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D E Hruby

Publications and source records attributed to D E Hruby.

At least 73 records · Page 4Linked to original sources
PubMed1990

Quaternary structure of vaccinia virus thymidine kinase.

Thymidine kinase enzymes isolated from a variety of sources are generally considered to have a native molecular weight of 80-90 kDa composed of two 40-45 kDa subunits. Although these parameters may accurately describe the atypical deoxypyrimidine kinases expressed by members of the Herpesviridae, the nucleotide sequences of thymidine kinase genes isolated from human, mouse, chicken and variety of poxviruses (vaccinia virus, monkeypox virus, variola virus, fowlpox virus and capripoxvirus) predict molecular weights on the order of 20-25 kDa for the derived primary translation products. To resolve this apparent dilemma, velocity sedimentation centrifugation, gel filtration chromatography and protein cross-linking procedures were employed to provide experimental evidence that enzymatically-active vaccinia virus thymidine kinase is a homotetrameric complex of 20 kDa monomers with a native Mr of 80 kDa.

Chromatography, Gel↗
PubMed1990

Anchoring a vaccinia virus promoter in the nucleus prevents its trans-activation by viral infection.

The vaccinia virus 7.5 kDa constitutive promoter, when fused to a reporter gene and recombined into the genome of L cells, is not activatable upon subsequent infection with vaccinia virus. However, the same promoter is actively transcribed during transient cytoplasmic transfection procedures or within the context of the viral genome. This suggests that the intact vaccinia transcriptional machinery either does not enter the nucleus or, if it does, is unable to interact with cellular chromatin.

Cell Nucleus↗
PubMed1990

Insertional inactivation of the large subunit of ribonucleotide reductase encoded by vaccinia virus is associated with reduced virulence in vivo.

To assess whether a fully functional VV ribonucleotide reductase enzyme is required during both in vitro and in vivo replication of VV, three mutant viruses were constructed by marker transfer techniques: M1 lambda, an M1 insertion mutant; TK-, an insertion mutant of the VV thymidine kinase (tk) gene; and M1 lambda/TK-, a double mutant. Extracts of cells infected with the M1 lambda or M1 lambda/TK- mutant viruses were assayed for ribonucleotide reductase activity and it was found that insertional inactivation of the M1 gene abolished the induction of viral enzyme activity in VV-infected cells. Each of the three mutant viruses replicated to levels comparable to the wild-type (WT) virus in BSC40 (monkey), growing A549 (human lung carcinoma) cells, and serum-starved A549 cells, indicating that a functional M1 gene was not required for viral replication in tissue culture. In contrast, in vivo studies indicate that the loss of viral ribonucleotide reductase activity leads to a mild attenuation of VV. By the intracranial route of inoculation, approximately 10-fold more of the M1 lambda recombinant than the WT virus was required to produce the average lethal dose for 50% of the population of injected mice.

Animals↗
PubMed1990

Vaccinia virus: a versatile tool for molecular biologists.

Continued advances in genetic engineering have made possible the high-level expression of correctly processed cellular, viral and bacterial polypeptides. This article focuses on viral expression vectors and, more specifically, the vaccinia virus expression system. Vaccinia virus has been used to express a variety of proteins with useful immunogenic, catalytic or pharmaceutical properties. We discuss briefly the biology of vaccinia and its significance in the use of vaccinia as an expression vector, the variety of vaccinia systems currently in use and, finally, we summarize some recent developments which bode well for future applications of vaccinia virus technology.

Animals↗
PubMed1990

Vaccinia virus vectors: new strategies for producing recombinant vaccines.

The development and continued refinement of techniques for the efficient insertion and expression of heterologous DNA sequences from within the genomic context of infectious vaccinia virus recombinants are among the most promising current approaches towards effective immunoprophylaxis against a variety of protozoan, viral, and bacterial human pathogens. Because of its medical relevance, this area is the subject of intense research interest and has evolved rapidly during the past several years. This review (i) provides an updated overview of the technology that exists for assembling recombinant vaccinia virus strains, (ii) discusses the advantages and disadvantages of these approaches, (iii) outlines the areas of outgoing research directed towards overcoming the limitations of current techniques, and (iv) provides some insight (i.e., speculation) about probable future refinements in the use of vaccinia virus as a vector.

Animals↗
PubMed1990

Use of a cell-free system to identify the vaccinia virus L1R gene product as the major late myristylated virion protein M25.

A 25-kDa vaccinia virus (VV) virion protein, designated M25, is modified in vivo by covalent addition of myristic acid. The predicted amino acid sequences of all VV open reading frames which have been reported were searched for the sequence M-G-X-X-X-(S/T/A), which has been proposed to be the consensus recognition signal for cotranslational modification of proteins by N-myristyltransferase. This conserved signal was found at the amino terminus of a single locus, which corresponded to the leftmost rightward-reading open reading frame (L1R) initiating within the VV HindIII L DNA fragment. By using synthetic oligonucleotides in concert with polymerase chain reaction techniques, a chimeric gene consisting of open reading fram L1R fused to a bacteriophage T7 promoter was constructed and cloned into a plasmid vector. Transcripts derived from the wild-type expression plasmid (designated pL1G1) were translated in vitro in a wheat germ extract to yield a polypeptide with an apparent molecular mass of 25 kDa. This polypeptide was labeled with either [35S]methionine or [3H]myristic acid and comigrated with in vivo-labeled protein M25 on sodium dodecyl sulfate-polyacrylamide gels. Polyclonal antiserum generated in rabbits against a trpE:L1R fusion protein immunoprecipitated a 25-kDa protein labeled either in vitro (the L1R gene product, designated protein L1) or in vivo (from purified VV, protein M25), identifying the M25 protein as the gene product of open reading frame L1R. Chromatographic analysis of the protein L1-bound fatty acid moieties liberated after acid methanolysis resulted in recovery of greater than 99% of the fatty acid as myristate-associated label. Cell-free translation of proteins derived from a set of deletions from the carboxy terminus of the open reading frame L1R suggested that the site of myristylation maps near the amino terminus of protein L1. This hypothesis was supported by cell-free translation of mutant L1R transcripts in which the penultimate glycine codon had been altered by site-directed mutagenesis to encode either an aspartic acid (pL1D1) or alanine (pL1A1) residue. In both cases, the mutant transcripts were translated into a 25-kDa protein which could be labeled in vitro with [35S]methionine but not with [3H]myristic acid. These data demonstrate that VV open reading frame L1R encodes a myristylated protein and provide evidence that the site of modification of protein L1 is the amino-terminal glycine residue.

Amino Acid Sequence↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1988

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↗