Palmitoylation of Semliki Forest virus glycoproteins in insect cells (C 6/36) occurs in an early compartment and is coupled to the cleavage of the precursor p 62

Schärer, C. G.; Naim, Hassan Y.; Koblet, H.

doi:10.1007/bf01309536

Cited by 11 publications

(6 citation statements)

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“…Acylation of E1 and E2 is also involved in alphavirus budding [46], [47], but was not investigated in this work due to the documented ability of insect cells to palmitoylate the glycoproteins of AcMNPV, Marburg virus, and SFV [48],[49], [50]. Taken together, these observations suggested that Sf 21 cellular or culture conditions were influencing the conformation and stability of the E1/E2 complex thought to be involved in budding [21].…”

Section: Resultsmentioning

confidence: 99%

Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line

Wagner¹,

Pajerowski²,

Daniels³

et al. 2014

PLoS ONE

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Chikungunya virus-like particles (VLPs) have potential to be used as a prophylactic vaccine based on testing in multiple animal models and are currently being evaluated for human use in a Phase I clinical trial. The current method for producing these enveloped alphavirus VLPs by transient gene expression in mammalian cells presents challenges for scalable and robust industrial manufacturing, so the insect cell baculovirus expression vector system was evaluated as an alternative expression technology. Subsequent to recombinant baculovirus infection of Sf21 cells in standard culture media (pH 6.2–6.4), properly processed Chikungunya structural proteins were detected and assembled capsids were observed. However, an increase in culture pH to 6.6–6.8 was necessary to produce detectable concentrations of assembled VLPs. Since this elevated production pH exceeds the optimum for growth medium stability and Sf21 culture, medium modifications were made and a novel insect cell variant (SfBasic) was derived by exposure of Sf21 to elevated culture pH for a prolonged period of time. The high-pH adapted SfBasic insect cell line described herein is capable of maintaining normal cell growth into the typical mammalian cell culture pH range of 7.0–7.2 and produces 11-fold higher Chikungunya VLP yields relative to the parental Sf21 cell line. After scale-up into stirred tank bioreactors, SfBasic derived VLPs were chromatographically purified and shown to be similar in size and structure to a VLP standard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG and neutralizing titers from guinea pigs vaccinated with SfBasic derived VLPs or HEK293 derived VLPs were not significantly different with respect to production method, suggesting that this adapted insect cell line and production process could be useful for manufacturing Chikungunya VLPs for use as a vaccine. The adaptation of Sf21 to produce high levels of recombinant protein and VLPs in an elevated pH range may also have applications for other pH-sensitive protein or VLP targets.

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Section: Resultsmentioning

confidence: 99%

Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line

Wagner¹,

Pajerowski²,

Daniels³

et al. 2014

PLoS ONE

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“…For example, the SFV glycoprotein E2 is palmitoylated close to its transmembrane domain (Scharer et al, 1993) but is not transported to MLMs. Palmitoylation might therefore be overruled by additional sorting signals in proteins that are not destined for myelin.…”

Section: Discussionmentioning

confidence: 99%

Palmitoylation is a sorting determinant for transport to the myelin membrane

Schneider

Länder

Schulz

et al. 2005

Journal of Cell Science

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IntroductionSpecialized glial cells, oligodendrocytes and Schwann cells, synthesize myelin as a multilamellar, compact membrane that ensheathes and insulates axons (Pedraza et al., 2001;Salzer, 2003). The structure and molecular composition of myelin is unique. In contrast to most plasma membranes, myelin is enriched in glycosphingolipids. The major glycosphingolipids in myelin are galactosylceramide and its sulfated derivative sulfatide (20% of lipid dry weight) (Morell and Jurevics, 1996). Myelin contains a restricted set of proteins, which are in most cases exclusively found in myelin. The major central-nervoussystem myelin proteins, the myelin basic protein (MBP) and the proteolipid proteins (PLP/DM20), are low-molecularweight proteins found in compact myelin and constitute 80% of total myelin proteins. PLP and its alternatively spliced isoform DM20 are acylated integral-membrane proteins, containing four transmembrane α-helices with cytoplasmic Nand C-termini, and molecular masses of 26 kDa and 20 kDa, respectively (Griffiths et al., 1998).The mechanisms by which oligodendrocytes target myelin components to myelin are not known. Studies performed in epithelial cells demonstrated that polarized protein transport to distinct apical and basolateral membrane compartments, which are separated by tight junctions, occurs after sorting in the trans-Golgi network into distinct vesicular carriers (Kreitzer et al., 2003). A polarized membrane composition and distinct trafficking routes have also been proposed for oligodendrocytes, because asymmetric membrane growth (oligodendroglial plasma membrane versus myelin membrane) requires polarized transport (de Vries et al., 1998). This hypothesis has been supported by the identification of claudin11/OSP-based tight junctions in the interlamellar strands of myelin sheaths at the border between compact and non-compact myelin. Tight junctions could serve as a diffusion barrier and maintain the asymmetric protein distribution between the different myelin subdomains and the plasma membrane (Bronstein et al., 2000;Gow et al., 1999;Morita et al., 1999). For epithelial cells, two major sorting principles have been described. Transport to the basolateral surface requires the interaction of a cytoplasmic protein transport machinery with transmembrane proteins (Mellman, 1996;Mostov et al., 2003;Nelson and Yeaman, 2001). Transport to the apical membrane involves the association of proteins with glycosphingolipid-and cholesterol-enriched membrane 2415Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a restricted set of proteins. The mechanisms by which oligodendrocytes target myelin components to myelin are not known. To identify the sorting determinants for protein transport to myelin, we used a primary oligodendrocyte culture system in which terminal differentiation is synchronized and there is excessive deposition of myelinlike membranes (MLMs). Because several myelin proteins are palmitoylated, we explored the role of acylation in protein ...

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“…7). As acylation of E~ takes place in the ER of C6/36 cells even at 8 °C [38] the two bands of E1 were tentatively identified as follows: Lower band: newly synthesized Ea containing a high mannose type glycan; upper band: acylated E1 with a high mannose type glycan. At higher temperatures, additional bands appear corresponding to the glycosylated and trimmed virion forms [29].…”

Section: Discussionmentioning

confidence: 98%

“…For the analysis of intracellular protein complex formation the viral protein complexes were isolated by sucrose density gradient centrifugation as described recently [38]. Gradient fractions were then analysed by SDS-PAGE and evaluated by densitometric scanning.…”

Section: Isolation Of Protein Complexesmentioning

confidence: 99%

Temperature-sensitive steps in the transport of Semliki Forest virus envelope proteins in mosquito C6/36 cells

Vallan

Schärer

Koblet

1994

Archives of Virology

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We have analysed the temperature dependence of the transport of Semliki Forest virus (SFV) envelope proteins in mosquito cells, the natural host cells of alphaviruses. These cells are cultivated at a lower temperature (28 degrees C) and have a different lipid composition as compared to mammalian cells. When the incubation temperature was reduced at early times after infection, the onset of virus shedding was delayed and the maximal titers decreased correspondingly to the temperature. No virus was shed at 12 degrees C. No evidence was observed for a block of virus release due to a shift of the sites of virus maturation. When the incubation temperature was reduced at later times after infection a critical temperature of 12 degrees C was again observed. At this temperature no transport of viral proteins took place, p62 remained uncleaved, the glycan processing of E1 did not occur and the envelope proteins accumulated in a pre-Golgi compartment. We suggest a mathematical formula which allows the extrapolation of transport data to the temperature at which intracellular protein transport becomes blocked.

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Palmitoylation of Semliki Forest virus glycoproteins in insect cells (C 6/36) occurs in an early compartment and is coupled to the cleavage of the precursor p 62

Cited by 11 publications

References 48 publications

Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line

Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line

Palmitoylation is a sorting determinant for transport to the myelin membrane

Temperature-sensitive steps in the transport of Semliki Forest virus envelope proteins in mosquito C6/36 cells

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