A dynamin-related protein contributes to Trichomonas vaginalis hydrogenosomal fission.

Trichomonas vaginalis is a highly divergent, unicellular eukaryote of the phylum Metamonada, class Parabasalia, and the source of a common sexually transmitted infection. This parasite lacks mitochondria, but harbors an evolutionarily related organelle, the hydrogenosome. We explored the role of dynamin-related proteins (DRPs) in the division of the hydrogenosome. Eight DRP homologues [T. vaginalis DRPs (TvDRPs)], which can be grouped into 3 subclasses, are present in T. vaginalis. We examined 5 TvDRPs that are representative of each subclass, by introducing dominant negative mutations analogous to those known to interfere with mitochondrial division in yeast, worms, and mammals. Microscopic and cell fractionation analyses of parasites expressing one of the mutated TvDRPs (TVAG_350040) demonstrated that this protein localizes to hydrogenosomes. Moreover, these organelles were found to be increased in size and reduced in number in cells expressing this dominant negative protein, relative to parasites expressing the corresponding wild-type TvDRP, the other 4 mutant TvDRPs, or an empty vector control. Our data indicate a role for a TvDRP in the fission of T. vaginalis hydrogenosomes, similar to that described for peroxisomes and mitochondria. These findings reveal a conservation of core components involved in the division of diverse eukaryotic organelles across broad phylogenetic distances.

Multifaceted action of Fuzeon as virus-cell membrane fusion inhibitor.

The viral peptide fusion inhibitor Fuzeon (T-20/DP178/enfuvirtide) is an essential part of the drug combination that has significantly increased the quality of life and life span of many acquired immuno-deficiency syndrome (AIDS) patients. Its development as a drug preceded the elucidation of its precise inhibitory mechanism, as well as its molecular targets. The initial model was that Fuzeon inhibits human immunodeficiency virus (HIV) entry by targeting one site within the viral transmembrane envelope protein. Herein, we describe the emerging discoveries that extend this model towards a multifaceted mechanism for the drug in targeting HIV. This significantly advances the understanding of how viruses enter host cells and opens a new window of opportunity for designing future viral fusion inhibitors.

Viral envelope protein folding and membrane hemifusion are enhanced by the conserved loop region of HIV-1 gp41.

Fusion of human immunodeficiency virus (HIV-1) with target cells is mediated by the gp41 transmembrane envelope protein. The loop region within gp41 contains 2 crucial cysteines that play an unknown role in HIV-cell fusion. On the basis of cell-cell fusion assay, using human T-cell lines [Jurkat E6-1 and Jurkat HXBc2(4)], and virus-cell fusion assay, using fully infectious HIV-1 HXBc2 virus and TZM-bl human cell line, we provide evidence that the oxidation state of the disulfide bond within a loop domain peptide determines its activity. The oxidized (closed) form inhibits fusion, while the reduced (opened) form enhances hemifusion. These opposite activities reach 60% difference in viral fusion. Both forms of the loop domain interact with gp41: the opened form enhances gp41 folding into a bundle, whereas the closed form inhibits this folding. Therefore, the transformation of the cysteines from a reduced to an oxidized state enables the loop to convert from opened to closed conformations, which assists gp41 to fold and induces hemifusion. The significant conservation of the loop region within many envelope proteins suggests a general mechanism, which is exploited by viruses to enhance entry into their host cells.

Virus-cell and cell-cell fusion mediated by the HIV-1 envelope glycoprotein is inhibited by short gp41 N-terminal membrane-anchored peptides lacking the critical pocket domain.

The interactions between the N- and C-terminal heptad repeat (NHR and CHR) regions of the human immunodeficiency virus (HIV-1) glycoprotein gp41 create a structure comprising a 6-helix bundle (SHB). A sequence in the SHB named the "pocket" is crucial for the SHB's stability and for the fusion inhibitory activity of 36-residue NHR peptide N36. We report that a short 27-residue peptide, N27, which lacks the pocket sequence, exhibits potent inhibitory activity in both cell-cell and virus-cell fusion assays when fatty acids were conjugated to its N but not C terminus. Furthermore, mutations in the positions that prevent interaction with the CHR but not with the NHR resulted in a dramatic reduction in N27 activity. These data support a mechanism in which N27 mainly targets the CHR rather than the internal NHR coiled-coil, reveal the N-terminal edge of the endogenous core structure in situ and hence complement our recent findings of the C-terminal edge of the core, and provide a new approach for designing short inhibitors from the NHR region of other lentiviruses due to similarities in their envelope proteins.

Membrane-anchored HIV-1 N-heptad repeat peptides are highly potent cell fusion inhibitors via an altered mode of action.

Peptide inhibitors derived from HIV-gp41 envelope protein play a pivotal role in deciphering the molecular mechanism of HIV-cell fusion. According to accepted models, N-heptad repeat (NHR) peptides can bind two targets in an intermediate fusion conformation, thereby inhibiting progression of the fusion process. In both cases the orientation towards the endogenous intermediate conformation should be important. To test this, we anchored NHR to the cell membrane by conjugating fatty acids with increasing lengths to the N- or C-terminus of N36, as well as to two known N36 mutants; one that cannot bind C-heptad repeat (CHR) but can bind NHR (N36 MUTe,g), and the second cannot bind to either NHR or CHR (N36 MUTa,d). Importantly, the IC(50) increased up to 100-fold in a lipopeptide-dependent manner. However, no preferred directionality was observed for the wild type derived lipopeptides, suggesting a planar orientation of the peptides as well as the endogenous NHR region on the cell membrane. Furthermore, based on: (i) specialized analysis of the inhibition curves, (ii) the finding that N36 conjugates reside more on the target cells that occupy the receptors, and (iii) the finding that N36 MUTe,g acts as a monomer both in its soluble form and when anchored to the cell membrane, we suggest that anchoring N36 to the cell changes the inhibitory mode from a trimer which can target both the endogenous NHR and CHR regions, to mainly monomeric lipopetides that target primarily the internal NHR. Besides shedding light on the mode of action of HIV-cell fusion, the similarity between functional regions in the envelopes of other viruses suggests a new approach for developing potent HIV-1 inhibitors.

Demonstrating the C-terminal boundary of the HIV 1 fusion conformation in a dynamic ongoing fusion process and implication for fusion inhibition.

The core complex is a structure involved in the fusion mechanism of many viruses, as well as in intracellular vesicle fusion. A powerful approach for studying the dynamic stages of HIV-1-cell fusion utilizes DP178, a core complex inhibitory peptide derived from the known sequence of the virus. Strikingly, we show that fatty acids can replace the entire C-terminal region of DP178, known to play a crucial role in the activity of the peptide. The inhibitory activity correlated with the length of the fatty acid, with the direction of fatty acid attachment (N- or C-terminus) and, as envisioned by a new triple staining assay, with the concentration of the peptides on cells. Our findings indicate, for the first time, the C-terminal boundary of the endogenous core structure in situ and establish that the C-terminal region of DP178 functions mainly as an anchor to the cell membrane. Apart from the mechanistic implications, such short lipopeptides provide new, promising fusion inhibitors. Because the fusion mechanism of HIV-1 is shared by other pathogen-enveloped viruses and by intracellular vesicle fusion, our results might influence the research and therapeutic efforts in these systems as well.

Characterization of the HIV N-terminal fusion peptide-containing region in context of key gp41 fusion conformations.

Central to our understanding of human immunodeficiency virus-induced fusion is the high resolution structure of fragments of the gp41 fusion protein folded in a low energy core conformation. However, regions fundamental to fusion, like the fusion peptide (FP), have yet to be characterized in the context of the cognate protein regardless of its conformation. Based on conformation-specific monoclonal antibody recognition, we identified the polar region consecutive to the N36 fragment as a stabilizer of trimeric coiled-coil assembly, thereby enhancing inhibitory potency. This tertiary organization is retained in the context of the hydrophobic FP (N70 fragment). Our data indicate that the N70 fragment recapitulates the expected organization of this region in the viral fusion intermediate (N-terminal half of the pre-hairpin intermediate (N-PHI)), which happens to be the prime target for fusion inhibitors. Regarding the low energy conformation, we show for the first time core formation in the context of the FP (N70 core). The alpha-helical and coiled-coil stabilizing polar region confers substantial thermal stability to the core, whereas the hydrophobic FP does not add further stability. For the two key fusion conformations, N-PHI and N70 core, we find that the FP adopts a nonhelical structure and directs higher order assembly (assembly of coiled coils in N-PHI and assembly of bundles in the N70 core). This supra-molecular organization of coiled coils or folded cores is seen only in the context of the FP. This study is the first to characterize the FP region in the context of the folded core and provides a basic understanding of the role of the elusive FP for key gp41 fusion conformations.

Structurally altered peptides reveal an important role for N-terminal heptad repeat binding and stability in the inhibitory action of HIV-1 peptide DP178.

Human immunodeficiency virus 1 gp41 folds into a six-helix bundle whereby three C-terminal heptad repeat regions pack in an anti-parallel manner against the coiled-coil formed by three N-terminal heptad repeats (NHR). Peptides that inhibit bundle formation contributed significantly to the understanding of the entry mechanism of the virus. DP178, which partially overlaps C-terminal heptad repeats, prevents bundle formation through an undefined mechanism; additionally it has been suggested to bind other ENV regions and arrest fusion in an unknown manner. We used two structurally altered DP178 peptides; in each, two sequential amino acids were substituted into their d configuration, d-SQ in the hydrophilic N-terminal region and d-LW in the hydrophobic C-terminal. Importantly, we generated an elongated NHR peptide, N54, obtaining the full N-helix docking site for DP178. Interestingly, d-LW retained wild type fusion inhibitory activity, whereas d-SQ exhibited significantly reduced activity. In correlation with the inhibitory data, CD spectroscopy and fluorescence studies revealed that all the DP178 peptides interact with N54, albeit with different stabilities of the bundles. We conclude that strong binding of DP178 N-terminal region to the endogenous NHR, without significant contribution of the C-terminal sequence of DP178 to core formation, is vital for DP178 inhibition. The finding that d-amino acid incorporation in the C terminus did not affect activity or membrane binding as revealed by surface plasmon resonance correlates with an additional membrane binding site, or membrane anchoring role, for the C terminus, which works synergistically with the N terminus to inhibit fusion.

The role of the N-terminal heptad repeat of HIV-1 in the actual lipid mixing step as revealed by its substitution with distant coiled coils.

The gp41 glycoprotein of HIV-1 is considered to be responsible for the actual fusion process between the virus and the host membranes. According to a prevailing model, gp41 trimer organization, directed by the N-terminal coiled-coil region (NHR), is essential for steps involved in the actual merging of viral and cellular membranes. This study addresses a major question: Is the specific sequence of the NHR obligatory for the fusion process, or can it be replaced by distant coiled coils that form different oligomeric states in solution? For this purpose we synthesized three known GCN4 coiled-coil mutants that oligomerize in solution into either dimers, trimers, or tetramers. These peptides were chemically ligated to the fusion peptide thereby creating three chimera peptides with different oligomeric tendencies in solution. These peptides were investigated, together with the 70-mer wild-type peptide (N70), regarding their structure in solution and membrane by using circular dichroism (CD) and FTIR spectroscopies, their ability to induce vesicle fusion, and their ability to bind phospholipid membranes by using surface plasmon resonance (SPR). Our results suggest that local assembly of fusion peptides, facilitated by coiled-coil oligomers, increases lipid mixing ability, probably by facilitating stronger binding of the fusion peptides to the opposing membrane as revealed by SPR. However, N70 is significantly more active than the other chimeras. Overall, the data indicate a correlation between the distinct conformation of N70 in solution and in membranes and its enhanced lipid mixing relative to the GCN4 chimeras.