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.
Highlights
Foamy viruses (FVs) are complex animal retroviruses, which encode for structural (Gag, Env), enzymatic (Pol) and regulatory proteins such as the transactivator protein, Tas
The expression of the Gag mutant missing both the GRII and GRIII domains (Gag511) was restricted only to some cells. These results suggest that the prototypic foamy virus (PFV) Gag region harboring the GRII box is responsible for the intercellular trafficking of the protein
While the parental green fluorescent protein (GFP) remained confined to expressing cells, we found that the 23 amino acids peptide, spanning from aa 535 to aa 557 of PFV Gag, was sufficient to allow the extension of the GFP signal from some transfected cells to the nucleus of surrounding ones (Fig. 1C)
Summary
Foamy viruses (FVs) are complex animal retroviruses, which encode for structural (Gag, Env), enzymatic (Pol) and regulatory proteins such as the transactivator protein, Tas. The prototypic foamy virus (PFV) Gag precursor lacks the landmarks of orthoretrovirus Gag polyproteins, such as the major homology region and the Cys-His motif, and rather displays unique distinctive features. The N-terminus of PFV Gag harbors a coiled-coil motif that interacts with the light chain 8 of cellular dynein, allowing microtubule-dependent trafficking of incoming viral particles towards the microtubule organizing center [2]. Three short glycine-arginine-rich (GR) boxes were mapped in the C-terminus of PFV Gag and have been implicated in viral genomic RNA packaging, reverse transcription and viral particles morphogenesis. The GRI domain binds to viral nucleic acids in vitro [3], while the GRII box contains an essential chromatin-binding sequence (CBS) that, by interacting with histone proteins, allows tethering of incoming PFV pre-integration complex to host chromosomes prior to viral genome integration [4]. The GRIII box can functionally complement the GRI box and vice versa [7]
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