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Biomedical subjects

B Moss

Publications and source records attributed to B Moss.

At least 361 records · Page 20Linked to original sources
PubMed1986

Resolution of linear minichromosomes with hairpin ends from circular plasmids containing vaccinia virus concatemer junctions.

The junctions, separating unit-length genomes in intracellular concatemeric forms of vaccinia virus DNA, are duplex copies of the hairpin loops that form the ends of mature DNA molecules present in infectious virus particles. Circular E. coli plasmids with palindromic junction fragments were replicated in vaccinia virus-infected cells and resolved into linear minichromosomes with vector DNA in the center and vaccinia virus DNA hairpins at the two ends. Resolution did not occur when the concatemer joint was less than 250 bp or when plasmids were transfected into uninfected cells, indicating requirements for a specific DNA structure and viral trans-acting factors. These studies indicate that concatemers can serve as replicative intermediates and account for the generation of flip-flop sequence variation of the hairpins at the ends of the mature vaccinia virus genome.

Animals↗
PubMed1986

Recognition of cloned vesicular stomatitis virus internal and external gene products by cytotoxic T lymphocytes.

It has generally been assumed that most if not all CTL specific for vesicular stomatitis virus (VSV)-infected cells recognize the viral glycoprotein (G), an integral membrane protein abundantly expressed on infected cell surfaces. Using recombinant vaccinia viruses containing copies of cloned VSV genes to examine CTL recognition of VSV, we have confirmed that G is recognized by VSV-specific CTL. More interestingly, however, we have also found that nucleocapsid protein (N), an internal virion protein, can be detected on infected cell surfaces using mAb, and serves as a major target antigen for VSV-specific CTL. In contrast to the highly serotype-specific recognition of G, N is recognized by a major population of CTL able to lyse cells infected with either the Indiana or New Jersey VSV serotypes. Using target cells expressing a cloned MHC class I gene, we could directly show that CTL recognition of N occurs in the context of the MHC Ld molecule.

Animals↗
PubMed1986

Surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors.

In polarized epithelial cells, maturation sites of enveloped viruses that form by budding at cell surfaces are restricted to particular membrane domains. Recombinant vaccinia viruses were used to investigate the sites of surface expression in the Madin-Darby canine kidney (MDCK) cell line of the hemagglutinin (HA) of influenza virus, the G glycoprotein of vesicular stomatitis virus (VSV), and gp70/p15E of Friend murine leukemia virus (MuLV). These glycoproteins could be demonstrated by immunofluorescence on the surfaces of MDCK cells as early as 4 h post-infection. In intact MDCK monolayers, vaccinia recombinants expressing HA produced a pattern of surface fluorescence typical of an apically expressed glycoprotein. In contrast, cells infected with vaccinia recombinants expressing VSV-G or MuLV gp70/p15E exhibited surface fluorescence only when monolayers were treated with EGTA to disrupt tight junctions, as expected of glycoproteins expressed on basolateral surfaces. Immunoferritin labeling in conjunction with electron microscopy confirmed that MDCK cells infected with the HA recombinant exhibited specific labeling of the apical surfaces whereas the VSV-G and MuLV recombinants exhibited the respective antigens predominantly on the basolateral membranes. Quantitation of surface expression by [125I]protein A binding assays on intact and EGTA-treated monolayers confirmed the apical localization of the vaccinia-expressed HA and demonstrated that 95% of the VSV-G and 97% of the MuLV gp70/p15E glycoproteins were localized on the basolateral surfaces. These results demonstrate that glycoproteins of viruses that normally mature at basolateral surfaces of polarized epithelial cells contain all of the structural information required for their directional transport to basolateral plasma membranes.

Animals↗
PubMed1986

Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits.

We have determined the nucleotide sequence of a region of the vaccinia virus genome encoding RNA polymerase subunits of 22 and 147 kDa and have mapped the 5' and 3' ends of the two mRNAs. The predicted amino acid sequence of the vaccinia 147-kDa subunit shows extensive homology with the largest subunit of Escherichia coli RNA polymerase, yeast RNA polymerases II and III, and Drosophila RNA polymerase II. The regions of homology between the five RNA polymerases are subdivided into five separate domains that span most of the length of each. A sixth domain shared by the vaccinia and the eukaryotic polymerases is absent from the E. coli sequence. In all specified regions, the vaccinia large subunit has greater homology with eukaryotic RNA polymerases II and III than with the E. coli polymerase. Vaccinia virus and eukaryotic RNA polymerases may therefore have evolved from a common ancestral gene after the latter diverged from prokaryotes.

Amino Acid Sequence↗
PubMed1986

Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene.

A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker. A single nucleotide substitution changing glycine at position 347 to aspartic acid accounts for the drug resistance of the mutant vaccinia virus. The 5' end of the DNA polymerase mRNA was located 80 base pairs before the methionine codon initiating the open reading frame. Correspondence between the predicted Mr 108,577 polypeptide and the 110,000 purified enzyme indicates that little or no proteolytic processing occurs. Extensive homology, extending over 435 amino acids, was found upon comparing the DNA polymerase of vaccinia virus and DNA polymerase of Epstein-Barr virus. A highly conserved sequence of 14 amino acids in the carboxyl-terminal regions of the above DNA polymerases is also present at a similar location in adenovirus DNA polymerase. This structure, which is predicted to form a turn flanked by beta-pleated sheets, may form part of an essential binding or catalytic site that accounts for its presence in DNA polymerases of poxviruses, herpesviruses, and adenoviruses.

Adenoviridae↗
PubMed1986

Expression of the F glycoprotein of respiratory syncytial virus by a recombinant vaccinia virus: comparison of the individual contributions of the F and G glycoproteins to host immunity.

A cDNA clone representing the mRNA coding sequence of the fusion glycoprotein (F) gene of human respiratory syncytial virus (RSV) was constructed and inserted into the thymidine kinase gene of vaccinia virus (WR strain) under the control of a vaccinia virus promoter. The resulting recombinant vaccinia virus, vaccinia F, expressed the F1 and F2 cleavage products (48 and 20 kDa, respectively) of the F glycoprotein in cell culture. F1 and F2 were indistinguishable from their authentic RSV counterparts with respect to glycosylation, disulfide linkage, electrophoretic mobility, cell-surface expression, and antigenic specificity. Cotton rats infected intradermally with vaccinia F developed a high titer of serum F-specific antibodies, which neutralized infectivity of RSV. This neutralizing antibody response exceeded that induced by infection of the respiratory tract with RSV and was 6-fold higher than that induced by vaccinia G, a recombinant vaccinia virus that expressed the RSV G glycoprotein gene. Immunization with vaccinia F stimulated almost complete resistance to replication of RSV in the lower respiratory tract as well as significant resistance in the upper respiratory tract. The degree of resistance conferred by vaccinia F exceeded that induced by vaccinia G.

Animals↗
PubMed1986

Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.

DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.

Acetyltransferases↗
PubMed1986

Resistance to human respiratory syncytial virus (RSV) infection induced by immunization of cotton rats with a recombinant vaccinia virus expressing the RSV G glycoprotein.

A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed under control of a vaccinia virus promoter and inserted into the thymidine kinase locus of the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and expressed a 93-kDa protein that migrated with the authentic RSV G glycoprotein upon polyacrylamide gel electrophoresis. Glycosylation of the expressed protein and transport to the cell surface were demonstrated in the absence of other RSV proteins. Cotton rats that were inoculated intradermally with the infectious recombinant virus produced serum antibody to the G glycoprotein that neutralized RSV in vitro. Furthermore, the vaccinated animals were resistant to lower respiratory tract infection upon intranasal inoculation with RSV and had reduced titers of RSV in the nose.

Animals↗
PubMed1986

Recognition of influenza A virus nucleoprotein by human cytotoxic T lymphocytes.

A recombinant vaccinia virus (NP-VAC) containing cDNA corresponding to segment 5, the nucleoprotein (NP) gene of influenza A/PR/8/34 virus was used to examine the specificity of human influenza virus immune cytotoxic T lymphocytes (CTL). Effector cell preparations from two donors recognized autologous lymphocytes that had been infected with NP-VAC. Lysis was specific because cells infected with vaccinia virus were not killed and recognition was HLA-restricted. In one donor, the influenza virus-specific CTL response changed with time so that his effector cells no longer recognized autologous lymphocytes infected with NP-VAC. However, a component that was NP-specific remained because these CTL lysed the more sensitive autologous B lymphoblastoid cells that had been infected with NP-VAC. In four other donors, no NP-specific CTL response could be detected using autologous lymphocyte targets. Thus NP, an internal virus protein, is one antigen that is recognized by human influenza A virus-specific CTL, but it is likely that other individual virus components contribute to the total CTL response.

Cytotoxicity, Immunologic↗
PubMed1986

Recognition of cloned influenza virus hemagglutinin gene products by cytotoxic T lymphocytes.

The influenza A virus hemagglutinin (HA) is an integral membrane glycoprotein expressed in large quantities on infected cell surfaces and is known to serve as a target antigen for influenza virus-specific cytotoxic T lymphocytes (CTL). Despite the fact that HAs derived from different influenza A virus subtypes are serologically non-cross-reactive, the HA has been implicated by previous experiments to be a target antigen for the subset of T cells capable of lysing cells infected with any human influenza A subtype (cross-reactive CTL). To directly determine whether the HA is recognized by cross-reactive CTL, we used vaccinia virus recombinants containing DNA copies of the PR8 (A/Puerto Rico/8/34) (H1N1) or JAP (A/JAP/305) (H2N2) HA genes. When these viruses were used to stimulate HA-specific CTL and to sensitize target cells for lysis by HA-specific CTL, we found no evidence for HA recognition by cross-reactive CTL aside from a relatively small degree of cross-reactivity between H1 and H2 HAs. Results of unlabeled target inhibition studies were consistent with the conclusion that the HA is, at most, only a minor target antigen for cross-reactive CTL.

Animals↗
PubMed1986

Herpes simplex virus (HSV)-specific human T-cell clones recognize HSV glycoprotein D expressed by a recombinant vaccinia virus.

Human cytotoxic T-cell (CTL) clones that lyse autologous cells infected with herpes simplex virus (HSV) type 1 or 2 were generated by stimulating lymphocytes with a recombinant vaccinia virus (recombinant vaccinia-gD-1 virus) that expresses HSV type 1 glycoprotein D (gD-1). Furthermore, CTL clones generated with HSV type 1 or with cloned gD-1 lysed autologous cells infected with the recombinant vaccinia-gD-1 virus. Our findings thus showed that gD serves as a target antigen for human CTLs and that a recombinant vaccinia-gD virus activates HSV-specific human CTL.

Antigens, Viral↗
PubMed1986

Multiple 3' ends of mRNA encoding vaccinia virus growth factor occur within a series of repeated sequences downstream of T clusters.

Analysis of the 5' ends of six apparently full-length cloned cDNA copies of the vaccinia virus growth factor gene suggested precise transcriptional initiation at the first purine following a run of five pyrimidines in the noncoding strand. By contrast, the 3' ends exhibited heterogeneity and were distributed over a 46-base-pair region. In each of the six cDNAs, the nucleotide immediately preceding the retained copy of the poly(A) tail corresponded to the first or second T of a TATGT repeat. Clusters of Ts occurred upstream of the 3' ends, but the AATAAA polyadenylation consensus sequence of higher eucaryotes was absent. Determination of the complete sequence of one cDNA revealed exact correspondence with the vaccinia virus growth factor gene, indicating the absence of internal RNA processing.

Base Sequence↗
PubMed1986

Hepatitis B virus large surface protein is not secreted but is immunogenic when selectively expressed by recombinant vaccinia virus.

The envelope region of the hepatitis B virus (HBV) genome contains an open reading frame that begins upstream of the major surface protein gene. The two minor proteins that are initiated within this pre-s segment are immunogenic and may be involved in virus attachment to hepatocytes. We have constructed a recombinant vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus promoter. Cells infected with the recombinant virus synthesized HBV polypeptides of 39 and 42 kilodaltons, corresponding to the unglycosylated and glycosylated forms of LS, respectively. The presence of pre-s epitopes in the 39- and 42-kilodalton polypeptides was demonstrated by binding of antibody prepared against a synthetic peptide. Synthesis of the 42-kilodalton species was specifically inhibited by tunicamycin, suggesting that it is N-glycosylated. Despite apparent glycosylation, LS was not secreted into the medium of infected cells. Nevertheless, rabbits vaccinated with the purified recombinant virus made antibodies that recognized s and pre-s epitopes. Antibody to the NH2 terminus of LS appeared before or simultaneously with antibody that bound to the major surface protein. The additional immunogenicity provided by expression of LS may be advantageous for the development of an HBV vaccine.

Animals↗
PubMed1986

Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment.

The sequence of the 8,600-base-pair HindIII H fragment, located at the center of the vaccinia virus genome, was determined to analyze several late genes. Seven major complete open reading frames (ORFs) and two that started from or continued into adjacent DNA segments were identified. ORFs were closely spaced and present on both DNA strands. Some adjacent ORFs had oppositely oriented overlapping termination codons or contiguous stop and start codons. Nucleotide compositional analysis indicated that the A-T frequency was consistently lowest in the first codon position. The sizes of the polypeptides predicted from the DNA sequence were compared with those determined by polyacrylamide gel electrophoresis of cell-free translation products of mRNAs selected by hybridization to cloned single-stranded DNA segments or synthesized in vitro by bacteriophage T7 RNA polymerase. Six transcripts that initiated within the HindIII H DNA fragment were detected, and of these, four were synthesized only at late times, one was synthesized only early, and one was synthesized early and late. The sites on the genome corresponding to the 5' ends of the transcripts were located by high-resolution nuclease S1 analysis. For late genes, the transcriptional and translational initiation sites mapped within a few nucleotides of each other, and in each case the sequence TAAATGG occurred at the start of the ORF. The extremely short leader and the absence of A or G in the -3 position, relative to the first nucleotide of the initiation codon, distinguishes the majority of vaccinia virus late genes from eucaryotic and vaccinia virus early genes.

Amino Acid Sequence↗
PubMed1986

Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity.

We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens.

Amino Acid Sequence↗
PubMed1985

Vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D prevents latent herpes in mice.

In humans, herpes simplex virus causes a primary infection and then often a latent ganglionic infection that persists for life. Because these latent infections can recur periodically, vaccines are needed that can protect against both primary and latent herpes simplex infections. Infectious vaccinia virus recombinants that contain the herpes simplex virus type 1 (HSV-1) glycoprotein D gene under control of defined early or late vaccinia virus promoters were constructed. Tissue culture cells infected with these recombinant viruses synthesized a glycosylated protein that had the same mass (60,000 daltons) as the glycoprotein D produced by HSV-1. Immunization of mice with one of these recombinant viruses by intradermal, subcutaneous, or intraperitoneal routes resulted in the production of antibodies that neutralized HSV-1 and protected the mice against subsequent lethal challenge with HSV-1 or HSV-2. Immunization with the recombinant virus also protected the majority of the mice against the development of a latent HSV-1 infection of the trigeminal ganglia. This is the first demonstration that a genetically engineered vaccine can prevent the development of latency.

Animals↗