Competition between baculovirus polyhedrin and p10 gene expression during infection of insect cells

Chaabihi, Hassan; Ogliastro, M.; Martin, Marianne; Giraud, Catherine; Devauchelle, G.; Cérutti, Martine

doi:10.1128/jvi.67.5.2664-2671.1993

Cited by 64 publications

(25 citation statements)

References 46 publications

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“…Using the quadruple vector, bluetongue virus double shelled VLPs consisting of four proteins were originally synthesized [23]. However, competition presumably occurs among promoters when different heterologous proteins are simultaneously co-expressed by one recombinant baculovirus in the same cells [64], thus inhibiting, to some extent, the protein expression and VLP production. Additionally, a novel and versatile BES, MultiBac, has been developed to allow simultaneous expression of multiple proteins in a single cell, which could be used to produce protein complexes and to recapitulate metabolic pathways [65][66][67][68][69].…”

Section: Monocistronic and Polycistronic Structuresmentioning

confidence: 99%

Use of baculovirus expression system for generation of virus-like particles: Successes and challenges

Liu

et al. 2013

Protein Expression and Purification

104

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The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production. In this article, we critically review the pros and cons of using the BES as a platform to produce both enveloped and non-enveloped VLPs.

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Section: Monocistronic and Polycistronic Structuresmentioning

confidence: 99%

Use of baculovirus expression system for generation of virus-like particles: Successes and challenges

Liu

et al. 2013

Protein Expression and Purification

104

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“…The 1766-nt-long NcoI-KpnI fragment was cloned in the similarly restricted baculovirus transfer vector p10-119pst (kindly provided by M. Cerutti, Station de Recherches de Pathologie Compare! e CNRS-INRA, Saint Christol-lez-Ale' s, France), a modified version of pGm8022, which allows expression under the control of the late p10 gene promoter [22], to give the construct p17AE. The ATG codon in the engineered NcoI site serves as an artificial start codon for translation.…”

Section: Plasmid Constructionsmentioning

confidence: 99%

Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus‒insect cell system

Héricourt¹,

Blanc²,

Redeker³

et al. 2000

Biochem. J.

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All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence.

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“…The polyhedrin and p10 genes are, however, regulated independently. Deletion of the polyhedrin promoter does not affect the expression of the p10 (van Oers et al, 19921, whereas deletion of the p10promoter upregulates even polyhedrin production (Chaabihi et al, 1993). The strengths of the AcMNPV polyhedrin and p10 promoters are on the same order of magnitude, the polyhedrin promoter being about 30% stronger than the p10 promoter (Roelvink et al, 1992).…”

Section: Resultsmentioning

confidence: 99%

Continuous β‐Galactosidase Production in Insect Cells with a p10 Gene Based Baculovirus Vector in a Two‐Stage Bioreactor System

Lier

Duijnhoven

Vaan

et al. 1994

Biotechnology Progress

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Continuous production of polyhedra or baculovirus-expressed proteins in insect cell cultures is limited to about 4 weeks. The decrease in production has been ascribed to the interference of defective deletion mutants with wild-type baculoviruses. The deleted genome sequences include the polyhedrin gene (or the heterologous gene of interest); in the remaining part, the major late p10 gene is always maintained. In the present study, the productivity of a recombinant baculovirus with the lacZ gene from Escherichia coli cloned downstream of the p10 promoter at the p10 locus was investigated. It was hypothesized that this p10 promoter driven gene is preserved over a longer period of time in a continuously operated two-stage bioreactor system than foreign genes behind the polyhedrin promoter at the polyhedrin locus. In two separate runs, beta-galactosidase production with the p10-lacZ recombinant reached quasi-steady-state levels of 30 and 60 units/cm3. Polyhedron production was about 3 x 10(6) and 6 x 10(6) polyhedra/cm3, respectively. However, both polyhedron and beta-galactosidase production decreased after about 30 days of relatively constant production. In the infection reactor, deletion mutants of the virus, which contained both the polyhedrin and lacZ gene, were predominant. Therefore, the presence of the polyhedrin or p10 gene alone in deletion mutants is not sufficient for prolonged expression; other genes involved in major late gene expression and not present in the deleted virus are probably necessary.

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Competition between baculovirus polyhedrin and p10 gene expression during infection of insect cells

Cited by 64 publications

References 46 publications

Use of baculovirus expression system for generation of virus-like particles: Successes and challenges

Use of baculovirus expression system for generation of virus-like particles: Successes and challenges

Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus‒insect cell system

Continuous β‐Galactosidase Production in Insect Cells with a p10 Gene Based Baculovirus Vector in a Two‐Stage Bioreactor System

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