology of Simian Immunodeficiency Virus as Shown by Negative Staining Electron Microscopy
More LessSUMMARYNegative staining electron microscopy was used to study sucrose gradient-purified preparations of the simian immunodeficiency virus (SIVmac251). Both isolated and aggregated virus particles were observed together with some free-lying virus cores. The cores were 110 nm long and 25 to 50 nm wide and were mainly conical or wedge-like in shape. Surface projections were seen on the envelope membrane of many of the virus particles; the knobs were approximately 6 nm in length, 10 nm wide and from an end-on view they had a Y or triangular-shaped morphology.
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Modulation of Lentivirus Replication by Antibodies. Non-neutralizing Antibodies to Caprine Arthritis–Encephalitis Virus Enhance Early Stages of Infection in Macrophages, but Do Not Cause Increased Production of Virions
P. E. Jolly, D. Huso, G. Hart and O. NarayanSUMMARYNon-neutralizing antibodies to caprine arthritis–encephalitis virus (CAEV) enhance the early stages of the virus life cycle but do not potentiate enhanced production of virus particles by macrophages. In primary macrophages used for these studies, there was enhancement in binding, internalization and uncoating of virus pretreated with nonneutralizing sera in comparison to virus pretreated with a non-immune serum. However, this did not lead to enhanced production of virus particles. Failure of non-neutralizing sera to inactivate CAEV may be due in part to low avidity of the antibodies for the virus particles which contain sialic acids on their envelopes, because desialylation of the particles made them neutralizable. The non-neutralizing antibodies probably bound to most of the native virus particles which were then internalized via Fc receptor-mediated endocytosis and degraded. Sialylated particles that failed to bind antibodies probably caused the infection. Thus there was no true enhancement of infection. The previously reported increase in severity of lesions in animals immunized with inactivated CAEV particles prior to challenge with live virus suggested enhancement of infection but in the light of our finding this may have been caused by factors other than an increase in production in the number of infectious virus particles.
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Oestrogen Stimulates Differential Transcription of Human Papillomavirus Type 16 in SiHa Cervical Carcinoma Cells
More LessSUMMARYHuman papillomavirus (HPV) type 16 is highly associated with cervical cancer, but it seems that cofactors such as hormones affect its potential oncogenicity. We have analysed the HPV-16 gene expression in response to sex hormones and glucocorticoids in SiHa cells, a human cervical carcinoma cell line. An eightfold induction of HPV-16 transcripts was obtained in oestrogen-treated SiHa cells. Of the five HPV-16 transcripts detected in these cells only the two major ones, the 4·6 kb and the 4·1 kb mRNA species, were affected by oestrogen. Since the five transcripts span the E6 and E7 open reading frames of the HPV-16 genome, these results suggest that the expression of the various transcripts is differentially controlled, as oestrogen regulates only two of them. We have identified in the HPV-16 genome seven different regions with a high degree of similarity to the oestrogen-responsive element consensus sequence (GGTCANNNTGACC). These sequences are located throughout the entire HPV-16 genome. Progesterone or dexamethasone had no detectable stimulatory effect on the various transcripts of HPV-16 in SiHa cells, up to 24 h after treatment of the cells. Since the E6 and E7 open reading frames have been associated with the oncogenic potential of HPV-16, the effect of oestrogen on the transcription of these viral genes may be of biological relevance in the malignant transformation of HPV-16-infected cervical cells.
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Comparison of RNAs and Polypeptides of Infectious Pancreatic Necrosis Virus Isolates from Eel and Rainbow Trout
More LessSUMMARYThirteen isolates of infectious pancreatic necrosis virus (IPNV) from eel and rainbow trout in Taiwan were compared with the selected serotypes AB, SP and VR-299 by PAGE analysis of their RNA genomes and early polypeptides. All the IPNV isolates from eels (1984 to 1986) from Lu Kang and Ping Tung, and from rainbow trout eggs and fry from Dan Sway (1985 to 1986) were most closely related to the AB serotype. However four rainbow trout isolates from Dan Sway (1984) had a similar RNA pattern to that of VR-299, whereas their early polypeptides were different, showing evidence of some mutations. Both RNA and polypeptide PAGE patterns were used to distinguish the isolates from known selected IPNV strains showing that this approach can be used for epizootiological studies.
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- Plant
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‘Pathogenesis-related’ P1(p14) Protein. Vacuolar and Apoplastic Localization in Leaf Tissue from Tomato Plants Infected with Citrus Exocortis Viroid; in vitro Synthesis and Processing
More LessSUMMARYTomato ‘pathogenesis-related’ P1(p14) protein was synthesized in vitro. mRNAs were isolated from leaves showing characteristic symptoms of viroid infection, followed by chromatography on oligo(dT)-cellulose and the poly(A)+ fraction was translated with a rabbit reticulocyte lysate system. No significant differences were found between the levels of [35S]methionine incorporation directed by mRNA preparations from healthy and viroid-infected leaves. Only mRNA from infected leaves incorporated label into a protein that could be immunoprecipitated with rabbit IgG specific for tomato P1(p14) protein. Analysis by SDS-PAGE of the immunoprecipitated protein from the in vitro translation system revealed that P1 (p14) was translated as a precursor protein of 2K to 3K larger than the P1(p14) that accumulated in vivo. This protein was converted to the mature form when translation was carried out in the presence of canine pancreatic microsomal membranes. By using Protein A-gold immunocytochemistry we have detected P1(p14) concentrated in dense inclusion bodies within the vacuole as well as in association with electron-dense material present in the intracellular spaces. The presence of the additional polypeptide sequence in the newly synthesized protein indicates that P1(p14) undergoes post-translational processing. The additional sequence is probably the signal peptide that directs its transport through the endoplasmic reticulum into the vacuolar compartment of tomato leaf cells and/or the intercellular space.
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Partial Cleavage of Sweet Potato Feathery Mottle Virus Coat Protein Subunit by an Enzyme in Extracts of Infected Symptomless Leaves
More LessSUMMARYThe coat protein of particles of sweet potato feathery mottle potyvirus (SPFMV) extracted from Ipomoea spp. migrated in SDS–PAGE mainly as bands of M r 38 000 (38K), 36K, 32K and 30K. Trypsin treatment of the particles resulted in the appearance of only one 30K polypeptide. The inclusion of protease inhibitors in the extraction procedure did not alter the heterogeneity of SPFMV coat protein. A partially purified fraction of extracts from recovering, symptomless, but not from healthy leaves of I. nil had a proteolytic activity similar to that of trypsin. Amino acid sequencing showed that the trypsin-cleaved 30K polypeptide had some sequence homology with other potyvirus coat proteins. The site at which the Ipomea extract cleaved the protein was five amino acids nearer the N terminus than the trypsin cleavage site.
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Molecular Cloning of a Potato Virus Y Genome: Nucleotide Sequence Homology in Non-coding Regions of Potyviruses
More LessSUMMARYAn aphid-transmissible field isolate of potato virus Y, strain MM, was purified from a pepper host and cloned. All but 18 terminal nucleotides of the 9·7 kb genome are apparently contained in two overlapping cDNA clones. The library of cDNA clones is likely to be representative of the viral RNA population present in infected plants because restriction endonuclease maps derived from cloned and uncloned cDNA are collinear, each region of the genome is represented by several independent clones, and nucleic acid sequencing of 5′ and 3′-terminal regions revealed small AU-rich non-coding domains with blocks of nucleotide sequence homology between this strain of PVY and three other potyviruses. The efficient application of cDNA cloning techniques to a large positive-stranded RNA virus is described.
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Molecular Cloning, Sequencing and Expression in Escherichia coli of the Bean Yellow Mosaic Virus Coat Protein Gene
More LessSUMMARYThe sequence of 1015 nucleotides from the 3′ poly(A) tract of the potyvirus bean yellow mosaic virus (BYMV) RNA has been determined from two cDNA clones. This sequence contained a single long open reading frame (ORF) starting upstream of the cloned region. The ORF was expressed as a fusion protein in Escherichia coli, and the product was detected by antibodies specific for the coat protein of BYMV. The predicted length of the coat protein gene was 822 nucleotides, corresponding to a 273 amino acid coat protein of M r 30910. The deduced amino acid sequence of the BYMV coat protein was compared to the chemically determined amino acid composition of purified virion protein, and of protein prepared from trypsin-treated virions. The nucleotide and deduced amino acid sequences were compared to the sequences of the coat protein genes of other potyviruses. The BYMV coat protein gene was found to be 50 to 61% homologous to those of other potyviruses at both the nucleotide and amino acid levels; the greatest variation was between the 5′-proximal one-fifth of the genes. Amino acid sequences and hydrophilicity plots of the different potyvirus coat proteins showed similarities which indicated that the structure of the coat protein is highly conserved; a non-terminal region of variability was predicted to be exposed on the exterior of the virion. A putative cleavage site at a glutamine-serine dipeptide was identified by similarity in context to the cleavage sites of tobacco etch virus and tobacco vein mottling virus coat proteins from the viral polyproteins. The BYMV 3′-terminal non-coding region of 166 nucleotides is followed by a poly(A) tract.
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Infection of Triticum monococcum Protoplasts with Barley Yellow Dwarf Virus
More LessSUMMARYProtoplasts from a Triticum monococcum cell culture line were successfully infected with barley yellow dwarf virus. Both purified virions and extracted RNA were shown to be infectious using a polyethylene glycol inoculation procedure. Up to 20% of the protoplasts contained viral antigens as judged by immunofluorescence assay. ELISA analysis showed that virus antigen expression was both dose- and time-dependent. Synthesis of new viral RNAs was confirmed by Northern blot hybridization. Studies of the expression of this economically important aphid-borne, phloem-limited virus should be greatly facilitated using this graminaceous protoplast system. It is the first reproducible protoplast infection system for this virus which can not be transmitted mechanically at the whole plant level.
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Barley Yellow Dwarf Virus RNA Has a 5′-Terminal Genome-linked Protein
More LessSUMMARYThe 5′ terminus of barley yellow dwarf virus RNA did not become labelled with 32P after dephosphorylation was attempted with calf intestinal alkaline phosphatase and subsequent treatment with [γ-32P]ATP and T4 polynucleotide kinase. Treatment of BYDV RNA with 125I-Bolton-Hunter reagent yielded 125I-labelled BYDV RNA, as shown by acid precipitation analysis. Treatment with RNase yielded a protein of approximate M r 17000 that was destroyed by treatment with Pronase and was serologically distinct from BYDV coat protein. BYDV therefore appears to have a genome-linked protein.
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