Interactions between HIV-1 Gag Molecules in Solution: An Inositol Phosphate-mediated Switch - Analytical Methods & Tools for Lyophilization

Interactions between HIV-1 Gag Molecules in Solution: An Inositol Phosphate-mediated Switch
Received 9 June 2006; revised 5 October 2006; accepted 21 October 2006. Edited by J. O. Thomas. Available online 26 October 2006.
Siddhartha A.K. Datta1, Zhuojun Zhao2, Patrick K. Clark3, Sergey Tarasov4, Jerry N. Alexandratos5, Stephen J. Campbell1, 1, Mamuka Kvaratskhelia2, Jacob Lebowitz6 and Alan Rein1
Journal of Molecular Biology
Article in Press
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Copyright ? 2006 Elsevier Ltd All rights reserved.
1HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, MD 21702, USA
2Center for Retrovirus Research, College of Pharmacy, The Ohio State University Health Sciences Center, Columbus, OH 43210, USA
3Basic Research Laboratory, SAIC Frederick, Inc., NCI-Frederick, Frederick, MD 21702, USA
4Structural Biophysics Laboratory, National Cancer Institute-Frederick, Frederick, MD 21702, USA
5Macromolecular Crystallography Laboratory, National Cancer Institute-Frederick, Frederick, MD 21702, USA
6Division of Bioengineering and Physical Science, Office of Research Services, National Institutes of Health, Bethesda, MD 20892, USA
Abstract
Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the ?dimer interface? is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.
Keywords: HIV-1; virus assembly; analytical ultracentrifugation; inositol phosphates; protein-protein interaction
Abbreviations: MA, matrix; CA, capsid; NC, nucleocapsid; VLP, virus-like particle; IP, inositol phosphate; IP5, D-myo-inositol 1,3,4,5,6-pentakisphosphate; IP6, D-myo-inositol hexakisphosphate; NTD, N-terminal domain of capsid; CTD, C-terminal domain of capsid; SLS, static light scattering; SE, sedimentation equilibrium; ITC, isothermal titration calorimetry; NHS-biotin, N-hydroxysuccinimidobiotin; IP5/6, inositol pentakisphosphate or inositol hexakisphosphate; HPG, p-hydroxyphenylglyoxal

Corresponding author.
1 Present address: S. J. Campbell, James A. Baker Institute for Animal Health, Hungerford Hill Road, Cornell University, Ithaca, NY 14853, USA.
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