Tài liệu Báo cáo khoa học: Structure and membrane interaction of the internal fusion peptide of

Structure and membrane interaction of the internal fusion peptide

of avian sarcoma leukosis virus

Shu-Fang Cheng, Cheng-Wei Wu, Eric Assen B Kantchev and Ding-Kwo Chang

Institute of Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China

The structure and membrane interaction of the internal

fusion peptide (IFP) fragment of the avian sarcoma and

leucosis virus (ASLV) envelope glycoprotein was studied by

an array of biophysical methods. The peptide was found to

induce lipid mixing of vesicles more strongly than the fusion

peptide derived from the N-terminal fusion peptide of

influenza virus (HA2-FP). It was observed that the helical

structure was enhanced in association with the model

membranes, particularly in the N-terminal portion of the

peptide. According to the infrared study, the peptide inserted

into the membrane in an oblique orientation, but less deeply

than the influenza HA2-FP. Analysis of NMR data in

sodium dodecyl sulfate micelle suspension revealed that

Pro13 of the peptide was located near the micelle–water

interface. A type II b-turn was deduced from NMR data for

the peptide in aqueous medium, demonstrating a conforma￾tional flexibility of the IFP in analogy to the N-terminal FP

such as that of gp41. A loose and multimodal self-assembly

was deduced from the rhodamine fluorescence self-quench￾ing experiments for the peptide bound to the membrane

bilayer. Oligomerization of the peptide and its variants can

also be observed in the electrophoretic experiments, sug￾gesting a property in common with other N-terminal FP of

class I fusion proteins.

Keywords: membrane fusion; conformational change;

insertion depth; self-assembly; fluorescence self-quenching.

Entry of enveloped viruses into the host cells is mediated

by the viral envelope glycoproteins [1], which in most

cases are cleaved by proteolysis to yield the transmem￾brane (TM) [2,3] subunit responsible for membrane fusion

and the surface (SU) subunit for receptor binding. For a

majority of the class I fusion proteins, a region in the TM

protein crucial for binding to and destabilizing target

membranes, termed fusion peptide (FP), is located at the

N-terminal region, while others have the internal fusion

peptide (IFP) domain [4]. Avain sarcoma/leucosis virus

(ASLV) is a prototype retrovirus [5], the envelope

glycoprotein of which uses IFP for fusion to target cells

[6,7]. A proline is often found near the centre of many of

the viral IFP sequences [1]. Delos et al. [8] have shown

that the central proline of the FP of ASLV subtype A

plays important roles in forming a native envelope protein

(EnvA) structure and in membrane fusion. It is thought

that the envelope protein undergoes conformational

change triggered by its binding to the receptor on the

target cell surface (e.g. Tva for ASLV-A), exposing the

hydrophobic FP domain to destabilize the cell membrane

preceding the membrane fusion [9] similar to influenza

haemagglutinin and HIV-1 gp41. As the majority of

studies were performed on the N-terminal FP, it would

be of interest to compare the structure of the internal FP

and its interaction with membrane bilayer, including in

particular the structural influence of proline. Consistent

with other class I viral fusion proteins, the IFP of ASLV

inserts into the membrane primarily as a helix in contrast

to the IFP of class II fusion protein which uses a
cd loop

to insert into the target membrane in the fusion process

[10,11]. In the following, a variety of physical properties of

the putative IFP of ASLV are reported and differences

between N-terminal and internal FP are compared. The

pH dependence of some of the properties is discussed in

regard to the experimental observation that ASLV

induced hemifusion, but not complete fusion, at neutral

pH [12].

Experimental procedures

All chemicals and solvents were used without further

purification. N-a-(9-Fluorenylmethoxycarbonyl) (Fmoc)-

protected amino acids were products of Anaspec (San

Jose, CA, USA) or Bachem (Bubendorf, Switzerland).

1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and

Correspondence to D.-K. Chang, Institute of Chemistry, Academia

Sinica, Taipei, Taiwan 115, Republic of China.

Fax: + 886 2 27831237, Tel.: + 886 2 27898594,

E-mail: [email protected]

Abbreviations: ASLV, avain/sarcoma leucosis virus; ASLV-A, ASLV

subtype A; ATR-FTIR, attenuated total reflectance-FTIR; DG, dis￾tance geometry; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocho￾line; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; EnvA,

native envelope protein; FP, fusion peptide; HA2-FP, N-terminal

fusion peptide of influenza virus; IFP, internal fusion peptide; NBD,

7-nitrobenz-2-oxa-1,3-diazole; NBD-PE, N-(7-nitrobenz-2-oxa-1,3-

diazol-4-yl)-1,2-dihexadecanyol-sn-glycero-3-phosphoethanolamine;

rhodamine, 5(6)-carboxytetramethylrhodamine; Rh-PE, Lissa￾mineTM rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phospho￾ethanolamine, triethylammonium salt; SA, simulated annealing;

SU, surface; TM, transmembrane.

(Received 26 May 2004, revised 6 October 2004,

accepted 13 October 2004)

Eur. J. Biochem. 271, 4725–4736 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04436.x