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J M Vlak - One of the best experts on this subject based on the ideXlab platform.
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Specificity of Polyhedrin in the generation of baculovirus occlusion bodies.
The Journal of general virology, 1999Co-Authors: T Luijckx, D Zuidema, L C Van Dinten, M M Van Oers, J P Haj S, F J Bianchi, J W Van Lent, J M VlakAbstract:The role of Polyhedrin in the occlusion of virions was studied by substituting two heterologous Polyhedrin-coding sequences, one from a multiple-nucleocapsid (M) nucleopolyhedrovirus (NPV) of Spodoptera exigua (Se) and one from a single-nucleocapsid (S) NPV of Buzura suppressaria (BusuNPV), into the genome of Autographa californica (Ac) MNPV. Both heterologous Polyhedrin genes were highly expressed and polyhedra were produced in the nuclei of cells infected with the respective recombinant AcMNPVs. Polyhedra produced by the recombinant with BusuNPV Polyhedrin showed normal occlusion of multiple-nucleocapsid virions and were equally as infectious to S. exigua larvae as were wild-type AcMNPV polyhedra. This indicates that virion occlusion is not specific with respect to whether the virions or Polyhedrin are from an SNPV or MNPV. Polyhedra produced by the recombinant containing the SeMNPV Polyhedrin had an altered morphology, being pyramidal rather than polyhedral in shape, and many fewer virions were occluded. These occlusion bodies were less infectious to S. exigua larvae than were those of wild-type AcMNPV. These results indicate that virion occlusion is a finely controlled process that is to some extent specific to the Polyhedrin involved and may also require other viral or host factors for optimal morphogenesis.
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Expression of theAutographa californica nuclear polyhedrosis virus p10 gene: effect of Polyhedrin gene expression
Archives of Virology, 1992Co-Authors: M. M. Oers, D. Malarme, J. M. P. Jore, J M VlakAbstract:Two major late proteins, Polyhedrin and p10, are synthesized in large quantities in baculovirus infected insect cells. This and the fact that both proteins are dispensable for virus replication, form the basis for the use of these viruses as vector for foreign gene expression. To address the question whether the Autographa californica nuclear polyhedrosis virus p10 promotor-driven expression is influenced by the concurrent expression of the Polyhedrin gene, several recombinants were constructed with various deletions in the Polyhedrin gene. The Escherichia coli lacZ gene was used as a marker to allow direct comparison between p10 and Polyhedrin-driven expression. None of the deletions in the Polyhedrin gene did result in higher expression of the p10 promoter-controlled gene. The suggested that the transcriptional and/or translational activity of the p10 and Polyhedrin gene are independently regulated. To compare the level of Polyhedrin and p10 promoter driven expression, recombinants with the lacZ gene cloned behind either promoter were studied. No significant difference in level of expression was observed. In cells infected with a recombinant with the lacZ gene present behind both promoters a reduced level of expression was observed, whereas a considerable increase was expected. This may be due to instability of the viral genome, as two copies of the lacZ gene were present.
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biochemical and biological characterization of four isolates of spodoptera exigua nuclear polyhedrosis virus
Biocontrol Science and Technology, 1992Co-Authors: P Caballero, D Zuidema, Candido Santiagoalvarez, J M VlakAbstract:Biological and biochemical properties of four nuclear polyhedrosis virus isolates from beet armyworm, Spodoptera exigua, were investigated. The isolates originated from the United States (SeNPV‐US), Thailand (SeNPV‐TH) and from two locations in Spain (SeNPV‐SP1 and SeNPV‐SP2). Restriction endonuclease analysis of the viral genomes revealed limited restriction fragment length polymorphism and indicated that these viruses contained distinct, but closely related, genotypes (variants). One BglII fragment from each isolate can serve as a restriction fragment length polymorphism marker for the identification of each isolate. The estimated genome size of the SeNPVs is approximately 134 kilobase pairs. The mobility profiles of the occluded virion polypeptides and Polyhedrins of the four SeNPV isolates were very similar. Staphylococcus aureus V8 digestion of Polyhedrin suggested that the Polyhedrin from SeNPV‐US is distinct from the Polyhedrins of the other isolates. Bioassays of the isolates in second‐instar S. e...
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Nucleotide sequence and transcriptional analysis of the Polyhedrin gene of Spodoptera exigua nuclear polyhedrosis virus.
The Journal of general virology, 1992Co-Authors: E A Van Strien, D Zuidema, R W Goldbach, J M VlakAbstract:The nucleotide sequence of a 1.1 kbp fragment of the multiple nucleocapsid nuclear polyhedrosis virus (MNPV) of Spodoptera exigua (Se) containing the Polyhedrin gene was determined. An open reading frame (ORF) of 738 nucleotides (nt) was detected. This ORF encoded a protein of 246 amino acids with a predicted M(r) of 29K. The nucleotide and amino acid sequences were compared with the sequences of eight other NPV Polyhedrins. The SeMNPV Polyhedrin protein was most closely related to S. frugiperda MNPV Polyhedrin with differences in only five amino acids, and most distantly related to the Lymantria dispar MNPV Polyhedrin. The size of the mRNA was approximately 1,000 nt, as determined by Northern blot analysis. Using primer extension assays and S1 nuclease mapping the transcriptional start and stop sites of the Polyhedrin mRNA were located. The 5' regulatory sequence appeared to be 44 nt in length with the mRNA start site predominantly at the first A of the TAAG consensus start sequence. Two degenerate poly(A) signals were found immediately downstream of the translational stop signal. The transcriptional stop was located approximately 230 nt downstream from the translational stop signal, in an AT-rich sequence that appears to be common to all baculovirus Polyhedrin genes. The SeMNPV Polyhedrin mRNA does not appear to be polyadenylated.
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Expression of the Autographa californica nuclear polyhedrosis virus p10 gene: effect of Polyhedrin gene expression.
Archives of Virology, 1992Co-Authors: M M Van Oers, J. M. P. Jore, D. Malarme, J M VlakAbstract:Two major late proteins, Polyhedrin and p10, are synthesized in large quantities in baculovirus infected insect cells. This and the fact that both proteins are dispensable for virus replication, form the basis for the use of these viruses as vector for foreign gene expression. To address the question whether the Autographa californica nuclear polyhedrosis virus p10 promotor-driven expression is influenced by the concurrent expression of the Polyhedrin gene, several recombinants were constructed with various deletions in the Polyhedrin gene. The Escherichia coli lacZ gene was used as a marker to allow direct comparison between p10 and Polyhedrin-driven expression. None of the deletions in the Polyhedrin gene did result in higher expression of the p10 promoter-controlled gene. This suggested that the transcriptional and/or translational activity of the p10 and Polyhedrin gene are independently regulated. To compare the level of Polyhedrin and p10 promoter driven expression, recombinants with the lacZ gene cloned behind either promoter were studied. No significant difference in level of expression was observed. In cells infected with a recombinant with the lacZ gene present behind both promoters a reduced level of expression was observed, whereas a considerable increase was expected. This may be due to instability of the viral genome, as two copies of the lacZ gene were present.
Robert D. Possee - One of the best experts on this subject based on the ideXlab platform.
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superior expression of juvenile hormone esterase and β galactosidase from the basic protein promoter of autographa californica nuclear polyhedrosis virus compared to the p10 protein and Polyhedrin promoters
Journal of General Virology, 1994Co-Authors: Bryony C Bonning, Just M. Vlak, Robert D. Possee, P.w. Roelvink, B. D. HammockAbstract:The expression characteristics of the p10, Polyhedrin and basic protein promoters of Autographa californica nuclear polyhedrosis virus were compared using two reporter enzymes, juvenile hormone esterase (JHE) and β-galactosidase. In these systems, JHE is exported from the cell and β-galactosidase is localized to the cytosol. Expression of JHE from the basic, p10 and Polyhedrin promoters was first detected in the medium at 13, 19 and 27 h post-infection respectively. The basic protein promoter yielded the highest expression of the three promoters tested for both enzymes, as determined by protein and enzyme activity assays. In addition, yields of β-galactosidase and JHE under control of the p10 promoter are higher relative to expression under control of the Polyhedrin promoter. These data highlight the importance of investigation of viral promoters other than the Polyhedrin promoter for high yield protein expression in vitro, and for insecticidal use of recombinant baculoviruses requiring high levels of expression. The results support revision of the current concept that very late viral promoters are always optimal for high yield recombinant protein expression.
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analysis of very late gene expression by autographa californica nuclear polyhedrosis virus and the further development of multiple expression vectors
Journal of General Virology, 1990Co-Authors: Ulrike Weyer, Stuart Knight, Robert D. PosseeAbstract:The consequences of locating the Polyhedrin gene coding sequences and the p10 promoter at heterologous positions within the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome were investigated. Positioning the Polyhedrin or β-galactosidase coding sequences under the control of the p10 gene promoter via the use of the new transfer vector, pAcUW1, resulted in viable recombinant viruses able to produce high levels of each non-fused gene product at the appropriate time. Polyhedra were also produced by the virus with the p10 promoter-Polyhedrin hybrid gene and appeared normal in thin sections. Therefore the combination of Polyhedrin promoter and coding sequences is evidently not essential for efficient expression of this protein. The p10 promoter can serve this function equally well. Viruses with the p10 promoter and β-galactosidase coding sequences placed upstream from the Polyhedrin gene in either orientation produced large amounts of β-galactosidase protein in infected cells, thus demonstrating that the p10 promoter can function at an alternative position within the virus genome. A second transfer vector, pAc-UW2B, was constructed, with a copy of the p10 gene promoter placed upstream and in opposition to the Polyhedrin gene. This mediates the insertion of any foreign gene under the control of the p10 promoter while preserving normal p10 gene expression. The advantages of these constructs over the conventional vectors presently used to express foreign genes in insect cell systems and their utilization in the production of virus insecticides are discussed.
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Functional analysis of a 603 nucleotide open reading frame upstream of the Polyhedrin gene of Autographa californica nuclear polyhedrosis virus.
Journal of General Virology, 1990Co-Authors: K L Gearing, Robert D. PosseeAbstract:The sequence of the 2000 nucleotides immediately upstream of the Polyhedrin gene of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus has been determined. Comparative analysis of the data identified a 603 nucleotide open reading frame (ORF) separated from the Polyhedrin gene coding sequences by 156 nucleotides and in the opposite strand of DNA. Northern hybridization analysis of polyadenylated RNA from infected cells highlighted a 3⋅7 kb species produced maximally at 12h post-infection, but not in the presence of cycloheximide. Preliminary nuclease S1 analysis of the 5′ end of this RNA suggested that it initiated at a position very close to that of the Polyhedrin mRNA start site. Deletion of a portion of the ORF 603 from viruses containing the normal Polyhedrin gene and the lacZ gene in lieu of Polyhedrin did not affect replication in cell culture or the production of β-galactosidase protein. A virus which lacked the ORF 603 gene but produced Polyhedrin had similar infectivity in Trichoplusia ni larvae compared to the wild-type virus. The chloramphenicol acetyltransferase (CAT) gene was also inserted in lieu of the ORF 603 in a virus containing the lacZ gene instead of the Polyhedrin (Ac.CAT.lacZ). Analysis of CAT expression revealed that a maximum level was reached at 16 h p.i. and that transcription was initiated in Ac.CAT.lacZ at the same site as for the normal gene.
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Functional analysis of a 603 nucleotide open reading frame upstream of the Polyhedrin gene of Autographa californica nuclear polyhedrosis virus.
The Journal of general virology, 1990Co-Authors: K L Gearing, Robert D. PosseeAbstract:The sequence of the 2000 nucleotides immediately upstream of the Polyhedrin gene of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus has been determined. Comparative analysis of the data identified a 603 nucleotide open reading frame (ORF) separated from the Polyhedrin gene coding sequences by 156 nucleotides and in the opposite strand of DNA. Northern hybridization analysis of polyadenylated RNA from infected cells highlighted a 3.7 kb species produced maximally at 12 h post-infection, but not in the presence of cycloheximide. Preliminary nuclease S1 analysis of the 5' end of this RNA suggested that it initiated at a position very close to that of the Polyhedrin mRNA start site. Deletion of a portion of the ORF 603 from viruses containing the normal Polyhedrin gene and the lacZ gene in lieu of Polyhedrin did not affect replication in cell culture or the production of beta-galactosidase protein. A virus which lacked the ORF 603 gene but produced Polyhedrin had similar infectivity in Trichoplusia ni larvae compared to the wild-type virus. The chloramphenicol acetyltransferase (CAT) gene was also inserted in lieu of the ORF 603 in a virus containing the lacZ gene instead of the Polyhedrin (Ac.CAT.lacZ). Analysis of CAT expression revealed that a maximum level was reached at 16 h p.i. and that transcription was initiated in Ac.CAT.lacZ at the same site as for the normal gene.
Byung Rae Jin - One of the best experts on this subject based on the ideXlab platform.
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Hyper-Enhanced Production of Foreign Recombinant Protein by Fusion with the Partial Polyhedrin of
2016Co-Authors: Sung Min Bae, Tae-young Shin, Jae-bang Choi, Hee Jung Kim, Jun Beom Lee, Hyun Na Koo, Kwang Sik Lee, Young Choi, Byung Rae JinAbstract:To enhance the production efficiency of foreign protein in baculovirus expression systems, the effects of Polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with Polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). Fusion expressions with Polyhedrin amino acids 19 to 110 and 32 to 110 lead to localization of recombinant protein into the nucleus and mediate its assembly. The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production was shown by the mutation of the NLS within the fused Polyhedrin fragment. In addition, when the Polyhedrin fragment fused with EGFP was not localized in the nucleus, some fragments increased the production of protein. Among these fragments, some degradation of only the fused Polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The fusion of amino acids 32 to 85 may be more useful for the enhanced and intact production of recombinant protein. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with Polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. This study suggests a new option for higher expression of useful foreig
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Hyper-Enhanced Production of Foreign Recombinant Protein by Fusion with the Partial Polyhedrin of Nucleopolyhedrovirus
PLoS ONE, 2013Co-Authors: Sung Min Bae, Jae Young Choi, Tae-young Shin, Jae-bang Choi, Hee Jung Kim, Jun Beom Lee, Hyun Na Koo, Kwang Sik Lee, Byung Rae JinAbstract:To enhance the production efficiency of foreign protein in baculovirus expression systems, the effects of Polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with Polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). Fusion expressions with Polyhedrin amino acids 19 to 110 and 32 to 110 lead to localization of recombinant protein into the nucleus and mediate its assembly. The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production was shown by the mutation of the NLS within the fused Polyhedrin fragment. In addition, when the Polyhedrin fragment fused with EGFP was not localized in the nucleus, some fragments increased the production of protein. Among these fragments, some degradation of only the fused Polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The fusion of amino acids 32 to 85 may be more useful for the enhanced and intact production of recombinant protein. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with Polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. This study suggests a new option for higher expression of useful foreign recombinant protein by using the partial Polyhedrin in baculovirus.
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insecticidal activity of recombinant baculovirus expressing both spider toxin isolated from araneus ventricosus and bacillus thuringiensis crystal protein fused to a viral Polyhedrin
Entomological Research, 2012Co-Authors: Myungpyo Jung, Jae Young Choi, Byung Rae Jin, Xue Ying Tao, Honghyun ParkAbstract:A novel recombinant Autographa californica nucleopolyhedrovirus (AcMNPV), ApPolh5-3006AvTox2, co-expressing two insecticidal toxins, one isolated from the spider Araneus ventricosus, and the other from Bacillus thuringiensis, was constructed to improve the insecticidal activity of AcMNPV. The recombinant virus was designed to express insect-specific spider toxin, Av-Tox2, under control of the early promoter from Cotesia plutellae bracovirus (CpBV). In addition, the B. thuringiensis cry1-5 crystal protein gene was introduced into the genome of this recombinant virus by fusing it with the viral Polyhedrin gene, thus creating a hybrid Polyhedrin-cry1-5 gene under control of the Polyhedrin gene promoter. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that both Av-Tox2 and Polyhedrin-Cry1-5 fusion protein were successfully expressed in the infected cells. In addition, SDS-PAGE revealed that Polyhedrin-Cry1-5 fusion protein expressed by recombinant viruses occluded the polyhedra. ApPolh5-3006AvTox2 showed significantly reduced LD50 and ST50 values against both Plutella xylostella and Spodoptera exigua larvae. These results strongly suggested that coexpression of spider and B. thuringiensis insecticidal toxins could be successful in improving the insecticidal activity of baculoviruses.
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Characterization of the Helicoverpa assulta nucleopolyhedrovirus genome and sequence analysis of the Polyhedrin gene region.
Journal of Biosciences, 2006Co-Authors: Soo-dong Woo, Jae Young Choi, Byung Rae JinAbstract:A local strain ofHelicoverpa assulta nucleopolyhedrovirus (HasNPV) was isolated from infectedH. assulta larvae in Korea. Restriction endonuclease fragment analysis, using 4 restriction enzymes, estimated that the total genome size of HasNPV is about 138 kb. A degenerate polymerase chain reaction (PCR) primer set for the Polyhedrin gene successfully amplified the partial Polyhedrin gene of HasNPV. The sequencing results showed that the about 430 bp PCR product was a fragment of the corresponding Polyhedrin gene. Using HasNPV partial predicted Polyhedrin to probe the Southern blots, we identified the location of the Polyhedrin gene within the 6 kbEcoRI, 15 kbNcoI, 20 kbXhoI, 17 kbBgl II and 3 kbClaI fragments, respectively. The 3 kbClaI fragment was cloned and the nucleotide sequences of the Polyhedrin coding region and its flaking regions were determined. Nucleotide sequence analysis indicated the presence of an open reading frame of 735 nucleotides which could encode 245 amino acids with a predicted molecular mass of 29 kDa. The nucleotide sequences within the coding region of HasNPV Polyhedrin shared 73.7% identity with the Polyhedrin gene fromAutographa californica NPV but were most closely related toHelicoverpa andHeliothis species NPVs with over 99% sequence identity.
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Characterization of the Polyhedrin Gene of Spodoptera litura Nucleopolyhedrovirus Isolated in Korea
Journal of Asia-Pacific Entomology, 2005Co-Authors: Soo-dong Woo, Byung Rae JinAbstract:Abstract A local strain of Spodoptera litura nucleopolyhedrovirus (SINPV-K1) was isolated from infected larvae of a Korean population of S. litura. Degenerate PCR primer set for the Polyhedrin gene successfully amplified the partial Polyhedrin gene of SINPV-K1. The sequencing results showed that the about 430 bp PCR product was a fragment of Corresponding Polyhedrin gene. Southern blot analysis of SINPV-K1 restriction fragments was performed by using 430 bp Polyhedrin PCR product of SINPV-Kl as a probe. As the result, we identified the location of the Polyhedrin gene within the approximately 6 kb EcoR I-, 3.5 kb Hind III-, 20 kb Xho I- and 4 kb Cla I-digested fragments, respectively. The Hind III 3.5kb fragment was cloned for sequencing the complete Polyhedrin gene. Nucleotide sequence analysis indicated the presence of an open reading frame of 747 nucleotides, which could encode 249 amino acids with a predicted molecular mass of 31 kDa. The nucleotide sequences within the coding region of SINPV-K1 Polyhedrin shared maximum 94.0% similarity with the Polyhedrin gene from previous reported other SINPVs but were most closely related to Spodoptera littoralis NPV with 99.0% sequence identity. These suggest that the SINPV isolate from Korea is a different SINPV strain.
Just M. Vlak - One of the best experts on this subject based on the ideXlab platform.
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Nucleotide Sequence Analysis of the Polyhedrin Gene of Heliothis armigera Single Nucleocapsid Nucleopolyhedrovirus
Virologica Sinica, 1997Co-Authors: X. Chen, Just M. VlakAbstract:The Polyhedrin gene of the Heliothis armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) was localized by Southern blot hybridization with a probe from the Autographa cali-fornica MNPV (AcMNPV) Polyhedrin gene. Nucleotide sequencing showed the open reading frame of the HaSNPV Polyhedrin gene was 738 nt, encoded 246 amino acids with a predicted size of 29 kDa. Alignment with Polyhedrin sequences in GENE Band indicated the highest homology with the Polyhedrin gene of H. zea SNPV(HzSNPV). There were four different nucleotides and only one change (nt 197 C→A)resulted in an amino acid change in HzSNPV (Thr→Lys). Thischange did not alter the secondary structure of the molecule. The promoter regions of both poly-hedrins were identical including the location of the ATAAG core element, where the transcription starts. The amino acid homology with other SNPVs and with MNPVs Polyhedrins was much low-er. The results indicate that HaSNPV and HzSNPV are probably variants of a single baculovirus genotype.
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superior expression of juvenile hormone esterase and β galactosidase from the basic protein promoter of autographa californica nuclear polyhedrosis virus compared to the p10 protein and Polyhedrin promoters
Journal of General Virology, 1994Co-Authors: Bryony C Bonning, Just M. Vlak, Robert D. Possee, P.w. Roelvink, B. D. HammockAbstract:The expression characteristics of the p10, Polyhedrin and basic protein promoters of Autographa californica nuclear polyhedrosis virus were compared using two reporter enzymes, juvenile hormone esterase (JHE) and β-galactosidase. In these systems, JHE is exported from the cell and β-galactosidase is localized to the cytosol. Expression of JHE from the basic, p10 and Polyhedrin promoters was first detected in the medium at 13, 19 and 27 h post-infection respectively. The basic protein promoter yielded the highest expression of the three promoters tested for both enzymes, as determined by protein and enzyme activity assays. In addition, yields of β-galactosidase and JHE under control of the p10 promoter are higher relative to expression under control of the Polyhedrin promoter. These data highlight the importance of investigation of viral promoters other than the Polyhedrin promoter for high yield protein expression in vitro, and for insecticidal use of recombinant baculoviruses requiring high levels of expression. The results support revision of the current concept that very late viral promoters are always optimal for high yield recombinant protein expression.
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Dissimilar expression of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus Polyhedrin and p10 genes.
Journal of General Virology, 1992Co-Authors: P.w. Roelvink, M. M. M. Van Meer, C. A. D. De Kort, R. D. Possee, B. D. Hammock, Just M. VlakAbstract:The temporal expression of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus Polyhedrin and p10 genes in Spodoptera frugiperda cells was studied using virus recombinants in which either gene was replaced by the juvenile hormone esterase (JHE) gene of Heliothis virescens. The JHE served as a highly specific and sensitive reporter for gene expression. Activation of the p10 gene followed a pattern different to that of Polyhedrin. The p10 gene was activated a few hours earlier than the Polyhedrin gene, but its expression reached a lower maximum level. Northern blot analysis complemented and confirmed the results obtained from the JHE assays. Co-infection of sense recombinants and those containing an anti-sense copy of the JHE gene in place of the Polyhedrin or p10 gene resulted in reduced levels of JHE gene expression. These experiments independently supported the hypothesis that the p10 gene promoter is more active at earlier times post-infection than that of the Polyhedrin gene. The results also highlight the potential of the antisense strategy as an experimental approach for the study of baculovirus gene regulation and possibly insect metabolism.
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Expression of the Autographa californica nuclear polyhedrosis virus p10 gene: effect of Polyhedrin gene expression.
Archives of Virology, 1992Co-Authors: M M Van Oers, J. M. P. Jore, D. Malarme, Just M. VlakAbstract:Two major late proteins, Polyhedrin and p10, are synthesized in large quantities in baculovirus infected insect cells. This and the fact that both proteins are dispensable for virus replication, form the basis for the use of these viruses as vector for foreign gene expression. To address the question whether theAutographa californica nuclear polyhedrosis virus p10 promotor-driven expression is influenced by the concurrent expression of the Polyhedrin gene, several recombinants were constructed with various deletions in the Polyhedrin gene. TheEscherichia coli lacZ gene was used as a marker to allow direct comparison between p10 and Polyhedrin-driven expression. None of the deletions in the Polyhedrin gene did result in higher expression of the p10 promoter-controlled gene. The suggested that the transcriptional and/or translational activity of the p10 and Polyhedrin gene are independently regulated. To compare the level of Polyhedrin and p10 promoter driven expression, recombinants with the lacZ gene cloned behind either promoter were studied. No significant difference in level of expression was observed. In cells infected with a recombinant with the lacZ gene present behind both promoters a reduced level of expression was observed, whereas a considerable increase was expected. This may be due to instability of the viral genome, as two copies of the lacZ gene were present.
James E Maruniak - One of the best experts on this subject based on the ideXlab platform.
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the anticarsia gemmatalis nuclear polyhedrosis virus Polyhedrin gene region sequence analysis gene product and structural comparisons
Journal of General Virology, 1992Co-Authors: Paolo Marinho De Andrade Zanotto, Maria Jose A Sampaio, Dave W Johnson, Talles L Rocha, James E MaruniakAbstract:The genomic region of the Anticarsia gemmatalis multiple nucleocapsid nuclear polyhedrosis virus (AgMNPV) strain 2D encoding the Polyhedrin gene was cloned and mapped, and a 2085 bp SphI-PstI fragment containing the gene was sequenced. The Polyhedrin polypeptide of the parental isolate AgMNPV was manually sequenced, and the amino acid sequence obtained agreed with that deduced from the DNA coding region sequence. AgMNPV and Orgyia pseudotsugata MNPV (OpMNPV) are similar in terms of promoter structure and Polyhedrin primary sequence, and the Polyhedrin gene of both viruses is transcribed in the anti-clockwise direction in relation to their physical maps. The region upstream from the Polyhedrin gene of AgMNPV, OpMNPV, Bombyx mori NPV and Autographa californica MNPV (AcMNPV) was compared and this showed that the open reading frame (ORF) common to all four viruses (ORF 5) has sequence homology with the AcMNPV 25K gene. The sequences between ORF 5 and the Polyhedrin gene were found to be variable among the Polyhedrin gene loci compared. Additionally, conserved elements in the promoters of the very late genes encoding Polyhedrin and granulin, and those encoding two p10 proteins were found to share sequence homology and positional similarity with consensus regions in the conserved boxes A and C, responsible for binding transcription factors to eukaryotic 5S ribosomal RNA genes, and to box C of tRNA genes.
