To infect target cells, HIV-1 employs a virally encoded transmembrane protein (gp41) to fuse its viral envelope with the target cell plasma membrane. We describe the gp41 ectodomain as comprised of N-and C-terminal subdomains, each containing a heptad repeat as well as a fusogenic region, whose organization is mirrored by the intervening loop region. Recent evidence indicates that the gp41 directed fusion reaction proceeds to initial pore formation prior to gp41 folding into its low energy hairpin conformation. This implies that exposed regions of the gp41 ectodomain are responsible for the bulk of the fusion work, probably through direct protein-membrane interactions. Prevalent fusion models contend that the gp41 ectodomain initially interacts with the target cell surface through its highly hydrophobic N terminus, which is believed to insert into the target membrane, thereby linking the virus to the target cell. This arrangement allows the N-terminal subdomain to interact with the target cell surface, whereas the Cterminal subdomain remains proximal to the virion, allowing interaction with the viral envelope. The composition of the viral envelope and the target cell surface differ due to the virus budding from raft microdomains. We show here that constructs corresponding to the Cterminal subdomain specifically destabilize ordered and cholesterol rich membranes (33 molar %), whereas the N-terminal subdomain is more effective in fusing both unordered cholesterol-free membranes and those containing lower amounts of cholesterol (10 molar %). Moreover we show that, in the context of the C-terminal subdomain, the heptad repeat contributes helical structure, which may describe the enhanced inhibitory effect of the C-terminal subdomain relative to the C-terminal heptad repeat (C34) alone. Our results are discussed in light of recent findings that showcase the role of exposed gp41 regions in effecting membrane fusion.
For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion.
Fusion between viral and host cell membranes is the initial step of HIV infection and is mediated by the gp41 protein which is embedded in the viral membrane. The ~20-residue N-terminal fusion peptide (FP) region of gp41 binds to the host cell membrane and plays a critical role in fusion catalysis. Key gp41 fusion conformations include an early pre-hairpin intermediate (PHI) characterized by extended coiled-coil structure in the region C-terminal of the FP and a final hairpin state with compact six-helix bundle (SHB) structure. The large "N70" (gp41 1-70) and "FP-Hairpin" constructs of the present study contained the FP and respectively modeled the PHI and hairpin conformations. Comparison was also made to the shorter "FP34" (gp41 1-34) fragment. Studies were done in membranes with physiologically relevant cholesterol content and in membranes without cholesterol. In either membrane type, there were large differences in fusion function among the constructs with little fusion induced by FP-Hairpin, moderate fusion for FP34, and very rapid fusion for N70. Overall, our findings support acceleration of gp41-induced membrane fusion by early PHI conformation and fusion arrest after folding to the final SHB structure. FP secondary structure at Leu-7 of the membrane-associated constructs was probed by solid-state NMR and showed populations of molecules with either β-sheet or helical structure with greater β-sheet population observed for FP34 than for N70 or FP-Hairpin. The large differences in fusion function among the constructs were not obviously correlated with FP secondary structure. Observation of cholesteroldependent FP structure for fusogenic FP34 and N70 and cholesterol-independent structure for nonfusogenic FP-Hairpin was consistent with membrane insertion of the FP for FP34 and N70 and with lack of insertion for FP-Hairpin. Membrane insertion of the FP may therefore be associated with the early PHI conformation and FP withdrawal with the final hairpin conformation.
Conformational changes in the HIV gp41 protein are directly correlated with fusion between the HIV and target cell plasma membranes which is the initial step of infection. Key gp41 fusion conformations include an early extended conformation termed pre-hairpin which contains exposed regions and a final low energy conformation termed hairpin which has compact six-helix bundle structure. Current fusion models debate the roles of hairpin and pre-hairpin conformations in the process of membrane merger. In the present work, gp41 constructs have been engineered which correspond to fusion relevant parts of both pre-hairpin and hairpin conformations, and have been analyzed for their ability to induce lipid mixing between membrane vesicles. The data correlate membrane fusion function with the pre-hairpin conformation and suggest that one of the roles of the final hairpin conformation is sequestration of membrane perturbing gp41 regions with consequent loss of the membrane disruption induced earlier by the pre-hairpin structure. To our knowledge, this is the first biophysical study to delineate the membrane fusion potential of gp41 constructs modeling key fusion conformations. KeywordsHIV; membrane fusion; gp41; fusion peptide; hairpin and pre-hairpin intermediate HIV initiates infection of target cells through a process of fusion between the viral envelope membrane and the cell plasma membrane at physiologic pH. The viral transmembrane (TM) protein gp41 catalyzes this process through direct interaction with the target cell membrane and through conformational changes (see fig. 1) (1,2). Gp41 is one of two subunits of the envelope (ENV) complex and is initially shrouded by the gp120 subunit on the virion surface ( fig. 1A). The putative oligomerization state of ENV is a trimer with three gp41 and three gp120 subunits (3). Activation of gp41 is triggered by sequential binding of gp120 to cell surface CD4 receptor (4) and co-receptor (5) which induces gp120 conformational changes. These changes allow the gp41 region outside the virus (ectodomain) to adopt an extended conformation (6,7) and anchor into the target cell membrane via its N-terminal apolar fusion peptide (FP) region (8,9). This early exposed gp41 conformation is termed the pre-hairpin † The work was supported by NIH awards AI47153 to D.P.W. and F32AI080136 to K.S. This study was partially supported by the Israel Science Foundation to Y.S. Address correspondence to: Dr. David Weliky, Department of Chemistry, Michigan State University, East Lansing, MI, 48824. Phone: 517-355-9715, Fax: 517-353-1793. weliky@chemistry.msu.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 March 31. The gp41 ectodomain is comprised of FP, N-terminal heptad repeat (NHR), disulfide loop, Cterminal heptad repeat (CHR), and membrane proximal external region (MPER) (figs. 1B and 2A). Development of fusion models has relied on high resolution structures of gp41 constructs in the hairpin conformation. These structures reveal compact six-hel...
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