Developing soluble co-receptor mimetics for the study of HIV Env/receptor interactions

Tuesday, September 23, 2014 — Poster Session III
12:00 p.m. – 2:00 p.m.	FAES Academic Center	NIAID	VIROL-3

* FARE Award Winner

Authors

• A. Hajduczki
• V.G. Bundoc
• J.E. Seedorff
• E.A. Berger

Abstract

The HIV envelope glycoprotein (Env) is a dynamic molecule undergoing a series of stepwise conformational changes during interactions with the primary receptor CD4, and co-receptors, CCR5/CXCR4. Obtaining structural information on the intermediates during viral entry is a key focus of antiviral and vaccine research and could open doors for more effective treatment and prevention. Due to the inherent insolubility of membrane proteins, working with the intact co-receptors outside the context of the membrane is not a viable option. This project aims to develop recombinant soluble co-receptor mimetics featuring critical determinants of CCR5 using two approaches: fusing the CCR5 moieties to soluble CD4 by flexible polypeptide linkers and using a globular scaffold protein to orient the co-receptor determinants in an optimal conformation to recreate the interaction with Env. We have successfully overexpressed and purified both variants from mammalian cells and ELISAs confirmed that sulfotyrosines, necessary for co-receptor function, are present. Experiments from a cell-fusion assay system suggest that the CD4-fused co-receptor mimetic competes with cell-surface CCR5 for binding to Env. The soluble variants of CCR5 will be used to elucidate the conformational changes in Env that immediately precede membrane fusion, and potentially for collaborative high-resolution structural analyses of the Env-coreceptor complex.

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