A Trimeric HIV-1 Fusion Peptide Construct Which Does Not Self-Associate in Aqueous Solution and Which Has 15-Fold Higher Membrane Fusion Rate

Yang, Rong; Prorok, Mary; Weliky, David P.

doi:10.1021/ja045612o

Cited by 55 publications

(162 citation statements)

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“…The highresolution structures of the soluble ectodomain of gp41 which lacks the HFP are trimeric and suggest that HFP trimers insert into the target cell membrane (1). The putative functional significance of trimers is supported by the 15-40-fold higher vesicle fusion rates of the chemically cross-linked HFP trimer (HFPtr) relative to HFPmn (7). Thus, the fusion rates are ordered HFPmn_mut Ͻ HFPmn Ͻ HFPtr and the present study examines the structures and membrane locations of these constructs with correlation to their very different fusogenicities.…”

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confidence: 99%

“…For example, membraneassociated HFP can adopt either helical or ␤ strand conformation and there has been effort to determine a correlation between conformation and fusogenicity. However, this work has resulted in conflicting models such as: (i) the helical conformation is fusogenic and the ␤ strand conformation is nonfusogenic (4); (ii) the ␤ strand conformation is fusogenic and the helical conformation is nonfusogenic (5); (iii) a transient random coil conformation is fusogenic (6); and (iv) both the helical and ␤ strand conformations are fusogenic (7). In the present study, the structural focus is on HFP membrane location rather than conformation and a clear correlation is observed between depth of membrane insertion and fusogenicity.…”

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confidence: 99%

“…There are large differences in the rates and extents of vesicle fusion induced by these constructs. The wild-type HFP monomer (HFPmn) induces fusion with moderate rate (7). HFPmn with the V2E mutation denoted HFPmn_mut does not induce vesicle fusion and was chosen because viruses and cells expressing gp41 V2E mutant have greatly impaired fusion and infectivity (2,11).…”

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

Wang

Sun

Weliky³

2009

Proc. Natl. Acad. Sci. U.S.A.

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Fusion between the membrane of HIV and the membrane of a host cell is a crucial step in HIV infection and is catalyzed by the binding of the fusion peptide domain (HFP) of the HIV gp41 protein to the host cell membrane. The HFP by itself induces vesicle fusion and is a useful model system to understand the fusion peptide/host cell membrane interaction. This article reports an experimental correlation between the membrane locations of different HFP constructs and their fusogenicities. The constructs were the HFP monomer with Val-2 to Glu-2 mutation (HFPmn_mut), wild type HFP monomer (HFPmn), and wild type HFP trimer (HFPtr). All constructs have predominant ␤ sheet structure in membranes with physiologically relevant cholesterol content. HFPmn_mut does not fuse vesicles, HFPmn has moderate fusion rate, and HFPtr has the putative oligomerization state of HIV gp41 and a very rapid fusion rate. The HFP membrane locations were probed with solid-state NMR measurements of distances between labeled carbonyl ( 13 CO) nuclei in the HFP backbone and lipid nuclei in the surface or interior regions of the membrane bilayer. HFPmn_mut is located at the membrane surface, HFPmn is inserted into a single membrane leaflet, and HFPtr is the most deeply inserted construct with contact with the center of the membrane. These results show a clear positive correlation between the insertion depths and the fusion activities of the HFP constructs. Other disease-causing enveloped viruses contain fusion peptides and this correlation may be a general structure-function model for these peptides.L ike many viruses that cause human disease, HIV is enveloped by a membrane obtained during virus budding from a previously infected host cell and infection of a new cell requires fusion between the viral membrane and the cell membrane. Fusion is catalyzed by the HIV fusion protein gp41, which has Ϸ170 ectodomain residues outside the virus including a Ϸ20-residue N-terminal fusion peptide (HFP) that binds to target cell membranes (1). Studies of HIV with a truncated or mutated HFP showed that the HFP is crucial in the fusion process (2, 3). Functionally critical fusion peptides are also found in fusion proteins of other enveloped viruses such as influenza and Ebola (1). Chemically synthesized peptides with HFP sequences have been developed as fusion model systems and provide information about HFP perturbation of target membranes. Free HFPs induce vesicle fusion and there are strong correlations between the mutation/activity relationships of HFP-induced fusion and HIV/host cell fusion (3).There have been some number of HFP structural studies, but in our view, there have not yet been clear correlations between HFP structure and fusogenic function. For example, membraneassociated HFP can adopt either helical or ␤ strand conformation and there has been effort to determine a correlation between conformation and fusogenicity. However, this work has resulted in conflicting models such as: (i) the helical conformation is fusogenic and the ␤ strand conformation i...

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

Wang

Sun

Weliky³

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Oligomerization/aggregation has also been detected by other biophysical methods (15,30). There is evidence that at least the lipid mixing step of membrane fusion can occur with the HFP in either helical or β strand conformation although there is some controversy in the literature about this conclusion (14,16,18,(31)(32)(33)(34)(35).HFP location in membranes has been primarily probed using a HFP-F8W mutant and by variation of the tryptophan fluorescence of this mutant with changes in environment (36,37). Key results have included: (1) fluorescence was higher for membrane-associated HFP-F8W than for HFP-F8W in buffered saline solution; (2) greater fluorescence quenching by acrylamide was observed for a soluble tryptophan analog than for membrane-associated HFP-F8W; and (3) similar fluorescence quenching of membrane-associated HFP-F8W was observed in samples containing either 1-palmitoyl-2-stearoyl-phosphocholine brominated at the 6, 7 carbons of the stearoyl chain or the corresponding lipid brominated at the 11, 12 carbons of the chain.…”

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Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide to Lipid Distances Reveal the Intimate Contact of β Strand Peptide with Membranes and the Proximity of the Ala-14−Gly-16 Region with Lipid Headgroups

2007

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Human immunodeficiency virus (HIV) infection begins with fusion between viral and host cell membranes and is catalyzed by the HIV gp41 fusion protein. The ~20 N-terminal apolar residues of gp41 are called the HIV fusion peptide (HFP), interact with the host cell membrane, and play a key role in fusion. In this study, the membrane location of peptides which contained the HFP sequence AVGIGALFLGFLGAAGSTMGARS was probed in samples containing either only phospholipids or phospholipids and cholesterol. Four HFPs were examined which each contained 13 CO labeling at three sequential residues between G5 and G16. The 13 CO chemical shifts indicated that HFP had predominant β strand conformation over the labeled residues in the samples. The internuclear distances between the HFP 13 COs and the lipid 31 Ps were measured using solid-state nuclear magnetic resonance rotational-echo double resonance experiments. The closest 13 CO-31 P distances of 5-6 Å were observed for HFP labeled between A14 and G16 and correlated with intimate association of β strand HFP and membranes. These results were confirmed with measurements using HFPs singly 13 CO labeled at A6 or A14. To our knowledge, these data are the first measurements of distances between HIV fusion peptide nuclei and lipid P and qualitative models of membrane location of oligomeric β strand HFP are presented which are consistent with the experimental data. Observation of intimate contact between β strand HFP and membranes provides rationale for further investigation of the relationship between structure and fusion activity for this conformation. KeywordsHIV; fusion peptide; membrane; carbonyl; phosphorus; REDOR; NMR The infection of enveloped viruses such as human immunodeficiency virus (HIV) begins with fusion between the viral and host cell membranes (1-4). Fusion may be catalyzed by fusion proteins and several models of fusion protein catalysis have been proposed (2,(5)(6)(7). For HIV, fusion is catalyzed by a "gp160" glycoprotein complex which is incorporated in the virus membrane and is composed of two non-covalently associated subunits "gp120" and "gp41". The gp120 subunit lies outside the virus and binds to receptors in the target cell membrane and the gp41 subunit contains a region inside HIV as well as a single-pass transmembrane domain *To whom correspondence should be addressed. Telephone: 517-355-9715. Fax: 517-353-1793. Email: weliky@chemistry.msu.edu. † This work was supported by NIH award AI47153 to D. P. W. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 January 27. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (8,9). 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 apolar and fairly conserved. The HFP is believed to interact with the target cell membrane after gp120 binds to cellular receptors and fusion is greatly disrupted by mutation or deletion of the H...

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“…The resin was introduced in the ABI-431A peptide synthesizer and all Fmoc-amino acids were coupled using the DCC/HOBt protocol from the company (4-fold excess of the Fmoc-amino acid, 90 min per coupling). The peptide was cleaved from the resin with a mixture of TFA: TIS: H 2 O (95%: 2.5%: 2.5%) (5 ml) yielding the crude DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala peptide which was precipitated and washed with cold ether (5 ml X 5). The crude DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala (385 mg) was purified by preparative HPLC (gradient b) and lyophilized to give 170 mg of the peptide product as a white solid.…”

Section: Dup-1 (1-12) 2 Ala (Fapnraqdyntn-amide) Peptidementioning

confidence: 99%

A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

Khandadash¹,

Partouche²,

Weiss³

et al. 2011

TOOPTSJ

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Herein we present a new application of a recently demonstrated fully synthetic "phage-like" system for screening of combinatorial mixtures in a live cell assay. The new application includes the direct synthesis of peptides and combinatorial libraries on 2 µm cross-linked mono-dispersed microspheres bearing a panel of fluorescence tags. Their characterization using classical chemical analysis as well as biological recognition of the synthesized sequences on the microspheres by specific antibodies, demonstrate the robustness of the system. Two biased positional combinatorial peptide libraries derived from peptide DUP-1 were synthesized and screened for affinity to prostate cancer PC-3 cell line as compared to DUP-1, a peptide with good affinity for this cell line. The best sub-library was deconvoluted and mixtures of deconvoluted peptides on microspheres were screened for identifying the sequences with the best affinity for cells. The best peptide, DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala with high affinity for PC-3 cells, was individually synthesized and validated in an independent binding assay. Finally, the cellular fate of DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala in PC-3 cell line is demonstrated using confocal laser scanning microscopy. Overall, these results demonstrate that the synthetic phage-like system recently assessed for screening mixtures of small organic molecules, can be also used for synthesis and screening of combinatorial libraries of peptides in live cell assays.

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A Trimeric HIV-1 Fusion Peptide Construct Which Does Not Self-Associate in Aqueous Solution and Which Has 15-Fold Higher Membrane Fusion Rate

Cited by 55 publications

References 14 publications

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

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

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide to Lipid Distances Reveal the Intimate Contact of β Strand Peptide with Membranes and the Proximity of the Ala-14−Gly-16 Region with Lipid Headgroups

A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

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