Search PubMed⌕ Search

Biomedical subjects

G W Both

Publications and source records attributed to G W Both.

At least 55 records · Page 3Linked to original sources
PubMed1990

Sequences in rotavirus glycoprotein VP7 that mediate delayed translocation and retention of the protein in the endoplasmic reticulum.

Glycosylation and translocation of the simian rotavirus protein VP7, a resident ER protein, does not occur co-translationally in vivo. In pulse-chase experiments in COS cells, nonglycosylated VP7 was still detectable after a 25-min chase period, although the single glycosylation site was only 18 residues beyond the signal peptide cleavage site. After labeling, glycosylated and nonglycosylated VP7 was recovered in microsomes but the latter was sensitive to trypsin (i.e., the nascent protein became membrane associated) but most of it entered the ER posttranslationally because of a rate-limiting step early in translocation. In contrast with the simian protein, bovine VP7 was glycosylated and translocated rapidly. Thus, delayed translocation per se was not required for retention of VP7 in the ER. By constructing hybrid proteins, it was further shown that the signal peptide together with residues 64-111 of the simian protein caused delayed translocation. The same sequences were also necessary and sufficient for retention of simian VP7 in the ER. The data are consistent with the idea that certain proteins are inserted into the ER membrane in a loop configuration.

Amino Acid Sequence↗
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↗
PubMed1990

Distribution of mutations around rearranged heavy-chain antibody variable-region genes.

The mechanism of somatic hypermutation in the variable region of immunoglobulin genes expressed in mammalian B cells is a major unexplained phenomenon in the generation of diversity in the immune system. To evaluate possible mechanisms, the distribution of somatic mutations was examined for a group of five cloned, rearranged, somatically mutated VH genes generated in C57BL/6j mice. These mutated VH genes were sequenced and compared with their germ line counterparts from a point approximately 550 base pairs upstream of the transcription start site to an EcoRI site some 1,200 base pairs downstream of JH-4. The location of the transcription start (cap) sites was also precisely determined. Most (greater than or equal to 94%) of the 118 mutations scored occurred between the transcription start site and the distal end of JH-4. However, seven mutations occurred upstream of the transcribed region, and at least four were found downstream of JH-4. The target region for the mutator mechanism therefore clearly extends into the 3' nontranslated and 5' nontranscribed regions. Thus, models which propose the transcribed region of the DNA as the sole substrate for the mutation process are not ruled out but are inadequate to explain the upstream distribution of somatic mutations.

Animals↗
PubMed1989

Influence of the sequence-dependent flexure of DNA on transcription in E. coli.

In order to study the effects of DNA structure on cellular processes such as transcription, we have made a series of plasmids that locate several different kinds of DNA structure (stiff, flexible or curved) near the sites of cleavage by commonly-used restriction enzymes. One can use these plasmids to place any DNA region of interest (e.g., promoter, operator or enhancer) close to certain kinds of DNA structure that may influence its ability to work in a living cell. In the present example, we have placed a promoter from T7 virus next to the special DNA structures; the T7 promoter is then linked to a gene for a marker protein (chloramphenicol acetyl transferase). When plasmids bearing the T7 promoter are grown in cells of E. coli that contain T7 RNA polymerase, the special DNA structures seem to have little or no influence over the activity of the T7 promoter, contrary to our expectations. Yet when the same plasmids are grown in cells of E. coli that do not contain T7 RNA polymerase, some of the DNA structures show a surprising promoter activity of their own. In particular, the favourable flexibility or curvature of DNA, in the close vicinity of potential -35 and -10 promoter regions, seems to be a significant factor in determining where E. coli RNA polymerase starts RNA chains. We show directly, in one example, that loss of curvature between -35 and -10 regions is associated with a nearly-complete loss of promoter activity. These results, and others of their kind, show that the structural and/or vibrational properties of DNA play a much more important role in determining E. coli promoter activity than has previously been supposed.

Base Sequence↗
PubMed1989

The signal peptide of the rotavirus glycoprotein VP7 is essential for its retention in the ER as an integral membrane protein.

The rotavirus glycoprotein VP7 has a cleavable signal peptide and is normally resident as an integral membrane protein in the ER of infected cells. A gene was constructed in which the VP7 H2 signal peptide was replaced by one from influenza hemagglutinin. COS cells transfected with this gene produced VP7 with the correct amino terminus, but the protein was rapidly secreted. Uncleaved VP7 from either precursor was not detected in cells after brief pulse-labeling, suggesting that the signal peptide was not acting as a temporary anchor; rather, it exerted its effect despite rapid cleavage. By splicing the H2 signal peptide onto another reporter protein, the malaria S-antigen, we demonstrated that H2 was necessary, but not itself sufficient, for targeting and retention. We propose that an interaction between the cleaved signal peptide and other downstream sequences in VP7 is required for retention of this protein in the ER as an integral membrane polypeptide.

Animals↗
PubMed1987

Fowlpox virus thymidine kinase: nucleotide sequence and relationships to other thymidine kinases.

The thymidine kinase (TK) gene of fowlpox virus (FPV) is located in a 2.2-kb HindIII-ClaI fragment derived from a 5.5-kb EcoR1 fragment of the FPV genome. The TK gene was mapped to the region of a 700-bp XbaI fragment contained within this HindIII-ClaI fragment. Nucleotide sequence analysis of this region revealed an open reading frame of 183 codons. Identification of this region as the FPV TK gene was confirmed by its homology with the vaccinia virus TK at both the nucleotide and amino acid levels. The derived FPV TK polypeptide has a calculated molecular weight of 20,380 and is six amino acids larger than the vaccinia virus TK gene product. We have reported previously that the FPV TK gene operates in vaccinia virus without the requirement for a vaccinia virus promoter. The sequence homologies between the two TK promoters substantiated this observation. Northern blot analysis of RNAs from cells infected with a vaccinia virus recombinant expressing the FPV TK gene showed major (700 nucleotide) and minor (1000 nucleotide) transcripts from the FPV TK gene. The deduced amino acid sequence of the FPV TK has significant homology with the TKs from chicken, man, and three other poxviruses, but shows no homology with herpes simplex virus TK. Comparisons of the homologous sequences indicated that the "core" of the enzyme has probably evolved in poxviruses four times as quickly as in vertebrates. Characterization of the FPV TK gene may facilitate the construction of recombinant FPVs as vehicles for the delivery of vaccine antigens to poultry and other avian species.

Amino Acid Sequence↗
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

Effect of in vitro mutations in a vaccinia virus early promoter region monitored by herpes simplex virus thymidine kinase expression in recombinant vaccinia virus.

The location of a promoter (PF) in the HindIII F region of the vaccinia virus genome was mapped by introducing deletions into this region of the DNA. Modified promoters were fused to the herpes simplex virus (HSV) thymidine kinase (TK) gene in plasmids facilitating the construction of recombinant vaccinia viruses, and promoter function was monitored by the ability of such plasmids to rescue TK+ vaccinia viruses from cells infected with TK- virus. Deletions from the 3' end of the promoter region produced mutants for which function was either not inhibited or abolished, allowing the 3' promoter boundary to be defined to within 13 nucleotides. As indicated by the presence of the PF transcript in early RNA and the kinetics of HSV TK expression in recombinant vaccinia viruses, transcription from PF occurred primarily at early times during infection. The major transcript was initiated at a site within 20 nucleotides of the 3' end of the promoter and nine bases upstream of the probable translation initiation codon. In one mutant for which a small but reproducible increase in promoter function was detected, the transcription start site was deleted. Nevertheless, transcription still appeared to begin at the equivalent position with respect to the promoter, despite the altered nucleotide sequence. The location of the start site for the PF transcript indicated that the HSV TK gene, inserted at the BamHI site following the promoter, was preceded by an initiation codon which could potentially attenuate expression of the inserted gene. Conversion of this ATG codon to TAG did not significantly improve HSV TK expression.

Base Sequence↗
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↗
PubMed1986

Temporal regulation of influenza hemagglutinin expression in vaccinia virus recombinants and effects on the immune response.

Regulation of the expression of influenza A/PR/8/34 hemagglutinin (HA) by the vaccinia virus promoters PF (early), P7.5 (early and late) and PL11 (late) has been demonstrated using HA-vaccinia recombinant viruses VV-PR8-HA3, VV-PR8-HA6 and VV-PR8-HA, respectively. Levels of HA on the surface of VV-PR8-HA3 (PF)-infected cells were lower than with either VV-PR8-HA6 (P7.5) or VV-PR8-HA8 (PL11). Expression of HA under the control of the late promoter PL11 was inhibited in the absence of DNA replication. All three recombinant viruses stimulated a specific antibody response in mice which was dependent on the presence of infectious virus. Recognition of HA by cytotoxic T lymphocytes (CTL) was assessed by the ability of the viruses to stimulate naive precursors in vivo, to restimulate primed CTL in vitro and by target cell recognition. HA expressed under the control of either of the promoters with early function (PF or P7.5) was recognized by CTL when VV-PR8-HA3 or VV-PR8-HA6 were used to prime or restimulate splenocytes or to infect target cells. On the other hand, HA expressed by VV-PR8-HA8 (PL11) failed to prime for a CTL response in naive CBA/H mice, was ineffective at restimulation of primed splenocytes and failed to produce target cells for recognition by specific CTL. However, in BALB/c mice VV-PR8-HA8 did prime for a specific CTL response. These studies show that HA synthesized early in infection was recognized by both B and T cells while HA expressed after DNA replication was not generally recognized by T cells. The implications of the observations with the late promoter with respect to the use of late promoters in potential vaccinia virus-based vaccines are considered.

Animals↗
PubMed1986

Responses of cattle, sheep and poultry to a recombinant vaccinia virus expressing a swine influenza haemagglutinin.

Groups of cattle, sheep and poultry were inoculated with a recombinant vaccinia virus expressing the haemagglutinin of the swine influenza virus A/NJ/11/76. No adverse clinical responses were recorded and none of the animals developed a viraemia when inoculated with the recombinant or wild-type vaccinia virus. Recombinant virus reisolated from lesions in cattle was stable, maintaining its thymidine kinase negative phenotype and ability to express the swine influenza haemagglutinin. Antibodies to the influenza haemagglutinin were detected in cattle, sheep and poultry inoculated with the recombinant virus. While no animals inoculated with wild-type virus developed these antibodies, there was no detectable spread of either recombinant or wild-type virus from the inoculation sites or to in-contact uninoculated animals. The results indicate that recombinant vaccinia viruses can induce immune responses in cattle, sheep and poultry demonstrating their potential as vaccine vectors in a variety of important veterinary species.

Animals↗
PubMed1985

Multiple-cloning-site plasmids for the rapid construction of recombinant poxviruses.

Plasmid vectors containing multiple cloning sites suitable for the rapid insertion of protein-coding sequences into poxviruses have been constructed. They are based on pUC plasmids and carry the thymidine kinase (TK) gene of vaccinia virus interrupted by a vaccinia virus promoter. Six unique restriction enzyme sites (BamHI, SalI/HincII, PstI, HindIII, EcoRI), located within 40 bp of vaccinia virus promoters transposed from the HindIII-F or HindIII-C fragment of the vaccinia virus genome, allow rapid insertion of foreign-protein-coding sequences into these plasmids. Such plasmids can be used to construct recombinant poxviruses expressing foreign proteins using marker-rescue recombination techniques and selection for TK negative viruses. Vaccinia viruses expressing the haemagglutinin (HA) gene of swine influenza virus, A/NJ/11/76 (H1N1), have been constructed.

Base 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↗
PubMed1985

Rotavirus neutralizing protein VP7: antigenic determinants investigated by sequence analysis and peptide synthesis.

The rotavirus neutralizing antigen, VP7, is a 37,000-molecular-weight glycoprotein which is a major component of the outer shell of the virion. The amino acid sequence of VP7 for strain S2 (human serotype 2) and Nebraska calf diarrhea virus (bovine serotype) has been inferred from the nucleic acid sequence of cloned copies of genomic segment nine. Comparison of the amino acid sequences of these two VP7 proteins with those already determined for other rotavirus strains reveals extensive sequence conservation between serotypes with clusters of amino acid differences sited predominantly in hydrophilic domains of the protein. Six peptides have been synthesized that span the hydrophilic regions of the molecule. Antisera to these peptides both recognize the respective homologous peptides in a solid-phase radioimmunoassay and bind to denatured VP7 in a Western blot. However, none of the antisera either recognize virus or exhibit significant neutralizing activity, indicating that these peptide sequences are not available on the surface of the virus.

Amino Acid Sequence↗