Comparative proteomics analysis of Spodoptera frugiperda cells during Autographa californica multiple nucleopolyhedrovirus infection
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Increasing evidence sugggest that in addition of balculovirus controling insect host, host cells also responds to balculovirus infection. However, compared to existing knowledge on virus gene, host cell responses are relatively poorly understood.In this study, Spodoptera frugiperda (Sf9) cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The protein composition and protein changes of Spodoptera frugiperda (Sf9) cells of different infection stages were analysed by isobaric tag for relative and absolute quantification (iTRAQ) techniques.A total of 4004 Sf9 proteins were identified by iTRAQ and 413 proteins were found as more than 1.5-fold changes in abundance. The 413 proteins were categorised according to GO classification for insects and were categorised into: biological process, molecular function and cellular component.The determination of the protein changes in infected Sf9 cells would help to better understanding of host cell responses and facilitate better design of this virus-host cell interaction in pest insect control and other related fields.Keywords:
Sf9
Autographa californica
Baculoviridae
Full-length and truncated human BCL2 lacking the entire C-terminal hydrophobic domain have been overexpressed in Spodoptera frugiperda insect cells with the baculovirus expression system. Immunoblot analysis with BCL2-specific antibodies revealed that both full-length and truncated BCL2 are expressed as multiple immunoreactive species, suggesting posttranslational modifications. The expression of the full-length but not the truncated BCL2 extended the survival of baculovirus-infected cells by preventing virus-induced DNA cleavage. This result is consistent with the reported protective effect of BCL2 against apoptosis in mammalian lymphocytes and suggests a conserved function in evolution. Subcellular fractionation and indirect immunofluorescence studies in intact cells demonstrated that the recombinant full-length and truncated BCL2 proteins were expressed predominantly as nuclear membrane-associated proteins. These results imply that BCL2 must utilize hydrophobic domains other than the deleted domain for its association with the subcellular membranes. Metabolic labeling of insect cells expressing the full-length and the truncated form of BCL2 with 32P(i) demonstrated that BCL2 is a phosphoprotein.
Sf9
Baculoviridae
Phosphoprotein
Cell fractionation
Infectivity
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The baculovirus expression vector system (BEVS), utilizing the Autographa californica nuclear polyhedrosis virus (AcNPV), has turned out to be an attractive alternative for high-level expression (<600 mg/l) of recombinant proteins. However, there is a shortage of reliable methods for monitoring the infection process in situations where marker proteins cannot be used.Three recombinant baculoviruses, FastBac1-wtGFP, VTBac-GFP, and VL1392-hIL-2Ralpha, all having the genes inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV), were used to infect Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-MB-0503) insect cells. The infection process of the recombinant baculoviruses was monitored by flow cytometric side-scatter and fluorescence intensity analyses over a period of 6-96 h.A clear correlation between the side-scatter (SSC) signal and the relative fluorescence was observed for both of the infected cell lines, compared to noninfected cells. Comparison of SSC histograms from noninfected insect cells with cells infected with the nonfluorescent recombinant baculovirus VL1392-hIL-2Ralpha showed a clear increase of SSC for the infected cells.The SSC parameter can therefore be utilized for flow cytometric monitoring of a baculovirus infection process in situations where suitable markers are not available.
Polyhedrin
Autographa californica
Sf9
Nuclear Polyhedrosis Virus
Baculoviridae
Recombinant virus
mCherry
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Exigua
Autographa californica
Sf9
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Increasing evidence sugggest that in addition of balculovirus controling insect host, host cells also responds to balculovirus infection. However, compared to existing knowledge on virus gene, host cell responses are relatively poorly understood.In this study, Spodoptera frugiperda (Sf9) cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The protein composition and protein changes of Spodoptera frugiperda (Sf9) cells of different infection stages were analysed by isobaric tag for relative and absolute quantification (iTRAQ) techniques.A total of 4004 Sf9 proteins were identified by iTRAQ and 413 proteins were found as more than 1.5-fold changes in abundance. The 413 proteins were categorised according to GO classification for insects and were categorised into: biological process, molecular function and cellular component.The determination of the protein changes in infected Sf9 cells would help to better understanding of host cell responses and facilitate better design of this virus-host cell interaction in pest insect control and other related fields.
Sf9
Autographa californica
Baculoviridae
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It is thought that insect haemocytes, or blood cells, play an important role in baculovirus pathogenesis by amplifying and helping to spread the infection within the insect. Here, infection is described of the lepidopteran noctuid species Spodoptera frugiperda with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV). Late instar S. frugiperda larvae were infected by intrahaemocoelic injection using a recombinant of AcMNPV expressing the enhanced green fluorescent protein gene to visualize infected cells. Approximately 1000-fold higher doses of injected virus were required to initiate infection in S. frugiperda larvae than in another permissive noctuid species, Trichoplusia ni . Infected S. frugiperda larvae survived twice as long as T. ni larvae and exhibited a slower build-up of virus in the haemolymph. In S. frugiperda , infection of fat body and epithelium was observed prior to significant infection of haemocytes, even though the virus was delivered by intrahaemocoelic injection. Expression of eGFP was first detected 12–18 h post-injection within the fat body and, by 24 h, infection had spread to the tracheal and body wall epithelium. In contrast, only 5% of haemocytes were infected at 24 h and the proportion of infected haemocytes increased slowly to only around 50% at 5 days post-infection, when most larval death occurred. Thus, in S. frugiperda , haemocytes do not appear to have a primary role in AcMNPV pathogenesis. This relative lack of infection of haemocytes may in part explain why S. frugiperda larvae are more resistant to AcMNPV infection than T. ni larvae.
Autographa californica
Hemolymph
Baculoviridae
Sf9
Nuclear Polyhedrosis Virus
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Abstract Autographa californica multiple Nucleopolyhedrovirus (AcMNPV) is the archetypal species of the alphabaculovirus . Sf9 are the ovarian cells of their host —Spodoptera frugiperda . In this study, a total of 3,463 pieces of time-series differentially expressed RNA are identified, including 1,200 mRNA and 2,263 lncRNA, with high-throughput sequencing technology using samples collected from Sf9 cells at different time points after AcMNPV infection. The result could provide a reference for the further study of the interaction and regulatory mechanism between the virus and the host.
Sf9
Autographa californica
Identification
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In this study, the kinetic behavior of Sf9 and Sf21 cells used in the production of a baculovirus biopesticide to control the pest of corn Spodoptera frugiperda was analyzed. Kinetic variables such as maximum specific growth rate, cell productivity, mean rate of infection, as well as the mean rate of occlusion body production were determined during the infection of these cell-lines with the extracellular virus of the S. frugiperda nucleopolyhedrovirus (SfMNPV). The Sf9 cell-line resulted in better viral production results (5.0 x 10(8) OB/mL) than the Sf21 cell-line (2.5 x 10(8) OB/mL).
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Baculoviridae
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We described a baculovirus expression system for high level production of secreted murine recombinant IL-4. We have constructed a recombinant baculovirus based on Autographa californica polyhedrosis virus, containing both a synthetic PCR-derived murine IL-4 cDNA under the control of the polyhedrin promoter and the lacZ gene under the control of the P10 promoter to allow an easy detection of recombinant virus. The baculovirus IL-4 was fully functional in biological assay and was present under two glycosylated forms in the supernatants of infected Sf9 cells. We also detected a third unglycosylated intracytoplasmic form resulting from a fusion between the 35 first amino acids of polyhedrin and the murine IL-4. Finally, confocal microscopy showed that this recombinant protein was secreted along a classical pathway like in mammalian cells.
Polyhedrin
Autographa californica
Sf9
Baculoviridae
Nuclear Polyhedrosis Virus
Recombinant virus
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Abstract A novel baculovirus‐based protein expression strategy was developed to produce recombinant proteins in insect cells without contaminating baculovirus virions. This novel strategy greatly simplifies the downstream processing of biopharmaceuticals produced in insect cells. The formation of these virions is prevented by deletion of a baculovirus gene essential for virion formation. The deletion is trans‐ complemented in a transgenic insect cell line in which the baculovirus seed stock is produced. The Autographa californica multicapsid nucleopolyhedrovirus vp80 gene was selected for this purpose, as absence of VP80 prevented the formation of budded virus as well as occlusion‐derived virus, while foreign gene expression was not affected. Sf9 insect cells were engineered to functionally complement the vp80 deletion in the expression vector virus during seed stock production. The trans ‐complemented vp80 ‐deletion baculovirus seed produced an amount of recombinant protein similar to that produced with conventional baculovirus vectors but without contaminating virions. This novel expression method obviates the need to purify the virions away from the biopharmaceuticals. Bioeng. 2011; 108:1056–1067. © 2010 Wiley Periodicals, Inc.
Sf9
Autographa californica
Baculoviridae
Recombinant virus
Nuclear Polyhedrosis Virus
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