A novel fluorescence resonance energy transfer assay demonstrates that the human immunodeficiency virus type 1 Pr55Gag I domain mediates Gag-Gag interactions.
Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55(Gag) polyprotein (Gag). One of the essential steps in the assembly process is the multimerization of Gag. We have developed a novel fluorescence resonance energy transfer (FRET) assay for the detection of protein-protein interactions between Gag molecules. We demonstrate that Gag multimerization takes place primarily on cellular membranes, with the majority of these interactions occurring on the plasma membrane. However, distinct sites of Gag-Gag interaction are also present at punctate intracellular locations. The I domain is a functional assembly domain within the nucleocapsid region of Gag that affects particle density, the subcellular localization of Gag, and the formation of detergent-resistant Gag protein complexes. Results from this study provide evidence that the I domain mediates Gag-Gag interactions. Using Gag-fluorescent protein fusion constructs that were previously shown to define the minimal I domain within HIV-1 Pr55(Gag), we show by FRET techniques that protein-protein interactions are greatly diminished when Gag proteins lacking the I domain are expressed. Gag-Tsg101 interactions are also seen in living cells and result in a shift of Tsg101 to the plasma membrane. The results within this study provide direct evidence that the I domain mediates protein-protein interactions between Gag molecules. Furthermore, this study establishes FRET as a powerful tool for the detection of protein-protein interactions involved in retrovirus assembly.
Derdowski
Aaron
A
Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2581, USA.
Ding
Lingmei
L
Spearman
Paul
P
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D019065:Virus Assembly; D000099177:Tsg101 Protein
D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D015683:Gene Products, gag; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000099177:Tsg101 Protein; C059767:p55 gag precursor protein, Human immunodeficiency virus 1