Use of a fluorescent chloramphenicol derivative as a substrate for CAT assays.
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Acetylation↗
Biomedical subjects
D E Hruby
Publications and source records attributed to D E Hruby.
Protection against streptococcal pharyngeal colonization with a vaccinia: M protein recombinant.
Phagocytosis of group A streptococci requires type-specific antibodies directed against the variable determinants of the bacterial surface M protein molecule. As a step toward developing a broadly protective anti-streptococcal vaccine, a vaccinia virus (VV) recombinant was constructed that expresses the conserved region of the structural gene encoding the M6 molecule (VV:M6'). Mice immunized intranasally with the VV:M6' virus showed markedly reduced pharyngeal colonization by streptococci after intranasal and oral challenge with these bacteria. M protein-specific serum immunoglobulin G was significantly elevated in vaccinated animals and absent in controls. A similar approach may prove useful for the identification of protective determinants present on other bacterial and viral pathogens.
Animals↗
Expression vector pT7:TKII for the synthesis of authentic biologically active RNA encoding vaccinia virus thymidine kinase.
A transcription vector, pT7: TKII, was constructed by a novel application of the polymerase chain reaction. Chimeric oligodeoxynucleotides were used to direct the synthesis of a DNA fragment which consisted of a truncated bacteriophage T7 promoter element fused to the vaccinia virus (VV) thymidine kinase gene (tk). This fragment was cloned into a pUC118 plasmid and sequenced to ensure no mutations had occurred during its synthesis. When linearized at the 3' end of the VV tk gene at the BamHI site located in the polylinker region of the vector, pT7:TKII was efficiently transcribed by T7 RNA polymerase into a 595 nucleotide transcript whose 5' end was identical to that found on authentic nascent VV tk mRNA. When translated in a rabbit reticulocyte lysate system, the synthetic VV tk RNA was shown to be biologically active in that it directed the synthesis of a 20-kDa protein which assembled into an enzymatically active 80-kDa tetrameric complex which was indistinguishable from VV thymidine kinase (TK) enzyme isolated from VV-infected cells. The pT7:TKII vector provides a powerful approach with which: (i) to investigate the translational and posttranslational regulation of the VV tk gene; (ii) to use directed genetics to identify potential cis-acting regulatory sequences or structures present within the VV tk RNA; and (iii) to apply protein engineering procedures to identify the catalytic, allosteric and subunit interactive domains of the VV TK enzyme. As an example, the translational effects of adding a m7G cap structure to the pT7:TKII-derived VV tk RNA are presented.
Cell-Free System↗
Nucleotide sequence and transcriptional studies of the vaccinia virus KpnI I DNA fragment.
The nucleotide sequence of the vaccinia virus (VV) KpnI I DNA fragment has been determined. This central, highly conserved portion of the VV genome corresponds to the right portion of the HindIII E, all of the HindIII O and P, and the left portion of the HindIII I DNA fragments. Computer-assisted analysis of this data indicated the presence of five tandemly oriented, leftward-reading open reading frames (ORFs) I-4, I-3, I-2, I-1, and O-1, with the I-4 ORF being an immediate early gene encoding the large M1 subunit of VV ribonucleotide reductase. Transcriptional analyses suggested that the I-3 and O-1 genes were constitutive genes, being expressed both before and after viral DNA synthesis. The I-1 and I-2 genes were late genes, expressed only after the initiation of viral DNA synthesis. Cell-free translation was used to confirm that the I-3, I-1, and O-1 ORFs were bonafide messages encoding proteins with molecular weights of 30, 35, and 71 kD, respectively. When the predicted amino acid sequences of the proteins encoded by the I-3, I-2, I-1, and O-1 genes were compared to the Genbank data base, no significant alignments were detected. Therefore, the biological functions of these proteins in the VV life cycle remain to be established.
Amino Acid Sequence↗
Rifampicin prevents virosome localization of L65, an essential vaccinia virus polypeptide.
In contrast to its irreversible effect on the Escherichia coliRNA polymerase beta-subunit, the antibiotic rifampicin reversibly inhibits vaccinia virus morphogenesis at a step during the formation of immature viral particles. The protein affected by the presence of rifampicin is L65, a major late vaccinia polypeptide to which mutations that confer rifampicin resistance have been mapped. We now provide evidence using a monospecific anti-L65 serum in concert with immunofluorescence and sucrose gradient analysis that the mechanism of action of rifampicin on vaccinia virus replication involves the inhibition of localization of L65 to the viral factories (virosomes) thereby blocking further development. Studies on the expression and distribution of L65 during the infection cycle reveal that L65 is a stable, nonglycosylated late protein associated with virions. These results are discussed in relationship to the possible in vivo functions of the L65 protein.
Blotting, Western↗
Fatty acid acylation of vaccinia virus proteins.
Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.
Acylation↗
DNA sequences that regulate expression of a vaccinia virus late gene (L65) and interact with a DNA-binding protein from infected cells.
To be efficiently expressed in vivo, the vaccinia virus late gene, L65, requires 5'-proximal cis-acting elements which bind a factor from infected cells. Deletion mutagenesis and vaccinia virus helper-dependent transient expression procedures were used to demonstrate that two distinct late promoter elements direct transcription from two different start sites (proximal [+1] and distal [-92]). The -128 to -112 region was essential for L65 distal promoter function, while sequences between -59 and +50 were sufficient for L65 proximal promoter function. The proximal DNA sequences interact with a protein, binding factor I (BF-I), which was isolated and partially purified from vaccinia virus-infected cells at late times postinfection. This activity is not detectable in uninfected cells or in purified virions. This factor binds specifically to two different sites within the proximal promoter, one 5' and one 3' to the transcription start site, but does not bind to the distal promoter element.
Animals↗
Yeast KEX2 endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells.
Mammalian cell lines (BSC-40, NG108-15, and GH4C1) that cannot process the murine neuroendocrine peptide precursor prepro-opiomelanocortin (mPOMC) when its synthesis is directed by a vaccinia virus vector were coinfected with a second recombinant vaccinia virus carrying the yeast KEX2 gene, which encodes an endopeptidase that cleaves at pairs of basic amino acid residues. mPOMC was cleaved intracellularly to a set of product peptides normally found in vivo, including mature gamma-lipotropin and beta-endorphin1-31. In GH4C1 cells (a rat pituitary line), product peptides were incorporated into stored secretory granules. These results suggest that the inability of any particular cell line to process a prohormone precursor is due to the absence of a suitable endogenous processing enzyme.
Cell Line↗
Use of the gene encoding neomycin phosphotransferase II to convect linked markers into the vaccinia virus genome.
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Genes↗
Characterization of a binding factor that interacts with the sequences upstream of the vaccinia virus thymidine kinase gene.
A small 176 base-pair cloned DNA fragment, representing the nucleotide sequences proximal to the 5'-end of the vaccinia virus thymidine kinase (VV TK) gene, was radiolabeled and used in concert with gel retention assays to detect, partially purify, and characterize a promoter binding factor (PBF) extracted from vaccinia virions. The VV TK PBF was purified from solubilized virus particles by a combination of ion-exchange and DNA-affinity chromatographic procedures. The interaction between VV TK PBF and VV TK promoter sequences was relatively specific in that binding to the radiolabeled probe could be effectively inhibited by unlabeled VV TK promoter or VV TK promoter-specific oligonucleotides, but not by similar-sized fragments of control plasmid DNA. The VV TK PBF did, however, bind to other VV early-promoter elements. Glycerol gradient sedimentation provided an estimate of 130-140 kD for the native molecular weight of VV PBF. This correlated well with data from the purification of VV PBF from radiolabeled VV particles that revealed 2 polypeptides, with molecular weights of 70 and 68 kD that co-purified with VV TK PBF activity. Taken together, these results suggest that a heterodimeric promoter-binding factor, which is present within the cytoplasm of VV-infected cells, is capable of specifically interacting with VV early-promoter elements.
Chromatography, Affinity↗
Nucleotide sequence and molecular genetic analysis of the large subunit of ribonucleotide reductase encoded by vaccinia virus.
We have mapped the vaccinia virus (VV) gene encoding the large subunit of ribonucleotide reductase (VV M1) within the HindIII I restriction fragment by using an oligonucleotide probe. Nucleotide sequencing revealed a 2340-bp open reading frame (orf), 1-3, whose amino acid sequence is highly homologous to the mouse M1 protein. The 1-3 gene was expressed as an immediate-early gene product, being transcribed in a leftward direction into a 2.7-kb polyadenylated transcript. Hybrid-selected translation of cycloheximide-amplified immediate-early viral RNA demonstrated that this mRNA encoded an 86-kd protein, which agrees with the expected size of the reductase large subunit. The 5'- and 3'-boundaries of the 1-3 transcriptional unit were determined by primer extension and S1-nuclease analysis, respectively, and shown to contain sequence elements typical of other VV early genes. Surprisingly, the predicted amino acid sequence of the VV enzyme subunit shares 72.5% homology with the mouse large subunit, M1.
Amino Acid Sequence↗
Nucleotide sequence and molecular genetic analysis of the vaccinia virus HindIII N/M region encoding the genes responsible for resistance to alpha-amanitin.
The genomic location of the gene(s) which provides vaccinia virus (VV) alpha-amanitin-resistant mutants with a drug-resistant phenotype have been mapped to the HindIII N/M region of the genome by the use of marker rescue techniques [E. C. Villarreal and D. E. Hruby (1986) J. Virol. 57, 65-70]. Nucleotide sequencing of a 2356-bp HindIII-Sau3A fragment of the vaccinia virus genome encompassing this region reveals the presence of two complete leftward-reading open reading frames (ORFs, N2 and M1) and two incomplete ORFs (N1 and M2). By computer analysis the N2 and M1 ORFs would be predicted to encode soluble VV polypeptides with molecular weights of approximately 20 and 48 kDa, respectively. The N2 and M1 ORFs have extremely A-T-rich 5'-proximal sequences, consistent with previous data regarding the location and A-T-richness of viral early promoters. Likewise, the consensus signal believed to be involved in terminating VV early gene transcription, TTTTTNT, was evident at the 3'-boundary of both the N2 and M1 ORFs suggesting that these genes may be VV early genes. The in vivo transcriptional activity, orientation, and limits of these putative transcriptional units were investigated by Northern blot, nuclease S1, and primer extension analysis. Both N2- and M1-specific transcripts were detected in the cytoplasm of VV-infected cells, suggesting that these loci are bonafide viral genes. Time-course nuclease S1 experiments revealed that the N2 gene was transcribed exclusively prior to VV DNA replication. In contrast, the M1 gene was transcribed throughout infection, although different start sites were used at early versus late times postinfection. These results are discussed in relation to the drug-resistant phenotype and future experiments to identify the viral gene product responsible.
Amanitins↗
Inhibition of vaccinia virus replication by nicotinamide: evidence for ADP-ribosylation of viral proteins.
Replication of vaccinia virus (VV) in monolayers of BSC40 cells was inhibited 99.9% in the presence of 60 mM nicotinamide (NIC), a competitive inhibitor of ADP-ribosylation reactions. Dot-blot hybridization analysis of infected cell extracts utilizing a VV DNA-specific probe indicated that the drug had only minimal effects on viral DNA synthesis. SDS-polyacrylamide gel electrophoresis of newly synthesized VV proteins pulse-labeled at early (2 h) or late (8 h) times post-infection revealed that although the full spectrum of expected viral polypeptides was evident, quantitative differences in the levels of expression of a distinct subset of viral proteins were observed in the presence of the drug. Velocity sedimentation of virus-infected cell lysates established that no mature particles were assembled in drug treated cells. Additional evidence suggesting that VV morphogenesis was abortive in the presence of NIC was obtained by pulse-chase labeling experiments that demonstrated that the two VV major late core polypeptide precursors P94 and P65, whose proteolytic processing to VP62 and VP60 is intimately associated with viral assembly, were not cleaved in the presence of NIC. Interestingly, growth of VV in the presence of [3H]adenosine resulted in the metabolic labeling of eight proteins that were associated with purified virions. These proteins co-migrated with proteins labeled with [3H]adenosine that were present in extracts of VV-infected, but not uninfected, cells. These analyses also revealed that the [3H]adenosine-labeling of a subset of cellular proteins (MW 18-20 kDa, possibly histones) was increased 4-fold by VV infection. The observed induction of either increased synthesis or hyper-modification of these 18-20 kDa proteins was inhibited by NIC. These results are discussed with respect to whether one or more VV polypeptides are subject to obligatory ADP-ribosylation modification reactions in order to attain their active configuration, and if so, whether the enzymes catalyzing these reactions are specified by the virus or host cell.
Adenosine Diphosphate Ribose↗
Present and future applications of vaccinia virus as a vector.
The use of live recombinant vaccinia virus strains, which are capable of expressing immunoreactive epitopes from pathogenic agents, holds great promise as a means of immunoprophylaxis against a variety of human and animal diseases. This review will outline the basic methodology employed by this approach, summarize some recent technological advancements which should facilitate the construction of genetically-engineered recombinant viruses, and consider potential future applications of this avenue of research.
Animals↗
Expression of streptococcal M protein in mammalian cells.
The M protein encoded by group A streptococci is a cell-wall polypeptide that has the property of enabling these organisms to evade the phagocytic cells of the human host. Therefore, the M protein plays a major role in the pathogenesis of streptococcal diseases. As an initial step toward the use of this protein as a target antigen for the production of protective anti-streptococcal immunity, a live vaccinia virus recombinant containing the M-protein gene has been constructed (VV:M6 delta). The bacterial M-protein DNA sequence is stable within this genetic context and is actively transcribed by viral RNA polymerase. Furthermore, high levels of immunoreactive M protein were detected in vivo when the VV:M6 delta recombinant was used to infect mammalian cells in culture. Thus, in addition to providing a powerful approach for dissecting the immunodominant domains of the M protein, the VV:M6 delta recombinant appears to be an excellent candidate vaccine for animal trials.
Animals↗
Molecular dissection of cis-acting regulatory elements from 5'-proximal regions of a vaccinia virus late gene cluster.
Promoter elements responsible for directing the transcription of six tightly clustered vaccinia virus (VV) late genes (open reading frames [ORFs] D11, D12, D13, A1, A2, and A3) from the HindIII D/A region of the viral genome were identified within the upstream sequences proximal to each individual locus. These regions were identified as promoters by excising them from the VV genome, abutting them to the bacterial chloramphenicol acetyl transferase gene, and demonstrating their ability to drive expression of the reporter gene in transient-expression assays in an orientation-specific manner. To delineate the 5' boundary of the upstream elements, two of the VV late gene (A1 and D13) promoter: CAT constructs were subjected to deletion mutagenesis procedures. A series of 5' deletions of the ORF A1 promoter from -114 to -24 showed no reduction in promoter activity, whereas additional deletion of the sequences from -24 to +2 resulted in the complete loss of activity. Deletion of the ORF A1 fragment from -114 to -104 resulted in a 24% increase in activity, suggesting the presence of a negative regulatory region. In marked contrast to previous 5' deletion analyses which have identified VV late promoters as 20- to 30-base-pair cap-proximal sequences, 5' deletions to define the upstream boundary of the ORF D13 promoter identified two positive regulatory regions, the first between -235 and -170 and the second between -123 and -106. Background levels of chloramphenicol acetyltransferase expression were obtained with deletions past -88. Significantly, this places the ORF D13 regulatory regions within the upstream coding sequences of the ORF A1. A high-stringency computer search for homologies between VV late promoters that have been thus far characterized was carried out. Several potential consensus sequences were found just upstream from RNA start sites of temporally related promoter elements. Three major conclusions are drawn from these experiments. (i) The presence of promoters preceding each late ORF supports the hypothesis that each is expressed as an individual transcriptional unit. (ii) Promoter elements can be located within the coding portion of the upstream gene. (iii) Sequence homologies between temporally related promoter elements support the notion of kinetic subclasses of late genes.
Base Sequence↗
Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector.
To study posttranslational mechanisms for the control of nerve growth factor (NGF), we used a recombinant vaccinia virus vector to independently express the two major NGF transcripts in a variety of mammalian cell lines. The two major transcripts contain NGF (12.5 kilodaltons [kDa]) at the C-terminus and differ by alternative splicing of an N-terminal exon, so that the large precursor (34 kDa) had 67 amino acids upstream of an internal signal peptide and the smaller precursor (27 kDa) had this signal peptide at its N-terminus. In L929 cells, expression of either NGF transcript with the vaccinia virus vector gave rise to an apparently identical intracellular 35-kDa glycosylated precursor formed by cleavage of the primary gene product after the signal peptide. These cells also secreted biologically active NGF. To determine whether NGF processing is restricted by cell type, we infected a variety of mammalian cell lines with both recombinant viruses; all accumulated the same 35-kDa precursor and secreted NGF. Thus, many types of cells have the machinery to process and secrete NGF. However, NGF accumulated intracellularly (presumably in secretory granules) in cells with a regulated pathway of secretion (e.g., AtT-20 and HIT cells). In these cells, a membrane-permeable cyclic AMP analog, 8-bromo-cyclic AMP, stimulated NGF secretion. This suggests a mechanism for the regulation of NGF levels in which specific secretagogues, e.g., neurotransmitters, control NGF secretion.
Animals↗
Gene transfer techniques to study neuropeptide processing.
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Neuropeptides↗