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Biomedical subjects

B Moss

Publications and source records attributed to B Moss.

At least 145 records · Page 8Linked to original sources
PubMed1994

Antigenic implications of human immunodeficiency virus type 1 envelope quaternary structure: oligomer-specific and -sensitive monoclonal antibodies.

A majority of monoclonal antibodies (mAbs) raised against soluble oligomeric human immunodeficiency virus type 1 isolate IIIB (HIV-1IIIB) envelope (env) glycoprotein reacted with conformational epitopes within the gp120 or gp41 subunits. Of 35 mAbs directed against gp41, 21 preferentially reacted with oligomeric env. A subset of these mAbs reacted only with env oligomers (oligomer-specific mAbs). In contrast, only 1 of 27 mAbs directed against the gp120 subunit reacted more strongly with env oligomers than with monomers, and none were oligomer-specific. However, 50% of anti-gp120 mAbs preferentially recognized monomeric env, suggesting that some epitopes in gp120 are partially masked or altered by intersubunit contacts in the native env oligomer. Two mAbs to oligomer-dependent epitopes in gp41 neutralized HIV-1IIIB and HIV-1SF2, and binding of these mAbs to env was blocked by preincubation with HIV-1-positive human serum. Thus, immunization with soluble, oligomeric env elicits antibodies to conserved, conformational epitopes including a newly defined class of neutralizing antibodies that bind to oligomer-specific epitopes in gp41, and may also minimize the production of antibodies that preferentially react with monomeric env protein.

Animals↗
PubMed1994

Vaccinia virus expresses a novel profilin with a higher affinity for polyphosphoinositides than actin.

We expressed in Escherichia coli the vaccinia virus gene for a protein similar to vertebrate profilins, purified the recombinant viral profilin, and characterized its interactions with actin and polyphosphoinositides. Compared with cellular profilins, this viral profilin has a low affinity (Kd > or = 35 microM) for human platelet actin monomers, a weak effect on the exchange of the nucleotide bound to the actin, and no detectable affinity for poly(L-proline). Vaccinia profilin binds to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate in micelles and large unilamellar vesicles, but not to phosphatidylserine or phosphatidylcholine. Kinetic analysis by surface plasmon resonance showed that both vaccinia and amoeba profilins bind slowly to polyphosphoinositides, with association rate constants in the range of (1-4) x 10(4) M-1 s-1. The higher affinity of vaccinia profilin for polyphosphoinositides (Kd = 0.2-8.5 microM) than for actin or poly(L-proline) and the concentration of vaccinia profilin expressed in infected HeLa cells (approximately 20 microM) suggest that vaccinia profilin binds preferentially to PIP and PIP2 in vivo. Consequently, vaccinia profilin is more likely to influence phosphoinositide metabolism than actin assembly. Expression of 7-105 microM vaccinia profilin in a Saccharomyces cerevisiae profilin null mutant did not rescue the null phenotype, so that the affinity of vaccinia profilin for phosphoinositides alone is insufficient for normal profilin function in yeast.

Actins↗
PubMed1994

Mutational analysis of a multifunctional protein, with mRNA 5' cap-specific (nucleoside-2'-O-)-methyltransferase and 3'-adenylyltransferase stimulatory activities, encoded by vaccinia virus.

The vaccinia virus-encoded protein VP39 is a poly(A) polymerase subunit that stimulates the formation of long poly(A) tails as well as a cap-specific mRNA (nucleoside-2'-O-)-methyltransferase. We have carried out mutagenesis studies aimed at locating regions of VP39 which are important for these activities. The open reading frame encoding VP39 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. The affinity-purified protein had both mRNA modification activities, before and after removal of the glutathione S-transferase domain. Truncation, charge cluster-->Ala scanning, and Cys-->Ser substitution mutations of VP39 were made, and the proteins were synthesized, purified, and analyzed. Deletion of the RNA binding domain, experimentally localized within the carboxyl-terminal 112 amino acids, resulted in the loss of both mRNA modification activities. Eleven of the 21 charge cluster-->Ala mutated proteins had low to nondetectable methyltransferase activity. Four of those 11 also lacked adenylyl-transferase stimulatory function, whereas the remainder had amino acid substitutions that selectively affected methyltransferase activity. However, no mutated proteins lacking adenylyltransferase stimulatory function but possessing methyltransferase activity were isolated by the procedures used. Neither of the 2 cysteine residues in VP39 was necessary for either mRNA modification activity.

Amino Acid Sequence↗
PubMed1994

Purification and identification of a vaccinia virus-encoded intermediate stage promoter-specific transcription factor that has homology to eukaryotic transcription factor SII (TFIIS) and an additional role as a viral RNA polymerase subunit.

Enzymes and factors, required for in vitro transcription of templates regulated by vaccinia virus intermediate stage promoters, are present in HeLa cells infected with vaccinia virus in the presence of an inhibitor of DNA replication. Previous studies indicated that in vitro transcription could be reconstituted by adding a partially purified transcription factor to the viral RNA polymerase and capping enzyme. By using an independent purification procedure, we isolated two vaccinia virus intermediate were necessary for transcription of several different intermediate stage promoter templates but not for early or late stage promoter templates. VITF-1 was purified to homogeneity, and the sequences of two tryptic peptides were mapped to the fourth open reading frame within the HindIII E fragment (E4L) of the vaccinia virus genome, which had previously been shown to encode an RNA polymerase subunit of 30 kDa (RPO30) with homology to eukaryotic transcription elongation factor SII. Co-chromatography of VITF-1 with the E4L-derived protein was demonstrated using specific antiserum. In addition, transcriptionally active recombinant VITF-1 was made by expressing the E4L open reading frame in Escherichia coli. Thus, E4L encodes a multifunctional protein, serving as a RNA polymerase subunit and a stage-specific transcription factor. The stepwise binding of capping enzyme, VITF-1, and VITF-2 to a DNA/viral RNA polymerase complex was demonstrated.

Amino Acid Sequence↗
PubMed1994

A cellular factor is required for transcription of vaccinia viral intermediate-stage genes.

The cytoplasmic location of vaccinia virus replication and evidence that the multisubunit DNA-dependent RNA polymerase, early and late stage transcription factors, capping and methylating enzymes, and poly(A) polymerase are virus encoded raised the possibility that all of the proteins needed for viral mRNA synthesis are of viral origin. Previous studies showed that four components from infected cells, the viral RNA polymerase and capping enzyme and two factors called vaccinia virus intermediate transcription factors (VITFs) 1 and 2, can reconstitute transcription of vaccinia virus intermediate-stage genes in vitro. Here, we demonstrate that VITF-2 can be isolated from the nuclei of uninfected HeLa cells as well as from the cytoplasm of infected cells. The proteins with VITF-2 activity from uninfected and infected cells cochromatographed and cosedimented, suggesting that they are identical. VITF-2 activity was found in extracts of other uninfected human and monkey cells but not in nonpermissive Trichoplusia ni insect cells or in conditionally permissive rabbit kidney 13 cells. VITF-2 activity was present, however, in a permissive line of rabbit kidney 13 cells that had been stably transfected with the vaccinia virus K1L host range gene. We suggest that the VITF-2 level acts as a gauge of the permissive state of the cell and thereby regulates the length of the early prereplicative phase of the infection.

Cell Cycle Proteins↗
PubMed1994

RNA polymerase-associated protein Rap94 confers promoter specificity for initiating transcription of vaccinia virus early stage genes.

The association of a 94,000-Da viral polypeptide, called Rap94, with 30-40% of the multisubunit DNA-dependent RNA polymerase molecules purified from infectious vaccinia virus particles was established by immunoaffinity chromatography. The submolar amount of Rap94, relative to RNA polymerase, was confirmed by quantitative immunoblotting of total virion extracts. Only the RNA polymerase molecules containing Rap94 could functionally interact with VETF, the vaccinia virus early transcription factor, to transcribe a double-stranded DNA template regulated by a viral early stage promoter. Rap94 was required for the synthesis of short oligoribonucleotides and for the formation of stable ternary transcription complexes. With a nonspecific single-stranded DNA template, however, the Rap94-deficient polymerase had greater catalytic activity than the Rap94-containing polymerase. These data support a model in which Rap94 confers specificity to the RNA polymerase for promoters of early stage genes.

Base Sequence↗
PubMed1994

A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus.

The immunogenicity of a recombinant virus derived from modified vaccinia virus Ankara (MVA), a host range-restricted, highly attenuated and safety-tested strain, was investigated. Plasmid transfer vectors that provide strong synthetic early/late promoters for the simultaneous expression of two genes as well as a transient or stable selectable marker and flanking sequences for homologous recombination with the MVA genome were constructed. A recombinant MVA containing influenza virus haemagglutinin and nucleoprotein genes was isolated in avian cells and shown to express both proteins efficiently upon infection of human or mouse cells in which abortive replication occurs. Mice, inoculated by various routes with recombinant MVA, produced antibody and cytotoxic T-lymphocyte responses to influenza virus proteins and were protected against a lethal influenza virus challenge as effectively as mice immunized with a recombinant derived from the replication-competent WR strain of vaccinia virus.

Animals↗
PubMed1994

Removal of the cleavage site of recombinant feline immunodeficiency virus envelope protein facilitates incorporation of the surface glycoprotein in immune-stimulating complexes.

Recombinant vaccinia viruses were constructed that expressed the complete env gene of feline immunodeficiency virus with or without the nucleotide sequence encoding the cleavage site between the surface (SU) protein and the transmembrane (TM) protein. The removal of this cleavage site resulted in the expression of a 150K protein that was processed into a 130K protein and was not cleaved into the SU and the TM proteins. Removal of the cleavage site also facilitated incorporation of the SU protein in immune-stimulating complexes (iscoms). Antibody responses to both an SU and a TM peptide representing two immunodominant B cell epitopes were measured. These were higher in cats immunized with iscoms prepared from the cleavage site-deleted envelope protein than in cats immunized with iscoms prepared from the native envelope protein or immunized with the envelope protein and the adjuvant Quil A.

Animals↗
PubMed1994

Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.

The cytoplasmic assembly of vaccinia virus is reversibly blocked by the antibiotic rifampin, leading to the accumulation of partially membrane-delineated rifampin bodies in infected cells. Rifampin-resistant vaccinia virus mutants have point mutations in the D13L gene, which is controlled by a late promoter and expresses a 65-kDa protein, designated p65. To further characterize the mechanism of rifampin inhibition and the function of p65 in virus assembly, we raised antibodies to this protein. Immunoreactive p65 was expressed at late times of infection, and neither its expression nor its turnover was affected by rifampin. Virus-associated p65 could be extracted only with denaturing detergents from purified virions, suggesting that it is an integral viral component. Immunofluorescence studies showed that p65 is localized to the sites of virus assembly. Also, immunoelectron microscopy showed p65 to be associated with viral crescents as well as spherical, immature virions, in both cases predominantly on the inner or concave surface. In the presence of rifampin, p65 was found in large, cytoplasmic inclusion bodies that were distinct from rifampin bodies. The rifampin bodies themselves were labeled with p65 antibodies only after reversal of the rifampin block, predominantly on the viral crescents which rapidly formed following removal of the drug. We propose that p65 functions as an internal scaffold in the formation of viral crescents and immature virions, analogously to the matrix proteins of other viruses.

Amino Acid Sequence↗
PubMed1994

Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles requires RAP94, an RNA polymerase-associated protein.

When expression of the vaccinia virus gene encoding RAP94 (a protein that is associated with the viral multisubunit RNA polymerase and confers transcriptional specificity for early promoters) was repressed, the infectious virus yield was reduced by more than 99%. Nevertheless, intermediate- and late-stage viral gene expression and formation of ultrastructurally mature, membrane-enveloped virions occurred under the nonpermissive conditions. The RAP94-deficient particles contained the viral genome, structural proteins, early transcription factor, and certain enzymes but, unlike normal virions, had low or undetectable amounts of the viral RNA polymerase, capping enzyme/termination factor, poly(A) polymerase, DNA-dependent ATPase, RNA helicase, and topoisomerase. The presence of these viral enzymes in the cytoplasm indicated that RAP94 is required for targeting a complex of functionally related proteins involved in the biosynthesis of mRNA.

Base Sequence↗
PubMed1994

Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities.

We synthesized and purified a recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein, lacking the gp120/gp41 cleavage site as well as the transmembrane domain, that is secreted principally as a stable oligomer. Mice were immunized with separated monomeric and oligomeric HIV-1 Env glycoproteins to analyze the repertoire of antibody responses to the tertiary and quaternary structure of the protein. Hybridomas were generated and assayed for reactivity by immunoprecipitation of nondenatured Env protein. A total of 138 monoclonal antibodies (MAbs) were generated and cloned, 123 of which were derived from seven animals immunized with oligomeric Env. Within this group, a significant response was obtained against the gp41 ectodomain; 49 MAbs recognized epitopes in gp41, 82% of which were conformational. The influence of conformation on gp120 antigenicity was less pronounced, with 40% of the anti-gp120 MAbs binding to conformational epitopes, many of which blocked CD4 binding. Surprisingly, less than 7% of the MAbs derived from mice immunized with oligomeric Env recognized the V3 loop. In addition, MAbs to linear epitopes in the C-terminal domain of gp120 were not obtained, suggesting that this region of the protein may be partially masked in the oligomeric molecule. A total of 15 MAbs were obtained from two mice immunized with monomeric Env. Nearly half of these recognized the V3 loop, suggesting that this region may be a less predominant epitope in the context of oligomeric Env than in monomeric protein. Thus, immunization with oligomeric Env generates a large proportion of antibodies to conformational epitopes in both gp120 and gp41, many of which may be absent from monomeric Env.

Amino Acid Sequence↗
PubMed1994

Modification of the cascade model for regulation of vaccinia virus gene expression: purification of a prereplicative, late-stage-specific transcription factor.

In vivo and in vitro studies have provided evidence that vaccinia virus late gene transcription factors are intermediate gene products synthesized exclusively after DNA replication. Here, we describe an additional transcription factor (P3 factor) that stimulates late gene transcription between 10- and 40-fold but is made in the absence of viral DNA replication. P3 factor activity was not detected either in uninfected cells or in purified virions. A > 1,500-fold purification of P3 factor was achieved by column chromatography of cytoplasmic extracts prepared from cells infected with vaccinia virus in the presence of a DNA replication inhibitor. P3 factor was stage specific, since it could not substitute for early or intermediate transcription factors. Evidence that late stage-specific transcription factors are made both before and after DNA replication necessitates a modification of the cascade model for vaccinia virus gene regulation.

Gene Expression Regulation, Viral↗
PubMed1994

Stable expression of the vaccinia virus K1L gene in rabbit cells complements the host range defect of a vaccinia virus mutant.

Modified vaccinia virus Ankara (MVA), having acquired genomic deletions during passage in chicken embryo fibroblasts, is highly attenuated and unable to productively infect most mammalian cell lines. Multiplication in rabbit kidney-derived RK13 cells, but not other nonpermissive cells, can be restored by insertion of the vaccinia virus K1L gene into the MVA genome. During nonproductive infection of RK13 cells by MVA, transcription of representative viral early genes was revealed by Northern (RNA) blotting, whereas synthesis of an intermediate mRNA and replication of viral DNA could not be detected. Despite the persistence of viral early mRNA for at least several hours, synthesis of virus-induced polypeptides occurred only during the first hour and was followed by abrupt inhibition of all protein synthesis. Transfection of RK13 cells with a eukaryotic expression plasmid that contained the K1L gene allowed MVA infection to proceed to late stages of viral protein synthesis. Moreover, RK13 cell lines that stably expressed the K1L gene were permissive for MVA as well as a K1E deletion mutant of the WR strain of vaccinia virus. This is the first description of the complementation of a poxvirus mutant by cells that stably express a viral gene.

Animals↗
PubMed1994

Ordered assembly of a functional preinitiation transcription complex, containing vaccinia virus early transcription factor and RNA polymerase, on an immobilized template.

A functional preinitiation transcription complex was formed by incubating vaccinia virus early transcription factor VETF and RAP94+ RNA polymerase with an early promoter template immobilized on paramagnetic particles. A preferred order of assembly, VETF followed by RNA polymerase, was demonstrated by stepwise addition experiments. ATP was unnecessary for preinitiation transcription complex formation, but divalent cations were required specifically for the association of RNA polymerase.

Base Sequence↗