Investigating the intercellular spreading properties of the foamy virus Gag protein

• Published in: PloS one Vol. 7; no. 2; p. e31108
• Main Authors: Tobaly-Tapiero, Joelle, Zamborlini, Alessia, Bittoun, Patricia, Saïb, Ali
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 29.02.2012; Public Library of Science (PLoS)
• Subjects: Animals; Arginine; Biological membranes; Biological Transport; Biology; C-Terminus; Cell division; Chlorocebus aethiops; Chromatin; Clonal deletion; COS Cells; Cytoplasm; Cytoplasm - metabolism; Diffusion; Fixation; Fluorescence; Fluorescence microscopy; Fusion protein; Gag protein; Gene Deletion; Gene Products, gag - metabolism; Glutaral - chemistry; Glutaraldehyde; Glycine; Green fluorescent protein; Green Fluorescent Proteins - metabolism; Herpes viruses; Infections; Investigations; Luciferase; Membranes; Methanol; Methanol - chemistry; Microscopy; Microscopy, Fluorescence - methods; Models, Biological; Monomolecular films; Nuclei; Nuclei (cytology); Open Reading Frames; Peptides; Plasmids - metabolism; Protein Structure, Tertiary; Protein transport; Proteins; Spreading; Spumavirus - metabolism; Transfection; Trichloroacetic acid; Viral proteins; Viruses; France
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0031108

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.