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Steve S.-L. Chen and Woan-Eng Chan Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan

Role of the Highly Conserved LWYIK Motif of the HIV-1 Transmembrane Protein gp41 in Env-mediated Membrane Fusion. Steve S.-L. Chen and Woan-Eng Chan Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan R.O.C. August 14, 2006 2006 International AIDS Conference. FP. NHR. CHR.

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Steve S.-L. Chen and Woan-Eng Chan Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan

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  1. Role of the Highly Conserved LWYIK Motif of the HIV-1 Transmembrane Protein gp41 in Env-mediated Membrane Fusion Steve S.-L. Chen and Woan-Eng Chan Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan R.O.C. August 14, 2006 2006 International AIDS Conference

  2. FP NHR CHR TM The Tryptophan-rich domain Ectodomain Cytoplasmic domain 705 684 587 628 663 527 559 512 N C C34 DP178 (638-673) 663 705 679 683 ELDKWASWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIV 2F5 4E10 Membrane-spanning domain (684-705)

  3. The LWYIK motif (residues 679~683): located immediately proximal to the TM region Li and Papadopoulos: Cholesterol-binding motif: L/V-(X)1-5-Y-(X)1-5-R/K HIV-1: 92BR025-9WQNLWTWFGITNWLWYIK GB8.C4WANLWNWFDITNWLWYIK NL4-3WASLWNWFNITNWLWYIK MNWASLWNWFDITNWLWYIK HXB2WASLWNWFNITNWLWYIK SIV: SIV cpzantWSSLWNWFDITQWLWYIK SIV cpzLNSWDVFGNWFDLASWIR SIV macLNSWDVFGNWFDLTSWIK SIV agmLNSWDVFGNWFDLASWIK HIV-2 HIV2CBL24LNSWDVFGNWFDLASWIK HIV-2STLNSWDVFGNWFDLTSWIK HIV2CBL21LNSWDVFGNWFDLTSWIR

  4. 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.

  5. Construction of LWYIK motif-mutant proviruses 679 683 TM region WT ----ITNWLWYIKLFIMI---- --------.....--------- DLWYIK ----------..---------- DYI -----------..--------- DIK KE ------------E--------- WA ---------A------------ YA ----------A-----------

  6. Replication kinetics of LWYIK motif-mutant viruses in CD4+ CEM-SS cells

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

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

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

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

  11. Env trans-complementation assay rev gag CAT vif tat SV40 pHXB D env CAT pol D env LTR LTR Bgl Bgl rev env LTR LTR SV40 env plasmid gp120 gp41 45 753 314 363 517 Bgl Bgl Transfection 293 T cells 2days Infection + CD4 T cells CAT assay

  12. All of mutant proteins inhibit their trans-complementation abilities

  13. A quantitative Env-mediated membrane fusion assay CMV promoter Tat pA Tat HIV-1 5’-LTR Cat gene 3’-LTR Tat 293T H938 Env CD4

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

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

  16. Three-color dye transfer assay CEM-SS Env-expressing 293T cells Dil andCalcein-AMlabeled CMAClabeled Hemifusion Fusion pore enlargement

  17. Lipid and cytosolic mixing

  18. Deletions in the LWYIK motif inhibit cytosolic dye transfer

  19. 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 mixing YI Normal - 10% Reduced cytosolic mixing IK Normal - 10% Reduced cytosolic mixing ___________________________________________________________________________

  20. 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.

  21. Acknowledgements Institute of Biomedical Sciences, Academia Sinica: Woan-Eng Chan Hsiao-Fen Li Yu Tsai Shu-Chen Huang Chia-Hung Chang Animal Technology Institute Taiwan: Chin-Kai Chuang Supported by : Academia Sinica and National Health Research Institute Taiwan, R.O.C.

  22. 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.

  23. 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, 2000 A 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, 1999 The 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.

  24. Membrane fusion- a series of cascade events: Fusion of outer leafets Fusion of inner leaflets Fusion pore formation and enlargement

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