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J. Biol. Chem., Vol. 282, Issue 32, 23104-23116, August 10, 2007

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Functional Links between the Fusion Peptide-proximal Polar Segment and Membrane-proximal Region of Human Immunodeficiency Virus gp41 in Distinct Phases of Membrane Fusion^*

Anna K. Bellamy-McIntyre¹, Chan-Sien Lay^¶1, Séverine Baär^||2, Anne L. Maerz, Gert H. Talbo^**, Heidi E. Drummer^¶, and Pantelis Poumbourios^¶3

From the Macfarlane Burnet Institute for Medical Research and Public Health, Prahran, Victoria 3004, Australia, the Department of Microbiology, Monash University, Clayton, Victoria 3070, Australia, the ^¶Department of Microbiology and Immunology, the University of Melbourne, Parkville, Victoria 3058, Australia, the ^||Department of Cell Biology, Institut Cochin, Paris, France 75014, and ^**Proteomics Centre, Baker Heart Research Institute, Melbourne, Victoria 3004, Australia

The binding of CD4 and chemokine receptors to the gp120 attachment glycoprotein of human immunodeficiency virus triggers refolding of the associated gp41 fusion glycoprotein into a trimer of hairpins with a 6-helix bundle (6HB) core. These events lead to membrane fusion and viral entry. Here, we examined the functions of the fusion peptide-proximal polar segment and membrane-proximal Trp-rich region (MPR), which are exterior to the 6HB. Alanine substitution of Trp⁶⁶⁶, Trp⁶⁷², Phe⁶⁷³, and Ile⁶⁷⁵ in the MPR reduced entry by up to 120-fold without affecting gp120-gp41 association or cell-cell fusion. The L537A polar segment mutation led to the loss of gp120 from the gp120-gp41 complex, reduced entry by 10-fold, but did not affect cell-cell fusion. Simultaneous Ala substitution of Leu⁵³⁷ with Trp⁶⁶⁶, Trp⁶⁷², Phe⁶⁷³, or Ile⁶⁷⁵ abolished entry with 50–80% reductions in cell-cell fusion. gp120-gp41 complexes of fusion-defective double mutants were resistant to soluble CD4-induced shedding of gp120, suggesting that their ability to undergo receptor-induced conformational changes was compromised. Consistent with this idea, a representative mutation, L537A/W666A, led to an 80% reduction in lipophilic fluorescent dye transfer between gp120-gp41-expressing cells and receptor-expressing targets, indicating a block prior to the lipid-mixing phase. The L537A/W666A double mutation increased the chymotrypsin sensitivity of the polar segment in a trimer of hairpins model, comprising the 6HB core, the polar segment, and MPR linked N-terminally to maltose-binding protein. The data indicate that the polar segment and MPR of gp41 act synergistically in forming a fusion-competent gp120-gp41 complex and in stabilizing the membrane-interactive end of the trimer of hairpins.

Received for publication, April 26, 2007 , and in revised form, May 24, 2007.

^* This work was supported by the National Health and Medical Research Council of Australia Grants 296200 and 345413, American Foundation for AIDS Research Grant 106610-36-RGNT, and Sidaction. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

^{1 1} Both authors contributed equally to this work.

² Present address: Program Infection and Cancer, Abt. F010 and INSERM U701, Deutsches 10 Krebsforschungszentrum, Heidelberg, Germany.

³ To whom correspondence should be addressed: 85 Commercial Rd., Melbourne, Victoria 3004, Australia. Fax: 613-9282-2100; E-mail: apoumbourios{at}burnet.edu.au.

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