Steve S.-L. Chen and Woan-Eng Chan Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan...
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Transcript of Steve S.-L. Chen and Woan-Eng Chan Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan...
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-----------
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
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
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
CEM-SS
Dil and Calcein-AM labeled CMAC labeled
Three-color dye transfer assay
Env-expressing 293T cells
Fusion pore enlargement
Hemifusion
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.