PMID:22393357

Citation	Tobaly-Tapiero, J, Zamborlini, A, Bittoun, P and Saïb, A (2012) Investigating the intercellular spreading properties of the foamy virus Gag protein. PLoS ONE 7:e31108
Abstract	Small regions called protein transduction domains (PTDs), identified in cellular and viral proteins, have been reported to efficiently cross biological membranes. Here we show that the structural Gag protein of the prototypic foamy virus (PFV) is apparently able to move from cell to cell and to transport the green fluorescent protein (GFP) from few transfected cells to the nuclei of the entire monolayer. Deletion studies showed that this property lies within the second glycine/arginine (GRII) box in the C-terminus of the protein. We also found that uptake and nuclear accumulation of Gag GRII expressed as GFP-fusion protein in recipient cells was observed only following methanol fixation, but never in living cells or when cells were fixed with glutaraldehyde or treated with trichloroacetic acid prior to methanol fixation. Absence of intercellular spreading in vivo was further confirmed using a sensitive luciferase activity assay based on transactivation of the PFV long terminal repeats. Thus, we conclude that intercellular spreading of PFV Gag represents an artificial diffusion event occurring during cell fixation, followed by nuclear retention mediated by the chromatin-binding sequence within the Gag GRII box. In light of these results, we advise caution before defining a peptide as PTD on the basis of intercellular spreading observed by fluorescence microscopy.
Links	PubMed PMC3290618 Online version:10.1371/journal.pone.0031108
Keywords	Animals; Biological Transport; COS Cells; Cercopithecus aethiops; Cytoplasm/metabolism; Diffusion; Gene Deletion; Gene Products, gag/metabolism; Glutaral/chemistry; Green Fluorescent Proteins/metabolism; Methanol/chemistry; Microscopy, Fluorescence/methods; Models, Biological; Open Reading Frames; Plasmids/metabolism; Protein Structure, Tertiary; Spumavirus/metabolism; Transfection
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
FOAMV:GAG		GO:0039715: nuclear viral factory	ECO:0000314:		C		Figure1.C. After methanol fixation, cells that were expressing GFP or GFP-GagGRII were examined for intrinsic fluorescence. Nuclei are stained with DAPI. Original magnification ×40. Red arrows indicate a cell undergoing mitosis	complete CACAO 12681
FOAMV:GAG		GO:0044094: host cell nuclear part	ECO:0000314:		C		Figure1.C. After methanol fixation, cells that were expressing GFP or GFP-GagGRII were examined for intrinsic fluorescence. Nuclei are stained with DAPI. Original magnification ×40. Red arrows indicate a cell undergoing mitosis	complete CACAO 12682
SFVCP:GAG		GO:0042025: host cell nucleus	ECO:0000314:		C		Figure1.C. After methanol fixation, cells that were expressing GFP or GFP-GagGRII were examined for intrinsic fluorescence. Nuclei are stained with DAPI. Original magnification ×40. Red arrows indicate a cell undergoing mitosis GAG_SFVCP for Simian foamy virus (isolate chimpanzee) (SFVcpz)	complete CACAO 12683

References

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