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Journal of Virology, November 2003, p. 11651-11660, Vol. 77, No. 21
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.21.11651-11660.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Murine Leukemia Virus Particle Assembly Quantitated by Fluorescence Microscopy: Role of Gag-Gag Interactions and Membrane Association

Mariam Andrawiss,1 Yasuhiro Takeuchi,1 Lindsay Hewlett,2 and Mary Collins1*

Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Science,1 MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom2

Received 25 March 2003/ Accepted 23 July 2003

In order to track the assembly of murine leukemia virus (MLV), we used fluorescence microscopy to visualize particles containing Gag molecules fused to fluorescent proteins (FPs). Gag-FP chimeras budded from cells to produce fluorescent spots, which passed through the same pore-size filters and sedimented at the same velocity as authentic MLV. N-terminal myristylation of Gag-FPs was necessary for particle formation unless wild-type Gag was coexpressed. By labeling nonmyristylated Gag with yellow FP and wild-type Gag with cyan FP, we could quantitate the coincorporation of two proteins into single particles. This experiment showed that nonmyristylated Gag was incorporated into mixed particles at approximately 50% the efficiency of wild-type Gag. Mutations that inhibit Gag-Gag interactions (K. Alin and S. P. Goff, Virology 216:418-424, 1996; K. Alin and S. P. Goff, Virology 222:339-351, 1996) were then introduced into the capsid (CA) region of Gag-FPs. The mutations P150L and R119C/P133L inhibited fluorescent particle formation by these Gag-FPs, but Gag-FPs containing these mutations could be efficiently incorporated into particles when coexpressed with wild-type Gag. When these mutations were introduced into nonmyristylated Gag-FPs, no incorporation into particles in the presence of wild-type Gag was detected. These data suggest that two independent mechanisms, CA interactions and membrane association following myristylation, cooperate in MLV Gag assembly and budding.

* Corresponding author. Mailing address: Windeyer Institute, 46 Cleveland St., London W1T 4JF, United Kingdom. Phone: 44-207-679-9301. Fax: 44-207-679-9301. E-mail: mary.collins{at}ucl.ac.uk.

Journal of Virology, November 2003, p. 11651-11660, Vol. 77, No. 21
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.21.11651-11660.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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