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Biomedical subjects

L A Ball

Publications and source records attributed to L A Ball.

At least 55 records · Page 3Linked to original sources
PubMed1987

Expression and glycosylation of the respiratory syncytial virus G protein in Saccharomyces cerevisiae.

A cDNA encoding the entire amino acid sequence of the G glycoprotein of respiratory syncytial virus (RSV) was inserted into a yeast-Escherichia coli shuttle vector such that expression of the virus gene was regulated by the yeast GAL1 promoter. Transformation of Saccharomyces cerevisiae with the vector led to the formation of the G protein when cells were grown in the presence of galactose. Under these conditions the RSV G appeared as a 60- to 65-kDa glycosylated protein. Expression of the G cDNA in secretory mutants of S. cerevisiae yielded a protein of 35 kDa in a mutant unable to glycosylate secreted proteins and a 65-kDa polypeptide in a mutant unable to transport proteins beyond the endoplasmic reticulum. The RSV protein formed in the latter mutant was converted to a 60-kDa protein by endoglycosidase H. Our results show that yeast can recognize the internal signal sequence of RSV G protein and add glycosyl groups to the polypeptide in the endoplasmic reticulum. Evidence is presented for both N- and O-linked glycosylation of the virus glycoprotein.

Acetylglucosaminidase↗
PubMed1987

Cytotoxic T cell specificity for respiratory syncytial virus proteins: fusion protein is an important target antigen.

We examined the specificity of BALB/c cytotoxic T (Tc) cells for respiratory syncytial virus (RSV) components, using recombinant vaccinia viruses (VV) coding for several individual RSV proteins. We found that immunization with the different VVs yielded the following Tc memory cell populations: high levels of RSV-specific Tc cells were induced with the fusion protein VV, but low levels were induced with VV coding for the RSV nucleoprotein. Tc cell recognition of attachment glycoprotein, part of the matrix molecule or 1A internal protein was poor. While high levels of fusion protein-specific Tc cells were induced by the fusion protein VV, they showed poor cross-reactivity between the A2 and 8/60 RSV strains compared with Tc cells primed by RSV infection.

Animals↗
PubMed1987

Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus.

Previous reports have established that vaccinia virus (VV) recombinants expressing G, F, or N protein of respiratory syncytial (RS) virus protect small animals against intranasal challenge with live RS virus. This work demonstrates that a variety of parameters affect the protection induced by recombinant viruses. The route of vaccination, the subtype of challenge virus, and the species used influenced the antibody titers and extent of protection. During these studies, observations were also made on the subclass of antibody generated, and pulmonary histopathological changes induced by challenge after vaccination were noted. The effect of route of inoculation on host response was examined by vaccinating mice intranasally, intraperitoneally, or by scarification with a recombinant VV expressing the RS virus G glycoprotein. Intranasal vaccination induced 25-fold-higher titers of antibody to RS virus in the lung than the intraperitoneal route did, but both routes resulted in complete suppression of virus replication after intranasal challenge 21 days after vaccination. Scarification was a less effective method of vaccination. The antibody induced by recombinant VV in mice was mostly immunoglobulin G2a (IgG2a) with some IgG2b. No antibody to RS virus was detected in the IgA, IgM, IgG1, or IgG3 subclass irrespective of the vaccination route. The G and F glycoproteins were shown to elicit similar subclasses of antibody. However, animals vaccinated with the G and F vectors differed strikingly in their response to challenge by heterologous virus. Mice or cotton rats vaccinated with recombinant VV carrying the G gene of RS virus were protected against challenge only with homologous subtype A virus. Vaccination with a recombinant VV expressing the F glycoprotein induced protection against both homologous and heterologous subtype B virus challenge. The protection induced in mice was greater than that detected in cotton rats, indicating that the host may also affect immunity. Finally, this report describes histological examination of mouse lungs after vaccination and challenge. Vaccinated mice that were subsequently challenged had significantly greater lung lesion scores than unvaccinated challenged mice. The lesions were primarily peribronchiolar and perivascular infiltrations of polymorphonuclear cells and lymphocytes. Further work will establish whether these pulmonary changes are a desirable immune response to virus invasion or a potential immunopathogenic hazard. The results have important implications for planning a strategy of vaccination against RS virus and emphasize potential dangers that may attend the use of recombinant VV as vaccines.

Administration, Cutaneous↗
PubMed1987

Expression of the fusion protein of human respiratory syncytial virus from recombinant vaccinia virus vectors and protection of vaccinated mice.

Vaccinia virus (VV) recombinants were constructed that contained full-length cDNA copies of the fusion (F) protein gene of human respiratory syncytial (RS) virus. The F protein gene was placed next to the strong early-late VV 7.5-kilodalton promoter and was located within the VV thymidine kinase (tk) gene. Full-length recombinant transcripts that initiated at both the tk and the 7.5-kilodalton promoters accumulated in cells early in infection, and one or more of these transcripts was translated to yield a glycoprotein which comigrated with Fo, the fusion protein precursor. This precursor was processed by proteolytic cleavage to produce the two disulfide-linked subunits F1 and F2, which were both glycosylated and of the same electrophoretic mobility as authentic F1 and F2. Immunofluorescence studies demonstrated that the mature F protein was transported to and expressed on the surface of recombinant VV-infected cells. Inoculation of rabbits with a recombinant vector expressing F resulted in the production of antiserum specific for the RS virus F protein. This antiserum neutralized virus infectivity and was capable of preventing fusion in RS virus-infected cells. Mice were vaccinated with recombinants expressing the F protein. At 3 weeks postinoculation, these animals had serum antibody against RS virus F protein. At 5 days after intranasal challenge with RS virus, the lungs of the mice previously vaccinated with recombinants expressing F protein were free of detectable RS virus, whereas the lungs of unvaccinated mice contained 10(4.2) PFU of virus per g.

Cell Line↗
PubMed1987

High-frequency homologous recombination in vaccinia virus DNA.

A recombinant vaccinia virus genome was constructed in which the viral thymidine kinase (tk) gene was placed between direct repeats of a 1.5-kilobase-pair DNA sequence of heterologous origin. When forced to replicate in tk- cells in the presence of methotrexate (i.e., under tk+-selective conditions), the recombinant maintained its tk+ phenotype. Under nonselective conditions, however, the tk gene was frequently excised by both inter- and intramolecular recombination events because the repeated sequences provided substantial targets for homologous DNA recombination. Unique DNA products of intramolecular recombination were detected in the cytoplasm of infected cells soon after the onset of viral DNA replication, and their appearance was blocked by inhibitors of DNA synthesis. During repeated passage of the virus under nonselective conditions, the tk+ fraction decreased with first-order kinetics at a rate that reflected the frequency of recombination per cycle of virus replication. Eventually, a residual population of stable tk+ viruses remained, and analyses of the genome structures of individual members of this population showed that some of them appeared to be the products of nonhomologous DNA recombination.

DNA Replication↗
PubMed1987

Recombinant vaccinia viruses carrying the N gene of human respiratory syncytial virus: studies of gene expression in cell culture and immune response in mice.

The construction and characterization of vaccinia virus recombinants carrying the nucleocapsid (N) protein gene of human respiratory syncytial (RS) virus are described. Recombinant viruses were constructed that contained the N gene oriented either positively or negatively with respect to the 7.5-kilodalton vaccinia virus promoter. In addition, a positively oriented recombinant was constructed that lacked an out-of-frame AUG codon in the 5'-terminal noncoding region. In HEp-2 cells, both positive-orientation recombinants induced the synthesis of a protein which comigrated with N protein and was precipitated by antisera to RS virus. Sera from mice immunized with these recombinants specifically precipitated the RS virus N protein. Analysis of mRNA and protein expressed from the recombinant N genes showed that deletion of the upstream AUG codon markedly improved the efficiency of protein synthesis. Mice were vaccinated with the high-expressing recombinant and subsequently challenged with live RS virus. The results of these experiments demonstrated that the immune response to N protein afforded a significant degree of protection against RS virus disease.

Animals↗
PubMed1986

Human and murine cytotoxic T cells specific to respiratory syncytial virus recognize the viral nucleoprotein (N), but not the major glycoprotein (G), expressed by vaccinia virus recombinants.

The viral antigens recognized by cytotoxic T cells (CTL) have not been defined in most viruses infecting mouse or man. Natural or artificial virus recombinants can be used to determine the antigen specificity of CTL directed against viruses with segmented genomes, such as influenza, but this technique is more difficult to apply to the study of unsegmented viruses. We describe here the use of recombinant vaccinia viruses, containing cDNA corresponding to either the nucleoprotein (N) gene or the major surface glycoprotein (G) gene of human respiratory syncytial virus (RSV), to examine the antigen specificity of anti-RSV cytotoxic T cells from humans and mice. The results demonstrate that the RSV N protein is one of the target antigens for CTL in man and mouse, whereas the G protein was not recognized and can at best represent a minor target antigen for CTL.

Animals↗
PubMed1986

Expression of the major glycoprotein G of human respiratory syncytial virus from recombinant vaccinia virus vectors.

The major glycoprotein, G, of human respiratory syncytial (RS) virus is a Mr 84,000-90,000 species that has about 60% of its mass contributed by carbohydrate, most of which is in the form of O-linked oligosaccharides. The G protein contains neither a hydrophobic N-terminal signal sequence nor a hydrophobic C-terminal anchor region. Instead, its amino acid sequence reveals only one region with significant hydrophobic character, which is between residues 38 and 66. In order to study the synthesis, processing, and functions of this unusual viral glycoprotein, full-length cDNA copies of the G protein mRNA were inserted into the DNA genome of vaccinia virus (VV) in a position that was adjacent to a strong VV promoter and within the VV gene for thymidine kinase (TK). The resulting TK- recombinant viruses were selected, plaque-purified, and characterized by Southern blot analysis of restriction enzyme digests of the viral DNA. Recombinant RNA transcripts that contained both G-specific and VV-specific sequences accumulated in cells infected with recombinant viruses having the G protein gene in the positive orientation. The translation product of these transcripts in infected cells was a Mr 84,000-90,000 glycoprotein that was indistinguishable from authentic RS virus G protein. It could be detected in cell lysates after metabolic labeling with [3H]glucosamine and was immunoprecipitated by anti-RS-virus antiserum. Immunofluorescence studies showed that the G protein accumulated intracellularly with the perinuclear distribution that is characteristic of newly synthesized glycoproteins. Furthermore, the protein was also clearly detectable on the surface of recombinant-infected cells, showing that it was transported to and inserted into the plasma membrane.

Animals↗
PubMed1986

Modulation of 2',5'-oligoadenylate synthetase in patients treated with alpha-interferon: effects of dose, schedule, and route of administration.

The interferon (IFN)-induced intracellular enzyme 2',5'-oligoadenylate (2-5A) synthetase was measured in extracts of peripheral mononuclear cells isolated from patients receiving a 300-fold range of doses of alpha interferon (IFN-alpha). The range of enzyme induction was 2.3- to 5.7-fold. The maximum fold increase varied from individual to individual as did the dose required for maximum enzyme stimulation. The magnitude and endurance of the enzyme response was a function of IFN dose and was unrelated to the duration of treatment or number of injections or to the route of administration. The enzyme assay was a more sensitive indicator of IFN administration than was measurement of the level of circulating IFN. These results substantiate the potential of a clinical 2-5A synthetase assay for monitoring IFN treatment.

2',5'-Oligoadenylate Synthetase↗
PubMed1986

Human respiratory syncytial virus glycoprotein G expressed from a recombinant vaccinia virus vector protects mice against live-virus challenge.

Recombinant vaccinia virus vectors were constructed which expressed the major surface glycoprotein G of human respiratory syncytial (RS) virus. The biological activity of the G protein expressed from these vectors was assayed. Inoculation of rabbits with live recombinant virus induced high titers of antibody which specifically immunoprecipitated RS virus G protein and was capable of neutralizing RS virus infectivity. Immunization of mice by either the intranasal or the intraperitoneal route with recombinant virus that expressed only the G protein resulted in complete protection of the lower respiratory tract upon subsequent challenge with live RS virus.

Animals↗
PubMed1985

Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein.

The major surface glycoprotein (G) of human respiratory syncytial (RS) virus has an estimated mature Mr of 84,000-90,000. Among a library of cDNA clones prepared from RS virus mRNAs, we identified clones that hybridized to a message that encoded a Mr 36,000 polypeptide that was specifically immunoprecipitated with anti-G antiserum. The amino acid sequence of the G protein backbone was determined by nucleotide sequence analysis of several of the cDNA clones. It contains a combination of structural features that make it unique among the known viral glycoproteins. The G mRNA is 918 nucleotides long and contains a single major open reading frame that encodes a polypeptide having 298 amino acid residues with a Mr of 32,587, a finding consistent with the Mr 36,000 estimate for the in vitro translation product of the G mRNA. This suggests that greater than 50% of the molecular weight of the mature glycoprotein may be contributed by carbohydrate. Glycosylation of G is largely resistant to tunicamycin, an inhibitor of the attachment of N-linked oligosaccharides, suggesting that the majority of the carbohydrate residues are attached via O-glycosidic bonds. In accordance with this, serine and threonine residues, the acceptor sites for O-linked oligosaccharides, comprise 30.6% of the total amino acid composition. There are also four potential acceptor sites for N-linked oligosaccharides. The amino acid sequence lacks both an NH2-terminal hydrophobic signal sequence and a COOH-terminal hydrophobic region. Instead, a strongly hydrophobic region is located between amino acid residues 38 and 66. This region may serve as both the signal to insert the nascent polypeptide through the membrane and as the membrane anchor site.

Amino Acid Sequence↗
PubMed1985

Measurement of 2',5'-oligoadenylate synthetase in patients receiving interferon-alpha.

The interferon-induced intracellular enzyme, 2',5'-oligoadenylate (2,5A) synthetase, was measured in extracts of Ficoll-purified human peripheral mononuclear cells from 28 normal healthy individuals and 14 patients receiving injections of interferon-alpha. Basal and stimulated levels could be measured reproducibly in 2 X 10(6) cells. In both groups, mononuclear cell levels of 2,5A synthetase varied widely, but measurements were reproducible in each individual. In most instances, increases in enzyme activity were detected within 8 h of interferon-alpha administration. Elevated levels persisted for at least 24 h and were maintained with daily treatment. In two of the 14 patients, the enzyme level failed to respond to multiple interferon injections. Interestingly, these two patients had pretreatment enzyme levels that were markedly elevated relative to those of a healthy population. Measurement of 2,5A synthetase levels is a valuable addition to clinical interferon studies, since the results may help to resolve questions of the optimal dose, route, and schedule of interferon administration.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

2-5A synthetase activity in patients with metastatic carcinoma and its response to interferon treatment.

The interferon-induced enzyme 2-5A synthetase was measured in extracts of ficoll-purified peripheral mononuclear cells from normal healthy volunteers and a large group of patients with metastatic carcinoma. The range of values in both populations was very large and overlapped considerably. On average, individuals with disseminated cancer have a two-fold increase in 2-5A synthetase activity. In addition, several patients have been identified who appear to have fully induced 2-5A synthetase activity with no detectable serum interferon and no history of prior interferon injections. This indicates that the interferon regulated 2-5A system is activated in metastatic carcinoma. Treatment of patients with interferon-alpha caused a dose-dependent increase in enzyme activity. The maximum fold increase varied from individual to individual, as did the dose required for maximum enzyme stimulation. A rigorous attempt must now be made to correlate enzyme induction with the clinical outcome of interferon treatment in order to assess the clinical value of such measurements.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Comparison of the biologic effects of two recombinant human interferons alpha (rA and rD) in humans.

IFN-alpha (rD) was investigated to determine the relationship between antiviral activity in vitro and the modulation of biologic effects in vivo. Eight patients with malignancy were given 15 and 45 micrograms weekly injections of IFN-alpha (rA) and IFN-alpha (rD). The frequency of side effects was much lower with IFN-alpha (rD) injections. This was objectively documented both in incidence of side effects (20 versus 45, p less than 0.01) and mean maximum temperature (1 degree C lower with IFN-alpha (rD), p less than 0.002). A bovine cell line, MDBK, was used to measure interferon concentrations in the serum. Geometric mean peak titers and time-to-peak titers were similar with the two recombinant interferon preparations. Although IFN-alpha (rD) has relatively less antiviral activity on human cells, its effect on the total granulocyte count, natural killer (NK) cell cytotoxicity, and 2'5'-A activity was comparable to IFN-alpha (rA). Mean NK cell percent specific 51Cr release was enhanced by both interferons (after 15 micrograms doses, mean percent NK cell cytotoxicity IFN-alpha (rD) preinjection, 10.5% +/- 2.3%; post-injection, 27.2% +/- 4.5%; IFN-alpha (rA), preinjection, 14.4% +/- 3.2%, postinjection, 25.1% +/- 5%). Species-specific antiviral activity of an interferon does not necessarily predict other biologic properties following in vivo administration.

2',5'-Oligoadenylate Synthetase↗
PubMed1983

Fine structure analysis and nucleotide sequence of the vaccinia virus thymidine kinase gene.

The thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene of vaccinia virus has previously been mapped near the middle of the viral DNA, within the 4.85-kilobase HindIII J fragment, and shown to encode a Mr 19,000 polypeptide [Hruby, D. E. & Ball, L. A. (1982) J. Virol. 43, 403-409]. To locate the gene more precisely and to determine the structure of the basic transcriptional unit, the positions of cleavage sites for several restriction endonucleases were mapped within the HindIII J DNA fragment. Four appropriate subfragments of HindIII J DNA were inserted into plasmid pBR322 derivatives and cloned in Escherichia coli. These recombinant plasmid DNAs were tested for their ability to inhibit the cell-free synthesis of active thymidine kinase and to retain the mRNA for this enzyme when immobilized on nitrocellulose filters. The data showed that the gene spanned an EcoRI cleavage site that lies 850 base pairs from the left-hand end of the HindIII J fragment (the HindIII L-J boundary). Because hybridization of vaccinia virus DNA to partially purified thymidine kinase mRNA detected only a single 670-nucleotide RNA species capable of hybridizing to this region of the genome, nuclease S1 mapping experiments were carried out with thymidine kinase mRNA to protect DNA fragments that were terminally labeled at this EcoRI site. The results indicated that the gene extended from about 550 to 1,150 base pairs from the left end of HindIII J, was transcribed in a rightward direction, and contained no intervening sequences. Hence, a 1.04-kilobase Ava II-Hpa II restriction fragment containing this region of DNA was isolated and subjected to nucleotide sequence analysis. An examination of this nucleotide sequence revealed the presence of an open reading frame of 531 nucleotides capable of encoding a protein of 177 amino acids with a Mr of 20,077.

Amino Acid Sequence↗
PubMed1982

Mapping and identification of the vaccinia virus thymidine kinase gene.

The thymidine kinase gene of vaccinia virus (VV) was mapped on the viral genome by using cloned fragments of the viral DNA to hybridize to early viral mRNA. Individual DNA fragments that represented about half of the viral genome were assayed, both for their ability to arrest the cell-free synthesis of active VV thymidine kinase and for their ability to select functional mRNA for the viral enzyme. Both activities were located in HindIII fragment J, which maps near the middle of VV DNA and contains about 2.6% of the genome (4,800 base pairs). This DNA fragment encodes four known early polypeptides, and to determine which of these was thymidine kinase, early VV mRNA was fractionated by sucrose gradient centrifugation and used to direct cell-free synthesis of the active enzyme. The thymidine kinase mRNA cosedimented with several species that encoded polypeptides in the molecular weight range 15,000 to 25,000. Hybridization of these mRNAs to HindIII-J DNA selected a message that directed the synthesis of thymidine kinase and a single polypeptide with an apparent molecular weight of 19,000. The native molecular weight of VV thymidine kinase is about 80,000, so these data indicate that, unlike thymidine kinase from several other sources, the active VV enzyme is probably a tetramer of 19,000-molecular-weight subunits.

DNA Restriction Enzymes↗