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Published 29 September 2003. doi:10.1083/jcb.200303200
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© The Rockefeller University Press, 0021-9525/2003/9/1233 $8.00
The Journal of Cell Biology, Volume 162, Number 7, 1233-1244

Article

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



Daniel R. Larson1, Yu May Ma2, Volker M. Vogt2 and Watt W. Webb1

1 School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853
2 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853

Address correspondence to Watt W. Webb, 212 Clark Hall, Cornell University School of Applied and Engineering Physics, Ithaca, NY 14853. Tel.: (607) 255-3331. Fax: (607) 255-7658. email: www2{at}cornell.edu

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.

Key Words: Rous sarcoma virus; two photon; fluorescence resonance energy transfer; protein–protein transfer

M. Ma's present address is Dept. of Cell Biology, Harvard Medical School, Boston, MA 02115.

Abbreviations used in this paper: 2PE, 2-photon excitation; CA, capsid domain; FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy transfer; MA, matrix domain; NC, nucleocapsid domain; PR, protease domain; RSV, Rous sarcoma virus; VLP, virus-like particle.


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