Journal
JOURNAL OF CELL BIOLOGY
Volume 162, Issue 7, Pages 1233-1244Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.200303200
Keywords
Rous sarcoma virus; two photon; fluorescence resonance energy transfer; protein-protein transfer
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Funding
- NCI NIH HHS [R01 CA020081, CA20081] Funding Source: Medline
- NCRR NIH HHS [P41-RR04224] Funding Source: Medline
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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.
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