A second SARS-CoV S2 glycoprotein internal membrane-active peptide. Biophysical characterization and membrane interaction.

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. The S2 domain of protein S has been suggested to have two fusion peptides, one located at its N-terminus, downstream of the furin cleavage, and another, more internal, located immediately upstream of the HR1. Therefore, we have carried out a study of the binding and interaction with model membranes of a peptide corresponding to segment 873-888 of the SARS-CoV S glycoprotein, peptide SARS IFP, as well as the structural changes taking place in both the phospholipid and the peptide induced by the binding of the peptide to the membrane. We demonstrate that SARS IFP peptide binds to and interacts with phospholipid model membranes and shows a higher affinity for negatively charged phospholipids than for zwitterionic ones. SARS IFP peptide specifically decreases the mobility of the phospholipid acyl chains of negatively charged phospholipids and adopts different conformations in the membrane depending upon their composition. These data support its role in SARS-mediated membrane fusion and suggest that the regions where this peptide resides might assist the fusion peptide and/or the pretransmembrane segment of the SARS-CoV spike glycoprotein in the fusion process.

The pre-transmembrane region of the HCV E1 envelope glycoprotein: interaction with model membranes.

The previously identified membranotropic regions of the HCV E1 envelope glycoprotein, a class II membrane fusion protein, permitted us to identify different sequences which might be implicated in viral membrane fusion, membrane interaction and/or protein-protein binding. HCV E1 glycoprotein presents a membrano-active region immediately adjacent to the transmembrane segment, which could be involved in membrane destabilization similarly to the pre-transmembrane domains of class I fusion proteins. Consequently, we have carried out a study of the binding and interaction with the lipid bilayer of a peptide corresponding to segment 309-340, peptide E1PTM, as well as the structural changes which take place in both the peptide and the phospholipid molecules induced by the binding of the peptide to the membrane. Here we demonstrate that peptide E1(PTM) strongly partitions into phospholipid membranes, interacts with negatively-charged phospholipids and locates in a shallow position in the membrane. These data support its role in HCV-mediated membrane fusion and suggest that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.

Interaction of the most membranotropic region of the HCV E2 envelope glycoprotein with membranes. Biophysical characterization.

The previously identified membrane-active regions of the hepatitis C virus (HCV) E1 and E2 envelope glycoproteins led us to identify different segments that might be implicated in viral membrane fusion, membrane interaction, and/or protein-protein binding. HCV E2 glycoprotein contains one of the most membranotropic segments, segment 603-634, which has been implicated in CD81 binding, E1/E2 and E2/E2 dimerization, and membrane interaction. Through a series of complementary experiments, we have carried out a study of the binding and interaction with the lipid bilayer of a peptide corresponding to segment 603-634, peptide E2(FP), as well as the structural changes induced by membrane binding that take place in both the peptide and the phospholipid molecules. Here, we demonstrate that peptide E2(FP) binds to and interacts with phospholipid model membranes, modulates the polymorphic phase behavior of membrane phospholipids, is localized in a shallow position in the membrane, and is probably oligomerized in the presence of membranes. These data support the role of E2(FP) in HCV-mediated membrane fusion, and sustain the notion that this segment of the E2 envelope glycoprotein, together with other segments of E2 and E1 glycoproteins, provides the driving force for the merging of the viral and target cell membranes.

Biophysical characterization and membrane interaction of the most membranotropic region of the HIV-1 gp41 endodomain.

The membrane fusion protein of HIV-1 is the envelope transmembrane gp41 glycoprotein, which is the responsible of the membrane fusion between the virus and the target cell. Gp41 has an unusual cytoplasmic tail, the endodomain, containing highly helicoidal segments with large hydrophobic moments, the so called lentivirus lytic peptides or LLPs. According to our previous work, one of the most membranotropic regions along the whole gp41 glycoprotein was located in the LLP3 region of the gp41. In order to get new insights into the viral membrane fusion mechanism, a peptide pertaining to the LLP3 domain has been studied by infrared, fluorescence and calorimetry regarding its structure, its ability to induce membrane rupture and aggregation, as well as its affinity towards specific phospholipids. Our results demonstrate that this peptide interacts with phospholipid-containing model membranes, affects the phase-behavior of membrane phospholipids and induces leakage and aggregation of liposomes. The membrane-perturbing properties of LLP3, together with the possibility that the Kennedy sequence could be part of an external loop, open the possibility that these domains might function in modulating viral membrane fusion or budding, synergistically with other membranotropic regions of the gp41 glycoprotein.

Identification of the membrane-active regions of hepatitis C virus p7 protein: biophysical characterization of the loop region.

We have identified the membrane-active regions of the hepatitis C virus p7 protein by performing an exhaustive study of membrane rupture, hemifusion, and fusion induced by a p7-derived peptide library on model membranes having different phospholipid compositions. We report the identification in p7 of a highly membranotropic region located at the loop domain of the protein. Here, we have investigated the interaction of a peptide patterned after the p7 loop (peptide p7(L)), studying its binding and interaction with the lipid bilayer, and evaluated the binding-induced structural changes of the peptide and the phospholipids. We show that positively rich p7(L) strongly binds to negatively charged phospholipids and it is localized in a shallow position in the bilayer. Furthermore, peptide p7(L) exhibits a high tendency to oligomerize in the presence of phospholipids, which could be the driving force for the formation of the active ion channel. Therefore, our findings suggest that the p7 loop could be an attractive candidate for antiviral drug development, because it could be a target for antiviral compounds that may lead to new vaccine strategies.

Interaction of a peptide from the pre-transmembrane domain of the severe acute respiratory syndrome coronavirus spike protein with phospholipid membranes.

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a Class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. In order to gain new insight into the protein membrane alteration leading to the viral fusion mechanism, a peptide pertaining to the putative pre-transmembrane domain (PTM) of the S glycoprotein has been studied by infrared and fluorescence spectroscopies regarding its structure, its ability to induce membrane leakage, aggregation, and fusion, as well as its affinity toward specific phospholipids. We demonstrate that the SARS-CoV PTM peptide binds to and interacts with phospholipid model membranes, and, at the same time, it adopts different conformations when bound to membranes of different compositions. As it has been already suggested for other viral fusion proteins such as HIV gp41, the region of the SARS-CoV protein where the PTM peptide resides could be involved in the merging of the viral and target cell membranes working synergistically with other membrane-active regions of the SARS-CoV S glycoprotein to heighten the fusion process and therefore might be essential for the assistance and enhancement of the viral and cell fusion process.

Characterization of the interaction of two peptides from the N terminus of the NHR domain of HIV-1 gp41 with phospholipid membranes.

The HIV-1 gp41 envelope glycoprotein is responsible for the membrane fusion between the virus and the target cell. According to recent models, the N-terminal coiled-coil (NHR) region of gp41 is involved in forming the interfaces between neighboring helices in the six-helix bundle, as well as in membrane binding and perturbation. In order to get new insights into the viral membrane fusion mechanism, two peptides, pFP15 and pFP23, pertaining to the first part of the gp41 NHR domain were studied regarding their structure and their ability to induce membrane leakage, aggregation, and fusion, as well as their affinity toward specific phospholipids by a variety of spectroscopic methods. Our results demonstrate that the first part of the NHR domain interacts with negatively charged phospholipid-containing model membranes, modifies the phase behavior of membrane phospholipids, and induces leakage and aggregation of liposomes, suggesting that it could be involved directly in the merging of the viral and target cell membranes working synergistically with other membrane-active regions of the gp41 glycoprotein to boost the fusion process. On the other hand, we suggest that this region of the NHR domain could be involved in the first steps of the destabilization of the HIV-1 gp41 six-helix bundle after its interaction with negatively charged phospholipid headgroups.

Structure of the C-terminal domain of the pro-apoptotic protein Hrk and its interaction with model membranes.

The protein harakiri (Hrk) is a pro-apoptotic BH3-only protein which belongs to the Bcl-2 family. Hrk appears associated to the mitochondrial outer membrane, apparently by a putative transmembrane domain, where it exerts its function. In this work we have identified a 27mer peptide supposed to be the putative membrane domain of the protein at the C-terminal region, and used infrared and fluorescence spectroscopies to study its secondary structure as well as to characterize its effect on the physical properties of phospholipid model membranes. The results presented here showed that the C-terminal region of Hrk adopts a predominantly alpha-helical structure whose proportion and destabilization capability varied depending on phospholipid composition. Moreover it was found that the orientation of the alpha-helical component of this C-terminal Hrk peptide was nearly perpendicular to the plane of the membrane. These results indicate that this domain is able of inserting into membranes, where it adopts a transmembrane alpha-helical structure as well as it considerably perturbs the physical properties of the membrane.

The membrane-active regions of the hepatitis C virus E1 and E2 envelope glycoproteins.

We have identified the membrane-active regions of the full sequences of the HCV E1 and E2 envelope glycoproteins by performing an exhaustive study of membrane leakage, hemifusion, and fusion induced by 18-mer peptide libraries on model membranes having different phospholipid compositions. The data and their comparison have led us to identify different E1 and E2 membrane-active segments which might be implicated in viral membrane fusion, membrane interaction, and/or protein-protein binding. Moreover, it has permitted us to suggest that the fusion peptide might be located in the E1 glycoprotein and, more specifically, the segment comprised by amino acid residues 265-296. The identification of these membrane-active segments from the E1 and E2 envelope glycoproteins, as well as their membranotropic propensity, supports their direct role in HCV-mediated membrane fusion, sustains the notion that different segments provide the driving force for the merging of the viral and target cell membranes, and defines those segments as attractive targets for further development of new antiviral compounds.

The membranotropic regions of the endo and ecto domains of HIV gp41 envelope glycoprotein.

We have identified the membranotropic regions of the full sequence of the HIV gp41 envelope glycoprotein by performing an exhaustive study of membrane rupture, phospholipid-mixing and fusion induced by two 15-mer gp41-derived peptide libraries from HIV strains HIV_MN and HIV_consensus_B on model membranes having different phospholipid compositions. The data obtained for the two strains and its comparison have led us to identify different gp41 membranotropic segments in both ecto- and endodomains which might be implicated in viral membrane fusion and/or membrane interaction. The membranotropic segments corresponding to the gp41 ectodomain were the fusion domain, a stretch located on the N-heptad repeat region adjacent to the fusion domain, part of the immunodominant loop, the pre-transmembrane domain and the transmembrane domain. The membranotropic segments corresponding to the gp41 endodomain were mainly located at some specific parts of the previously described lentivirus lytic sequences. Significantly, the C-heptad repeat region and the Kennedy sequence located in the ectodomain and in the endodomain, respectively, presented no membranotropic activity in any model membrane assayed. The identification of these gp41 segments as well as their membranotropic propensity sustain the notion that different segments of gp41 provide the driving force for the merging of the viral and target cell membranes as well as they help us to define those segments as attractive targets for further development of new anti-viral compounds.

A peptide pertaining to the loop segment of human immunodeficiency virus gp41 binds and interacts with model biomembranes: implications for the fusion mechanism.

The human immunodeficiency virus gp41 envelope protein mediates the entry of the virus into the target cell by promoting membrane fusion. In order to gain new insights into the viral fusion mechanism, we studied a 35-residue peptide pertaining to the loop domain of gp41, both in solution and membrane bound, by using infrared and fluorescence spectroscopy. We show here that the peptide, which has a membrane-interacting surface, binds and interacts with phospholipid model membranes and tends to aggregate in the presence of a membranous medium and induce the leakage of vesicle contents. The results reported in this work, i.e., the destabilization and fusion of negatively charged model membranes, suggest an essential role of the loop domain in the membrane fusion process induced by gp41.

Identification of the membrane-active regions of the severe acute respiratory syndrome coronavirus spike membrane glycoprotein using a 16/18-mer peptide scan: implications for the viral fusion mechanism.

We have identified the membrane-active regions of the severe acute respiratory syndrome coronavirus (SARS CoV) spike glycoprotein by determining the effect on model membrane integrity of a 16/18-mer SARS CoV spike glycoprotein peptide library. By monitoring the effect of this peptide library on membrane leakage in model membranes, we have identified three regions on the SARS CoV spike glycoprotein with membrane-interacting capabilities: region 1, located immediately upstream of heptad repeat 1 (HR1) and suggested to be the fusion peptide; region 2, located between HR1 and HR2, which would be analogous to the loop domain of human immunodeficiency virus type 1; and region 3, which would correspond to the pretransmembrane region. The identification of these membrane-active regions, which are capable of modifying the biophysical properties of phospholipid membranes, supports their direct role in SARS CoV-mediated membrane fusion, as well as facilitating the future development of SARS CoV entry inhibitors.

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan.

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.