The envelope glycoproteins of HIV/SIV, assembled as a (gp120/gp41)3 trimeric complex, mediate viral attachment and membrane fusion. Biochemical and structural studies have provided a general picture of how binding to viral receptor and co-receptor triggers a cascade of fusion-promoting conformational changes. Understanding of HIV/SIV Env conformations at the atomic level has come mainly from three sets of crystal structures: that of an unliganded SIV gp120 core fragment, those of receptor-induced conformations of the HIV gp120 core, and those of the postfusion form of HIV and SIV gp41. The conformation of the native (gp120/gp41)3 on the surface of the virion, especially that of its gp41 component, remains unknown, except for some low-resolution images from electron microscopy. We also lack a proper picture of an important intermediate in the pathway from the prefusion to postfusion conformation of gp41 - the so-called "prehairpin intermediate" that is the target of T-20/Entfuvirtide (the first approved fusion-inhibiting antiviral drug) and of certain broadly neutralizing antibodies, as we describe in the Preliminary Data. Filling these gaps in our knowledge of envelope protein structures will guide development of vaccines and therapeutics. Moreover, production of stable, homogeneous preparations of recombinant gp41 in defined conformations will help us understand structural correlates for neutralization, even in the absence of high-resolution structures. In this proposal, we will explore the hypothesis that the various conformational states of gp41 hold key to our understanding of fusion mechanism and antibody neutralization by biochemical and structural approaches. We propose here to study gp41 in its prefusion, fusion-intermediate, and postfusion conformations, with structural information as our ultimate goal. We will build upon preliminary results that show we can express and characterize suitable forms of each. In particular, we will pursue the following specific aims: 1. To carry out biochemical and structural studies of the "prehairpin intermediate" of gp41;2. To characterize gp41 in the prefusion conformation and determine its structure;3. To study the conformation of the membrane-interacting segments of gp41 in the postfusion state.

Public Health Relevance

Nearly 40 million people are living with human immunodeficiency virus (HIV) and 4 million people are newly infected with HIV in 2006 alone (UNAIDS/WHO AIDS epidemic update, 2006). An effective vaccine is urgently needed to stop this epidemic. The proposed studies will allow us to visualize in great detail the organization of the viral envelope glycoprotein, which is the main target by host immune responses and by some new-generation antiviral drugs, called fusion inhibitors. The information obtained from these experiments is expected to guide development of both AIDS vaccine and antiviral therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083680-04
Application #
8243563
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sakalian, Michael
Project Start
2009-04-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
4
Fiscal Year
2012
Total Cost
$353,866
Indirect Cost
$150,495
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
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Frey, Gary; Chen, Jia; Rits-Volloch, Sophia et al. (2010) Distinct conformational states of HIV-1 gp41 are recognized by neutralizing and non-neutralizing antibodies. Nat Struct Mol Biol 17:1486-91
Freeman, Michael M; Seaman, Michael S; Rits-Volloch, Sophia et al. (2010) Crystal structure of HIV-1 primary receptor CD4 in complex with a potent antiviral antibody. Structure 18:1632-41
Nkolola, Joseph P; Peng, Hanqin; Settembre, Ethan C et al. (2010) Breadth of neutralizing antibodies elicited by stable, homogeneous clade A and clade C HIV-1 gp140 envelope trimers in guinea pigs. J Virol 84:3270-9