A single-stranded insertion vector (SSIV) for introducing foreign genes into the vaccinia virus genome.
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DNA, Single-Stranded↗
Biomedical subjects
D E Hruby
Publications and source records attributed to D E Hruby.
Association of non-viral proteins with recombinant vaccinia virus virions.
Evidence is presented which suggests that recombinant vaccinia virus particles (VV:CAT), containing the bacterial chloramphenicol acetyl transferase gene, are capable of encapsidating both the foreign protein which they encode (CAT) as well as cellular enzymes such as thymidine kinase. These results are discussed with respect to using VV to passively introduce biologically-active proteins into cells or organisms.
Acetyltransferases↗
Cell-free translation of a chimeric eucaryotic-procaryotic message yields functional chloramphenicol acetyltransferase.
A vaccinia virus (VV) recombinant containing the DNA sequences encoding the bacterial chloramphenicol acetyltransferase (CAT) gene was constructed. The ability of the chimeric VV:CAT transcript to be translated in vitro into enzymatically active enzyme was assessed. Addition of mRNA isolated from the cytoplasm of VV:CAT infected cells to a mRNA-dependent reticulocyte lysate resulted in the synthesis of high levels of enzymatically active CAT. These results suggest that this assay may be used in concert with physical assays to study the expression and stability of chimeric transcripts in virus-infected cells.
Acetyltransferases↗
Quantitative assay of recombinant vaccinia virus-encoded neomycin phosphotransferase in infected eukaryotic cell lysates.
A method for the detection and quantitation of neomycin phosphotransferase (NPT II) activity in recombinant vaccinia virus (VV)-infected eukaryotic cell lysates is described. The assay is linear with respect to both protein concentration and time of incubation. Cytoplasmic extracts of cells infected with a recombinant VV expressing the bacterial neo gene exhibited NPT II levels more than 50-fold higher than those detected in extracts from either uninfected or VV-infected cells. These results indicate that interference from cellular or viral-induced ATPase activities is sufficiently low that NPT II enzyme activity can be measured in crude cell lysates without employing additional protein purification procedures.
Acetyltransferases↗
Use of vaccinia virus to express biopharmaceutical products.
Recent technological advancements have fostered the continued development of vaccinia virus as an efficient eukaryotic cloning and expression vector system. Genetically engineered vaccinia virus strains have been constructed for use (i) as recombinant vaccines for the prophylaxis of infectious disease, (ii) in producing significant quantities of biologically active polypeptide factors or enzymes, and (iii) as basic research tools with which to investigate primary structure-function relationships between proteins and their catalytic activities. This review examines the basic vaccinia vector system, its advantages and limitations, and current areas of research. As a specific example of the power and utility of this approach, attention is focused on the application of this technology to the field of neurobiology, specifically the use of recombinant vaccinia to study the expression, processing, and transport of cellular neuropeptides.
Cloning, Molecular↗
Noncoordinate regulation of a vaccinia virus late gene cluster.
Identification of a tightly spaced and tandemly oriented late gene cluster within the central conserved region of the vaccinia virus genome suggested the possibility of coordinate regulation of the genes within this domain (S.L. Weinrich and D.E. Hruby, Nucleic Acids Res. 14:3003-3016, 1986). To test this hypothesis, the steady-state levels of transcripts derived from the individual late genes were examined. Cytoplasmic RNA was isolated from infected cells at hourly intervals throughout infection and was used in concert with 5' S1 nuclease mapping procedures to detect transcripts from specific late genes. Among the set of six closely linked late genes, marked differences were observed in both the levels of transcription and the kinetic patterns of expression, providing direct evidence for the existence of differentially regulated gene subsets within the late gene class. Furthermore, these experiments identified one of the genes (encoding a 33,000-molecular-weight polypeptide) as being expressed both early and late postinfection. Interestingly, although transcripts from the constitutively expressed gene were initiated at the same start sites throughout infection, a discrete terminus for these transcripts was detected only at early times. These data suggest that the lack of cis-acting termination signals is not the reason for the late gene transcript heterogeneity observed in vaccinia virus-infected cells.
Chromosome Mapping↗
Nucleotide sequence and transcript organization of a region of the vaccinia virus genome which encodes a constitutively expressed gene required for DNA replication.
A vaccinia virus (VV) gene required for DNA replication has been mapped to the left side of the 16-kilobase (kb) VV HindIII D DNA fragment by marker rescue of a DNA- temperature-sensitive mutant, ts17, using cloned fragments of the viral genome. The region of VV DNA containing the ts17 locus (3.6 kb) was sequenced. This nucleotide sequence contains one complete open reading frame (ORF) and two incomplete ORFs reading from left to right. Analysis of this region at early times revealed that transcription from the incomplete upstream ORF terminates coincidentally with the complete ORF encoding the ts17 gene product, which is directly downstream. The predicted proteins encoded by this region correlate well with polypeptides mapped by in vitro translation of hybrid-selected early mRNA. The nucleotide sequences of a 1.3-kb BglII fragment derived from ts17 and from two ts17 revertants were also determined, and the nature of the ts17 mutation was identified. S1 nuclease protection studies were carried out to determine the 5' and 3' ends of the transcripts and to examine the kinetics of expression of the ts17 gene during viral infection. The ts17 transcript is present at both early and late times postinfection, indicating that this gene is constitutively expressed. Surprisingly, the transcriptional start throughout infection occurs at the proposed late regulatory element TAA, which immediately precedes the putative initiation codon ATG. Although the biological activity of the ts17-encoded polypeptide was not identified, it was noted that in ts17-infected cells, expression of a nonlinked VV immediate-early gene (thymidine kinase) was deregulated at the nonpermissive temperature. This result may indicate that the ts17 gene product is functionally required at an early step of the VV replicative cycle.
Amino Acid Sequence↗
Expression and cell type--specific processing of human preproenkephalin with a vaccinia recombinant.
The posttranslational maturation of a complex precursor polyprotein, human proenkephalin, was assessed by infection of a wide spectrum of cell types with a recombinant vaccinia virus that expressed human proenkephalin. The infected cells rapidly produced both cellular and secreted Met-enkephalin immunoreactivity. Although each cell line could secrete intact proenkephalin, only a mouse pituitary line was capable of processing proenkephalin to mature enkephalin peptides. The quantity of intact proenkephalin secreted from BSC-40 cells (derived from African Green monkey kidney) was sufficient to establish the value of vaccinia virus as a mammalian cell expression vector.
Animals↗
A tandemly-oriented late gene cluster within the vaccinia virus genome.
The nucleotide sequence of a 5.1 kilobase-pair fragment from the central portion of the vaccinia virus genome has been determined. Within this region, five complete and two incomplete open reading frames (orfs) are tightly-clustered, tandemly-oriented, and read in the leftward direction. Late mRNA start sites for the five complete orfs and one incomplete orf were determined by S1 nuclease mapping. The two leftmost complete orfs correlated with late polypeptides of 65,000 and 32,000 molecular weight previously mapped to this region. When compared with each other and with sequences present in protein data banks, the five complete orfs showed no significant homology matches amongst themselves or any previously reported sequence. The six putative promoters were aligned with three previously sequenced late gene promoters. While all of the nine are A-T rich, the only apparent consensus sequence is TAA immediately preceeding the initiator ATG. Identification of this tandemly-oriented late gene cluster suggests local organization of the viral genome.
Amino Acid Sequence↗
Use of vaccinia virus as a neuropeptide expression vector.
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Cloning, Molecular↗
Construction of recombinant vaccinia virus strains using single-stranded DNA insertion vectors.
The ability of single-stranded (ss) DNA, isolated from recombinant M13 bacteriophage, to direct the insertion of foreign genetic elements into the vaccinia virus (VV) genome was examined. An identical chimeric transcriptional unit [VV promoter/chloramphenicol acetyl transferase (CAT) gene embedded in DNA sequences encoding vaccinia virus thymidine kinase (TK)] was inserted into either the previously characterized plasmid insertion vector, pGS20, or into M13mp18. It was found that the ss vector (M13mp18:TK/CAT) was four times more efficient than the plasmid vector (pGS20:CAT) in catalyzing homologous recombination of the cat gene by marker transfer into the VV genome. Furthermore, Southern blot analyses and CAT enzymatic activity assays confirmed that the structure of the M13-derived recombinant genomes were as expected and that the chimeric genes were fully active. Although the precise mechanism responsible for the ss DNA-catalyzed insertion event is not known, these results are discussed with respect to the advantages of using M13-based vectors with which to manipulate and insert genetic information into infectious VV recombinants.
Cloning, Molecular↗
Mapping the genomic location of the gene encoding alpha-amanitin resistance in vaccinia virus mutants.
To facilitate the determination of the genomic location of the vaccinia virus gene(s) encoding alpha-amanitin resistance (alpha r) (Villarreal et al., J. Virol. 51:359-366, 1984), a collection of alpha r, temperature-sensitive (ts) mutants were isolated. The premise of these experiments was that mutants might be found whose dual phenotypes were the result of a single or two closely linked mutations. Genetic analyses of the alpha rts mutant library revealed two mutants, alpha rts7 and alpha rts12, that apparently fit this criterion; in alpha rts7 the two lesions were indistinguishable, whereas in alpha rts12 the two mutations were closely linked but separable. Cloned vaccinia virus HindIII DNA fragments were used to marker rescue the temperature-sensitive phenotype of these two dual mutants. The temperature-sensitive lesion of alpha rts7 was rescued by the HindIII N fragment (1.5 kilobases), whereas alpha rts12 was rescued by the neighboring HindIII M fragment (2.0 kilobases). The progeny virions of the alpha rts7 HindIII-N rescue reverted to an alpha-amanitin-sensitive phenotype, whereas the alpha rts12 HindIII-M progeny were still resistant to the drug. Taken together, these data indicate that the gene encoding alpha-amanitin resistance maps to the HindIII N fragment and provides evidence for the existence of essential vaccinia virus genes in a region of the genome previously believed to be nonessential for replication in tissue culture. Biochemical analyses revealed that both mutants were capable of synthesizing DNA as well as early and late viral proteins at the permissive and nonpermissive temperatures. At the nonpermissive temperature alpha rts12 and alpha rts7 were unable to process the major core precursors P94 and P65 into VP62 and VP60.
Amanitins↗
Expression and regulation of the vaccinia virus thymidine kinase gene in non-permissive cells.
The expression and regulation of the vaccinia virus (VV) thymidine kinase (tk) gene was examined in two non-permissive cell lines, CHO and MDBK, which restrict VV development at different stages of the viral replication cycle. The VV tk gene was expressed in these two cell lines with kinetics similar to a fully permissive cell line BSC40. These results are consistent with the hypothesis that inhibition of tk mRNA translation by another viral early gene product is a normal component of the overall strategy employed to express and regulate the VV tk gene during a productive infection.
Animals↗
Inhibition of vaccinia virus thymidine kinase by the distal products of its own metabolic pathway.
Vaccinia virus thymidine kinase activity is inhibited by low concentrations (10 microM) of dTDP or dTTP, but not by dTMP. This inhibition is specified for the thymidine nucleotides as dATP, dGTP, and dCTP have no effect. The viral enzyme phosphorylates thymidine to dTMP with typical first-order kinetics. However, evidence was obtained to indicate that the observed dTTP inhibition was noncompetitive in nature. This suggests that thymidine and dTTP interact with different sites in the native viral enzyme.
Animals↗
Expression of recombinant vaccinia virus-derived alphavirus proteins in mosquito cells.
A recombinant vaccinia virus strain which contains and expresses a 26S cDNA insert encoding Sindbis virus structural proteins (VV:3S) was used to infect a continuous line of Aedes albopictus mosquito cells. There were not visible cytopathic effects due to the virus infection and the cells continued to grow normally. However, examination of the proteins present in the cytoplasm of the infected cells with Sindbis virus-specific antisera revealed that Sindbis virus proteins were being synthesized and processed. These results are discussed with respect to vaccinia virus as a non-lethal expression vector to deliver and express eukaryotic genetic information in insect cell systems and using this system (VV:3S) to dissect various facets of togavirus-insect cell interactions.
Aedes↗
Transcriptional and translational analysis of the vaccinia virus late gene L65.
Among the products of vaccinia virus genes which are expressed late in infection is a major polypeptide (Mr, 65,000) designated L65. Pulse-chase analyses indicated that L65 is not subject to posttranslational cleavage as is the core polypeptide p4b which migrates to a similar position in sodium dodecyl sulfate-polyacrylamide gels. A polypeptide of 65,000 molecular weight produced in reticulocyte lysates programmed with viral mRNA isolated late in infection was identified as L65 by peptide mapping. L65 mRNA was purified by hybridization selection to restriction fragments of the viral genome and translated in vitro. This allowed the gene encoding L65 to be mapped to the rightmost 4.5 kilobase pairs of the HindIII D fragment. Transcriptional mapping of this region of the genome detected a late mRNA which was initiated at 450 base pairs to the right of the HindIII D-A junction, was transcribed in the leftward direction, and was terminated in the nondescript manner typical of vaccinia virus late mRNAs.
DNA Restriction Enzymes↗
Expression of Sindbis virus structural proteins via recombinant vaccinia virus: synthesis, processing, and incorporation into mature Sindbis virions.
We have obtained a vaccinia virus recombinant which contains a complete cDNA copy of the 26S RNA of Sindbis virus within the thymidine kinase gene of the vaccinia virus genome. This recombinant constitutively transcribed the Sindbis sequences throughout the infectious cycle, reflecting the dual early-late vaccinia promoter used in this construction. The Sindbis-derived transcripts were translationally active, giving rise to both precursor and mature structural proteins of Sindbis virus, including the capsid protein (C), the precursor of glycoprotein E2 (PE2), and the two mature envelope glycoproteins (E1 and E2). These are the same products translated from the 26S mRNA during Sindbis infection, and thus these proteins were apparently cleaved, glycosylated, and transported in a manner analogous to that seen during authentic Sindbis infections. By using epitope-specific antibodies, it was possible to demonstrate that recombinant-derived proteins were incorporated into Sindbis virions during coinfections with monoclonal antibody-resistant Sindbis variants. These results suggest that all the information necessary to specify the proper biogenesis of Sindbis virus structural proteins resides within the 26S sequences and that vaccinia may provide an appropriate system for using DNA molecular genetic manipulations to unravel a variety of questions pertinent to RNA virus replication.
Biological Transport↗
Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants.
The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.
Animals↗