Direct measurement of Gag-Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy

被引:90
作者
Larson, DR
Ma, YM
Vogt, VM
Webb, WW
机构
[1] Cornell Univ, Sch Appl & Engn Phys, Ithaca, NY 14853 USA
[2] Cornell Univ, Dept Mol Biol & Genet, Ithaca, NY 14853 USA
关键词
Rous sarcoma virus; two photon; fluorescence resonance energy transfer; protein-protein transfer;
D O I
10.1083/jcb.200303200
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
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.
引用
收藏
页码:1233 / 1244
页数:12
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