The human immunodeficiency virus (HIV) employs an array of properties that enable it to evade antibody neutralization. Rational vaccine design has been limited due to the insufficient structural understanding of the HIV envelope glycoprotein (Env), the primary target for neutralizing antibodies. Advances in structural determination have been hindered due to the presence of flexible hypervariable loops and the high carbohydrate content of this protein and the arrangement of the full Env complex remains controversial. The nature of the glycan shield, thought to mask conserved sites, is also of key interest as it mediates immune escape. Furthermore the binding of host cell CD4 receptor is known to induce massive conformational changes in Env, but a detailed examination of these changes has yet to be addressed. This proposal aims to combine two complementary approaches, small-angle X-ray scattering (SAXS) with ab initio shape reconstruction and hydrogen-deuterium exchange (HXMS) with mass spectrometry analysis to address several questions regarding the architecture of trimeric Env. The extensive catalog of constructs available from the Hu lab enables comparative studies of Env ranging from monomeric subunits to trimeric complexes, in the context of the same isolate. Identification of the trimeric interface, conformational changes induced upon receptor binding, and epitope occlusion by variable loops and glycans can be addressed with these techniques. The interaction of several well-characterized monoclonal antibodies directed against the CD4 binding site will also be compared to address the question of how conformational changes induced by antibody binding differ from those resulting from CD4 binding, and whether there are potentially correlates with neutralizing potency. Understanding these properties of Env will assist current efforts to identify targets for development of broadly neutralizing antibodies, necessary for an effective vaccine.

Public Health Relevance

The poor understanding of the primary surface protein of the Human Immunodeficiency Virus (HIV) has been a major hindrance to vaccine development. A thorough understanding of the organization of this protein is critical for designing next generation immunogens to induce an effective immune response. Using two emerging techniques we will address uncertainties regarding the architecture of this surface protein, identify how critical antigenic regions are protected, and identify ways to improve vaccine design.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-AARR-J (22))
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Flicker, Paula F
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University of Washington
Schools of Pharmacy
United States
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Guttman, Miklos; Lee, Kelly K (2016) Isotope Labeling of Biomolecules: Structural Analysis of Viruses by HDX-MS. Methods Enzymol 566:405-26
Guttman, Miklos; Cupo, Albert; Julien, Jean-Philippe et al. (2015) Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env. Nat Commun 6:6144
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Guttman, Miklos; Lee, Kelly K (2013) A functional interaction between gp41 and gp120 is observed for monomeric but not oligomeric, uncleaved HIV-1 Env gp140. J Virol 87:11462-75
Guttman, Miklos; Weis, David D; Engen, John R et al. (2013) Analysis of overlapped and noisy hydrogen/deuterium exchange mass spectra. J Am Soc Mass Spectrom 24:1906-12
Guttman, Miklos; Weinkam, Patrick; Sali, Andrej et al. (2013) All-atom ensemble modeling to analyze small-angle x-ray scattering of glycosylated proteins. Structure 21:321-31
Guttman, Miklos; Kahn, Maria; Garcia, Natalie K et al. (2012) Solution structure, conformational dynamics, and CD4-induced activation in full-length, glycosylated, monomeric HIV gp120. J Virol 86:8750-64