HIV Fusion Peptide and Its Cross-Linked Oligomers: Efficient Syntheses, Significance of the Trimer in Fusion Activity, Correlation of β Strand Conformation with Membrane Cholesterol, and Proximity to Lipid Headgroups

Abstract

For enveloped viruses such as HIV, a ~20-residue N-terminal fusion peptide domain in the envelope protein binds to target cell membranes and plays a key role in fusion between the viral and cellular membranes during infection. The chemically synthesized HIV fusion peptide or "HFP" catalyzes fusion between membrane vesicles and is a useful model system to understand some aspects of HIV fusion. Previous studies have shown a common trimeric state for envelope protein from several different viruses including HIV and in this study, practical high-yield syntheses are reported for HFP monomer (HFPmn) and chemically cross-linked HFP dimer (HFPdm), trimer (HFPtr), and tetramer (HFPte). The vesicle fusion rates per strand were ordered HFPmn < HFPdm < HFPtr ≈ HFPte and suggested that HFPtr is the smallest catalytically efficient oligomer. Solid-state NMR measurements of 13 CO chemical shifts were carried out in constructs labeled at either Ala-6 or Ala-15. For all constructs associated with cholesterol-containing membranes, the chemical shifts of both residues correlated with β strand conformation while association with membranes without cholesterol resulted in a mixture of helical and β strand conformations. The dependence of fusion rate on oligomer size is independent of membrane cholesterol content, so one interpretation of the data is fusion activity of both helical and β strand conformations. Membrane location may be a determinant of fusion activity and for all constructs in both conformations, a large fraction of the Ala-15 13 COs were 5-6 Å from the 31 Ps in the lipid headgroups, while the Ala-6 13 COs were more distant.Keywords fusion peptide; HIV; NMR; membrane location; oligomer; cholesterol; synthesis Infection by the human immunodeficiency virus (HIV) begins with fusion between the viral and host cell membranes and leads to deposition of the viral nucleocapsid in the cytoplasm and viral replication (1,2). The fusion process is mediated by the HIV gp41 fusion protein which contains a region inside HIV as well as a single-pass transmembrane domain. The ~170-residue ectodomain of gp41 lies outside HIV and is subdivided into a more C-terminal "soluble ectodomain" and a ~20-residue N-terminal fusion peptide (HFP) which is hydrophobic and fairly conserved. Peptides with the HFP sequence catalyze fusion between membrane vesicles and there is good correlation between the mutation/fusion activity relationships of HFPinduced vesicle fusion and gp41-induced membrane fusion (3)(4)(5)(6)(7)(8). These data suggest that studies of HFP will provide information about HIV/target cell fusion. † This work was supported by NIH grant AI47153 to D.P.W. *To whom correspondence should be addressed. Email: E-mail: weliky@chemistry.msu.edu, Phone: 517-355-9715, Fax: 517-353-1793. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 January 20. Published in final edited form as:Biochemistry. 2009 January 20; 48(2): 289-301. doi:10.1021/bi8015668. NIH-PA Author ManuscriptNIH-PA Author Manusc...

A strong correlation between fusogenicity and membrane insertion depth of the HIV fusion peptide

“…In addition, an EPR study of HFPmn was consistent with an Ala-1 location close to the aqueous phase (8). A solid-state NMR study of HFPmn and HFPtr showed that for a large fraction of both constructs, the Ala-15 carbonyl ( 13 CO) nuclei are 5-6 Å from 31 P nuclei in the lipid headgroups (15). This result was obtained both for helical HFPs in membranes which lacked cholesterol and for ␤ strand HFPs in membranes with cholesterol.…”

“…For HFPmn and HFPtr associated with membranes that do not contain cholesterol, there are significant populations of molecules with helical conformation (15). Helical conformation is also observed for HFPmn in detergent micelles (4,12).…”

“…Insertion models for ␤ strand HFPs will be discussed in the context of previous and present work. Earlier solid-state NMR studies on HFPmn showed that relative to the Gly-5 to Gly-13 13 COs, the Ala-1 to Gly-3 and Ala-14 to Gly-16 13 COs were closer to the lipid 31 Ps (15,19). Two insertion models were proposed with either insertion into a single leaflet or membrane traversal of both leaflets.…”

“…The 5-19 F-DPPC lipid was synthesized by Avanti Polar Lipids using the 5-fluoropalmitic acid. The general protocols for peptide synthesis and NMR sample preparation have been described and a brief summary is provided in SI Text (15,19). Solid-state NMR experiments were conducted on a 9.4 T spectrometer.…”

“…Solid-state NMR experiments were conducted on a 9.4 T spectrometer. Most of the experimental setup has been described previously and the new parts of the setup are detailed in SI Text (15,19). Spectra were processed with 200-Hz Gaussian line broadening and polynomial baseline correction.…”