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HIV gp41 six-helix bundle constructs induce rapid vesicle fusion at pH 3.5 and little fusion at pH 7.0: understanding pH dependence of protein aggregation, membrane binding, and electrostatics, and implications for HIV-host cell fusion

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Abstract

The HIV gp41 protein catalyzes fusion between HIV and target cell membranes. The fusion states of the gp41 ectodomain include early coiled-coil (CC) structure and final six-helix bundle (SHB) structure. The ectodomain has an additional N-terminal apolar fusion peptide (FP) sequence which binds to target cell membranes and plays a critical role in fusion. One approach to understanding gp41 function is study of vesicle fusion induced by constructs that encompass various regions of gp41. There are apparent conflicting literature reports of either rapid or no fusion of negatively charged vesicles by SHB constructs. These reports motivated the present study, which particularly focused on effects of pH because the earlier high and no fusion results were at pH 3.0 and 7.2, respectively. Constructs include “Hairpin,” which has SHB structure but lacks the FP, “FP-Hairpin” with FP + SHB, and “N70,” which contains the FP and part of the CC but does not have SHB structure. Aqueous solubility, membrane binding, and vesicle fusion function were measured at a series of pHs and much of the pH dependences of these properties were explained by protein charge. At pH 3.5, all constructs were positively charged, bound negatively charged vesicles, and induced rapid fusion. At pH 7.0, N70 remained positively charged and induced rapid fusion, whereas Hairpin and FP-Hairpin were negatively charged and induced no fusion. Because viral entry occurs near pH 7 rather than pH 3, our results are consistent with fusogenic function of early CC gp41 and with fusion arrest by final SHB gp41.

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Abbreviations

Chol:

Cholesterol

CHR:

C-terminal heptad repeat

FP:

Fusion peptide

LUV:

Large unilamellar vesicle

MPER:

Membrane-proximal external region

NCL:

Native chemical ligation

NHR:

N-terminal heptad repeat

N-dansyl-DOPE:

N-(5-dimethylamino-1-naphthalenesulfonyl) (ammonium salt) dioleoylphosphatidylethanolamine

N-NBD-DPPE:

N-(7-nitro-2,1,3-benzoxadiazol-4-yl) (ammonium salt) dipalmitoylphosphatidylethanolamine

N-Rh-DPPE:

N-(lissamine rhodamine B sulfonyl) (ammonium salt) dipalmitoylphosphatidylethanolamine

N-PHI:

N-terminal half of the pre-hairpin intermediate

PC:

Phosphatidylcholine

PE:

Phosphatidylethanolamine

PG:

Phosphatidylglycerol

PHI:

Pre-hairpin intermediate

POPC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPG:

1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt)

PS:

Phosphatidylserine

RP-HPLC:

Reverse phase–high performance liquid chromatography

SHB:

Six-helix bundle

Sph:

Sphingomyelin

TCEP:

Tris(2-carboxyethyl) phosphine hydrochloride

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Acknowledgments

Dr. Lisa Lapidus is acknowledged for use of the fluorescence spectrometer and the MSU Mass Spectrometry facility is also acknowledged. The work was supported by NIH awards R01AI047153 to D.P.W. and F32AI080136 to K.S.

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Authors and Affiliations

  1. Department of Chemistry, Michigan State University, East Lansing, MI, 48824, USA

    Kelly Sackett & David P. Weliky

  2. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, 48824, USA

    Allan TerBush

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  1. Kelly Sackett
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  2. Allan TerBush
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  3. David P. Weliky
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Correspondence to David P. Weliky.

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Membrane-active peptides: 455th WE-Heraeus Seminar and AMP 2010 Workshop.

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Sackett, K., TerBush, A. & Weliky, D.P. HIV gp41 six-helix bundle constructs induce rapid vesicle fusion at pH 3.5 and little fusion at pH 7.0: understanding pH dependence of protein aggregation, membrane binding, and electrostatics, and implications for HIV-host cell fusion. Eur Biophys J 40, 489–502 (2011). https://doi.org/10.1007/s00249-010-0662-3

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  • DOI: https://doi.org/10.1007/s00249-010-0662-3

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