Interaction of a peptide corresponding to the loop domain of the S2 SARS-CoV virus protein with model membranes

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein is responsible for the fusion between the membranes of the virus and the target cell. In the case of the S2 domain of protein S, it has been found a highly hydrophobic and interfacial domain flanked by the heptad repeat 1 and 2 regions; significantly, different peptides pertaining to this domain have shown a significant leakage effect and an important plaque formation inhibition, which, similarly to HIV-1 gp41, support the role of this region in the fusion process. Therefore, we have carried out a study of the binding and interaction with model membranes of a peptide corresponding to segment 1073–1095 of the SARS-CoV S glycoprotein, peptide SARS_L in the presence of different membrane model systems, as well as the structural changes taking place in both the lipid and the peptide induced by the binding of the peptide to the membrane. Our results show that SARS_L strongly partitions into phospholipid membranes and organizes differently in lipid environments, displaying membrane activity modulated by the lipid composition of the membrane. These data would support its role in SARS-CoV mediated membrane fusion and suggest that the region where this peptide resides could be involved in the merging of the viral and target cell membranes.     

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 and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a newly identified member of the family Coronaviridae and poses a serious public health threat. Recent studies indicated that the SARS-CoV viral spike glycoprotein is a class I viral fusion protein. A fusion peptide present at the N-terminal region of class I viral fusion proteins is believed to initiate viral and cell membrane interactions and subsequent fusion. Although the SARS-CoV fusion protein heptad repeats have been well characterized, the fusion peptide has yet to be identified. Based on the conserved features of known viral fusion peptides and using Wimley and White interfacial hydrophobicity plots, we have identified two putative fusion peptides (SARS(WW-I) and SARS(WW-II)) at the N terminus of the SARS-CoV S2 subunit. Both peptides are hydrophobic and rich in alanine, glycine, and/or phenylalanine residues and contain a canonical fusion tripeptide along with a central proline residue. Only the SARS(WW-I) peptide strongly partitioned into the membranes of large unilamellar vesicles (LUV), adopting a beta-sheet structure. Likewise, only SARS(WW-I) induced the fusion of LUV and caused membrane leakage of vesicle contents at peptide/lipid ratios of 1:50 and 1:100, respectively. The activity of this synthetic peptide appeared to be dependent on its amino acid (aa) sequence, as scrambling the peptide rendered it unable to partition into LUV, assume a defined secondary structure, or induce both fusion and leakage of LUV. Based on the activity of SARS(WW-I), we propose that the hydrophobic stretch of 19 aa corresponding to residues 770 to 788 is a fusion peptide of the SARS-CoV S2 subunit.     

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.     

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 E1_PTM, 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.     

Membrane recognition by vesicular stomatitis virus involves enthalpy-driven protein-lipid interactions

Vesicular stomatitis virus (VSV) infection depends on the fusion of viral and cellular membranes, which is mediated by virus spike glycoprotein G at the acidic environment of the endosomal compartment. VSV G protein does not contain a hydrophobic amino acid sequence similar to the fusion peptides found among other viral glycoproteins, suggesting that membrane recognition occurs through an alternative mechanism. Here we studied the interaction between VSV G protein and liposomes of different phospholipid composition by force spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Force spectroscopy experiments revealed the requirement for negatively charged phospholipids for VSV binding to membranes, suggesting that this interaction is electrostatic in nature. In addition, ITC experiments showed that VSV binding to liposomes is an enthalpically driven process. Fluorescence data also showed the lack of VSV interaction with the vesicles as well as inhibition of VSV-induced membrane fusion at high ionic strength. Intrinsic fluorescence measurements showed that the extent of G protein conformational changes depends on the presence of phosphatidylserine (PS) on the target membrane. Although the increase in PS content did not change the binding profile, the rate of the fusion reaction was remarkably increased when the PS content was increased from 25 to 75%. On the basis of these data, we suggest that G protein binding to the target membrane essentially depends on electrostatic interactions, probably between positive charges on the protein surface and negatively charged phospholipids in the cellular membrane. In addition, the fusion is exothermic, indicating no entropic constraints to this process.     

Cloaked similarity between HIV-1 and SARS-CoV suggests an anti-SARS strategy

Background

Severe acute respiratory syndrome (SARS) is a febrile respiratory illness. The disease has been etiologically linked to a novel coronavirus that has been named the SARS-associated coronavirus (SARS-CoV), whose genome was recently sequenced. Since it is a member of the Coronaviridae, its spike protein (S2) is believed to play a central role in viral entry by facilitating fusion between the viral and host cell membranes. The protein responsible for viral-induced membrane fusion of HIV-1 (gp41) differs in length, and has no sequence homology with S2.

Results

Sequence analysis reveals that the two viral proteins share the sequence motifs that construct their active conformation. These include (1) an N-terminal leucine/isoleucine zipper-like sequence, and (2) a C-terminal heptad repeat located upstream of (3) an aromatic residue-rich region juxtaposed to the (4) transmembrane segment.

Conclusions

This study points to a similar mode of action for the two viral proteins, suggesting that anti-viral strategy that targets the viral-induced membrane fusion step can be adopted from HIV-1 to SARS-CoV. Recently the FDA approved Enfuvirtide, a synthetic peptide corresponding to the C-terminal heptad repeat of HIV-1 gp41, as an anti-AIDS agent. Enfuvirtide and C34, another anti HIV-1 peptide, exert their inhibitory activity by binding to a leucine/isoleucine zipper-like sequence in gp41, thus inhibiting a conformational change of gp41 required for its activation. We suggest that peptides corresponding to the C-terminal heptad repeat of the S2 protein may serve as inhibitors for SARS-CoV entry.     

Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is not only responsible for receptor binding and virus fusion, but also a major Ag among the SARS-CoV proteins that induces protective Ab responses. In this study, we showed that the S protein of SARS-CoV is highly immunogenic during infection and immunizations, and contains five linear immunodominant sites (sites I to V) as determined by Pepscan analysis with a set of synthetic peptides overlapping the entire S protein sequence against the convalescent sera from SARS patients and antisera from small animals immunized with inactivated SARS-CoV. Site IV located in the middle region of the S protein (residues 528-635) is a major immunodominant epitope. The synthetic peptide S(603-634), which overlaps the site IV sequence reacted with all the convalescent sera from 42 SARS patient, but none of the 30 serum samples from healthy blood donors, suggesting its potential application as an Ag for developing SARS diagnostics. This study also provides information useful for designing SARS vaccines and understanding the SARS pathogenesis.     

Biophysical characterization of the fusogenic region of HCV envelope glycoprotein E1

We have studied the binding and interaction of the peptide E1_FP with various model membranes. E1_FP is derived from the amino acid segment 274-291 of the hepatitis C virus envelope glycoprotein E1, which was previously proposed to host the peptide responsible for fusion to target membranes. In the present study we addressed the changes which take place upon E1_FP binding in both the peptide and the phospholipid bilayer, respectively, through a series of complementary experiments. We show that peptide E1_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 interacts preferentially with cholesterol. The capability of modifying the biophysical properties of model membranes supports its role in HCV-mediated membrane fusion and suggests that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.     

Solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.     

Membrane insertion of the three main membranotropic sequences from SARS-CoV S2 glycoprotein

In order to complete the fusion process of SARS-CoV virus, several regions of the S2 virus envelope glycoprotein are necessary. Recent studies have identified three membrane-active regions in the S2 domain of SARS-CoV glycoprotein, one situated downstream of the minimum furin cleavage, which is considered the fusion peptide (SARSFP), an internal fusion peptide located immediately upstream of the HR1 region (SARSIFP) and the pre-transmembrane domain (SARSPTM). We have explored the capacity of these selected membrane-interacting regions of the S2 SARS-CoV fusion protein, alone or in equimolar mixtures, to insert into the membrane as well as to perturb the dipole potential of the bilayer. We show that the three peptides interact with lipid membranes depending on lipid composition and experiments using equimolar mixtures of these peptides show that different segments of the protein may act in a synergistic way suggesting that several membrane-active regions could participate in the fusion process of the SARS-CoV.     

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.     

NMR structures and localization of the potential fusion peptides and the pre-transmembrane region of SARS-CoV: Implications in membrane fusion

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) poses a serious public health hazard. The S2 subunit of the S glycoprotein of SARS-CoV carries out fusion between the virus and the host cells. However, the exact mechanism of the cell fusion process is not well understood. Current model suggests that a conformational transition, upon receptor recognition, of the two heptad core regions of S2 may expose the hydrophobic fusogenic peptide or fusion peptide for membrane insertion. Three regions of the S2 subunit have been proposed to be involved in cell–cell fusion. The N-terminal fusion peptide (FP, residues 770–788), an internal fusion peptide (IFP, residues 873–888) and the pre-transmembrane region (PTM, residues 1185–1202) demonstrated interactions with model lipid membranes and potentially involved in the fusion process. Here, we have determined atomic resolution structures of these three peptides in DPC detergent micelles by solution NMR. FP assumes α-helical conformation with significant distortion at the central Gly residues; enabling a close packing among sidechains of aromatic residues including W, Y and F. The 3-D structure of PMT is characterized by a helix–loop–helix with extensive aromatic interactions within the helices. IFP adopts a rather straight α-helical conformation defined by packing among sidechains of aromatic and aliphatic residues. Paramagnetic spin labeled NMR has demonstrated surface localization of PMT whereas FP and IFP inserted into the micelles. Collectively, data presented in this study will aid in understanding fusion mechanism of SARS-CoV.     

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.     

Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor

A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S(1190)). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S(1190) binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S(1190) maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S(1190) glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.     

Membrane destabilization induced by the human immunodeficiency virus type-1 fusion peptide

Summary The human immunodeficiency virus type-1 (HIV-1) fusion peptide, corresponding to a sequence of 23 amino acid residues at the N-terminus of the spike transmembrane subunit gp41, has the capacity to destabilize negatively charged and neutral large unilamellar vesicles, representing, respectively, the acidic and the neutral fraction of the plasma membrane lipids of viral target cells. As revealed by infrared spectroscopy, the peptide associated with the vesicles may exist in different conformations. In negatively charged membranes the structure is mainly an α-helix, while in Ca²⁺-neutralized negatively charged membranes the conformation switches to a predominantly extended conformation. In membranes composed of zwitterionic phospholipids and cholesterol, the peptide also adopts a predominant extended structure. The α-helical structure permeabilizes negatively charged vesicles but does not induce membrane fusion. The peptide in β-type conformation, on the other hand, permeabilizes neutral membranes and triggers fusion. As seen by³¹P NMR, the latter structure also exhibits the capacity to alter the lamellar organization of the membrane.     

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.     

Structures and polymorphic interactions of two heptad-repeat regions of the SARS virus S2 protein

Entry of SARS coronavirus into its target cell requires large-scale structural transitions in the viral spike (S) glycoprotein in order to induce fusion of the virus and cell membranes. Here we describe the identification and crystal structures of four distinct alpha-helical domains derived from the highly conserved heptad-repeat (HR) regions of the S2 fusion subunit. The four domains are an antiparallel four-stranded coiled coil, a parallel trimeric coiled coil, a four-helix bundle, and a six-helix bundle that is likely the final fusogenic form of the protein. When considered together, the structural and thermodynamic features of the four domains suggest a possible mechanism whereby the HR regions, initially sequestered in the native S glycoprotein spike, are released and refold sequentially to promote membrane fusion. Our results provide a structural framework for understanding the control of membrane fusion and should guide efforts to intervene in the SARS coronavirus entry process.     

Structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80R

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV). The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 A resolution, as well as the structure of the uncomplexed S1 RBD at 2.2 A resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor- and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity.     

Characterization of neutralizing monoclonal antibodies recognizing a 15-residues epitope on the spike protein HR2 region of severe acute respiratory syndrome coronavirus (SARS-CoV)

Summary The spike (S) glycoprotein is thought to play a complex and central role in the biology and pathogenesis of SARS coronavirus infection. In this study, a recombinant protein (rS268, corresponding to residues 268–1255 of SARS-CoV S protein) was expressed in Escherichia coli and was purified to near homogeneity. After immunization with rS268, S protein-specific BALB/c antisera and mAbs were induced and confirmed using ELISA, Western blot and IFA. Several BALB/c mAbs were found to be effectively to neutralize the infection of Vero E6 cells by SARS-CoV in a dose-dependent manner. Systematic epitope mapping showed that all these neutralizing mAbs recognized a 15-residues peptide (CB-119) corresponding to residues 1143–1157 (SPDVDLGDISGINAS) that was located to the second heptad repeat (HR2) region of the SARS-CoV spike protein. The peptide CB-119 could specifically inhibit the interaction of neutralizing mAbs and spike protein in a dose-dependent manner. Further, neutralizing mAbs, but not control mAbs, could specifically interact with CB-119 in a dose-dependent manner. Results implicated that the second heptad repeat region of spike protein could be a good target for vaccine development against SARS-CoV.