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Biomedical subjects

A R Bellamy

Publications and source records attributed to A R Bellamy.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1989

Interaction of rotavirus cores with the nonstructural glycoprotein NS28.

The nonstructural rotavirus receptor glycoprotein NS28 is 175 amino acids long and oriented in the RER membrane with the NH2 terminus on the luminal side and approximately 131 amino acids accessible from the cytoplasmic side. Au et al. (1988) have demonstrated that NS28 is able to interact with rotavirus single-shelled particles (cores) in a receptor:ligand interaction in which NS28 appears to act as the receptor and the rotavirus core as the ligand. This interaction appears to model the events that occur in the infected cell in which virus maturation involves budding of the core into the lumen of the RER. We have investigated the nature of the interaction between cores and NS28 in vitro using membranes derived from SA11 rotavirus-infected MA104 cells and membranes from cells where NS28 and other rotavirus proteins have been expressed using a series of recombinant vaccinia viruses that incorporate appropriate cloned rotavirus genes. The interaction between the core and the receptor is enhanced by the presence of Ca2+ and Mg2+ and Scatchard analysis yields a dissociation constant (Kd) of 5 x 10(-11) M. The major core protein VP6 is the ligand involved because (i) a monoclonal antibody specific for VP6 blocks the reaction, (ii) membranes prepared from cells infected with a double recombinant vaccinia virus which expresses both NS28 and VP6 exhibit a reduced capacity to bind cores, and (iii) VP6 prepared from virus blocks the ability of membranes to bind cores. When VP6, VP7, VP4, and NS28 are expressed singly as the sole viral proteins present in the cell, only membranes from cells expressing NS28 mediate receptor function, indicating that the presence of NS28 is sufficient to mediate the interaction between cores and the membrane and that other viral proteins probably are not involved in the initial receptor:ligand interaction.

Capsid↗
PubMed1989

Topology of the non-structural rotavirus receptor glycoprotein NS28 in the rough endoplasmic reticulum.

The rotavirus non-structural glycoprotein (NS28), the receptor for the virus core during budding into the lumen of the rough endoplasmic reticulum (RER), is 175 amino acids long and possesses an uncleaved signal sequence and two amino-terminal glycosylation sites. Utilizing one of three potential hydrophobic domains, the protein spans the membrane only once, with the glycosylated amino-terminal region oriented to the luminal side of the ER and the carboxy-terminal region to the cytoplasmic side. To localize sequences involved in translocation of NS28, we constructed a series of mutations in the coding regions for the hydrophobic domains of the protein. Mutant protein products were studied by in vitro translation and by transfection in vivo. In transfected cells, all mutant forms localize to the ER, and none are secreted. In vitro, each of the three hydrophobic domains is able to associate with microsomes. However, glycosylation and proteolysis of wild-type and mutant forms of NS28 indicates that the wild-type protein is anchored in the membrane only by the second hydrophobic domain, leaving approximately 131 residues exposed on the cytoplasmic side for receptor - ligand interaction.

Amino Acid Sequence↗
PubMed1988

Infantile gastroenteritis in Western Samoa.

Gastroenteritis has been identified traditionally as a major health problem in Western Samoa. In order to establish the extent of the problem, and to identify the viral and bacterial pathogens involved, stool specimens were collected from all children less than 2 years of age admitted to Apia Hospital, Western Samoa, over a 2 year period. Rotavirus was recovered from 5.5% of all admissions (20.5% of those labelled gastroenteritis) and bacterial pathogens were isolated from 2.1% of all admissions (0.9% of all those labelled gastroenteritis). Thus in Apia, as elsewhere, rotavirus is the most commonly identified enteric pathogen associated with infantile diarrhoea.

Bacteria↗
PubMed1988

Two forms of VP7 are involved in assembly of SA11 rotavirus in endoplasmic reticulum.

Two pools of the glycoprotein VP7 were detected in the endoplasmic reticulum (ER) of SA11 rotavirus-infected cells. One portion of the newly synthesized protein with VP3 composed the virus outer capsid, while the rest remained associated with the membrane. The two populations could be separated biochemically by fluorocarbon extraction or by immunological methods which used two classes of antibodies. A monoclonal antibody with neutralizing activity recognized VP7 only as displayed on intact virus particles, while a polyclonal antiserum precipitated predominantly the unassembled ER form of the protein and precipitated virus-assembled VP7 poorly. Virus-associated VP7 was localized by immunofluorescence to small punctate structures, presumably corresponding to accumulated virus particles, and to regions of the ER surrounding viroplasmic inclusions, whereas the membrane-associated molecules were distributed in an arborizing reticular pattern throughout the ER. VP3 and the nonstructural glycoprotein NCVP5 displayed a localization similar to that of virus-associated VP7. Intracellular virus particles were isolated from infected cells to determine the kinetics of assembly of VP7 and of the other structural proteins into virions. It was found that incorporation of the inner capsid proteins into single-shelled particles occurred rapidly, while VP7 and VP3 appeared in mature double-shelled particles with a lag time of 10 to 15 min. In addition, the alpha-mannosidase processing kinetics of virus-associated VP7 oligosaccharides showed a 15-min lag compared with that of the membrane-associated form, suggesting that the latter is the precursor to virion VP7. This lag may represent the time required for virus budding and outer capsid assembly.

Animals↗
PubMed1987

cDNA probes of individual genes of human rotavirus distinguish viral subgroups and serotypes.

The use of cDNA probes for detection of rotaviruses has been investigated using plasmids containing inserts specific for each of the eleven genes of human rotavirus strain Wa. In a dot-blot detection system in which radioactive DNA probes were hybridized to viral RNA extracted from cultivatable rotavirus strains, cDNAs of genes 7, 8, 10 and 11, were found to be the most reliable probes for detecting a range of rotavirus strains. Unexpectedly, rotaviruses could be distinguished with respect to subgroup and subtype specificities when cDNAs of genes 6 and 9, which encode the immunologically relevant proteins VP6 (group-specific antigen) and VP7 (type-specific antigen), were used as probe, even though the nucleic acid sequences of these genes are known to have a high degree of sequence homology.

Child, Preschool↗
PubMed1987

Processing of rotavirus glycoprotein VP7: implications for the retention of the protein in the endoplasmic reticulum.

Rotaviruses are icosahedral particles that assemble in the lumen of the endoplasmic reticulum (ER). The viral glycoprotein, VP7, is also directed into this compartment and is retained for assembly onto the surface of viral cores. VP7 is therefore a resident ER glycoprotein with a luminal orientation. The VP7 gene possesses two potential in-frame initiation codons, each preceding a hydrophobic domain. Mature VP7 is derived from a precursor by cleavage but the site of cleavage has not been determined because viral VP7 has a blocked amino terminus. Using site-directed mutagenesis of the gene and in vitro transcription and translation systems, we have investigated the synthesis and processing of the primary products synthesized from each initiation codon. Proteins translated from either codon were processed in vitro to yield products indistinguishable in size. The primary translation products therefore appeared to be cleaved at the same site. The site was located empirically between Ala50 and Gln51 and mutation of the gene to convert Ala50----Val prevented processing. Amino-terminal sequence analyses of proteins synthesized in vitro, and characterization of an amino-terminal fragment of VP7 purified from virus unequivocally established Gln51 as the amino-terminal residue. Pyroglutamic acid was tentatively identified as the blocking group. Processing of VP7 therefore removes both amino-terminal hydrophobic domains from the protein. Some other mechanism not requiring the presence of these hydrophobic sequences must account for the retention of this novel glycoprotein in the ER.

Cell Compartmentation↗
PubMed1987

Vaccinia virus recombinants expressing the SA11 rotavirus VP7 glycoprotein gene induce serotype-specific neutralizing antibodies.

A cDNA copy of the gene coding for the major outer neutralizing protein (VP7) of simian 11 rotavirus was incorporated into the vaccinia virus genome under the control of the vaccinia promoter (molecular weight, 7,500). A deletion mutant of this gene which codes for a secreted form of VP7 when expressed under the control of the simian virus 40 late promoter (M. S. Poruschynsky, C. Tyndall, G. W. Both, F. Sato, A. R. Bellamy, and P. H. Atkinson, J. Cell Biol. 101:2199-2209, 1985) was also inserted. Each recombinant vaccinia virus directed the synthesis of a rotavirus protein in infected cells, and the product encoded by the mutated gene was secreted. Rabbits immunized with the two types of recombinant vaccinia virus generated antibodies that were able both to recognize simian 11 rotavirus in an enzyme-linked immunosorbent assay and to neutralize the virus in a plaque-reduction test. Antibodies induced by the recombinant vaccinia viruses expressing either form of VP7 were serotype specific.

Antibodies, Viral↗
PubMed1987

Location of sequences within rotavirus SA11 glycoprotein VP7 which direct it to the endoplasmic reticulum.

The Simian 11 rotavirus glycoprotein VP7 is directed to the endoplasmic reticulum (ER) of the cell and retained as an integral membrane protein. The gene coding for VP7 predicts two potential initiation codons, each of which precedes a hydrophobic region of amino acids (H1 and H2) with the characteristics of a signal peptide. Using the techniques of gene mutagenesis and expression, we have determined that either hydrophobic domain alone can direct VP7 to the ER. A protein lacking both hydrophobic regions was not transported to the ER. Some polypeptides were directed across the ER membrane and then into the secretory pathway of the cell. For a variant retaining only the H1 domain, secretion was cleavage dependent, since an amino acid change which prevented cleavage also stopped secretion. However, secretion of two other deletion mutants lacking H1 and expressing truncated H2 domains was unaffected by this mutation, suggesting that these proteins were secreted without cleavage of their NH2-terminal hydrophobic regions or secreted after cleavage at a site(s) not predicted by current knowledge.

Amino Acid Sequence↗
PubMed1985

Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein.

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.

Amino Acid Sequence↗