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Biomedical subjects

B Moss

Publications and source records attributed to B Moss.

At least 235 records · Page 13Linked to original sources
PubMed1991

Sequence analysis, expression, and deletion of a vaccinia virus gene encoding a homolog of profilin, a eukaryotic actin-binding protein.

A 4,500-bp BamHI fragment, located within the HindIII A segment of the vaccinia virus genome, was found to contain eight potential coding regions for polypeptides of 78 to 346 amino acids. The open reading frames with 133, 346, and 125 codons were homologous to profilin (an actin-binding protein), 3-beta-hydroxysteroid dehydrogenase, and Cu-Zn superoxide dismutase, respectively. Sequence alignments indicated that the vaccinia virus and mammalian profilins were more closely related to each other than to known profilins of other eukaryotes. The expression and possible role of the profilin homolog in the virus replicative cycle were therefore investigated. Antibody raised to Escherichia coli expressed vaccinia virus profilin was used to demonstrate the synthesis of the 15-kDa polypeptide at late times after vaccinia virus infection of mammalian cells. The protein accumulated in the cytoplasm, but only trace amounts remained associated with highly purified virions. The isolation of vaccinia virus mutants (in strains WR and IHD-J), with nearly the entire profilin gene replaced by the E. coli gpt gene, indicated that the protein is not essential for infectivity. The characteristic vaccinia virus-induced changes in actin fibers, seen by fluorescence microscopy, occurred in cells infected with the mutant. Moreover, the virus-encoded profilin homolog was not required for actin-associated events, including intracellular virus movement to the periphery of the cell, formation of specialized microvilli, or release of mature virions, as shown by electron microscopy and yields of infectious intra- and extracellular virus.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Inhibition of the complement cascade by the major secretory protein of vaccinia virus.

The complement system contributes to host defenses against invasion by infectious agents. A 35-kilodalton protein, encoded by vaccinia virus and secreted from infected cells, has sequence similarities to members of a gene family that includes complement control proteins. Biochemical and genetic studies showed that the viral protein binds to derivatives of the fourth component of complement and inhibits the classical complement cascade, suggesting that it serves as a defense molecule to help the virus evade the consequences of complement activation.

Amino Acid Sequence↗
PubMed1990

Mapping the fine specificity of a cytolytic T cell response to HIV-1 nef protein.

Epitope mapping of a MHC class I-restricted cytotoxic T cell response to nef, a regulatory protein of HIV, was performed with fresh PBMC from HIV-seropositive donors and target cells pulsed with a panel of overlapping peptides of the nef protein. These nef-specific CTL recognized a synthetic peptide of 10 residues derived from a nonamphipathic, highly conserved region of the nef protein in association with the HLA A3.1 molecule. Using human cell transfectants expressing mutations of the A3 molecule, we demonstrated that the amino acid at position 152 of the A3.1 molecule appears to be critical for detection of this response. Thus, rapid analysis of the epitopes of HIV proteins stimulating CTL responses can be achieved using a combination of fresh donor PBMC and target cells pulsed with synthesized peptides.

Amino Acid Sequence↗
PubMed1990

Role of DNA replication in vaccinia virus gene expression: a naked template is required for transcription of three late trans-activator genes.

The DNA replication requirement for vaccinia virus late gene expression was mimicked by transfecting a late promoter-controlled reporter gene into infected cells in the presence of a DNA synthesis inhibitor. This late promoter activation block was overcome by cotransfecting either naked linear vaccinia virion DNA or three cloned viral genes encoding trans-activator polypeptides of 17, 26, and 30 kd. These newly identified trans-activator genes were independently transcribed only from replicated or transfected DNA. These data suggest a regulatory cascade in which the parental viral genome serves as a template for the RNA polymerase and early promoter-specific transcription factors that are packaged in the infectious particle; the newly replicated DNA is accessible to sequentially synthesized intermediate promoter- and late promoter-specific trans-activators.

Amino Acid Sequence↗
PubMed1990

Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice.

The role of the immune response to hepatitis B virus (HBV)-encoded antigens in the pathogenesis of liver cell injury has not been defined because of the absence of appropriate experimental models. HBV envelope transgenic mice were used to show that HBV-encoded antigens are expressed at the hepatocyte surface in a form recognizable by major histocompatibility complex (MHC) class I-restricted, CD8+ cytotoxic T lymphocytes specific for a dominant T cell epitope within the major envelope polypeptide and by envelope-specific antibodies. Both interactions led to the death of the hepatocyte in vivo, providing direct evidence that hepatocellular injury in human HBV infection may also be immunologically mediated.

Amino Acid Sequence↗
PubMed1990

Anchor sequence-dependent endogenous processing of human immunodeficiency virus 1 envelope glycoprotein gp160 for CD4+ T cell recognition.

Human CD4+ T cell clones and cell lines were shown to lyse recombinant vaccinia virus-infected cells that synthesize the HIV-1 envelope glycoprotein gp160. The processing of endogenously synthesized gp160 for recognition by CD4+ T cells required that the protein, after synthesis on the rough endoplasmic reticulum and during subsequent cellular transport, remain attached to the luminal/extracellular membrane face by a hydrophobic anchor sequence.

Amino Acid Sequence↗
PubMed1990

An immunodominant class I-restricted cytotoxic T lymphocyte determinant of human immunodeficiency virus type 1 induces CD4 class II-restricted help for itself.

We have observed that a peptide corresponding to an immunodominant epitope of the HIV-1 envelope protein recognized by class I MHC-restricted CD8+ CTL can also induce T cell help for itself. The help is necessary for restimulation of CTL precursors in vitro with peptide alone in the absence of exogenous lymphokines, can be removed by depletion of CD4+ T cells, and can be replaced by exogenous IL-2. Whereas the CTL in BALB/c or B10. D2 mice are restricted by the class I molecule Dd, the Th cells are restricted by the class II molecule Ad, and the help can be blocked by anti-Ad mAb. To examine the genetic regulation of the induction of help, we studied B10.A mice that share the class I Dd molecule, but have different class II molecules, Ak and Ek. Spleen cells of immune B10.A mice behave like CD4-depleted BALB/c spleen cells in that they cannot be restimulated in vitro by the peptide alone, but can with peptide plus IL-2. Therefore, in the absence of exogenous lymphokines, peptide-specific help is necessary for restimulation with this immunodominant CTL epitope peptide, and in H-2d mice, this peptide stimulates help for itself as well as CTL. We speculate on the implications of these findings for the immunodominance of this peptide in H-2d mice, and for the selective advantage of pairing certain class I and class II molecules in an MHC haplotype.

Animals↗
PubMed1990

A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation.

A synthetic peptidemimetic substrate of the human immunodeficiency virus 1 (HIV-1) protease with a nonhydrolyzable pseudodipeptidyl insert at the protease cleavage site was prepared. The peptide U-81749 inhibited recombinant HIV-1 protease in vitro (inhibition constant Ki of 70 nanomolar) and HIV-1 replication in human peripheral blood lymphocytes (inhibitory concentration IC50 of 0.1 to 1 micromolar). Moreover, 10 micromolar concentrations of U-81749 significantly inhibited proteolysis of the HIV-1 gag polyprotein (p55) to the mature viral structural proteins p24 and p17 in cells infected with a recombinant vaccinia virus expressing the HIV-1 gag-pol genes. The HIV-1 like particles released from inhibitor-treated cells contained almost exclusively p55 and other gag precursors, but not p24. Incubation of HIV-like particles recovered from drug-treated cultures in drug-free medium indicated that inhibition of p55 proteolysis was at least partially reversible, suggesting that U-81749 was present within the particles.

Amino Acid Sequence↗
PubMed1990

Reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses.

We have developed a procedure for the selection of recombinant vaccinia viruses with applicability to poxvirus mutagenesis studies and to the use of vaccinia virus as an expression vector. The method depends on the specific inability of a recombinant vaccinia virus expressing the Escherichia coli guanine phosphoriboxyltransferase gene (gpt) to form plaques on a hypoxanthine-guanine phosphoribosyltransferase-negative line of mouse fibroblasts in the presence of 6-thioguanine. Recombinant viruses that have the gpt removed can form plaques under selection conditions, thus providing a simple and efficient selection protocol. We have demonstrated the method by isolating a pseudo-wild type revertant virus and a simple deletion mutant virus from a recombinant vaccinia virus with gpt inserted into the vaccinia virus gene encoding the major 35,000-Da secretory protein.

Animals↗
PubMed1990

Synthesis, oligomerization, and biological activity of the human immunodeficiency virus type 2 envelope glycoprotein expressed by a recombinant vaccinia virus.

The full-length envelope gene from an infectious human immunodeficiency virus type 2 (HIV-2) molecular clone was expressed in CD4+ and CD4- cells by a recombinant vaccinia virus vector. Pulse-chase experiments indicated that gp160 was processed into gp120 and gp41 subunits. Although large amounts of gp120 were shed into the medium, the recombinant vaccinia virus-infected cells fused with uninfected CD4+ cells. The receptor binding of HIV-2 gp120 was further analyzed using a panel composed of nine soluble CD4 mutants containing insertions of 2 amino acids within the first and second immunoglobulin-like domains. Of three mutations previously shown to interfere with HIV-1 gp120 binding, two also interfered with binding of the HIV-2 glycoprotein indicating use of the same binding site. Chemical crosslinking, sucrose gradient sedimentation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were employed to study the oligomerization of the envelope protein. The data indicated that gp160 assembles posttranslationally into dimers and higher oligomers that are probably tetramers.

Animals↗
PubMed1990

Attenuation and immunogenicity in primates of vaccinia virus recombinants expressing human interleukin-2.

Vector-directed lymphokine expression represents a novel approach to the attenuation of live recombinant viruses which might be used as vaccines. Expression of interleukin-2 (IL-2) by recombinant vaccinia virus has been shown to significantly attenuate virus virulence in rodent species without diminishing immunogenicity. Skin lesion formation and immunogenicity of vaccinia/IL-2 recombinants in three species of primates was examined. IL-2 expression was associated with a 15-fold reduction in the area of induration after intradermal inoculation of recombinant viruses in patas monkeys. Wild type and a control vaccinia recombinant produced large (greater than 5000 mm2) skin ulcers in this species, but the IL-2 expressing recombinant produced no ulceration. Production of antibodies to vaccinia virus and to influenza A virus haemagglutinin expressed by recombinant vectors was examined in rhesus and squirrel monkeys. IL-2 expression accelerated the resolution of skin lesions in rhesus but not squirrel monkeys. Despite this, antibody production was equivalent in the presence or absence of IL-2. IL-2 expression can greatly reduce the skin lesions formed by live recombinant vaccinia vectors in primates, indicating significant attenuation, without reducing the immunogenicity of the vaccine.

Animals↗
PubMed1990

Interaction and mutual stabilization of the two subunits of vaccinia virus mRNA capping enzyme coexpressed in Escherichia coli.

The genes D1 and D2, predicted to encode the 95- and 31-kDa subunits of the vaccinia virus mRNA capping enzyme, were coexpressed from the same plasmid in Escherichia coli. Induction with low concentrations of isopropyl beta-D-thiogalactoside was necessary to obtain soluble enzyme. The active heterodimer was purified by column chromatography and was shown to have both RNA guanylyltransferase and mRNA (guanine-N7-)-methyltransferase activities. Formation of the m7G(5')pppG cap structure was verified by enzyme digestion and thin-layer chromatography. Each subunit was also expressed individually in E. coli. Without the large subunit, the small one was very unstable in some bacterial strains and could only be detected by pulse labeling with radioactive amino acids. The individually expressed large subunit contained the guanylyltransferase domain, but the activity from E. coli was less than 2% of that obtained with both subunits. Two other products of the D1 open reading frame were formed: a 55-kDa subfragment with the GMP binding site and a 38-kDa C-terminal fragment that started at amino acid 498. Expression of this heterodimeric enzyme in E. coli may facilitate the analysis of its functional domains and provide a useful reagent for the specific 5' labeling of uncapped or capped RNA and for enhancing RNA translatability in eukaryotic systems.

Cloning, Molecular↗