Peter Krell
University of Guelph, Molecular and Cellular Biology, Faculty Member
Research Interests: Biochemistry, Bacteriology, Catalysis, Biology, Medicine, and 15 moreSequence Analysis, Biological Sciences, Xylanase, Enzyme, Substrate Specificity, Xylans, Xylose, Amino Acid Sequence, Structure activity Relationship, Recombinant Proteins, Hydrolysis, Oligosaccharides, Molecular Sequence Data, DNA mutational analysis, and Medical and Health Sciences
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Virus-like particles have been isolated from the oviducts of a parasitoid wasp, Hyposoter exiguae (Hymenoptera: Ichneumonidae). Particles are readily purified by centrifugation on either Ficoll or sucrose gradients. Double-stranded... more
Virus-like particles have been isolated from the oviducts of a parasitoid wasp, Hyposoter exiguae (Hymenoptera: Ichneumonidae). Particles are readily purified by centrifugation on either Ficoll or sucrose gradients. Double-stranded circular DNA isolated from purified particles is heterodisperse in terms of molecular weight; none of the molecules are sufficiently large to code for the aggregate of structural proteins comprising the particles. Preliminary Southern blot hybridization data suggest that there is minimal sequence homology between the different size classes of DNA examined.
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Research Interests: Biochemistry, Bacteriology, Catalysis, Biology, Medicine, and 13 moreBiological Sciences, Xylanase, Escherichia coli, Molecular cloning, Amino Acid Sequence, Base Sequence, Recombinant Proteins, Molecular Sequence Data, DNA mutational analysis, Azotobacter Vinelandii, Restriction Mapping, binding sites, and Medical and Health Sciences
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We identified four CfMNPV DNA fragments with autonomously replicating sequences (ARS) functional in Saccharomyces cerevisiae. A 0.9-kb fragment which, mapped to 54.5 to 55.3 map units within EcoRI HI of the CfMNPV genome, showed the... more
We identified four CfMNPV DNA fragments with autonomously replicating sequences (ARS) functional in Saccharomyces cerevisiae. A 0.9-kb fragment which, mapped to 54.5 to 55.3 map units within EcoRI HI of the CfMNPV genome, showed the strongest ARS activity of the four. Sequence analysis of this 0.9-kb DNA segment revealed an A+T-rich region separated from a G+C-rich region by 320 bp. Although no sequence matched exactly the ARS core-consensus sequence, 13 near-matches differing by only one or two nucleotides from the core-consensus sequence, were identified. Ten near-matches were clustered within a 105-bp A+T-rich region, and were arranged as inverted repeats. A section of bent DNA structure was predicted within this region. The bent DNA, which showed temperature-dependent retardation during polyacrylamide gel electrophoresis, was unique as its sequence was arranged as a symmetrical 'tilde' (approximately) structure. The second (1.0 kb) and third (1.6 kb) ARS-bearing fragments mapped within EcoRI-E and -B fragments which contain homologous repeat sequences. The fourth (1.5 kb) fragment had the weakest ARS activity and mapped to the EcoRI-D or -B regions of the genome.
Research Interests: Genetics, Biology, DNA replication, Medicine, Lepidoptera, and 12 moreBiological Sciences, DNA, Virus, Genome, Plasmids, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Genome Organization, Replication, Base Sequence, Nucleic Acid Conformation, Molecular Sequence Data, and Restriction Mapping
Female parasitic wasps of the species Diadegma terebrans produce a polydnavirus, DtPV, in cells of the calyx. The virus morphology is similar to that of other polydnaviruses. It has a segmented superhelical DNA genome with an estimated... more
Female parasitic wasps of the species Diadegma terebrans produce a polydnavirus, DtPV, in cells of the calyx. The virus morphology is similar to that of other polydnaviruses. It has a segmented superhelical DNA genome with an estimated size range of 2.3 to 5.5 kilobases. DtPV replication starts within large nuclei where nucleocapsids assemble and become enveloped. They bud through the nuclear envelope and are then "secreted" through the apical microvilli into either the calyx lumen directly or into a channel which is continuous with the lumen. DtPV-producing cells are distributed throughout the length of the calyx and form a discrete, one cell thick "tissue" within it. The large, sector-shaped, DtPV-producing cells form a semicircle around the calyx lumen near the ovariole end of the calyx. This semicircle of DtPV-infected cells increases in circumference towards the lateral oviduct end of the calyx until they completely encircle the lumen and form the major component of the calyx wall.
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Research Interests: Microbiology, Molecular Biology, Biology, Medicine, Lepidoptera, and 15 moreBiological Sciences, Nucleic acid hybridization, Promoter, Specific Activity, Chitinase, Enzyme activity, Amino Acid Sequence, Temporal Analysis, Nucleotides, Gene expression profiling, Molecular Sequence Data, Total protein, reverse transcriptase polymerase chain reaction, Medical and Health Sciences, and Nucleopolyhedrovirus
Poxviruses carry the enzyme, nucleoside triphosphate phosphohydrolase I (NPH I), required for early viral transcription in the cytoplasm of infected cells. The gene (nph I) encoding this enzyme from Choristoneura fumiferana entomopoxvirus... more
Poxviruses carry the enzyme, nucleoside triphosphate phosphohydrolase I (NPH I), required for early viral transcription in the cytoplasm of infected cells. The gene (nph I) encoding this enzyme from Choristoneura fumiferana entomopoxvirus (CfEPV) has been located in the viral genome, cloned and characterized. It has an open reading frame of 1941 nucleotides, potentially encoding a protein with a predicted molecular mass of 76.04 kDa and a pI of 8.83. It has a TAAATG motif where the trinucleotide ATG represents the translational start signal an AT-rich (88%) sequence and an early transcription termination signal (TTTTTAT) upstream of the ATG codon. Northern blot analysis of mRNA from infected larvae showed that a single 4.0 kb transcript which appeared late at day 20 post infection (p.i.) and its transcription continued till day 37 p.i.. Primer extension experiments suggested that the main transcripts started at 15 bases upstream of AUG codon. NPH I homologues have been found in the genomes of other entomopoxviruses and vertebrate poxviruses. Alignment of their amino acid sequences suggested three conserved domains, two of which are considered as ATP binding domains. The most similar homologue is from the closely related entomopoxvirus. Choristoneura biennis EPV (CbEPV) where 98.2% of nucleotide and 97.2% of amino acid identities are observed, respectively. A single nucleotide difference in CfEPV nph I was sufficient to distinguish it from CbEPV by PCR amplification and digestion with a restriction enzyme.
Research Interests: Molecular Biology, Biology, Medicine, Biological Sciences, Phylogeny, and 15 moreSequence alignment, Virus, Polymerase Chain Reaction, Gene, Enzyme, Molecular cloning, Time Factors, Larva, Molecular Mass, Amino Acid Sequence, Base Sequence, Open Reading Frame, Nucleotides, Molecular Sequence Data, and restriction enzyme
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Research Interests: Molecular Biology, Biology, Virology, Medicine, Biological Sciences, and 15 moreCell line, South Africa, Gene, Green Fluorescent Protein, Recombinant DNA, Protein Sequence Analysis, Zinc Finger, Amino Acid Profile, Molecular Mass, Virus infection, Open Reading Frame, Nucleotides, Spodoptera exigua, Reporter gene, and Medical and Health Sciences
Research Interests: Molecular Biology, Biology, Virology, Medicine, Sequence Analysis, and 14 moreBiological Sciences, Cell line, Animals, Gene, Green Fluorescent Protein, Glycosylation, Inclusion Bodies, Moths, Amino Acid Sequence, Base Sequence, Recombinant Proteins, Immunoblotting, Medical and Health Sciences, and Nucleopolyhedrovirus
A β‐N‐acetylglucosaminidase cDNA (CfGlcNAcase) was cloned from the spruce budworm, Choristoneura fumiferana. Western blotting analysis of developmental CfGlcNAcase expression revealed high levels of expression of the gene on the last day... more
A β‐N‐acetylglucosaminidase cDNA (CfGlcNAcase) was cloned from the spruce budworm, Choristoneura fumiferana. Western blotting analysis of developmental CfGlcNAcase expression revealed high levels of expression of the gene on the last day of the 5th instar larvae and the first day in the 6th instar larvae, followed by a decrease to background levels during the intermolt of the 6th instar. CfGlcNAcase was detected again from the last day of the 6th instar to day 2 of pupal stage. CfGlcNAcase expression was induced by tebufenozide at 24 h post treatment and remained at high levels until 72 h. Immunohistochemical localization analysis of CfGlcNAcase indicated that CfGlcNAcase was present in the molting fluid, epidermis, trachea, and hemolymph in prepupae during the transformation from larva to pupa. CfGlcNAcase cDNA was expressed into a recombinant protein in bacterial and baculovirus systems and the protein expressed in the baculovirus system had a higher chitinolytic activity than in the bacterial system and appeared to be secreted. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley‐Liss, Inc.
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Research Interests: Genetics, Biology, Medicine, Biological Sciences, Cell line, and 15 moreHumans, Animals, Genome, Gene, Protein Sequence Analysis, Moths, Amino Acid Profile, Eukaryotic Cells, Amino Acid Sequence, Base Sequence, DNA primase, Molecular Sequence Data, conserved sequence , Medical and Health Sciences, and Nucleopolyhedrovirus
Abstract The polypeptides in TIV and in TIV-infected hemocytes were studied by polyacrylamide gel electrophoresis (PAGE). TIV consists of 28 polypeptides ranging in molecular weight from 17,500 to 300,000. The lowest molecular weight... more
Abstract The polypeptides in TIV and in TIV-infected hemocytes were studied by polyacrylamide gel electrophoresis (PAGE). TIV consists of 28 polypeptides ranging in molecular weight from 17,500 to 300,000. The lowest molecular weight polypeptide appears to be a lipoprotein, and none appear to be glycoproteins. Proteins, banding in the same area as TIV polypeptides, are seen in gels after PAGE of hemocytes from larvae 4 hr after viral injection. Also present in infected cells are 6 noncapsid viral polypetides. Infection by TIV is accompanied by an immediate depression of leucine incorporation into hemocyte and plasma proteins and a switch in synthesis from host-specific to virus-specific polypeptides.
Research Interests: Biology, Virology, Medicine, DNA viruses, Biological Sciences, and 15 moreInsect Viruses, Animals, Insects, Peptides, Glycoproteins, Tritium, Larva, Lipoproteins, Hemolymph, Galleria Mellonella, Molecular weight, Polyacrylamide Gel Electrophoresis, Sodium Dodecyl Sulfate, Leucine, and Medical and Health Sciences
Research Interests: Genetics, Fluorescence Microscopy, Biology, Tobacco, Medicine, and 15 moreBiological Sciences, Plant diseases, Sequence alignment, Virus, Endoplasmic Reticulum, Genome, Gene, Subcellular Localization, Cell nucleus, Nicotiana Tabacum, Vitis, Amino Acid Sequence, Intracellular Space, Protein Transport, and Molecular Sequence Data
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Research Interests: Molecular Biology, Biology, Cell Biology, Insect Molecular Biology, Medicine, and 14 moreSignal Transduction, Biological Sciences, RNA interference, Animals, Phosphorylation, Transcription Factor, Molecular cloning, Moths, Amino Acid Sequence, DNA binding proteins, Molting, Protein Transport, Molecular Sequence Data, and Protein Kinase C
Bacteriocinogenic Clostridium perfringens, strain 28, harboured plasmid DNA detectable by dye-buoyant density-gradient centrifugation. This plasmid DNA was absent from an ultraviolet light cured variant which had simultaneously lost its... more
Bacteriocinogenic Clostridium perfringens, strain 28, harboured plasmid DNA detectable by dye-buoyant density-gradient centrifugation. This plasmid DNA was absent from an ultraviolet light cured variant which had simultaneously lost its immunity and ability to produce bacteriocin. Agarose gel electrophoresis of the plasmid DNA revealed at least six bands but denaturation experiments suggested three plasmids occurring in more than one conformation. Electron microscopy revealed three major size distributions of circular DNA of molecular weights 1,5.6, and 7.1 megadaltons. Some evidence suggests that the 5.6 megadalton plasmid may control bacteriocin 28 production.
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Research Interests: Fluorescence Microscopy, Biology, Virology, Confocal Microscopy, Medicine, and 11 moreBiological Sciences, Cell line, Animals, Insects, Endoplasmic Reticulum, Bimolecular Fluorescence Complementation, Chitinase, Cysteine endopeptidases, Protein Binding, Virus-Cell interactions, and Medical and Health Sciences
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Spheroidin (SPH) is the most abundant late protein in cells infected with the Amsacta moorei entomopoxvirus (AMEV). This locus can be used for expression of exogenous genes because it is not essential for virus replication. The sph... more
Spheroidin (SPH) is the most abundant late protein in cells infected with the Amsacta moorei entomopoxvirus (AMEV). This locus can be used for expression of exogenous genes because it is not essential for virus replication. The sph promoter contains a conserved TAAATG motif, which serves as the site of initiation for both transcription and translation. Additional sequences downstream of the conserved motif have been shown to be involved in high-level expression of the sph gene. As a first step towards developing a protein expression vector based on the sph locus, four recombinant AMEV viruses expressing either gfp or lacZ were constructed. Both reporter genes were expressed under the control of the sph promoter containing the TAAATG motif. An additional 6 bp or 21 bp of sph coding region was included in three of the recombinants, to be expressed as an N-terminal fusion protein of GFP or LacZ. GFP and beta-galactosidase expression was observed at 2 days post-infection and continued throughout the observation period. The highest level of reporter gene expression was observed in the recombinant containing 21 bp from the sph coding region. These results indicate that sph locus of AMEV can be used successfully to express exogenous genes.
Research Interests: Microbiology, Molecular Biology, Medical Microbiology, Biology, Medicine, and 14 moreGene expression, Lepidoptera, Genetic Engineering, Cell line, Animals, Method, Gene, Green Fluorescent Protein, Journal of Virological Methods, Protein Expression, Recombinant Proteins, Fusion Protein, Recombinant Virus, and Reporter gene
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Research Interests: Molecular Biology, Biology, Medicine, Gene expression, Biological Sciences, and 15 moreAnimals, CHEMICAL SCIENCES, Molecular cloning, Protein Sequence Analysis, Moths, Insect Biochemistry and Molecular Biology, Amino Acid Sequence, Base Sequence, Open Reading Frame, Plasma Membrane, Full Length Movies, Biochemistry and cell biology, Molecular Sequence Data, Protein Kinase C, and Medical and Health Sciences
Research Interests: Molecular Biology, Biology, Virology, DNA damage, Apoptosis, and 15 moreMedicine, Biological Sciences, DNA, Escherichia coli, Methylation, Animals, Viruses, Chlorella, Time Factors, Moths, Amino Acid Sequence, Base Sequence, Recombinant Proteins, Molecular Sequence Data, and Medical and Health Sciences
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The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered... more
The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production. The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral DNA synthesis and virus replication. Rather, we identified three features, including two nuclear localization signals and a highly conserved 10-amino-acid sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity, as measured by viral DNA synthesis and virus replication.
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A recombinant plasmid containing the Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus (CfMNPV) HindIII R fragment (m.u. 2.2-3.9) was shown to undergo CfMNPV infection-dependent DNA replication in Cf-124T cells.... more
A recombinant plasmid containing the Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus (CfMNPV) HindIII R fragment (m.u. 2.2-3.9) was shown to undergo CfMNPV infection-dependent DNA replication in Cf-124T cells. Replication of this DNA sequence was detectable by 24 hr p.i. and did not appear to have resulted as a consequence of recombination with the virus genome. Replication was inhibited by mimosine, an inhibitor of eukaryotic DNA replication. These data suggest that HindIII R of CfMNPV DNA contains an origin of DNA replication which we call ori1. HindIII R contains five GC-rich and three AT-rich regions and a 0.9-kb homologous repeat region 1 (hr1). Two short 440- and 740-bp contiguous sequences at the right end of the HindIII R fragment separately exhibited limited ori function. HindIII R subfragments with optimal ori activity contained a cluster of repeated and inverted sequences including nine copies of a 50-bp homologous repeat sequence (hr1a to hr1i) within hr1. The CfMNPV hr1 sequence was somewhat homologous with the homologous repeat (hr) of the putative Autographa californica MNPV (AcMNPV) replication origins. HindIII Y, another CfMNPV DNA fragment containing an hr sequence, hr3, also supported infection-dependent DNA replication, suggesting that it too contains an ori. Although replication of a putative AcMNPV origin (HindIII Q) was detectable in CfMNPV-infected Cf-124T cells, replication of CfMNPV HindIII R was not detectable in AcMNPV-infected Spodoptera frugiperda cells.
Research Interests: Molecular Biology, Biology, DNA replication, Medicine, Biological Sciences, and 15 moreCell line, Animals, Plasmids, Capsid, Moths, Spodoptera Frugiperda, Transfection, Base Sequence, Nucleic Acid Conformation, DNA fragmentation, DNA sequence, Molecular Sequence Data, Restriction Mapping, Medical and Health Sciences, and Nucleopolyhedrovirus
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Genomic plasticity is the hallmark of all biological processes and for viruses is more apparent because of their short generation times and high number of "progeny" produced during each replication cycle or passage. Despite the... more
Genomic plasticity is the hallmark of all biological processes and for viruses is more apparent because of their short generation times and high number of "progeny" produced during each replication cycle or passage. Despite the inherent fidelity of DNA replication, DNA genomes, including those of baculoviruses, continuously undergo variations in their nucleotide sequences. Due to recombination and errors in DNA replication there is a certain probability of error at each passage which for the baculovirus genome leads to point mutations, deletions, rearrangements, inversions, reiterations and acquisition of host cell DNA. It is in this background that baculoviruses replicate either in cells of insect larvae or of insect tissue cultures. Whether the baculovirus replicates in its insect host as a "natural" pathogen, or in cultured cells for the production of microbial insecticides and foreign proteins of economic or scientific interest, the potential for such genomic alterations is omnipresent. Faulkner (1981) first suggested that the production of variants, atypical morphogenesis and lower virulence of baculovirus preparations occurred as a consequence of repeated serial passage, herein referred to as the "passage effect". The result of prolonged (undiluted) passage of baculovirus in vitro, was first shown by MacKinnon et al. (1974) for Trichoplusia ni NPV (TnNPV) derived from "late passage" (up to 50) in T. ni TN-368 cells. At late passage fewer polyhedra per cell and less virulent polyhedra were produced. Moreover polyhedral morphology mutants and aberrant and shorter nucleocapsids resulted and the number of normal occluded virions per polyhedral inclusion body (PIB; the form of baculoviruses responsible for insect to insect horizontal transmission) also decreased. Payne (1988) noted the importance of passage effect in the production of effective viral pathogens for insect control. Tramper & Vlak (1986) first recognized the importance of the passage effect in bioreactor production of baculoviruses and foreign protein in the context of decreased productivity. The accumulation of genomic alterations through several replication cycles either through individual insects or tissue culture flasks or through the several cycles of replication in bioreactors can be referred to as the passage effect. Some of these new genotypic variants could replicate at the expense of the original parental virus and could, with sufficient numbers of passages become dominant. The major problems with the passage effect on baculovirus replication and expression is the loss of virulence of PIB for virus grown in tissue culture and, for viruses used as expression vectors, a reduced ability to produce the foreign protein of interest. For growth of PIB in tissue culture there is no selection pressure for virus capable of production of infectious polyhedra. Similarly for foreign genes in recombinant baculoviruses there is often no selection to maintain and express those genes.
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Viruses are described from several genera of ichneumonid parasitoids. New morphologic categories have been observed, one of which is similar to typical baculoviruses. Calyx particles from several species were found to contain polydisperse... more
Viruses are described from several genera of ichneumonid parasitoids. New morphologic categories have been observed, one of which is similar to typical baculoviruses. Calyx particles from several species were found to contain polydisperse DNA's. An electrophoretic method for screening individual field-collected wasps for the presence of such DNA's is reported. DNA profiles obtained by this procedure were sufficiently consistent, within any particular affected species, to suggest that they could provide useful taxonomic information.
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The CMV immediate early promoter from the EGFP expression plasmid pEGFP-N1 was replaced with the very left end of the fowl adenovirus 9 (FAdV-9) genome (ntds 73-574) to demonstrate and delineate the promoter function of this sequence.... more
The CMV immediate early promoter from the EGFP expression plasmid pEGFP-N1 was replaced with the very left end of the fowl adenovirus 9 (FAdV-9) genome (ntds 73-574) to demonstrate and delineate the promoter function of this sequence. Expression of an EGFP ORF which replaced ORF1 and ORF2 demonstrated that the native promoter can drive down stream foreign gene expression. Replacement of ORF1 and ORF2 with a bicistronic cassette, incorporating a 493 bp IRES from an Ontario strain of avian encephalomyelitis virus (AEV) separating an EGFP ORF and mCherry ORF allowed for expression of both ORFs from a recombinant FAdV. These results provide an additional platform for multivalent vaccines development based on a native FAdV-9 promoter and an avian virus IRES.
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Fowl adenovirus (FAdV) type 1, CELO strain has no homologies to mastadenovirus E1A, E1B, E3 and E4, which regulate virus gene expression, DNA replication and virus-host interaction. Similarly, the right 5 kb and left 15 kb ends of CELO... more
Fowl adenovirus (FAdV) type 1, CELO strain has no homologies to mastadenovirus E1A, E1B, E3 and E4, which regulate virus gene expression, DNA replication and virus-host interaction. Similarly, the right 5 kb and left 15 kb ends of CELO virus DNA are non-homologous to mastadenoviruses. To compare CELO virus with another FAdV, 7.5 kb of the left and 17 kb of the right ends of FAdV type 8 (strain A-2A) were sequenced and nine and 17 open reading frames (ORFs), respectively, were found. This FAdV-8 genome was similar to CELO virus in that (1) the central region contained the major structural protein genes including the fibre, pVIII, 100K, late 33K and pIVa2 genes, which were in the same order as in mastadenoviruses, (2) no homologues of mastadenovirus E1A, E1B, E3 and E4 were found in the ends, and (3) the left 6 kb and the right 13 kb ends showed no homology to mastadenoviruses. Several genomic features were unique to FAdV-8 compared to CELO virus. FAdV-8 contained one fibre gene in contrast to two in CELO virus. Three of eight unassigned ORFs in the left and five of 13 unassigned ORFs in the right ends were unique compared to CELO virus. Two sets of tandem repeats, one with five identical 33 bp repeats and the other with more than ten identical 135 bp repeats, mapped between 4.5 and 7.5 kb from the right terminus. No virus-associated RNA gene was found. Fifteen of 16 unique FAdV-8 ORFs tested were, as determined by RT-PCR, transcribed early.
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Viruses within the family Baculoviridae have large (80–180 kbp), circular, double-stranded DNA (dsDNA) genomes and are divided into the genera Nucleopolyhedrovirus and Granulovirus, producing large refractile bodies termed polyhedra or... more
Viruses within the family Baculoviridae have large (80–180 kbp), circular, double-stranded DNA (dsDNA) genomes and are divided into the genera Nucleopolyhedrovirus and Granulovirus, producing large refractile bodies termed polyhedra or granules. The nucleocapsids are enveloped either singly (SNPV or granulovirus (GV); NPV = nucleopolyhedrovirus) or in multiples (MNPV) into membrane-bound virions. Viruses occur in two morphologies. Budded viruses, produced early in infection, contain nucleocapsids surrounded by a membrane acquired during budding through the cell membrane. Occluded viruses, produced late in infection contain one or more nucleocapsids surrounded by a membrane derived de novo inside the nucleus and become embedded within a crystalline, alkaline-sensitive matrix of polyhedrin (NPV) or granulin (GV) with NPVs containing multiple virions and GVs containing only a single virion. Transcription is temporally regulated into immediate early, early, late, and very late phases, with viral DNA replication initiating between the early and late phases. These insect viruses have widely varying host ranges and can modulate the cellular apoptotic pathway and insect metamorphosis to their advantage. Furthermore, baculoviruses are of interest as biological pesticides, expression vectors, and in gene therapy.
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DNA polymerase (DNApol) is highly conserved in all baculoviruses and plays an essential role in viral DNA replication. It determines the fidelity of baculovirus DNA replication by inserting the correct nucleotides into the primer terminus... more
DNA polymerase (DNApol) is highly conserved in all baculoviruses and plays an essential role in viral DNA replication. It determines the fidelity of baculovirus DNA replication by inserting the correct nucleotides into the primer terminus and proofreading any mispaired nucleotides. DNApols between both Groups I and II of the Alphabaculovirus genus in the family Baculoviridae share many common structural features. However, it is not clear whether a group I AcMNPV DNApol can be substituted by a group II NPV DNApol. Here we report the successful generation of AcMNPV dnapol null virus being rescued by a Group II Spodoptera litura NPV dnapol (Bac-AcΔPol:Slpol). Viral growth curves and quantitative real-time PCR showed that the dnapol replacement reduced the level of viral production and DNA replication of Bac-AcΔPol:SlPol compared with WTrep, a native dnapol insertion in an AcMNPV dnapol null virus. Light microscopy showed that production of occlusion bodies for Bac-AcΔPol:Slpol was reduced. We also identified a nuclear localization signal (NLS) for the SpltNPV DNApol C terminus at residues 827-838 by mutational analysis and confocal microscopy. Multiple point substitution of SpltNPV DNApol NLS abrogated virus production and viral DNA replication. Overall, these data suggested that the NLS plays an important role in SpltNPV DNApol nuclear localization and that SpltNPV DNApol cannot efficiently substitute the AcMNPV DNApol in AcMNPV.