Designing a Soluble Near Full-Length HIV-1 GP41 Trimer

The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has...

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Bibliographic Details
Main Authors: Gao, Guofen, Wieczorek, Lindsay, Peachman, Kristina K, Polonis, Victoria R, Alving, Carl R, Rao, Mangala, Rao, Venigalla B
Format: Report
Language:English
Subjects:
BACTERIOPHAGE T4 DISPLAY
BACTERIOPHAGES
Biochemistry
Genetic Engineering and Molecular Biology
GLYCOPROTEINS
GP41 TRIMER
HIV-1 ENVELOPE
HUMAN IMMUNODEFICIENCY VIRUSES
HYDROPHOBIC PROPERTIES
PREHAIRPIN FUSION INTERMEDIATE
T LYMPHOCYTES
VIROLOGY
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Summary:The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses were tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intra-molecular HR1-HR2 interactions but not inter-molecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-aa foldon at the C-terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into mechanism of entry and development of gp41-based HIV-1 vaccines. In JBC Papers in Press, published 26 Nov 2012.