Mechanism of HIV-1 Env-Mediated Membrane Fusion Project Description The persistence of the worldwide AIDS pandemic underscores the need to develop new therapeutic and vaccine strategies against its causative agent, HIV-1. As the primary viral component on the HIV-1 surface responsible for cellular attachment and entry, the glycoprotein Env is an attractive target. This protein assembles as a homotrimer, with each protomer composed of two subunits, gp120 and gp41. The gp120 subunits bind cellular CD4 and chemokine receptors, events that coordinate a complex series of gp41 structural changes that culminate in fusion of viral and cellular membranes. The molecular mechanisms that guide these transitions remain largely unknown, in part due to the homotrimeric nature of Env. The presence of three identical binding sites for receptors that trigger conformational changes and for inhibitors that block this structural evolution makes it difficult to parse allosteric regulation within and between subunits. To explore the mechanisms of Env allostery, we have recently developed a novel strategy that combines the use of fusion inhibitors with functional complementation. Fusion inhibitors bind intermediate conformational states and report on the spatial and temporal exposure of different regions of the Env ectodomain. Functional complementation allows for the analysis of the structure and activity of individual protomers in the context of the trimer. These techniques, employed in combination, have proved to be a powerful new method to reveal structural features of Env during HIV-1 entry. We propose to use the strategy to ask the following questions: how are the exposure and conformational transitions of the gp41 subunits allosterically coupled to CD4- and coreceptor-binding to gp120? What impact do the first, second and third CD4 and chemokine receptor binding events have on the structure of the Env trimer and efficiency of membrane fusion? What structural features enable Env to maintain the conformational metastability of its native state? The specific aims are: (1) To characterize an open-to-closed conformational switch in native Env regulated by the trimer apex; (2) To probe receptor-controlled exposure of the gp41 ectodomain and explore the impact of multiple CD4 and chemokine receptor binding events on Env trimer activity; and (3) To decipher molecular pathways of inter- and intra- protomer allosteric communication within the Env trimer using sequence-based statistical coupling analysis. The information resulting from these experiments will provide new insights into the dynamic properties of the Env complex critical for viral entry and will facilitate new directions for therapeutic and vaccine development for the treatment and prevention of AIDS.

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 surface protein on the virion responsible for viral attachment and entry. This application proposes to explore the molecular mechanisms that guide Env function by using a novel methodology to probe the conformational evolution of a single protomer in the context of the Env trimer. 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 #
5R01GM066682-12
Application #
9689461
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sakalian, Michael
Project Start
2002-09-01
Project End
2021-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
12
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Thomas Jefferson University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Danial, Maarten; Stauffer, Angela N; Wurm, Frederik R et al. (2017) Site-Specific Polymer Attachment to HR2 Peptide Fusion Inhibitors against HIV-1 Decreases Binding Association Rates and Dissociation Rates Rather Than Binding Affinity. Bioconjug Chem 28:701-712
Ahn, Koree W; Root, Michael J (2017) Complex interplay of kinetic factors governs the synergistic properties of HIV-1 entry inhibitors. J Biol Chem 292:16498-16510
Khasnis, Mukta D; Halkidis, Konstantine; Bhardwaj, Anshul et al. (2016) Receptor Activation of HIV-1 Env Leads to Asymmetric Exposure of the gp41 Trimer. PLoS Pathog 12:e1006098
Patton, John; Vuyyuru, Raja; Siglin, Amanda et al. (2015) Evaluation of the efficiency of human immune system reconstitution in NSG mice and NSG mice containing a human HLA.A2 transgene using hematopoietic stem cells purified from different sources. J Immunol Methods 422:13-21
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
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
Kahle, Kristen M; Steger, H Kirby; Root, Michael J (2009) Asymmetric deactivation of HIV-1 gp41 following fusion inhibitor binding. PLoS Pathog 5:e1000674
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

Showing the most recent 10 out of 12 publications