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A R Bellamy

Publications and source records attributed to A R Bellamy.

17 recordsLinked to original sources
PubMed1992

Cytoplasmic delivery of ribozymes leads to efficient reduction in alpha-lactalbumin mRNA levels in C127I mouse cells.

Ribozymes targeted to five sites along the alpha-lactalbumin (alpha-lac) mRNA were delivered to the cytoplasm of mouse C127I mammary cells using the T7-vaccinia virus delivery system and the amount of alpha-lac mRNA was monitored 24-48 h post-transfection. Three target sites were selected in the alpha-lac coding region (nucleotides 15, 145 and 361) and two were located in the 3' non-coding region (nucleotides 442 and 694). Acting in trans and at a target:ribozyme ratio of 1:1000, ribozymes targeting sites 361 and 694 reduced alpha-lac mRNA by > 80%; another two ribozymes (targeting nucleotides 442 and 145) reduced mRNA levels by 80 and 60% respectively; the fifth ribozyme (targeting nucleotide 15, near the AUG) was largely ineffective. The kinetic activity (kcat) of each ribozyme in vitro was somewhat predictive of the activity of the two ribozymes that targeted nucleotides 361 and 694, but was not predictive of the in vivo activity of the other three ribozymes. Down-regulation of the intracellular levels of alpha-lac paralleled the ribozyme-dependent reduction achieved for mRNA. For site 442, the reduction in both mRNA and protein was attributed to the catalytic activity of the ribozyme rather than to the antisense effects of the flanking arms, because delivery of an engineered (catalytically-inactive) variant had no effect on mRNA levels and a minimal effect on the level of alpha-lac present in the cell.

Animals↗
PubMed1992

Rotavirus VP6 modified for expression on the plasma membrane forms arrays and exhibits enhanced immunogenicity.

The major inner capsid protein of rotavirus is VP6, a 42-kDa polypeptide that forms the icosahedral surface of the rotavirus single-shelled particle. A chimeric form of VP6 (VP6sc) was constructed containing an upstream leader sequence derived from the influenza virus hemagglutinin and a downstream membrane-spanning (anchor) domain from a mouse immunoglobulin gene. When VP6sc was expressed in cells using a recombinant vaccinia virus, the protein was transported, glycosylated, and anchored in the plasma membrane as a trimer with the major domains of the protein orientated externally. Immunofluorescence and immunolabeling with colloidal gold indicated that VP6sc also localized in patches on the cell surface; electron microscopy revealed that the protein assembled into two-dimensional arrays which exhibited the same periodicity as the paracrystalline arrays formed by purified (viral) VP6. Mice inoculated with a recombinant vaccinia virus that expressed VP6sc produced rotavirus-specific antibodies at a titer 10 times higher than that achieved when wild-type, intracellular VP6 was delivered in the same way. Presentation at the cell surface therefore may represent a general method for enhancing the immunogenicity of rotavirus proteins.

Animals↗
PubMed1992

Vaccinia-rotavirus VP7 recombinants protect mice against rotavirus-induced diarrhoea.

Recombinant vaccinia viruses expressing wild type intracellular VP7 (VP7wt) from rotavirus SA11 or VP7sc, a cell surface-anchored variant, boosted antibody titres in SA11-immune mice. Pups born to these mice were protected from diarrhoea following challenge with SA11. In rotavirus-naive mice, two immunizations with recombinant vaccinia virus expressing VP7sc stimulated protective immunity that could be transferred to pups, whereas viruses expressing VP7wt did not stimulate protective immunity. Recombinant vaccinia viruses expressing intracellular or cell surface-anchored VP6, the rotavirus group-reactive antigen from the inner capsid, did not stimulate protective immunity. These experiments demonstrate that a live viral vector expressing cell surface anchored VP7 may represent a strategy for the development of safe, effective vaccines against rotavirus-induced diarrhoea.

Animals↗
PubMed1992

Relocation of antigens to the cell surface membrane can enhance immune stimulation and protection.

The major outer capsid glycoprotein of rotaviruses, VP7, is normally synthesized and directed to the ER, where it is required for virus assembly. By substituting a foreign signal sequence for the VP7 signal peptide, a secreted form of VP7 with an authentic amino terminus was produced. Secreted VP7 was further modified by the addition of a transmembrane anchor and cytoplasmic domain to its C-terminus. When the novel chimeric protein was expressed in transfected cells it became anchored in the cell surface membrane. The antigenicity of the chimeric protein was compared with that of the intracellular form of VP7 using recombinant vaccinia viruses to deliver the antigens in vivo. The novel antigen produced enhanced stimulation of both B and T lymphocytes of the immune system, and in mice it was able to induce protection against rotavirus-induced diarrhoeal disease. Other secreted and intracellular antigens show a similar improved level of antigenicity as a result of their relocation to the cell surface. Surface localization may therefore have general utility in the development of recombinant subunit vaccines.

Animals↗
PubMed1992

Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methionine residue is essential for ligand binding.

Maturation of rotavirus involves an intracellular membrane budding event in which the single-shelled icosahedral particle interacts with a virus-encoded receptor glycoprotein, NS28, that is located in the rough endoplasmic reticulum membrane. The receptor is a tetramer and is oriented with the C-terminal 131 amino acids on the cytoplasmic side of the membrane (A.R. Bellamy and G.W. Both, Adv. Virus Res. 38:1-48, 1990). We have used the T7-vaccinia virus transient expression system to deliver mutant variants of the NS28 gene to CV1 cells in order to assess the effects of site-specific modifications on receptor function. Three types of mutant proteins have been constructed by altering the extreme C-terminal methionine, cysteine residues within the third hydrophobic domain, and internal residues located within the cytoplasmic portion of the receptor, respectively. Deletion or conservative substitution of the C-terminal methionine completely abolishes receptor activity. Substitution of cysteine residues has no effect on receptor activity or on the ability of the receptor to adopt its native oligomeric state. Internal deletions result only in a reduction in the level of binding. An N-terminally truncated form of the receptor, containing only the cytoplasmic domain, retains full receptor activity and can form membrane-associated tetramers.

Animals↗
PubMed1991

Immunization with baculovirus-expressed recombinant rotavirus proteins VP1, VP4, VP6, and VP7 induces CD8+ T lymphocytes that mediate clearance of chronic rotavirus infection in SCID mice.

Clearance of chronic murine rotavirus infection in SCID mice can be demonstrated by adoptive transfer of immune CD8+ T lymphocytes from histocompatible donor mice immunized with a murine homotypic rotavirus (T. Dharakul, L. Rott, and H.B. Greenberg, J. Virol 64:4375-4382, 1990). The present study focuses on the protein specificity and heterotypic nature of cell-mediated clearance of chronic murine rotavirus infection in SCID mice. Heterotypic cell-mediated clearance was demonstrated in SCID mice infected with EDIM (murine) rotavirus after adoptive transfer of CD8+ T lymphocytes from BALB/c mice that were immunized with a variety of heterologous (nonmurine) rotaviruses including Wa (human, serotype 1), SA11 and RRV (simian, serotype 3), and NCDV and RF (bovine, serotype 6). This finding indicates the serotypic independence of T-cell-mediated rotavirus clearance. To further identify the rotavirus proteins that are capable of generating CD8+ T cells that mediate virus clearance, donor mice were immunized with SF-9 cells infected with a baculovirus recombinant expressing one of the following rotavirus proteins: VP1, VP2, NS53 (from RF), VP4, VP7, NS35 (from RRV), VP6, and NS28 (from SA11). SCID mice stopped shedding rotavirus after receiving CD8+ T cells from mice immunized with VP1, VP4, VP6, and VP7 but not with VP2, NS53, NS35, NS28, or wild-type baculovirus. These results suggest that heterotypic cell-mediated clearance of rotavirus in SCID mice is mediated by three of the major rotavirus structural proteins and by a putative polymerase protein.

Animals↗
PubMed1991

Rotavirus spike structure and polypeptide composition.

Negatively stained preparations of rotavirus imaged with a low dose of electrons provide sufficient contrast to reveal surface projections or spikes. The number of spikes found projecting from different particles indicates that not all 60 peripentonal sites are occupied. Treatment at pH 11.2 with 250 mM ammonium hydroxide specifically removes the spikes, yielding smooth double-shelled particles of the same diameter as that of the native virus. Protein analysis confirms that the released spikes are composed of polypeptide VP4 (or its two cleavage products VP5* and VP8*) and that the smooth particle retains the other major outer shell protein VP7. Spikeless particles can be decorated by a monoclonal antibody specific for the major immunodominant neutralizing domain of VP7, implying that removal of the spikes does not denature the VP7 that is retained on the surface of the smooth particle.

Ammonium Hydroxide↗
PubMed1990

The immunogenicity of VP7, a rotavirus antigen resident in the endoplasmic reticulum, is enhanced by cell surface expression.

The glycoprotein VP7, the major serotype antigen of rotaviruses, is localized to the endoplasmic reticulum (ER) of the cell, where it is retained as a membrane-associated protein before assembly into mature virus particles. Wild-type VP7 expressed by a recombinant vaccinia virus was also located internally and was poorly antigenic. Using recombinant techniques, a correctly processed, secreted form of VP7 (S.C. Stirzaker and G.W. Both, Cell 56:741-747, 1989) was modified by addition to its C terminus of the membrane anchor and cytoplasmic domains from the influenza virus hemagglutinin. The hybrid protein was directed to the surface of cells, where it was anchored in the plasma membrane. When expressed in mice and rabbits by a recombinant vaccinia virus, the surface-anchored antigen stimulated a level of rotavirus-specific antibodies that was greater than 100-fold above the level induced by wild-type VP7. T-cell responses to the novel antigen were also elevated in comparison with the wild-type, intracellular protein. Cell surface anchoring may provide a strategy to increase the immunogenicity of intracellular antigens from other parasites and viruses.

Amino Acid Sequence↗
PubMed1979

Two strains of human rotavirus in Auckland.

Polyacrylamide gel electrophoresis of human rotavirus RNA has identified at least two distinct strains of virus currently circulating in Auckland. The sensitivity of the method both for detecting rotavirus infection and for identifying variation in rotavirus strains is discussed.

Diarrhea↗
PubMed1978

Virus accumulation by the rock oyster Crassostrea glomerata.

The accumulation of virus by the New Zealand rock oyster Crassostrea glomerata has been studied in a static seawater system using radioactively labeled reovirus type III and Semliki Forest virus. The uptake of virus was found to be less rapid than for the bacterium Escherichia coli and to be unaffected by the presence of the marine alga Dunaliella primolecta in the seawater. Accumulation was dependent on virus concentration, with saturation achieved at 4 X 10(10) reovirus particles per oyster, implying that an oyster possesses a large but finite number of sites for virus adsorption. When the rates of uptake of two viruses of similar size but differing surface properties were compared, the rate of accumulation of the lipoprotein-enveloped Semliki Forest virus was found to be less than that for the protein-enclosed reovirus. This observation, together with the finding that the oyster shell has a strong affinity for virus, suggests that surface properties, rather than size, are the principal factors governing the accumulation of viruses by filter-feeding marine bivalves.

Adsorption↗
PubMed1976

Modulating effects of interferon preparations on an antibody response in vitro.

The effect of mouse interferon preparations on the primary in vitro antibody response of mouse spleen cells was studied. Concentrations of interferon greater than 8 units per ml significantly inhibited the antibody response while low concentrations of 0-08-0-8 units per ml could be shown to be mildly enhancing. Various treatments which affected the antiviral activity of the interferon preparations reduced the immunosuppressive activity to a similar extent. Interferon acts during the first few hours of a response but the effect is not apparent for at least 50 h. Interferon had no effect when added after 48 h. The kinetic data has been interpreted as demonstrating interferon-sensitive and interferon-resistant components of the in vitro response. The results from investigations of the polyclonal response to lipopolysaccharide support the view that interferon acts mainly on B cells alone, although effects on T-B interactions cannot be excluded.

Animals↗
PubMedDate not supplied

Procedures for characterisation of the genetic material of candidate vaccine strains.

Two procedures for characterising the genomes of recombinant influenza viruses are described. The first of these involves ribonuclease T4 oligonucleotide fingerpart analysis of separated viral RNAs labelled either in vivo or in vitro and the second utilises polyacrylamide gel electrophoresis to identify the double-stranded molecules formed by hybridisation between the complementary and virion RNAs of two viruses. Although the latter method is more suitable for routine screening purposes, both procedures are suitable for distinguishing between equivalent RNA components of closely related viruses.

Base Sequence↗