Identification of the Membrane-Active Regions of t
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病菌学杂志 2005年第3期
Instituto de Biología Molecular y Celular, Universidad "Miguel Hernández", Elche-Alicante, Spain
ABSTRACT
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.
INTRODUCTION
An infectious disease, designated severe acute respiratory syndrome (SARS), broke out in China in late 2002 and quickly spread to several countries. The infectious agent responsible for this epidemic outbreak was identified as a previously unknown member of the family of coronaviruses (CoV), SARS CoV (10, 21, 30, 31). Its phylogenetic analysis showed that it was neither a mutant nor a recombinant of previously characterized CoV (35). These viruses are a diverse group of enveloped, positive-strand RNA viruses, with three or four proteins embedded in the envelope, that cause respiratory and enteric diseases in humans and other animals (10, 21, 30, 31). CoV infection, similarly to other envelope viruses, is achieved through fusion of the lipid bilayer of the viral envelope with the host cell membrane.
The fusion of viral and cellular membranes, the critical early events in viral infection, are mediated by envelope glycoproteins located on the outer surfaces of the viral membranes (11, 17). SARS CoV membrane fusion is mediated by the viral spike glycoprotein located on the viral envelope, which is synthesized as a 180-kDa precursor and displayed in 200 copies on the viral membrane in a trimeric or dimeric structure (10, 21, 30, 35, 47). In some CoV strains, the spike glycoprotein is cleaved by a protease to yield two noncovalently associated subunits, S1 and S2 (Fig. 1A), which have different functions (16, 40). However, cleavage is not an absolute requirement for the mechanism of fusion, and the available data suggest that the SARS CoV spike glycoprotein is not cleaved into two subunits (9, 18, 35). S1, which forms the globular portion of the spike, contains the receptor-binding site and thus defines the host range of the virus (42), while S2, more conserved than S1, forms the membrane-anchored stalk region and mediates the fusion between the viral and cellular membranes (35, 47).
S2 contains two predicted -helical heptad repeat (HR) domains (HR1 and HR2) which form coiled-coil structures (5, 6, 20, 22, 35, 43, 50). These regions, separated by a stretch of 140 amino acid residues called the interhelical domain, are thought to play important roles in defining the oligomeric structure of the spike protein in its native state and its fusogenic ability (23). The presence of the HR regions, in conjunction with recent studies, indicates that CoV spike proteins can be classified as class 1 viral fusion proteins (5, 6, 20, 22, 43, 50). In the current paradigm of virus-host cell membrane fusion for class 1 viral fusion proteins, the HR domains form a six-helix bundle, where three HR1 helices fold into a central parallel triple-stranded -helical coiled coil, and wrapped antiparallel on the outside of this core is an outer layer of three antiparallel HR2 -helices, each HR1-HR2 pair connected by a loop that reverses the polypeptide chain (5, 15, 17, 20, 22, 43, 50). The HR1 and HR2 regions are believed to be important domains in this process and show different conformations in different fusion states (11, 49). Under the current model, there are at least three conformational states of the envelope fusion protein, the prefusion native state, the prehairpin intermediate state, and the postfusion hairpin state (11, 49). This trimeric helical hairpin structure is thought to form at a late stage during the membrane fusion process (15, 17). Formation of the six-helix coiled-coil bundle brings into close proximity the fusion peptide (FP) and the pretransmembrane (PTM) and transmembrane (TM) domains, thereby driving the viral and host cell membranes into close contact, making possible the formation of the fusion pore (11, 15, 17). In class 1 viral fusion proteins, the FP invariably occurs upstream of the HR1 region; however, no FP has been experimentally identified in any CoV spike protein, although a hydrophobic region has been predicted recently at the N terminus of the HR1 region (5).
Although much information has been gathered in recent years, we do not yet know the exact mechanism of membrane fusion and the processes which are behind it. The mechanism by which proteins facilitate the formation of fusion intermediates is a complex process involving several segments of fusion proteins (15, 32). These regions, either directly or indirectly, might interact with biological membranes, contributing to the viral envelope and cell membrane merging. Even though the detailed structures of different segments of the SARS CoV spike glycoprotein have been elucidated, there are still many questions to be answered regarding its mode of action in accelerating membrane fusion. Moreover, SARS CoV entry is an attractive target for anti-SARS therapy. To investigate the structural basis of SARS CoV membrane fusion and identify new fusion inhibitors, we carried out the analysis of the different regions of the SARS CoV spike glycoprotein that might interact with phospholipid membranes, using an approach similar to that used for studying the human immunodeficiency virus (HIV) gp41 ectodomain (25), i.e., the identification of membrane-active regions of SARS CoV spike glycoprotein by determining the effect on membrane integrity of a 16/18-mer spike glycoprotein-derived peptide library. By monitoring the effect of this peptide library on membrane integrity, i.e., leakage, we have identified different regions on the SARS CoV spike glycoprotein with membrane-interacting capabilities, which supports their direct role in membrane fusion and therefore might help in understanding the molecular mechanism of membrane merging, as well as making possible the future development of SARS entry inhibitors, which may lead to new vaccine strategies.
MATERIALS AND METHODS
Materials and reagents. Egg L--phosphatidylcholine (EPC), egg sphingomyelin (SM), and cholesterol (Chol), were obtained from Avanti Polar Lipids (Alabaster, Ala.). 5-Carboxyfluorescein (CF) (>95% by high-performance liquid chromatography) was from Sigma-Aldrich (Madrid, Spain). A set of 169 peptides 16 or 18 amino acids in length derived from the SARS spike glycoprotein, with 10-amino-acid overlap between sequential peptides, was obtained through the AIDS Research and Reference Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.). Porcine lungs were obtained from a local slaughterhouse. Plasma membranes from lung tissue pneumocytes were obtained according to the method of Müller et al. (26), and lipid extraction from porcine lungs was performed according to the procedure of Bligh and Dyer using a ratio of 1:1:0.9 (vol/vol/vol) between chloroform-methanol and the corresponding aqueous sample (4). All other reagents used were of analytical grade and were obtained from Merck (Darmstad, Germany). Water was deionized, distilled twice, and passed through a Milli-Q apparatus (Millipore Ibérica, Madrid, Spain) to a resistivity better than 18 M/cm.
Sample preparation. Aliquots containing the appropriate amount of lipid in chloroform-methanol (2:1 [vol/vol]) were placed in a test tube, the solvents were removed by evaporation under a stream of O2-free nitrogen, and finally, traces of solvents were eliminated under vacuum in the dark for >3 h. After that, 1 ml of buffer containing 10 mM Tris, 20 mM NaCl, pH 7.4, and CF at a concentration of 40 mM was added, and multilamellar vesicles were obtained. Large unilamellar vesicles (LUV) with a mean diameter of 90 nm were prepared from multilamellar vesicles by the extrusion method (19), using polycarbonate filters with a pore size of 0.1 μm (Nuclepore Corp., Cambridge, Calif.). Breakdown of the vesicle membrane leads to leakage of the contents, i.e., CF fluorescence. Nonencapsulated CF was separated from the vesicle suspension through a Sephadex G-75 filtration column (Pharmacia, Uppsala, Sweden) eluted with buffer containing 10 mM Tris-0.1 M NaCl-1 mM EDTA, pH 7.4.
Leakage measurement. Leakage of intraliposomal CF was assayed by treating the probe-loaded liposomes (final lipid concentration, 0.125 mM) with the appropriate amounts of peptide on microtiter p
ABSTRACT
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.
INTRODUCTION
An infectious disease, designated severe acute respiratory syndrome (SARS), broke out in China in late 2002 and quickly spread to several countries. The infectious agent responsible for this epidemic outbreak was identified as a previously unknown member of the family of coronaviruses (CoV), SARS CoV (10, 21, 30, 31). Its phylogenetic analysis showed that it was neither a mutant nor a recombinant of previously characterized CoV (35). These viruses are a diverse group of enveloped, positive-strand RNA viruses, with three or four proteins embedded in the envelope, that cause respiratory and enteric diseases in humans and other animals (10, 21, 30, 31). CoV infection, similarly to other envelope viruses, is achieved through fusion of the lipid bilayer of the viral envelope with the host cell membrane.
The fusion of viral and cellular membranes, the critical early events in viral infection, are mediated by envelope glycoproteins located on the outer surfaces of the viral membranes (11, 17). SARS CoV membrane fusion is mediated by the viral spike glycoprotein located on the viral envelope, which is synthesized as a 180-kDa precursor and displayed in 200 copies on the viral membrane in a trimeric or dimeric structure (10, 21, 30, 35, 47). In some CoV strains, the spike glycoprotein is cleaved by a protease to yield two noncovalently associated subunits, S1 and S2 (Fig. 1A), which have different functions (16, 40). However, cleavage is not an absolute requirement for the mechanism of fusion, and the available data suggest that the SARS CoV spike glycoprotein is not cleaved into two subunits (9, 18, 35). S1, which forms the globular portion of the spike, contains the receptor-binding site and thus defines the host range of the virus (42), while S2, more conserved than S1, forms the membrane-anchored stalk region and mediates the fusion between the viral and cellular membranes (35, 47).
S2 contains two predicted -helical heptad repeat (HR) domains (HR1 and HR2) which form coiled-coil structures (5, 6, 20, 22, 35, 43, 50). These regions, separated by a stretch of 140 amino acid residues called the interhelical domain, are thought to play important roles in defining the oligomeric structure of the spike protein in its native state and its fusogenic ability (23). The presence of the HR regions, in conjunction with recent studies, indicates that CoV spike proteins can be classified as class 1 viral fusion proteins (5, 6, 20, 22, 43, 50). In the current paradigm of virus-host cell membrane fusion for class 1 viral fusion proteins, the HR domains form a six-helix bundle, where three HR1 helices fold into a central parallel triple-stranded -helical coiled coil, and wrapped antiparallel on the outside of this core is an outer layer of three antiparallel HR2 -helices, each HR1-HR2 pair connected by a loop that reverses the polypeptide chain (5, 15, 17, 20, 22, 43, 50). The HR1 and HR2 regions are believed to be important domains in this process and show different conformations in different fusion states (11, 49). Under the current model, there are at least three conformational states of the envelope fusion protein, the prefusion native state, the prehairpin intermediate state, and the postfusion hairpin state (11, 49). This trimeric helical hairpin structure is thought to form at a late stage during the membrane fusion process (15, 17). Formation of the six-helix coiled-coil bundle brings into close proximity the fusion peptide (FP) and the pretransmembrane (PTM) and transmembrane (TM) domains, thereby driving the viral and host cell membranes into close contact, making possible the formation of the fusion pore (11, 15, 17). In class 1 viral fusion proteins, the FP invariably occurs upstream of the HR1 region; however, no FP has been experimentally identified in any CoV spike protein, although a hydrophobic region has been predicted recently at the N terminus of the HR1 region (5).
Although much information has been gathered in recent years, we do not yet know the exact mechanism of membrane fusion and the processes which are behind it. The mechanism by which proteins facilitate the formation of fusion intermediates is a complex process involving several segments of fusion proteins (15, 32). These regions, either directly or indirectly, might interact with biological membranes, contributing to the viral envelope and cell membrane merging. Even though the detailed structures of different segments of the SARS CoV spike glycoprotein have been elucidated, there are still many questions to be answered regarding its mode of action in accelerating membrane fusion. Moreover, SARS CoV entry is an attractive target for anti-SARS therapy. To investigate the structural basis of SARS CoV membrane fusion and identify new fusion inhibitors, we carried out the analysis of the different regions of the SARS CoV spike glycoprotein that might interact with phospholipid membranes, using an approach similar to that used for studying the human immunodeficiency virus (HIV) gp41 ectodomain (25), i.e., the identification of membrane-active regions of SARS CoV spike glycoprotein by determining the effect on membrane integrity of a 16/18-mer spike glycoprotein-derived peptide library. By monitoring the effect of this peptide library on membrane integrity, i.e., leakage, we have identified different regions on the SARS CoV spike glycoprotein with membrane-interacting capabilities, which supports their direct role in membrane fusion and therefore might help in understanding the molecular mechanism of membrane merging, as well as making possible the future development of SARS entry inhibitors, which may lead to new vaccine strategies.
MATERIALS AND METHODS
Materials and reagents. Egg L--phosphatidylcholine (EPC), egg sphingomyelin (SM), and cholesterol (Chol), were obtained from Avanti Polar Lipids (Alabaster, Ala.). 5-Carboxyfluorescein (CF) (>95% by high-performance liquid chromatography) was from Sigma-Aldrich (Madrid, Spain). A set of 169 peptides 16 or 18 amino acids in length derived from the SARS spike glycoprotein, with 10-amino-acid overlap between sequential peptides, was obtained through the AIDS Research and Reference Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.). Porcine lungs were obtained from a local slaughterhouse. Plasma membranes from lung tissue pneumocytes were obtained according to the method of Müller et al. (26), and lipid extraction from porcine lungs was performed according to the procedure of Bligh and Dyer using a ratio of 1:1:0.9 (vol/vol/vol) between chloroform-methanol and the corresponding aqueous sample (4). All other reagents used were of analytical grade and were obtained from Merck (Darmstad, Germany). Water was deionized, distilled twice, and passed through a Milli-Q apparatus (Millipore Ibérica, Madrid, Spain) to a resistivity better than 18 M/cm.
Sample preparation. Aliquots containing the appropriate amount of lipid in chloroform-methanol (2:1 [vol/vol]) were placed in a test tube, the solvents were removed by evaporation under a stream of O2-free nitrogen, and finally, traces of solvents were eliminated under vacuum in the dark for >3 h. After that, 1 ml of buffer containing 10 mM Tris, 20 mM NaCl, pH 7.4, and CF at a concentration of 40 mM was added, and multilamellar vesicles were obtained. Large unilamellar vesicles (LUV) with a mean diameter of 90 nm were prepared from multilamellar vesicles by the extrusion method (19), using polycarbonate filters with a pore size of 0.1 μm (Nuclepore Corp., Cambridge, Calif.). Breakdown of the vesicle membrane leads to leakage of the contents, i.e., CF fluorescence. Nonencapsulated CF was separated from the vesicle suspension through a Sephadex G-75 filtration column (Pharmacia, Uppsala, Sweden) eluted with buffer containing 10 mM Tris-0.1 M NaCl-1 mM EDTA, pH 7.4.
Leakage measurement. Leakage of intraliposomal CF was assayed by treating the probe-loaded liposomes (final lipid concentration, 0.125 mM) with the appropriate amounts of peptide on microtiter p