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Journal of Virology, July 2009, p. 7322-7336, Vol. 83, No. 14
0022-538X/09/$08.00+0     doi:10.1128/JVI.02545-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding{triangledown} ,{dagger}

Ian B. Hogue,1 Adam Hoppe,1,2,{ddagger} and Akira Ono1*

Department of Microbiology and Immunology,1 Center for Live-Cell Imaging, University of Michigan Medical School, Ann Arbor, Michigan 481092

Received 10 December 2008/ Accepted 21 April 2009

The human immunodeficiency virus type 1 structural polyprotein Pr55Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Michigan Medical School, 5736 Medical Science Bldg. II, 1150 W. Medical Center, Dr. Ann Arbor, MI 48109. Phone: (734) 615-4407. Fax: (734) 746-3562. E-mail: akiraono{at}umich.edu

{triangledown} Published ahead of print on 29 April 2009.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

{ddagger} Present address: Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007.

Journal of Virology, July 2009, p. 7322-7336, Vol. 83, No. 14
0022-538X/09/$08.00+0     doi:10.1128/JVI.02545-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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