Direct measurement of Gag–Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy
| Autor: | Volker M. Vogt, Watt W. Webb, Yu May Ma, Daniel R. Larson |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
viruses
Green Fluorescent Proteins Gene Products gag Fluorescence correlation spectroscopy 7. Clean energy Article Cell Line Cell membrane 03 medical and health sciences Cytosol Retrovirus Fluorescence Resonance Energy Transfer medicine Animals 030304 developmental biology 0303 health sciences Rous sarcoma virus biology Virus Assembly 030302 biochemistry & molecular biology RNA two photon fluorescence resonance energy transfer protein–protein transfer Cell Biology Fibroblasts biology.organism_classification Luminescent Proteins Retroviridae Spectrometry Fluorescence Förster resonance energy transfer medicine.anatomical_structure Biochemistry Mutagenesis Cytoplasm Biophysics Indicators and Reagents Chickens Subcellular Fractions |
| Zdroj: | The Journal of Cell Biology |
| ISSN: | 1540-8140 0021-9525 |
| DOI: | 10.1083/jcb.200303200 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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