The HIV-1 surface glycoprotein Env (gp120/gp41) mediates viral entry through a series of coordinated structural changes initiated when CD4 interacts with gp120. Ultimately, the gp41 ectodomain folds into a compact trimer-of-hairpins that brings the viral and cellular membranes into the close proximity required for efficient membrane fusion. These conformational transitions have been delineated, in part, through the use of C- peptide and 5-Helix inhibitors that target the HR1 and HR2 regions of gp41 and block trimer-of-hairpins formation. Our previous work has described the physical basis behind C-peptide and 5-Helix inhibition and quantitatively modeled the relationship between inhibitory potency and fusion kinetics. The experiments outlined in this application explore the structure and dynamics of Env in its native and intermediate conformations. The research will address three fundamental questions regarding the mechanism of viral membrane fusion: How does CD4 binding to gp120 trigger formation of the prehairpin intermediate state? What is the structure of Env in this intermediate conformation? What aspects of gp41 folding from the prehairpin state drive the fusion of viral and cellular membranes? Owing to the unstable nature of Env in its native and intermediate states, these questions are often difficult to address using standard experimental techniques, such as scanning mutagenesis, that require stable proteins in long lived conformations. Our basic approach will be to generate new inhibitors that target the HR1 and HR2 regions, and dissect the mechanism of resistance to them. This strategy takes advantage of the power of natural selection to generate well-behaved mutant Env variants with interesting structural or dynamic properties.
The specific aims are: 1) to explore the structure and exposure of the HR1 region using novel inhibitors engineered to overcome standard C-peptide resistance profiles;2) to probe the structure and exposure of the HR2 region using multivalent inhibitors, and to dissect the mechanisms of resistance to these agents;and 3) to explore the energetic requirements of Env- mediated membrane fusion, and to examine the structure of mutant Env variants trapped in intermediate conformations. Our findings will provide new insights into the dynamic properties of the gp120/gp41 complex critical to the viral entry mechanism and will aide the development of new viral entry inhibitors and HIV-1 vaccines.

Public Health Relevance

The long term objective of the proposed research is to increase our knowledge about the structure, function and inhibition of the HIV-1 Env, the major protein on the viral surface. These experiments involve inhibitor design and characterization in order to explore how Env promotes HIV-1 entry through viral membrane fusion. Analyzing of the mechanisms of viral escape from these inhibitors will provide insights into the energetics and kinetics of Env structural changes that promote membrane fusion. The research will ultimately facilitate therapeutic and vaccine development for the treatment and prevention of AIDS.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066682-09
Application #
8383456
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sakalian, Michael
Project Start
2002-09-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2014-11-30
Support Year
9
Fiscal Year
2013
Total Cost
$430,776
Indirect Cost
$152,856
Name
Thomas Jefferson University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Danial, Maarten; Root, Michael J; Klok, Harm-Anton (2012) Polyvalent side chain peptide-synthetic polymer conjugates as HIV-1 entry inhibitors. Biomacromolecules 13:1438-47
Welch, Brett D; Francis, J Nicholas; Redman, Joseph S et al. (2010) Design of a potent D-peptide HIV-1 entry inhibitor with a strong barrier to resistance. J Virol 84:11235-44
Kahle, Kristen M; Steger, H Kirby; Root, Michael J (2009) Asymmetric deactivation of HIV-1 gp41 following fusion inhibitor binding. PLoS Pathog 5:e1000674
Champagne, Kelly; Shishido, Akira; Root, Michael J (2009) Interactions of HIV-1 inhibitory peptide T20 with the gp41 N-HR coiled coil. J Biol Chem 284:3619-27
Sugaya, Makoto; Hartley, Oliver; Root, Michael J et al. (2007) C34, a membrane fusion inhibitor, blocks HIV infection of langerhans cells and viral transmission to T cells. J Invest Dermatol 127:1436-43
Steger, H Kirby; Root, Michael J (2006) Kinetic dependence to HIV-1 entry inhibition. J Biol Chem 281:25813-21
Root, Michael J; Steger, H Kirby (2004) HIV-1 gp41 as a target for viral entry inhibition. Curr Pharm Des 10:1805-25
Root, Michael J; Hamer, Dean H (2003) Targeting therapeutics to an exposed and conserved binding element of the HIV-1 fusion protein. Proc Natl Acad Sci U S A 100:5016-21