Direct Measurement of Gag-Gag Interaction during Retrovirus Assembly with FRET and Fluorescence Correlation Spectroscopy

• Published in: The Journal of cell biology 2003-09, Vol.162 (7), p.1233-1244
• Main Authors: Larson, Daniel R., Ma, Yu May, Vogt, Volker M., Webb, Watt W.
• Format: Article
• Language: English
• Subjects: Animals; Budding; Cell Line; Cell membranes; Cells; Cellular biology; Chickens; Cytoplasm; Cytosol; Cytosol - metabolism; Diffusion coefficient; Fibroblasts - cytology; Fibroblasts - virology; Fluorescence; Fluorescence Resonance Energy Transfer; Gag protein; Gene Products, gag - analysis; Gene Products, gag - genetics; Gene Products, gag - metabolism; Green Fluorescent Proteins; Indicators and Reagents - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Molecules; Mutagenesis - physiology; Plasma interactions; Proteins; Retroviridae - growth & development; Retroviridae - metabolism; Rous sarcoma virus; Spectrometry, Fluorescence; Spectrum analysis; Subcellular Fractions - metabolism; Virus Assembly - physiology; Viruses
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.200303200

During retrovirus assembly, the polyprotein Gag directs protein multimerization, membrane binding, and RNA packaging. It is unknown whether assembly initiates through Gag-Gag interactions in the cytosol or at the plasma membrane. We used two fluorescence techniques-two-photon fluorescence resonance energy transfer and fluorescence correlation spectroscopy-to examine Rous sarcoma virus Gag-Gag and -membrane interactions in living cells. Both techniques provide strong evidence for interactions between Gag proteins in the cytoplasm. Fluorescence correlation spectroscopy measurements of mobility suggest that Gag is present in large cytosolic complexes, but these complexes are not entirely composed of Gag. Deletion of the nucleocapsid domain abolishes Gag interactions and membrane targeting. Deletion of the membrane-binding domain leads to enhanced cytosolic interactions. These results indicate that Gag-Gag interactions occur in the cytosol, are mediated by nucleocapsid domain, and are necessary for membrane targeting and budding. These methods also have general applicability to in vivo studies of protein-protein and -membrane interactions involved in the formation of complex macromolecular structures.