The global responses of human bone marrow-derived mesenchymal stem cells to baculovirus transduction
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE15810 | GEO | 2009/04/30
SECONDARY ACCESSION(S): PRJNA116723
REPOSITORIES: GEO
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Project description:Baculovirus expression systems have been widely used to produce recombinant mammalian proteins owing to the lack of viral replication in vertebrates. Although several lines of evidence have demonstrated impacts of baculovirus infection in mammalian hosts, genome-wide effects have not been fully elucidated. Here, we provide comparative transcriptome profiles of baculovirus and host-immune response genes in recombinant baculovirus-infected mammalian and insect cells. Specifically, to decipher the impacts of baculovirus infection in mammalian cells, we conducted total RNA-seq on human 293 cells and insect Sf9 cells infected with recombinant baculovirus. We found that baculovirus genes were rarely expressed under the control of baculoviral promoters in 293 cells. Although some baculovirus early genes, such as PE38 and IE-01, showed limited expression in 293 cells, baculoviral late genes were mostly silent. We also found modest induction of a small number of mammalian immune response genes associated with Toll-like receptors, cytokine signaling, and complement in baculovirus-infected 293 cells. These comprehensive transcriptome data will contribute to improving recombinant baculovirus as tools for gene delivery, gene therapy, and vaccine development.
2020-09-28 | GSE158603 | GEO
Project description:The human Adeno-Associated Virus serotype 2 (WT AAV2) is a common non-pathological virus and its recombinant form (rAAV) is widely used as gene therapy vector. However, it has been shown that WT AAV2 and recombinant AAV display significantly different characteristics, especially regarding infection rate, with a near perfect infectivity and better encapsidation rate of WT AAV2. Even though rAAVs are routinely produced in the Baculovirus/Sf9 cell system, WT AAV2 has never been produced in this context. To understand the infectivity and encapsidation rate differences between WT AAV2 and rAAV, we tried to produce WT AAV2 in baculovirus/Sf9 cells system hypothesizing that the WT AAV2 may be considered as a normal recombinant AAV transgene. Through our attempts to produce WT AAV2 in Baculovirus/Sf9, we found that WT AAV2 p5 promoter, which controls the expression of large Rep proteins in mammalian cells, was active in this system. p5 promoter activity in the baculovirus/Sf9 cell system led to the expression of Rep78 that finally excises WT AAV2 genome from the baculovirus genome during the earliest phase of baculovirus stock production. The p5 promoter expression kinetics and the specific strand RNA-Seq analysis of the WT AAV2, rAAV Rep2/Cap2 cassettes in the baculovirus context was performed. We demonstrate that the WT AAV2 native promoters, p5, p19 and p40 are all active and lead to the expression of different proteins and peptides. In addition, this study demonstrates that the baculovirus brings at least some of the helper functions needed in the AAV replication/life cycle.
2018-06-21 | GSE96855 | GEO
Project description:Baculovirus-encoded proteins can be efficiently produced following infection of different Drosophila cell lines. Epitope-tagged proteins produced in baculovirus-infected cells provide genome-wide profiles of the histone variant H2Av that are comparable to those produced by stably transformed cell lines. Keywords: Chromatin affinity-purification on microarray
2010-07-12 | GSE21416 | GEO
Project description:MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host–virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 59 end of miRNA. The 59 ends of the miRNAs were more conserved than the 39 ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect’s miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host–virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs.
2015-06-11 | GSE60064 | GEO
Project description:Recombinant insect baculoviral vectors efficiently transduce several types of cells in the brain and can possibly be used for gene therapy for brain disorders. To verify the suitability of using these viral vectors to develop gene therapy strategies in the brain, we evaluated immune reactions upon acute administration of baculoviral vectors into the brain of the cynomolgus macaque using microarray global gene expression profiling. Adult male cynomolgus macaques (Macaca fascicularis) were administered with baculovirus BV-HSVtk purified by membrane chromatography + high-speed centrifugation (MC+HS) into the brain.
2015-11-21 | E-GEOD-75264 | biostudies-arrayexpress
Project description:Baculovirus-insect cell culture technology is applied for the production of veterinary and human vaccines‚ gene delivery vectors‚ and biopesticides. The current study investigated systemic cell – virus interactions under different cell culture conditions resulting in differing in virus occlusion bodies (OB)/cell yields for HearNPV infected Hzea cells. 22 microarrays were used. While mRNA levels for most genes decreased, a core set of 14 genes consistently increased in all 11 different infection conditions studied‚ suggesting a conserved up regulation pattern of these genes upon baculovirus infections. KEGG pathway analysis found strongest responses by the genes for the E3 ubiquitin-protein ligase Topors in the ubiquitin proteasome pathway‚ GAPDH in the glycolytic pathway‚ and host DNA ligase and DNA helicase in the replication process. Weighted co expression network analysis determined a module and 5 hub genes that strongly correlated to OB/cell yields. The endoplasmic reticulum functional group was particularly reduced in low producing systems. Total virus mRNA and viral genome levels had positive correlations to OB/cell yields. Virus genes were expressed in a similar temporal pattern regardless of the cell lines‚ media or infection cell densityies used. The virus Chitinase gene and Superoxide dismutase gene may be particularly important for high productivities. Overall this genome-scale expression study of different infection conditions provides insights into host insect cell responses to baculovirus infections and suggests gene expression changes that are likely important for high virus yields.
2014-11-25 | GSE63595 | GEO
Project description:MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host–virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 59 end of miRNA. The 59 ends of the miRNAs were more conserved than the 39 ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect’s miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host–virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs. Identification of miRNA and other small non coding RNA in NPV infected Sf9 cells
2015-06-11 | E-GEOD-60064 | biostudies-arrayexpress
Project description:Baculovirus-encoded proteins can be efficiently produced following infection of different Drosophila cell lines. Epitope-tagged proteins produced in baculovirus-infected cells provide genome-wide profiles of the histone variant H2Av that are comparable to those produced by stably transformed cell lines. Keywords: Chromatin affinity-purification on microarray All experiments were done using strepavidin or FLAG pulldown DNA cohybridized with total input DNA to the same array. Two channels per array, Cy5 and Cy3, were used in each experiment.
2010-07-12 | E-GEOD-21416 | biostudies-arrayexpress
Project description:System level knowledge of host alterations is crucial to elucidate the molecular events of viral pathogenesis and to develop strategies to block viral establishment and amplification. Here, we applied quantitative proteomics approach to study global proteome changes in the host; Spodoptera frugiperda upon infection by a baculovirus, Spodoptera litura NPV at two stages i.e. 12h and 72h post infection. At 12hpi, >95% of host proteins remained stable, however at 72hpi, 52% host proteins exhibited downregulation of 2-fold or more. Functional analysis revealed significant upregulation of transposition and proteasomal machinery while translation, transcription, protein export and oxidative phosphorylation pathways were adversely affected. An assessment of perturbed proteome after viral infection and viral miRNAs expression led to the identification of 117 genes that are potential targets of 10 viral miRNAs. Using miRNA mimics, we confirmed the down regulation of 9 host genes. The results comprehensively show dynamics of host responses after viral infection.
2017-09-25 | PXD005870 | Pride
Project description:Recombinant baculoviral vectors efficiently transduce several types of cells in the brain. To characterize host responses to viral challenge, thus verifying the suitability of using the virus for the development of gene therapy strategies in the central nervous system, we used cDNA microarray technology to examine in vitro and in vivo global cellular gene expression profiles after viral transduction. We demonstrated that the transduction induced host antiviral responses as a major reaction in all three types of samples profiled, including the rat brain, cultured human astrocytes and human neuronal cells. The related genes were mainly those associated with innate immunity. Several genes of the major histocompatibility complex molecules, an important component of the host adaptive immunity to exogenous pathogens, were up-regulated in the rat brain and human astrocytes, but not in neuronal cells. We also observed that genes related to cell death and apoptosis were up-regulated and genes related cell cycle regulation were down-regulated in neuronal cells, but not obviously affected in astrocytes. These findings should be useful in understating the molecular basis for neural cell response to baculoviral transduction and guiding rational applications of baculoviral vectors in the central nervous systems Experiment Overall Design: CMV-Luc baculovirus with a luciferase reporter gene under the control of hCMV promoter and CMV-GFP baculovirus with a gene encoding for a green fluorescence protein were constructed as described previously (Wang et al., 2006). Both vectors were used to confirm baculoviral transduction. Our pilot test, as well as previous studies (Stilwell and Samulski, 2003; Zhao et al., 2005), demonstrated no detectable difference between viral vectors with and without a reporter gene in affecting global gene expression profiles. Thus, CMV-Luc baculovirus was used throughout microarray experiments. Experiment Overall Design: Recombinant baculovirus vectors were produced and propagated in Spodoptera frugiperda (Sf9) insect cells according to the manual of the Bac-to-Bac baculovirus expression system (Invitrogen). Sf9 insect cells pre-adapted to Sf-900 II serum-free medium were purchased from Invitrogen (Carlsband, CA) and grown in spinner flasks at 27.5oC. Budded viruses in the insect cell culture medium were collected and filtered through a 0.45-μm pore size filter (Minipore, Bedford, MA, USA) to remove any contamination, and concentrated by ultracentrifugation at 28,000g for 60 min. Viral pellets were re-suspended in appropriate volumes of 0.1 M phosphate-buffered saline (PBS) and their infectious titers (plaque-forming units, pfu) were determined by plaque assay on Sf9 cells. Experiment Overall Design: For in vitro study, normal human astrocytes (Lonza, Basel, Switzerland) were cultured in Astrocyte Basal Medium (ABM) supplemented with the AGM SingleQuots. Cells were maintained according to manufacturerâs instructions. Human neuronal cells, transdifferentiated from human Cord Blood Stem Cell culture, were obtained from Celprogen (San Pedro, CA). The cells were cultured on flasks coated with neuronal expansion matrix in human neuronal expansion complete media and maintained according to the manufacturerâs instructions. Cells were transduced with baculoviruses in Optimem (Invitrogen) at an MOI of 50 and incubated at 37°C for 1 h. After the incubation, the medium containing the viruses was replaced by fresh growth medium, and the cells were collected 48 hours later for analysis.
2008-06-17 | E-GEOD-9834 | biostudies-arrayexpress