HIV assembles in a two step process in which an immature virion composed of the Gag polyprotein assembles at the plasma membrane, acquires the envelope glycoprotein, and buds from the infected cell. In the second step, a viral encoded protease cleaves the Gag polyprotein into its constituent matrix (MA), capsid (CA), and nucleocapsid (NC) structural domains. Cleavage results in a profound morphological rearrangement of th structural domains marked by the formation of a conical core of CA surrounding a complex of the NC and viral RNA. If the core is not well formed, either through blocked cleavage or the introduction of mutations, the resultant virus is non-infectious. This suggest that blocking the structural rearrangement is a potential therapeutic approach. To do so requires a detailed understanding of the immature and mature virions as well as the sequence of events driving the transformation. We have developed a mass spectrometry based hydrogen/deuterium exchange and crosslinking approach that allows us to the structural rearrangements that accompany maturation at the molecular level. In this application we propose to use that technology to:
Aim 1 - Determine whether CA exists in a domain swapped configuration in either the immature or mature virion Aim 2- Obtain a detailed understanding of the process of HIV maturation at the molecular level Aim 3 - Assemble stable hexamers of HIV-1 CA and peform a detailed biophysical and structural analysis to fully characterize the N-terminal domain/C-terminal domain interaction that is formed upon maturation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI044626-07
Application #
7383114
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sharma, Opendra K
Project Start
2000-04-01
Project End
2011-02-28
Budget Start
2008-03-01
Budget End
2009-02-28
Support Year
7
Fiscal Year
2008
Total Cost
$311,841
Indirect Cost
Name
University of Alabama Birmingham
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Cortines, Juliana R; Monroe, Eric B; Kang, Sebyung et al. (2011) A retroviral chimeric capsid protein reveals the role of the N-terminal ýý-hairpin in mature core assembly. J Mol Biol 410:641-52
Prevelige Jr, Peter E (2011) New approaches for antiviral targeting of HIV assembly. J Mol Biol 410:634-40
Monroe, Eric B; Kang, Sebyung; Kyere, Sampson K et al. (2010) Hydrogen/deuterium exchange analysis of HIV-1 capsid assembly and maturation. Structure 18:1483-91
Kang, Sebyung; Mou, Liyuan; Lanman, Jason et al. (2009) Synthesis of biotin-tagged chemical cross-linkers and their applications for mass spectrometry. Rapid Commun Mass Spectrom 23:1719-26
Lanman, Jason; Prevelige Jr, Peter E (2004) High-sensitivity mass spectrometry for imaging subunit interactions: hydrogen/deuterium exchange. Curr Opin Struct Biol 14:181-8
Douglas, Chanel C; Thomas, Dennis; Lanman, Jason et al. (2004) Investigation of N-terminal domain charged residues on the assembly and stability of HIV-1 CA. Biochemistry 43:10435-41
Lanman, Jason; Lam, TuKiet T; Emmett, Mark R et al. (2004) Key interactions in HIV-1 maturation identified by hydrogen-deuterium exchange. Nat Struct Mol Biol 11:676-7
Lanman, Jason; Lam, TuKiet T; Barnes, Stephen et al. (2003) Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry. J Mol Biol 325:759-72
Lam, TuKiet T; Lanman, Jason K; Emmett, Mark R et al. (2002) Mapping of protein:protein contact surfaces by hydrogen/deuterium exchange, followed by on-line high-performance liquid chromatography-electrospray ionization Fourier-transform ion-cyclotron-resonance mass analysis. J Chromatogr A 982:85-95
Lanman, Jason; Sexton, Jennifer; Sakalian, Michael et al. (2002) Kinetic analysis of the role of intersubunit interactions in human immunodeficiency virus type 1 capsid protein assembly in vitro. J Virol 76:6900-8