Steve S.-L. Chen and Woan-Eng Chan Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan R.O.C. August 14, 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 FPNHRCHRTM Cytoplasmic domain NC C34 DP178 ( ) F54E10Membrane-spanning domain ( ) ELDKWASWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIV

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 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: , 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 TM region WT ---- ITNWLWYIKLFIMI ----  LWYIK  YI  IK KE E WA A YA A

Replication kinetics of LWYIK motif-mutant viruses in 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 envplasmid CAT assay 293T cells Transfection Infection 2days CD4 + T cells LTR SV40 pol Bgl  env gag vif CAT tat rev SV40 rev Bgl gp120gp41 LTR env pHXB  envCAT

All of mutant proteins inhibit their trans-complementation abilities

HIV-1 5’-LTR CMV promoterpA Tat Env CD4 Cat gene 3’-LTR Tat H T 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 AbrogatedGreatly reduced - W(1~5)A Greatly Reduced AbrogatedGreatly reduced + W(1~3)A Greatly Reduced AbrogatedGreatly 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 ___________________________________________________________________________

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 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.

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: , 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: , 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: , 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.

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