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Biomedical subjects

D E Hruby

Publications and source records attributed to D E Hruby.

At least 73 records · Page 4Linked to original sources
PubMed1985

Immunisation of cattle with a recombinant togavirus-vaccinia virus strain.

Genetic engineering techniques have been used to construct a vaccinia virus recombinant which contains and expresses togavirus (Sindbis) genetic information. Intradermal inoculation of this recombinant strain into calves caused a transient pock-type lesion at the site of inoculation and elicited the production of substantial levels of anti-Sindbis virus neutralising antibodies. These results suggest that recombinant vaccinia virus vaccines may have potential for use in veterinary medicine.

Animals↗
PubMed1984

Novel codon utilization within the vaccinia virus thymidine kinase gene.

The nucleotide and predicted amino acid sequences of the thymidine kinase genes encoded by vaccinia virus and herpes simplex virus (type 1) were analyzed, and there was no evidence of any significant homology. The manner in which the triplet code was used by each virus was also examined. The frequencies of codon utilization by the herpes virus gene were very similar to those used by most human genes, whereas the vaccinia virus gene was quite distinct, suggesting novel evolutionary and regulatory mechanisms.

Codon↗
PubMed1984

Isolation of vaccinia virus mutants capable of replicating independently of the host cell nucleus.

alpha-Amanitin-resistant vaccinia virus mutants were isolated after serial viral passages in BSC-40 cells that were carried out in the presence of inhibitory levels (6 micrograms/ml) of alpha-amanitin. One such mutant, alpha-27, was highly refractory (greater than 95%) to alpha-amanitin-mediated inhibition and was selected for further study. In the absence of drug, the phenotypes of alpha-27 and wild-type vaccinia virus were indistinguishable with respect to growth kinetics. DNA synthesis, protein synthesis, and morphogenesis. Infections in the presence of alpha-amanitin revealed two striking differences, however. First, wild-type virus was unable to catalyze proteolytic processing of the two major capsid proteins VP62 and VP60, whereas alpha-27 was most efficient at this process. Second, wild-type viral morphogenesis within the infected cells was arrested by alpha-amanitin at an apparently analogous step to that previously described for enucleated cells. This observation was supported by the ability of alpha-27 virus to replicate in enucleated BSC-40 cells. Restriction enzyme analyses of alpha-27 versus wild-type genomes revealed that a XhoI cleavage site was altered in the alpha-27 DNA molecule, suggesting a possible location for the alpha-amanitin resistance locus.

Amanitins↗
PubMed1983

Fine structure analysis and nucleotide sequence of the vaccinia virus thymidine kinase gene.

The thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene of vaccinia virus has previously been mapped near the middle of the viral DNA, within the 4.85-kilobase HindIII J fragment, and shown to encode a Mr 19,000 polypeptide [Hruby, D. E. & Ball, L. A. (1982) J. Virol. 43, 403-409]. To locate the gene more precisely and to determine the structure of the basic transcriptional unit, the positions of cleavage sites for several restriction endonucleases were mapped within the HindIII J DNA fragment. Four appropriate subfragments of HindIII J DNA were inserted into plasmid pBR322 derivatives and cloned in Escherichia coli. These recombinant plasmid DNAs were tested for their ability to inhibit the cell-free synthesis of active thymidine kinase and to retain the mRNA for this enzyme when immobilized on nitrocellulose filters. The data showed that the gene spanned an EcoRI cleavage site that lies 850 base pairs from the left-hand end of the HindIII J fragment (the HindIII L-J boundary). Because hybridization of vaccinia virus DNA to partially purified thymidine kinase mRNA detected only a single 670-nucleotide RNA species capable of hybridizing to this region of the genome, nuclease S1 mapping experiments were carried out with thymidine kinase mRNA to protect DNA fragments that were terminally labeled at this EcoRI site. The results indicated that the gene extended from about 550 to 1,150 base pairs from the left end of HindIII J, was transcribed in a rightward direction, and contained no intervening sequences. Hence, a 1.04-kilobase Ava II-Hpa II restriction fragment containing this region of DNA was isolated and subjected to nucleotide sequence analysis. An examination of this nucleotide sequence revealed the presence of an open reading frame of 531 nucleotides capable of encoding a protein of 177 amino acids with a Mr of 20,077.

Amino Acid Sequence↗
PubMed1982

Mapping and identification of the vaccinia virus thymidine kinase gene.

The thymidine kinase gene of vaccinia virus (VV) was mapped on the viral genome by using cloned fragments of the viral DNA to hybridize to early viral mRNA. Individual DNA fragments that represented about half of the viral genome were assayed, both for their ability to arrest the cell-free synthesis of active VV thymidine kinase and for their ability to select functional mRNA for the viral enzyme. Both activities were located in HindIII fragment J, which maps near the middle of VV DNA and contains about 2.6% of the genome (4,800 base pairs). This DNA fragment encodes four known early polypeptides, and to determine which of these was thymidine kinase, early VV mRNA was fractionated by sucrose gradient centrifugation and used to direct cell-free synthesis of the active enzyme. The thymidine kinase mRNA cosedimented with several species that encoded polypeptides in the molecular weight range 15,000 to 25,000. Hybridization of these mRNAs to HindIII-J DNA selected a message that directed the synthesis of thymidine kinase and a single polypeptide with an apparent molecular weight of 19,000. The native molecular weight of VV thymidine kinase is about 80,000, so these data indicate that, unlike thymidine kinase from several other sources, the active VV enzyme is probably a tetramer of 19,000-molecular-weight subunits.

DNA Restriction Enzymes↗
PubMed1981

Translation of black beetle virus RNA and heterologous viral RNAs in cell-free lysates derived from Drosophila melanogaster.

A cell-free protein synthesizing system was prepared from cells of Drosophila melanogaster line 1 and made mRNA dependent by treatment with micrococcal nuclease. The system was tested with homologous RNA from black beetle virus propagated in Drosophila cells, with Drosophila heat shock mRNA, and with various heterologous viral mRNA's. Under optimal conditions amino acid incorporation programmed with black beetle virus RNAs was 30-fold higher than endogenous incorporation. RNAs 1 and 2 primarily directed the synthesis of proteins with approximately molecular weights of 120,000 and 46,000, respectively. mRNA's, prepared by transcription from vesicular stomatitis virus or vaccinia virus, were translated efficiently and yielded products that comigrated with authentic viral proteins. Brome mosaic virus RNA and encephalomyocarditis virus RNA were translated poorly. The system retained full activity after freezing.

Animals↗
PubMed1979

Vaccinia virus replication requires active participation of the host cell transcriptional apparatus.

The ability of vaccinia virus to replicate in BSC-40 monkey cells whose nuclei have been functionally inactivated was examined. Exposure of cell monolayers to ultraviolet radiation at doses that did not alter the cells' capacity to support a subsequent infection by a cytoplasmic virus (vesicular stomatitis virus) caused a reduction to less than 10% in the observed yield of infectious progeny from vaccinia virus and herpes simplex virus (type 1) infections. Similarly, replication of vaccinia virus was reduced to 5% by treatment of BCS-40 cells with alpha-amanitin (10 microgram/ml), a potent inhibitor of nuclear mRNA synthesis. In both situations, ultraviolet irradiation and alpha-amanitin treatment, early and late vaccinia viral genes were expressed at high levels, but the newly synthesized virion components were not assembled into mature infectious particles. Taken together, these data suggest that the active involvement of the host cell nuclear transcriptive system is obligatory in the vaccinia virus replicative cycle.

Amanitins↗
PubMed1979

Vaccinia virus replication. I. Requirement for the host-cell nucleus.

Using cytochalasin B-induced enucleation techniques, we examined the ability of vaccinia virus to replicate in the absence of the host-cell nucleus in several mammalian cell lines. It was found that virus-infected enucleated cells (cytoplasts) prepared from BSC-40, CVC, and L cells were incapable of producing infectious progeny virus. The nature of this apparent nuclear involvement was studied in detail in BSC-40 cells. Modulations designed to maximize cytoplast integrity and longevity, such as reduction of the growth temperature and initial multiplicity of infection, did not improve virus growth in cytoplasts. Sodium dodecyl sulfate-polyacrylamide gel analysis of the [(35)S]methionine pulse-labeled proteins synthesized in vaccinia virus-infected cytoplasts demonstrated that both early and late viral gene products were being expressed at high levels and with the proper temporal sequence. Vaccinia virus cytoplasmic DNA synthesis, as measured by [(3)H]thymidine incorporation, peaked at 3 h postinfection and was 70 to 90% of control levels in cytoplasts. However, in the cytoplasts this DNA was not converted to a DNase-resistant form late in infection, which was consistent with the failure to isolate physical particles from infected cytoplasts. Treatment of vaccinia virus-infected cells with 100 mug of rifampin/ml from 0 to 8 h to increase the pools of viral precursors, followed by subsequent removal of the drug, resulted in a threefold increase virus yield. This treatment had no effect on virus-infected cytoplasts. Finally, vaccinia virus morphogenesis was studied under an electron microscope in thin sections of virus-infected cells and cytoplasts which had been prepared at various times during a single-step growth cycle. It was apparent that, although early virus morphogenetic forms appeared, there was no subsequent DNA condensation or particle maturation in the cytoplasts. These results suggest that vaccinia virus requires some factor or function from the host-cell nucleus in order to mature properly and produce infectious progeny virus.

Animals↗
PubMed1977

An ATP-dependent inhibition of protein synthesis in ascites cell extracts by wheat germ protein.

A high speed supernatant fraction from wheat germ was shown to be a very effective inhibitor of protein synthesis in a cell-free protein synthesizing system from Ehrlich ascites cells. Low concentrations of the extract were equally effective in inhibiting the translation of EMC viral RNA, ascites cell mRNA (exogenous) and endogenous mRNA. The kinetics of inhibition in the presence and absence of pactamycin, as well as the observed inhibition of polyphenylalanine synthesis, indicate that the wheat germ inhibitor acts at the level of elongation. Preincubation of the ascites system with the wheat germ inhibitor in the presence and absence of ATP showed that ATP was required for the development of the inhibition. The inhibitor was partially purified and appears to be a basic protein with a molecular weight of 30 000--40 000. These results are discussed with respect to the hypothesis that this may be another example of a protein kinase-induced inhibition of protein synthesis.

Adenosine Triphosphate↗
PubMed1977

Isolation and characterization of mRNA from Paramecium aurelia.

Total cellular RNA was isolated from the ciliate protozoan Paramecium aurelia by pH 9.5 chloroform/octanol extraction. Passage of this RNA through an oligo(dT)-cellulose column in 0.5 M NaCl resulted in 2--3% binding, indicating the presence of polyadenylic acid sequences. These polyadenylic acid regions were estimated to be 250-500 nucleotides in length, based on their resistance to ribonuclease degradation. The oligo(dT)-cellulose bound RNA sedimented at 14--25 S in sodium dodecyl sulphate/sucrose gradients. The base composition of this RNA is similar to the base composition of the DNA. This RNA was also actively translated into protein by an in vitro protein synthesizing system isolated from wheat germ. Translation was optimal under conditions similar to those used for mammalian mRNA translation. In addition, translation of the P. aurelia oligo(dT)-cellulose bound RNA was inhibited 80% by the analog 7-methylguanosine-5'-phosphate, suggesting the presence of a 5'-capped terminus.

Animals↗
PubMed1977

Encephalomyocarditis virus RNA. II. Polyadenylic acid requirement for efficient translation.

Differentially polyadenylated subpopulatons of encephalomyocarditis (EMC) viral RNA were isolated by affinity chromatography on oligodeoxythymidylic acid-cellulose. Translation of these RNA fractions in several in vitro protein-synthesizing systems, isolated from Ehrlich ascites tumor cells, demonstrated that poly(A)+EMC viral RNA was translated two to three times more efficiently than poly(A)-EMC viral RNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the polypetides synthesized by the in vitro system in response to the different RNAs showed no detectable differences in the size or relative amount- of the translational products. mRNA saturation curves indicated that the in vitro systems were stimulated maximally by equivalent amounts of RNA, wheter it be poly(A)-or poly(A)+ EMC viral RNA. Time course experiments showed that the differences in translatability were more pronounced late in the reaction when reinitiation was required, and that by eliminating reinitiation with high salt the apparent effect of poly(A) on translation was diminished. Together, these results suggest that poly(A) may be required for efficient initiation and reinitiation of protein synthesis in the cell-free systems. This interpretation is discussed relative to earlier data.

Animals↗
PubMed1976

Encephalomyocarditis virus RNA: variations in polyadenylic acid content and biological activity.

Encephalomyocarditis (EMC) viral RNA was isolated from purified virus grown in Ehrlich ascites tumor cells. The viral RNA was found to contain polyadenylic acid [poly(A)] regions that were very heterogeneous in length. Chromatography of the EMC viral RNA on oligo(dT)-cellulose columns separated the RNA into three distinct fractions (peaks 1 to 3). Approximately 20% of the EMC viral RNA appeared as peak 1, 40% as peak 2, and 40% as peak 3. The RNA in each fraction appeared to be intact as shown by co-sedimentation with 35S unfractionated EMC viral RNA in SDS-sucrose density gradients. Approximately 95 to 100% of peaks 1 and 3, and 60 to 70% of peak 2, reappeared at the same elution position after rechromatography on oligo(dT)-cellulose. The RNA in peak 1 contained poly(A) with an average length of 16 nucleotides, peak 2 contained poly(A) with an average of 26 nucleotides, and peak 3 contained an average of 74 nucleotides in its poly(A) region. The distribution in the three fractions, as well as the average length of the poly(A) moieties, was relatively unaffected by changes in the cell suspension medium used during infection. Finally, each of the three viral RNA fractions was assayed for biological activity using an infectious RNA assay on L-cell monolayers. Infectivity of the viral RNA was found to increase with poly(A) length, with peak 3 viral RNA being approximately 10 times more infectious than peak 1 viral RNA.

Encephalomyocarditis virus↗