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   inhibits HIV-1 infectivity. We have defined the following
   characteristics of this inhibitory activity. First, the inhibition is
   not attributable to the isolated N34 and C28 peptides, because the
   N34(L6)C28 trimer is still highly stable to thermal denaturation at its
   IC[50], with an apparent T[m] of approximately 63°C ([210]40). Second,
   there is amino acid sequence-specific inhibition by N34(L6)C28. The
   inhibitory activity is dramatically increased by the fusion-defective
   Leu 568-to-Ala and Trp 571-to-Arg mutations; W571R, L568A, and
   L568A/W571R exhibit 5-, 16-, and 35-fold greater activity than the
   wild-type molecule, respectively. Third, the inhibition is also
   conformation specific; the presence of the Ile 573-to-Ser mutation in
   the double mutant essentially disrupts the six-helix bundle formation,
   while reducing its inhibitory activity 16-fold. Fourth, the enhanced
   inhibitory activity by these mutations correlates with local structural
   perturbations near the hydrophobic cavity which destabilize the
   N34(L6)C28 trimer. Further studies of the inhibition mechanism of the
   gp41 core should provide insights into the HIV-1 entry process and
   could open new perspectives in the search for effective antiviral
   therapies.
   Implications for membrane fusion.
   The hemagglutinin protein of influenza virus irreversibly switches from
   the native structure to the fusogenic conformation when exposed to the
   acidic environment of the cellular endosome ([211]2, [212]6, [213]26,
   [214]63). This structural dimorphism is the basis for conformational
   changes that are crucial for activation of membrane fusion. The HIV-1
   envelope protein is also thought to exist in two different
   conformations (for recent reviews, see references [215]8 and [216]52).
   It is generally accepted that the native conformation exists on the
   surface of free virions, while upon binding of gp120 to CD4 and
   particular coreceptors (e.g., CCR5 or CXCR4), the HIV-1 envelope
   protein undergoes a complex of structural changes to the fusogenic
   state. The current model for gp41-mediated membrane fusion suggests
   that formation of the six-helix bundle leads to colocalization of the
   viral and cellular membranes for fusion ([217]23, [218]27, [219]57).
   While relatively little is known about how membrane apposition leads to
   complete fusion, there is evidence for the higher-order assembly of
   envelope protein trimers and the formation of fusion pores, as proposed
   to be required for influenza virus fusion ([220]20, [221]53, [222]59).
   Since the gp41 ectodomain core structure, with a T[m] in excess of
   90°C, is too stable to be disrupted by exogenous peptide binding, only
   during the gp41 conformational change to the fusogenic state does one
   anticipate that the targets for the peptides are available ([223]11,
   [224]23, [225]34, [226]40, [227]41). This consideration has led to the
   proposal that gp41 can exist as a transiently populated intermediate
   after initiating the receptor-activated conformational change but prior
   to formation of the six-helix bundle ([228]8, [229]23, [230]45).
   According to this view, synthetic peptides derived from the gp41
   ectodomain inhibit membrane fusion in a dominant-negative manner by
   associating with their endogenous partners of viral gp41 at this
   intermediate stage.
   Earlier genetic studies indicate that mutations in the Leu 568 and Trp
   571 residues abolish membrane fusion activity, although the mutant
   HIV-1 envelope proteins appear to have no other defects, including cell
   surface expression, gp160 precursor processing, and CD4 binding
   ([231]5). Our results indicate that these fusion-defective mutations
   destabilize the gp41 core structure although they still confer the
   six-helix bundle fold. Since the Leu 568 and Trp 571 residues form the
   right wall of a conserved coiled-coil cavity that provides a binding
   pocket for three C-terminal helices ([232]9), our data suggest that the
   fusion-defective mutations introduce structural perturbations in the
   cavity that weaken helical packing interactions in the six-helix
   complex and thus inhibit its formation.
   These fusion-defective mutations also exert striking effects on the
   inhibitory activity of N34(L6)C28; the L568A and W571R mutants exhibit
   5- to 16-fold-greater activity than the wild-type molecule. Several
   lines of evidence suggest that this enhanced inhibitory activity
   results from the synergistic inhibition of the N34 and C28 peptides in
   the mutant molecules. First, while the L568A and W571R trimers are
   stable, with T[m] values of 56 and 61°C, respectively, in PBS (pH 7.0)
   at a peptide concentration of 10 μM, L568A and W571R are predominantly
   unfolded at their IC[50]s (0.1 μM for L568A and 0.3 μM for W571R) under
   physiological conditions. The monomeric forms of the L568A and W571R
   molecules readily interact bivalently with virus gp41. Second, the Ile
   573-to-Ser mutation that disrupts the N34 coiled-coil formation
   ([233]40) can reduce the potency of the double mutant (L568A/W571R) in
   inhibiting membrane fusion close to that of the isolated C28 peptide.
   The nature of the multivalency in the N34(L6)C28 variants is likely to
   be responsible for their enhanced inhibitory activity. Finally, this
   synergy is fully consistent with the hypothesis that there is a
   populated intermediate of gp41 during transition to the fusogenic
   structure ([234]8, [235]23, [236]45). Only in the intermediate state
   are the N- and C-terminal heptad-repeat regions of virus gp41 not
   associated, allowing the N34 and C28 peptides to bind to these regions
   with a high effective concentration.

   ACKNOWLEDGMENTS
   We thank Jun Dong for suggestions on structural refinement and Neville
   Kallenbach for critical reading of the manuscript.
   This research was funded by NIH grants (AI-42693 to S.J. and AI-42382
   to M.L.) and by the New York City Council Speaker’s Fund for Biomedical
   Research (to M.L.).
   [237]Top
   [238]Abstract
   [239]MATERIALS AND METHODS
   [240]RESULTS
   [241]DISCUSSION
   [242]REFERENCES
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