The HIV-1 exterior envelope glycoprotein, gp120, mediates binding to the viral receptors (CD4 and CCR5/CXCR) and is a major target for neutralizing antibodies. The gp41 transmembrane glycoprotein mediates fusion and, in the viral spike, is maintained in a metastable conformation by interactions with gp120. In the viral spike, the gp41 oligomerization domain retains gp120 in a trimeric configuration. Rich Wyatt?s laboratory in the Vaccine Research Center has replaced gp41 with a potentially more stable trimerization domain in order to generate a more stable gp120 trimer for the better design of gp120 immunogens that elicit broadly neutralizing antibodies as well as for structural analysis. Last year we validated using sedimentation equilibrium and light scattering analysis that the VRC construct was indeed trimeric. In the past year we used both sedimentation velocity and equilibrium analysis to characterize the stoichiometry of CD4 and antibody binding. HIV particle assembly is an essential step in the viral replication cycle and is a potential target for antiviral therapy. However, molecular mechanisms in assembly are not understood as yet. The major structural component in all retrovirus particles is the Gag protein. Gag is a multi-domain protein that is cleaved during virus maturation into a series of cleavage products. The immature virus particle contains full length Gag. Alan Rein?s laboratory has discovered that a virus like particle (VLP) can be generated by adding nucleic acid to Gag and assembly cofactors. We are currently performing a rigorous biophysical analysis of the molecular events underlying VLP assembly. This analysis will focus on (a) detection of Gag oligomers as potential assembly intermediates; (b) interactions of Gag protein with the assembly cofactors; and (c) conformational changes in Gag protein molecules. Paul Randazzo?s laboratory is studying ArfGAPs proteins that are multifunctional proteins regulating membrane traffic and actin remodeling. One of the ArfGAP proteins under investigation is ASAP1 which has multifunctional domain designated BAR. Both sedimentation velocity and equilibrium measurements establish that the BAR domain undergoes homodimerization. Bernie Moss? Laboratory of Viral Diseases has been studying all aspects of the vaccine virus replication cycle over an extended period of time. A protein designated HD13 appears to be involved in virus particle assembly. Using sedimentation velocity the HD13 oligomeric state is trimeric in agreement with other methodologies used in the Moss laboratory.

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
Intramural Research (Z01)
Project #
1Z01OD011081-02
Application #
7013050
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2004
Total Cost
Indirect Cost
Name
Office of the Director, NIH
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Garcia, Alonzo D; Otero, Joel; Lebowitz, Jacob et al. (2006) Quaternary structure and cleavage specificity of a poxvirus holliday junction resolvase. J Biol Chem 281:11618-26
Nie, Zhongzhen; Hirsch, Dianne S; Luo, Ruibai et al. (2006) A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor. Curr Biol 16:130-9
Shimp Jr, Richard L; Martin, Laura B; Zhang, Yanling et al. (2006) Production and characterization of clinical grade Escherichia coli derived Plasmodium falciparum 42 kDa merozoite surface protein 1 (MSP1(42)) in the absence of an affinity tag. Protein Expr Purif 50:58-67
Cole, Nelson B; Murphy, Diane D; Lebowitz, Jacob et al. (2005) Metal-catalyzed oxidation of alpha-synuclein: helping to define the relationship between oligomers, protofibrils, and filaments. J Biol Chem 280:9678-90
Szajner, Patricia; Weisberg, Andrea S; Lebowitz, Jacob et al. (2005) External scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice. J Cell Biol 170:971-81
Aldaz-Carroll, Lydia; Whitbeck, J Charles; Ponce de Leon, Manuel et al. (2005) Physical and immunological characterization of a recombinant secreted form of the membrane protein encoded by the vaccinia virus L1R gene. Virology 341:59-71
Pancera, Marie; Lebowitz, Jacob; Schon, Arne et al. (2005) Soluble mimetics of human immunodeficiency virus type 1 viral spikes produced by replacement of the native trimerization domain with a heterologous trimerization motif: characterization and ligand binding analysis. J Virol 79:9954-69
Center, Rob J; Lebowitz, Jacob; Leapman, Richard D et al. (2004) Promoting trimerization of soluble human immunodeficiency virus type 1 (HIV-1) Env through the use of HIV-1/simian immunodeficiency virus chimeras. J Virol 78:2265-76
Budge, Philip J; Lebowitz, Jacob; Graham, Barney S (2003) Antiviral activity of RhoA-derived peptides against respiratory syncytial virus is dependent on formation of peptide dimers. Antimicrob Agents Chemother 47:3470-7
Center, Rob J; Leapman, Richard D; Lebowitz, Jacob et al. (2002) Oligomeric structure of the human immunodeficiency virus type 1 envelope protein on the virion surface. J Virol 76:7863-7