Biomedical subjects

B Moss

Publications and source records attributed to B Moss.

At least 37 records · Page 2Linked to original sources

PubMed2001

Unique temperature-sensitive defect in vaccinia virus morphogenesis maps to a single nucleotide substitution in the A30L gene.

Marker rescue experiments demonstrated that the genetic lesion of a previously isolated vaccinia virus temperature-sensitive mutant which forms multilayered envelope structures with lucent interiors and foci of viroplasm with dense centers mapped to the A30L open reading frame. A single base change, resulting in a nonconservative Ser-to-Phe substitution at residue 17, was associated with degradation of the A30L protein at elevated temperatures.

Base Sequence↗

PubMed2001

Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails.

Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens Listeria and Shigella, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr(112) and Tyr(132) of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 microm/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.

Actins↗

PubMed2001

Role of vaccinia virus A20R protein in DNA replication: construction and characterization of temperature-sensitive mutants.

Previous analyses of randomly generated, temperature-sensitive vaccinia virus mutants led to the mapping of DNA synthesis negative complementation groups to the B1R, D4R, D5R, and E9L genes. Evidence from the yeast two-hybrid system that the D4R and D5R proteins can interact with the A20R protein suggested that A20R was also involved in DNA replication. We found that the A20R gene was transcribed early after infection, consistent with such a role. To investigate the function of the A20R protein, targeted mutations were made by substituting alanines for charged amino acids occurring in 11 different clusters. Four mutants were not isolated, suggesting that they were lethal, two mutants exhibited no temperature sensitivity, two mutants exhibited partial temperature sensitivity, and two mutants formed no plaques or infectious virus at 39 degrees C. The two mutants with stringent phenotypes were further characterized. Temperature shift-up experiments indicated that the crucial period was between 6 and 12 h after infection, making it unlikely that the defect was in virus entry, early gene expression, or a late stage of virus assembly. Similar patterns of metabolically labeled viral early proteins were detected at permissive and nonpermissive temperatures, but one mutant showed an absence of late proteins under the latter conditions. Moreover, no viral DNA synthesis was detected when cells were infected with either stringent mutant at 39 degrees C. The previous yeast two-hybrid analysis together with the present characterization of A20R temperature-sensitive mutants suggested that the A20R, D4R, and D5R proteins are components of a multiprotein DNA replication complex.

Amino Acid Sequence↗

PubMed2001

Elicitation of high-frequency cytotoxic T-lymphocyte responses against both dominant and subdominant simian-human immunodeficiency virus epitopes by DNA vaccination of rhesus monkeys.

Increasing evidence suggests that the generation of cytotoxic T-lymphocyte (CTL) responses specific for a diversity of viral epitopes will be needed for an effective human immunodeficiency virus type 1 (HIV-1) vaccine. Here, we determine the frequencies of CTL responses specific for the simian immunodeficiency virus Gag p11C and HIV-1 Env p41A epitopes in simian-human immunodeficiency virus (SHIV)-infected and vaccinated rhesus monkeys. The p11C-specific CTL response was high frequency and dominant and the p41A-specific CTL response was low frequency and subdominant in both SHIV-infected monkeys and in monkeys vaccinated with recombinant modified vaccinia virus Ankara vectors expressing these viral antigens. Interestingly, we found that plasmid DNA vaccination led to high-frequency CTL responses specific for both of these epitopes. These data demonstrate that plasmid DNA may be useful in eliciting a broad CTL response against multiple epitopes.

AIDS Vaccines↗

PubMed2001

Regulation of viral intermediate gene expression by the vaccinia virus B1 protein kinase.

The B1 gene of vaccinia virus encodes a serine/threonine protein kinase that is expressed early after infection. Under nonpermissive conditions, temperature-sensitive mutants (ts2 and ts25) that map to B1 fail to efficiently replicate viral DNA. Our goal was to extend studies on the function of B1 by determining if the kinase is required for intermediate or late gene expression, two events that ordinarily depend on viral DNA replication. First, we established that early viral gene expression occurred at the nonpermissive temperature. By using a transfection procedure that circumvents the viral DNA replication requirement, we found that reporter genes regulated by an intermediate promoter were transcribed only under conditions permissive for expression of active B1. To assay late gene expression, the T7 RNA polymerase gene was inserted into the genome of ts25 to form ts25/T7. A DNA replication-independent late gene transcription system was established by cotransfecting plasmids containing T7 promoter-driven late gene transcription factors and a late promoter reporter gene into ts25/T7-infected cells. Late genes, unlike intermediate genes, were expressed at the nonpermissive temperature. Last, we showed that overexpression of B1 stimulated intermediate but inhibited late gene expression in cells infected with wild-type virus.

Animals↗

PubMed2000

Vaccinia virus E10R protein is associated with the membranes of intracellular mature virions and has a role in morphogenesis.

This study provides the initial biochemical, microscopic, and genetic characterization of the product of the vaccinia virus E10R gene, which belongs to the ERV1/ALR family of eukaryotic proteins, is conserved in all poxviruses and has homologs in other cytoplasmic DNA viruses. DNA encoding a short epitope tag was appended to the C-terminus of the 95-amino-acid open-reading frame without affecting virus reproduction. The E10R protein was synthesized after DNA replication and was associated with purified intracellular mature virions (IMV), from which it could be extracted with a nonionic detergent. Antibody to the tag decorated the surface of IMV, consistent with the anchorage of the E10R protein to the membrane via its hydrophobic N-terminus. Immunoelectron microscopy revealed that the E10R protein was associated with crescent membranes, immature virions, IMV, and extracellular particles. To investigate the role of E10R in the virus life cycle, we constructed an inducer-dependent null mutant. In the absence of inducer, the formation of infectious virus was severely inhibited and electron microscopy revealed an assembly block with accumulation of crescent membranes and immature virions. Cysteines 43 and 46, comprising a putative redox motif common to all poxvirus E10R homologs, were essential for complementation of the mutant virus by transfected E10R DNA.

Animals↗

PubMed2000

A viral member of the ERV1/ALR protein family participates in a cytoplasmic pathway of disulfide bond formation.

Proteins of the ERV1/ALR family are encoded by all eukaryotes and cytoplasmic DNA viruses for which substantial sequence information is available. Nevertheless, the roles of these proteins are imprecisely known. Multiple alignments of ERV1/ALR proteins indicated an invariant C-X-X-C motif, but no similarity to the thioredoxin fold was revealed by secondary structure predictions. We chose a virus model to investigate the role of these proteins as thiol oxidoreductases. When cells were infected with a mutant vaccinia virus in which the E10R gene encoding an ERV1/ALR family protein was repressed, the disulfide bonds of three other viral proteins-namely, the L1R and F9L proteins and the G4L glutaredoxin-were completely reduced. The same outcome occurred when Cys-43 or Cys-46, the putative redox cysteines of the E10R protein, was mutated to serine. These two cysteines were disulfide bonded during a normal virus infection but not if the synthesis of other viral late proteins was inhibited or the E10R protein was expressed by itself in uninfected cells, suggesting a requirement for an upstream viral thiol oxidoreductase. Remarkably, the cysteine-containing domains of the E10R and L1R viral membrane proteins and the glutaredoxin are in the cytoplasm, in which assembly of vaccinia virions occurs, rather than in the oxidizing environment of the endoplasmic reticulum. These data indicated a viral pathway of disulfide bond formation in which the E10R protein has a central role. By extension, the ERV1/ALR family may represent a ubiquitous class of cellular thiol oxidoreductases that interact with glutaredoxins or thioredoxins.

Amino Acid Sequence↗

PubMed2000

Evaluation of recombinant vaccinia virus--measles vaccines in infant rhesus macaques with preexisting measles antibody.

Immunization of newborn infants with standard measles vaccines is not effective because of the presence of maternal antibody. In this study, newborn rhesus macaques were immunized with recombinant vaccinia viruses expressing measles virus hemagglutinin (H) and fusion (F) proteins, using the replication-competent WR strain of vaccinia virus or the replication-defective MVA strain. The infants were boosted at 2 months and then challenged intranasally with measles virus at 5 months of age. Some of the newborn monkeys received measles immune globulin (MIG) prior to the first immunization, and these infants were compared to additional infants that had maternal measles-neutralizing antibody. In the absence of measles antibody, vaccination with either vector induced neutralizing antibody, cytotoxic T cell (CTL) responses to measles virus and protection from systemic measles infection and skin rash. The infants vaccinated with the MVA vector developed lower measles-neutralizing antibody titers than those vaccinated with the WR vector, and they sustained a transient measles viremia upon challenge. Either maternal antibody or passively transferred MIG blocked the humoral response to vaccination with both WR and MVA, and the frequency of positive CTL responses was reduced. Despite this inhibition of vaccine-induced immunity, there was a reduction in peak viral loads and skin rash after measles virus challenge in many of the infants with preexisting measles antibody. Therefore, vaccination using recombinant vectors such as poxviruses may be able to prevent the severe disease that often accompanies measles in infants.

Animals↗

PubMed2000

Bacterial-type DNA holliday junction resolvases in eukaryotic viruses.

Homologous DNA recombination promotes genetic diversity and the maintenance of genome integrity, yet no enzymes with specificity for the Holliday junction (HJ)-a key DNA recombination intermediate-have been purified and characterized from metazoa or their viruses. Here we identify critical structural elements of RuvC, a bacterial HJ resolvase, in uncharacterized open reading frames from poxviruses and an iridovirus. The putative vaccinia virus resolvase was expressed as a recombinant protein, affinity purified, and shown to specifically bind and cleave a synthetic HJ to yield nicked duplex molecules. Mutation of either of two conserved acidic amino acids abrogated the catalytic activity of the A22R protein without affecting HJ binding. The presence of bacterial-type enzymes in metazoan viruses raises evolutionary questions.

Amino Acid Sequence↗

PubMed2000

Immunization of rhesus monkeys with a recombinant of modified vaccinia virus Ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge.

Dengue epidemics increasingly pose a public health problem in most countries of the tropical and subtropical areas. Despite decades of research, development of a safe and effective live dengue virus vaccine is still at the experimental stage. To explore an alternative vaccine strategy, we employed the highly attenuated, replication-deficient modified vaccinia Ankara (MVA) as a vector to construct recombinants for expression of the major envelope glycoprotein of one or more dengue virus serotypes. MVA recombinants expressing the highly immunogenic C-terminally truncated dengue type 2 virus (DEN2) or dengue type 4 virus (DEN4) envelope protein (E), approx. 80% of the full-length, were evaluated for their protective immunity in animal models. Each of these recombinants elicited an elevated antibody response to DEN2 or DEN4 E in mice following the booster inoculation, as detected by radio-immunoprecipitation. Recombinant MVA-DEN2 80%E, but not MVA-DEN4 80%E, induced a neutralizing antibody response. The MVA-DEN2 80%E recombinant was chosen to further evaluate its ability to induce resistance to wild type DEN2 challenge in monkeys. Monkeys immunized twice with recombinant MVA-DEN2 80%E developed a low to moderate antibody response and were partially protected against DEN2 challenge, as determined by the viremia pattern. Importantly, the subsequent study showed that all four monkeys immunized with the recombinant in a three dose schedule developed an increased level of antibodies and were completely protected against DEN2 challenge. The potential efficacy of recombinant MVA-DEN2 80%E to protect primates against dengue infection suggests that construction and evaluation of MVA recombinants expressing other serotypes of dengue virus E for use in a tetravalent vaccine strategy might be warranted.

Animals↗

PubMed2000

Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen.

The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispecific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine to induce CTL responses against a single SIVgag CTL epitope. As assessed using both 51Cr release assays and tetramer staining of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased p11C, C-->M (CTPYDINQM)-specific CD8+ T lymphocyte responses in six of six Mamu-A*01+ rhesus macaques. Tetramer staining of fresh, unstimulated PBMC from two of the DNA-vaccinated animals indicated that as much as 0.4% of all CD3+/CD8alpha+ T lymphocytes were specific for the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL epitope further boosted these responses, such that 0.8-20.0% of CD3+/CD8alpha+ T lymphocytes in fresh, unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays confirmed this high frequency of Ag-specific cells, and intracellular IFN-gamma staining demonstrated that the majority of these cells produced IFN-gamma after peptide stimulation. Moreover, direct ex vivo SIV-specific cytotoxic activity could be detected in PBMC from five of the six DNA/MVA-vaccinated animals, indicating that this epitope-based DNA prime/MVA boost regimen represents a potent method for inducing high levels of functionally active, Ag-specific CD8+ T lymphocytes in non-human primates.

Adjuvants, Immunologic↗

PubMed2000

Genome-wide analysis of vaccinia virus protein-protein interactions.

To detect interactions between proteins of vaccinia virus, we carried out a comprehensive two-hybrid analysis to assay every pairwise combination. We constructed an array of yeast transformants that contained each of the 266 predicted viral ORFs as Gal4 activation domain hybrid proteins. The array was individually mated to transformants containing each ORF as a Gal4-DNA-binding domain hybrid, and diploids expressing the two-hybrid reporter gene were identified. Of the approximately 70,000 combinations, we found 37 protein-protein interactions, including 28 that were previously unknown. In some cases, e.g., late transcription factors, both proteins were known to have related roles although there was no prior evidence of physical associations. For some other interactions, neither protein had a known role. In the majority of cases, however, one of the interacting proteins was known to be involved in DNA replication, transcription, virion structure, or host evasion, thereby providing a clue to the role of the other uncharacterized protein in a specific process.

Base Sequence↗

PubMed2000

Chemical and enzymatic stability as well as transport properties of a Leu-enkephalin analogue and ester prodrugs thereof.

The Leu-enkephalin analogue (Tyr-D-Ala-Gly-Phe-Leu-NH(2)) was synthesized together with three esters prodrugs hereof. The prodrugs synthesized were the O-acetyl, O-propionyl and O-pivaloyl99%) and in good yields (60-75%). The chemical and enzymatic stability of the prodrugs has been investigated in detail. The prodrugs studied are quite chemically stable and the degradation of the prodrugs follows the pattern previously shown for similar esters (U-shaped pH-profile; maximal stability at pH 4-5). The prodrugs are degraded quantitatively in plasma to the parent peptide with half-lives in the range 2.9 min-2.6 h. Type B esterases were shown to be involved in the degradation as the half-lives increased in the presence of paraoxon. No significant stabilization was seen in 10% porcine gut homogenate. Half-lives in the same order were seen for the analogue and the prodrugs in pure Leucine aminopeptidase solution. The analogue was stable in Carboxypeptidase A solution whereas a faster degradation of the prodrugs was seen in this media. Furthermore the transport properties of the compounds has been studied. A P(app) value of 0.284x10(-6) cm/s for the analogue was obtained for the transport across Caco-2 cell monolayers in the BL-AP direction. The P(app) values were increased by a factor of 2, 7 and 18 for the acetyl-, propionyl- and pivaloylprodrug. The increase could be explained by higher lipofilicities of the prodrugs compared to the analogue.

Biological Transport↗

PubMed2000

Immune-defense molecules of molluscum contagiosum virus, a human poxvirus.

Molluscum contagiosum virus encodes more than 150 proteins including some involved in host interactions that might contribute to prolonged viral replication in the skin. These include homologs of a selenocysteine-containing glutathione peroxidase, a death effector domain protein, a chemokine, a major histocompatibility complex class I molecule and an interleukin-18-binding protein.

Humans↗

PubMed2000

The human immunodeficiency virus type 1 gp120 V2 domain mediates gp41-independent intersubunit contacts.

The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.

Cell Line↗

PubMed2000

Characterization of the vaccinia virus H3L envelope protein: topology and posttranslational membrane insertion via the C-terminal hydrophobic tail.

The vaccinia virus H3L open reading frame encodes a 324-amino-acid immunodominant membrane component of virus particles. Biochemical and microscopic studies demonstrated that the H3L protein was expressed late in infection, accumulated in the cytoplasmic viral factory regions, and associated primarily with amorphous material near immature virions and with intracellular virion membranes. Localization of the H3L protein on the surfaces of viral particles and anchorage via the hydrophobic tail were consistent with its extraction by NP-40 in the absence of reducing agents, its trypsin sensitivity, its reactivity with a membrane-impermeable biotinylation reagent, and its immunogold labeling with an antibody to a peptide comprising amino acids 247 to 259. The H3L protein, synthesized in a coupled in vitro transcription/translation system, was tightly anchored to membranes as determined by resistance to Na(2)CO(3) (pH 11) extraction and cytoplasmically oriented as shown by sensitivity to proteinase K digestion. Further studies demonstrated that membrane insertion of the H3L protein occurred posttranslationally and that the C-terminal hydrophobic domain was necessary and sufficient for this to occur. These data indicated that the H3L protein is a member of the C-terminal anchor family and supported a model in which it is synthesized on free ribosomes and inserts into the membranes of viral particles during their maturation.

Animals↗

PubMed2000

Effects of deletion or stringent repression of the H3L envelope gene on vaccinia virus replication.

The C-terminal membrane anchor protein encoded by the H3L open reading frame of vaccinia virus is located on the surfaces of intracellular mature virions. To investigate the role of the H3L protein, we constructed deletion (vH3Delta) and inducible (vH3i) null mutants. The H3L protein was not detected in lysates of cells infected with vH3Delta or vH3i in the absence of inducer. Under these conditions, plaques were small and round instead of large and comet shaped, indicative of decreased virus replication or cell-to-cell spread. The mutant phenotype was correlated with reduced yields of infectious intra- and extracellular virus in one-step growth experiments. The defect in vH3i replication could not be attributed to a role of the H3L protein in virus binding, internalization, or any event prior to late gene expression. Electron microscopic examination of cells infected with vH3Delta or vH3i in the absence of inducer revealed that virion assembly was impaired, resulting in a high ratio of immature to mature virus forms with an accumulation of crescent membranes adjacent to granular material and DNA crystalloids. The absence of the H3L protein did not impair the membrane localization of virion surface proteins encoded by the A27L, D8L, and L1R genes. The wrapping of virions and actin tail formation were not specifically blocked, but there was an apparent defect in low-pH-mediated syncytium formation that could be attributed to decreased virus particle production. The phenotypes of the H3L deletion and repression mutants were identical to each other but differed from those produced by null mutations of genes encoding other vaccinia virus membrane components.

Animals↗

PubMed2000

A glutaredoxin, encoded by the G4L gene of vaccinia virus, is essential for virion morphogenesis.

Vaccinia virus encodes two glutaredoxins, O2L and G4L, both of which exhibit thioltransferase and dehydroascorbate reductase activities in vitro. Although O2L was previously found to be dispensable for virus replication, we now show that G4L is necessary for virion morphogenesis. RNase protection and Western blotting assays indicated that G4L was expressed at late times after infection and was incorporated into mature virus particles. Attempts to isolate a mutant virus with a deleted G4L gene were unsuccessful, suggesting that the protein was required for virus replication. This interpretation was confirmed by the construction and characterization of a conditional lethal recombinant virus with an inducible copy of the G4L gene replacing the original one. Expression of G4L was proportional to the concentration of inducer, and the amount of glutaredoxin could be varied from barely detectable to greater than normal amounts of protein. Immunogold labeling revealed that the induced G4L protein was associated with immature and mature virions and adjacent cytoplasmic depots. In the absence of inducer, the production of infectious virus was severely inhibited, though viral late protein synthesis appeared unaffected except for decreased maturation-dependent proteolytic processing of certain core components. Electron microscopy of cells infected under nonpermissive conditions revealed an accumulation of crescent membranes on the periphery of electron-dense globular masses but few mature particles. We concluded that the two glutaredoxin homologs encoded by vaccinia virus have different functions and that G4L has a role in virion morphogenesis, perhaps by acting as a redox protein.

Gene Expression Regulation, Viral↗