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G W Both

Publications and source records attributed to G W Both.

At least 37 records · Page 2Linked to original sources
PubMed1995

Sequence of ovine adenovirus homologs for 100K hexon assembly, 33K, pVIII, and fiber genes: early region E3 is not in the expected location.

Ovine adenovirus OAV287 was previously isolated from sheep in Western Australia. As a first step in characterizing the genome of this virus we have determined the sequence of its genome between map units 65 and 81. This region was expected to contain the nonessential E3 region which, in other adenoviruses, lies between the genes encoding the pVIII and fiber proteins, although its size and complexity varies. OAV287 genes coding for the hexon assembly, 33K, pVIII, and fiber proteins were identified by their homologies with human Ad2. These genes lie in the same relative positions in the OAV287 genome, but the intergenic region between the pVIII and the fiber genes is only 197 nucleotides and these appear to be incapable of coding for any protein. Thus, the ovine adenovirus E3 region is not present in the expected location. In addition, using cDNA synthesis, PCR amplification, and nucleotide sequencing we determined the location of splice junctions and transcription termination signals in mRNA species encoding these proteins. This showed that a family of variably spliced L4 RNAs is produced and that the region between the pVIII and the fiber genes contains several signals for RNA synthesis and processing. As the E3 region in human adenoviruses is nonessential for replication, in many instances it has been replaced with foreign DNA during the construction of recombinants. Because of this unexpected difference in the organization of the OAV287 genome further experimentation will be required to determine whether potential vaccine recombinants can be constructed for this adenovirus by making insertions into the pVIII/fiber intergenic region.

Amino Acid Sequence↗
PubMed1995

Investigation of promoter function in human and animal cells infected with human recombinant adenovirus expressing rotavirus antigen VP7sc.

Human adenovirus (Ad) vectors are being used increasingly for a variety of applications in vaccination and gene therapy. The ability of vectors to enter cells and the efficiency of promoters expressing the therapeutic gene or vaccine antigen are critical to the outcome of such experiments. To identify promoters which might be suitable for use under a variety of conditions we have investigated the expression of a rotavirus antigen, VP7sc, employing several commonly used promoters carried in E1-substituted Ad vectors both in cell types which support virus replication and in cells which do not. Although not all gene constructions were identical, wide variations in promoter function were evident even in human 293 cells which support virus replication. The simian virus type 40 (SV40) early and beta-actin promoters expressed poorly; the SV40 late promoter was somewhat better. The human IE94 cytomegalovirus (CMV) promoter and a modified Ad major late promoter were best, functioning equally well but with different kinetics. In other human cell lines the CMV promoter was more versatile, generally providing sustained expression at a significant level, in one case for at least 6 days. In addition, as mouse, rabbit and pig models of rotavirus infection are under investigation and VP7sc is a vaccine antigen, we also investigated the ability of the recombinant adenovirus to infect cells from these and other sources. VP7sc expression was detected in several heterologous cell types, illustrating the ubiquity of the human Ad receptor and the versatility of human Ad as vectors when suitable promoters are used.

Actins↗
PubMed1995

Membrane binding and endoplasmic reticulum retention sequences of rotavirus VP7 are distinct: role of carboxy-terminal and other residues in membrane binding.

The sequences responsible for binding rotavirus glycoprotein VP7 to the membrane of the endoplasmic reticulum (ER) have not been identified. Here we show that the sequences which promote membrane binding in vitro are distinct from the N-terminal sequences which promote retention of VP7 in the ER in vivo. The role of the C-terminal region in membrane binding was also examined by using truncation mutants. Membrane binding in vitro was reduced but not abolished by removing up to 102 residues from the C terminus. The data suggest that the last 36 residues of VP7 may be present in the membrane or translocation pore, possibly with the C terminus protruding into the cytoplasm, since these residues contribute to, but do not account for, membrane binding. Surprisingly, modified forms of VP7 which are secreted from transfected cells showed the same membrane-binding properties in vitro as the protein retained in the ER membrane. Thus, secreted VP7 may not be present as a soluble polypeptide in the ER. A model to explain these results is presented. Previously published data are consistent with the idea that the highly conserved C terminus of nascent VP7 could have a cytoplasmic orientation which is important for assembly of mature virus particles.

Amino Acid Sequence↗
PubMed1994

Characterisation of Australian ovine adenovirus isolates.

We have characterised two groups of adenoviruses isolated from sheep in Australia. Restriction endonuclease maps for enzymes BamHI, ClaI, SalI, SmaI and SphI have been determined for the genome of ovine adenoviruses related to bovine adenovirus serotype 7 (BAV 7) from sheep in Western Australia. Although previously serotyped as BAV 7 these isolates are different from bovine isolates of BAV 7 based on comparison with published restriction endonuclease profiles and maps of BAV 7 cattle isolates. Additional adenovirus isolates obtained from Victorian sheep have been serotyped as ovine adenovirus type 5 (OAV 5). On the basis of restriction endonuclease analysis these viruses are different from the sheep BAV 7 isolates. Following infection of sheep with ovine BAV 7 and OAV 5 isolates, virus was recovered from nasal and rectal swabs for several days. Antibodies detected by ELISA and serum neutralisation tests (SN) developed by 15 days after infection. Virus also spread from the infected sheep to an incontact control and one of ten sheep purchased for infection studies had SN antibodies to BAV 7 suggesting that BAV 7-like viruses naturally infect sheep in Victoria and Western Australia. With further development, these ovine adenoviruses may be suitable as vectors for the delivery of vaccine antigens to sheep and cattle.

Adenoviridae↗
PubMed1994

Induction of rotavirus-specific cytotoxic T lymphocytes by vaccinia virus recombinants expressing individual rotavirus genes.

We determined the capacity of vaccinia virus recombinants expressing individual rotavirus genes to induce virus-specific cytotoxic T lymphocytes (CTLs) in mice. Mice were orally inoculated with vaccinia virus recombinants containing genes which encode rotavirus outer capsid proteins vp4 or vp7, single-shelled virus proteins vp1, vp2, or vp6, or rotavirus nonstructural proteins NS53, NS35, NS28, or NS26/NS12. We found that (i) the greatest frequencies of virus-specific CTLs were induced by vaccinia virus recombinants expressing vp7, (ii) transport of vp7 beyond the endoplasmic reticulum was not necessary for induction of CTLs, (iii) recombinants expressing vp7 induced CTLs which reacted with different rotavirus serotypes, and (iv) CTLs were induced among both intestinal and nonintestinal lymphocytes after oral inoculation. These findings may be relevant to vaccine strategies which utilize vectors expressing individual rotavirus genes.

Administration, Oral↗
PubMed1994

Presentation of neutralizing epitopes by engineered rotavirus VP7's expressed by recombinant vaccinia viruses.

Previous studies showed that a calcium-dependent neutralization domain forms on the rotavirus glycoprotein VP7 during assembly into particles. Here, we demonstrate that expressed, recombinant VP7 is capable of forming this neutralization domain in the absence of other rotavirus proteins, but that the domain is unstable. High calcium environments, incorporation into particles, and binding of neutralizing antibodies stabilize the neutralization domain on expressed VP7. A chimeric, cell surface-anchored molecule, VP7sc, has an enhanced ability to react with neutralizing antibodies. This may explain why immunization of mice with expressed native VP7 has had limited success while immunization with VP7sc efficiently induced neutralizing antibodies and passively protected pups from diarrhea. A model of VP7 folding consistent with these results is presented.

Animals↗
PubMed1993

Somatic hypermutation in 5' flanking regions of heavy chain antibody variable regions.

The aim of this study has been to determine the distribution of somatic mutations in the 5' flanking regions of rearranged immunoglobulin heavy chain variable region genes (VDJ). We sequenced the 5' flanking region in 12 secondary immune response antibodies produced in C57BL/6j mice against the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken-gamma-globulin. In these and previously published sequences, almost 97% of the mutations occurred in the transcribed region of the gene, and only a minority of genes (5/29) contained mutations upstream of the transcription start (cap) site. No potential germ-line donor was found for a cluster of five base changes previously found in a single heavy chain gene, 3B62. However, the uniqueness of this mutational cluster and its distance from the normally mutated region suggests that the nucleotide changes may not be due to the normal mutator mechanism. Thus, as this was the only instance of somatic mutations that far upstream of the promoter/cap site region, the reverse transcriptase model for somatic hypermutation is still a possibility. The data are consistent with a mutational mechanism that requires transcription of the rearranged target V(D)J gene which appears to result in the generation of a positively skewed asymmetrical distribution of somatic mutations. A single mode is centered near the V(D)J and a long tail extends into the 3' non-translated region of the J-C intron. Two classes of model could explain this mutation distribution pattern: those where transcription products (RNA, cDNA) are the direct mutational substrates, or those that postulate local unfolding of the chromatin around a V(D)J rearrangement directly exposing the DNA of the transcribed region to specific mutational enzymes.

Animals↗
PubMed1993

Protective immunity to rotavirus-induced diarrhoea is passively transferred to newborn mice from naive dams vaccinated with a single dose of a recombinant adenovirus expressing rotavirus VP7sc.

VP7sc is a novel rotavirus antigen engineered for presentation at the cell surface. Several recombinant viruses were constructed in which VP7sc was inserted into the E3 region of the human type 5 adenovirus (Ad5) genome and expression and transport of the antigen was monitored in cultured 293 cells. The recombinant virus showing the greatest level of expression (Ad5/7.4) was then used to determine whether antibodies to VP7sc could be induced in a nonhuman host. BALB/c and CBA/H mice were inoculated with Ad5/7.4 by iv, ip, oral and intranasal routes and serum antibody levels were assayed by ELISA. All vaccinated animals seroconverted but, depending on the route of vaccination, not all animals showed a significant secondary response following re-inoculation. The ability of Ad5/7.4 to induce protective immunity in mice was also examined using several vaccination regimes. A single dose of Ad5/7.4 given intranasally to dams not previously exposed to rotavirus was sufficient to induce immunity which could be passively transferred to protect suckling neonates. Recombinant adenoviruses expressing protective antigens therefore may provide an alternative to the use of attenuated rotaviruses in the development of a vaccine against gastroenteritis.

Adenoviruses, Human↗
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

Defining the nucleic acid substrate for somatic hypermutation.

Recent reports have more precisely defined the distribution of somatic mutations around rearranged mouse V-D-J genes. The 5' boundary of mutation is most likely in the region of the transcription start site (cap) and/or the promoter (P), implying that transcription may be a prerequisite for mutations to be generated. As more than 95% of somatic mutations lie downstream of the cap site, the transcription unit itself is implicated as the target of the mutational machinery. For heavy chain genes, the 3' boundary can extend into the enhancer region (E). For kappa light chain genes, the 3' boundary extends to approximately 700 bp beyond J kappa-5 (approximately 700 bp upstream of E). In a single study on mutated derivatives of the rearranged mouse lambda 1 light chain V-J gene, it was claimed that the 3' boundary fell within the constant region (C) exon. Although more data are required, the frequency of mutations around V-D-J genes appears asymmetrical, being positively skewed with a single mode centred near the V-D-J coding region and a long tail extending into the 3' non-translated region of the J-C intron. Such a distribution may place constraints on possible molecular mechanisms. It is suggested that the apparent asymmetrical pattern of mutation is best explained by models that assume localized error-prone DNA synthesis generating variable fragment lengths of mutated DNA or cDNA retrotranscripts. The frequency distribution of these lengths of mutated DNA is positively skewed into the 3' J-C intron, with a common terminus at or near the cap site. It is then assumed that they can be integrated into the target V-D-J region via a gene conversion or homologous recombination process. The model invoking reverse transcription may be preferred as it best explains the data without too many ad hoc assumptions.

Animals↗
PubMed1991

Outer capsid glycoprotein vp7 is recognized by cross-reactive, rotavirus-specific, cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) generated in mice orally inoculated with rotaviruses lyse target cells infected with different rotavirus serotypes (cross-reactive CTLs). Using vaccinia virus recombinants expressing individual rotavirus proteins from two different rotavirus serotypes, we found that cross-reactive CTLs recognize target cells expressing outer capsid protein vp7 better than those expressing outer capsid protein vp4 or inner capsid protein vp6. These findings may be relevant to vaccine strategies which include immunization with reassortant rotaviruses or viral or bacterial vectors expressing individual rotavirus proteins. The region or regions of vp7 which are antigenically conserved among different rotavirus serotypes and recognized by cross-reactive CTLs remain to be determined.

Animals↗
PubMed1991

Expression of rotavirus proteins encoded by alternative open reading frames of genome segment 11.

The nucleotide sequence of rotavirus genome segment 11 shows that this gene contains three potential open reading frames. We used several approaches to determine whether any polypeptides other than NS26, the primary protein product, are expressed. In particular, we sought to determine whether the strong out-of-phase start codon present at nucleotides 80-82, which would encode a protein of 92 amino acids, is used in vivo or in cell-free systems. Several modifications of gene 11 were made and found to produce proteins from the different initiation codons in cell-free transcription-translation systems. The protein from the out-of-phase open reading frame was shown to be expressed in rotavirus-infected MA104 cells; this was demonstrated using monospecific sera prepared to this protein expressed in Spodoptera frugiperda insect cells infected with a baculovirus recombinant containing only the out-of-phase open reading frame. The origin of some of the lower-molecular-weight bands serologically related to the primary product of gene 11, NS26, was also studied by selective immunoprecipitation using two different sera made from recombinant baculovirus lysates. All of these polypeptides are present in infected cells in a complex which is still incompletely defined.

Amino Acid Sequence↗
PubMed1990

Conservation of a potential metal binding motif despite extensive sequence diversity in the rotavirus nonstructural protein NS53.

The nucleotide sequence for the simian rotavirus SA11 gene segment 5 has been determined. The gene is 1611 nucleotides in length and contains a single open reading frame of 1485 nucleotides. The segment codes for the nonstructural protein NS53 which is predicted to be a polypeptide of 495 amino acids with a molecular weight of 58,484. When compared to the sequence of bovine RF gene segment 5 there are homologies of only 49 and 36% at the nucleotide and amino acid levels, respectively. This is in marked contrast to the situation with other rotavirus nonstructural proteins which are highly conserved between isolates. Nevertheless, there is a conserved region between amino acids 37-81 which contains a generalized motif for a metal binding domain. All eight cysteine and two histidine residues in this short sequence are conserved between the simian and bovine NS53 proteins. The conservation of this domain despite extensive sequence diversity in the remainder of the protein suggests that this region is functionally important.

Amino Acid Sequence↗
PubMed1990

Completion of the genomic sequence of the simian rotavirus SA11: nucleotide sequences of segments 1, 2, and 3.

The nucleotide sequences for gene segments 1, 2, and 3 of the simian rotavirus SA11 genome, coding for the structural polypeptides VP1, VP2, and VP3, respectively, have been determined. Comparison of the VP1 and VP2 amino acid sequences with those determined for other strains indicates that certain features of these proteins are conserved. The possible functions of the viral polypeptides VP1, VP2, and VP3 are discussed in the light of enzyme functions known to be present in the rotavirus particle. The complete sequence of the entire SA11 genome, which consists of 11 segments of dsRNA totaling 18,555 nucleotides, has now been determined. This is the first complete sequence available for a rotavirus genome. Each genome segment appears to code for only one primary product; there are no significant, alternative open reading frames which are conserved between strains. Relevant data for each genome segment are tabulated.

Amino Acid Sequence↗