Steve S.-L. Chen and Woan-Eng Chan

Institute of Biomedical SciencesAcademia Sinica

Taipei, TaiwanR.O.C.

August 14, 20062006 International AIDS Conference

Role of the Highly Conserved LWYIK Motif of the HIV-1 Transmembrane Protein gp41 in Env-mediated Membrane Fusion

The Tryptophan-rich domain

Ectodomain FP NHR CHR TM

Cytoplasmicdomain

N CC34

DP178(638-673)

663 679 683 705

2F5 4E10 Membrane-spanning domain(684-705)

51

2

52

7

55

9

58

7

62

8

66

3

68

4

70

5

ELDKWASWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIV

HIV-1:92BR025-9 WQNLWTWFGITNWLWYIKGB8.C4 WANLWNWFDITNWLWYIKNL4-3 WASLWNWFNITNWLWYIKMN WASLWNWFDITNWLWYIKHXB2 WASLWNWFNITNWLWYIK

SIV:SIV cpzant WSSLWNWFDITQWLWYIKSIV cpz LNSWDVFGNWFDLASWIRSIV mac LNSWDVFGNWFDLTSWIKSIV agm LNSWDVFGNWFDLASWIK

HIV-2HIV2CBL24 LNSWDVFGNWFDLASWIKHIV-2ST LNSWDVFGNWFDLTSWIKHIV2CBL21 LNSWDVFGNWFDLTSWIR

Li and Papadopoulos: Cholesterol-binding motif: L/V-(X)1-5-Y-(X)1-5-R/K

The LWYIK motif (residues 679~683): located immediately proximal to the TM region

Vincent et al.Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups. Biochim. Biophys. Acta 1567: 157-164, 2002

• The LWYIK motif in the format of a MBP fusion protein was shown to bind to cholesteryl group in vitro.

• This motif was therefore proposed to play a role in Env association with lipid rafts and Env-mediated membrane fusion.

• However, the biological significance of this motif in virus replication is not known.

Construction of LWYIK motif-mutant proviruses

679 683TM region

WT ----ITNWLWYIKLFIMI----LWYIK --------.....---------YI ----------..----------IK -----------..---------KE ------------E---------WA ---------A------------YA ----------A-----------

Replication kinetics of LWYIK motif-mutant virusesin CD4+ CEM-SS cells

All mutant viruses inhibit their one-cycle virus infectivities: on an HIV-1 LTR-driven, cat gene-harboring reporter cell line, H938

Mutations in the LWYIK motif do not have significant effects on Env precursor synthesis, processing, or Env incorporation into the virus

Mutations in the LWYIK motif do not affect cell surface expression or CD4-binding ability of the Env

All of the mutant proteins are able to self-assemble into an oligomeric structure

Env trans-complementation assay

env plasmid

CAT assay

293 T cells

Transfection

Infection

2days

CD4+ T cells

LTR LTRSV40

pol

Bgl Bgl

env

gag vif CATtat

rev

SV40

rev

45 753517363314

Bgl Bgl

gp120 gp41

LTR LTRenv

pHXBenv CAT

All of mutant proteins inhibit their trans-complementation abilities

HIV-1 5’-LTR

CMV promoter pA Tat

Env

CD4

Cat gene 3’-LTR

Tat

Tat

H938

293T

A quantitative Env-mediated membrane fusion assay

Effects of mutations in the LWYIK motif on the Env membrane fusion ability

Mutations in the LWYIK motif have no apparent effects on Env association with lipid rafts

CEM-SS

Dil and Calcein-AM labeled CMAC labeled

Three-color dye transfer assay

Env-expressing 293T cells

Fusion pore enlargement

Hemifusion

Lipid and cytosolic mixing

Deletions in the LWYIK motif inhibit cytosolic dye transfer

Comparison of Env mutants

_________________________________________________________________ Env Membrane Virus

Incorporation fusion Infectivity Dye transfer_________________________________________________________________________WT Normal 100% 100% +

665~682 Greatly Reduced Abrogated Greatly reduced -

W(1~5)A Greatly Reduced Abrogated Greatly reduced +W(1~3)A Greatly Reduced Abrogated Greatly reduced

678~682 Greatly Reduced 20% Greatly reduced +666~670 Greatly Reduced 20% Greatly reduced

LWYIK Normal - 10% No cytosolic mixing no cytosolic mixingYI Normal - 10% Reduced cytosolic mixingIK Normal - 10% Reduced cytosolic mixing___________________________________________________________________________

Conclusion

• While the LWYIK motif is not critical for Env localization in lipid rafts, this motif is critical for membrane fusion.

• Env localization in lipid rafts does not necessarily warrant the membrane fusion ability of Env.

• The LWYIK motif acts as a unique and distinct membrane fusion determinant located in the gp41 C-terminal ectodomain in modulating the membrane fusion process.

Acknowledgements

Institute of Biomedical Sciences, Academia Sinica:

Woan-Eng ChanHsiao-Fen Li

Yu TsaiShu-Chen HuangChia-Hung Chang

Animal Technology Institute Taiwan:

Chin-Kai Chuang

Supported by : Academia Sinica and

National Health Research InstituteTaiwan, R.O.C.

The pre-transmembrane region

• The pre-transmembrane region has been proposed to serve as – a flexible extender; – a contributor to trimer formation and stability; and – an agent for membrane destabilization that fosters

membrane fusion.

• Although Eric Hunter’s group previously implicated this Trp-rich region in membrane fusion and viral infectivity, the molecular basis for the role of this Trp-rich region in viral replication is still not fully understood.

A. Saez-Cirion et al. Biophysical J. 85:3769-3780, 2-003

Only the functional pre-TM sequence: 1), adopts helical structures in solution and in membranes; 2), forms homo-oligomers in solution and membranes; and 3), inhibits gp41-induced cell-cell fusion. These data support two roles for gp41 aromatic-rich pretransmembrane sequence: 1), oligomerization of gp41; and 2), immersion into the viral membrane interface.

T. Suarez et al. FEBS Lett. 477:145-149, 2000A functional peptide can induce vesicle leakage and lipid mixing. A sequence representing a defective gp41 phenotype unable to mediate both cell–cell fusion and virus entry, is equally unable to induce vesicle fusion, and adopted a non-helical conformation in the membrane. Therefore, membrane perturbation and adoption of the α-helical conformation by this gp41 region might be functionally meaningful.

Salzwedel et al. J. Virol. 73:2469-2480, 1999The multiple effects of various mutations in this region on membrane fusion, Env incorporation into the virus, and virus infectivity suggest that different residues or sequences in this motif may still play disparate functions in HIV-1 infection and that multiple sequences in this motif may contribute synergistically to these functions.

Membrane fusion- a series of cascade events:Fusion of outer leafetsFusion of inner leaflets

Fusion pore formation and enlargement