Membrane-induced step in the activation of Sendai virus fusion protein

Peptides derived from conserved heptad-repeat regions of several viruses have been shown recently to inhibit virus-cell fusion. To find out their possible role in the fusion process, two biologically active heptad-repeat segments of the fusion protein (F) of Sendai virus, SV-150 (residues 150-186), and SV-473 (residues 473-495) were synthesized, fluorescently labeled and spectroscopically characterized for their structure and organization in solution and within the membrane. SV-150 was found to be 50-fold less active than SV-473 in inhibiting Sendai virus-cell fusion. Circular dichroism (CD) spectroscopy revealed that in aqueous solution, the peptides are self-associated and adopt low alpha-helical structure. However, when the two peptides are mixed together, their alpha-helical content significantly increases. Fluorescence studies, CD, and polarized attenuated total reflection infrared (ATR-FTIR) spectroscopy showed that both peptides, alone or as a complex, bind strongly to negatively charged and zwitterionic phospholipid membranes, dissociate therein into alpha-helical monomers, but do not perturb the lipid packing of the membrane. The ability of the peptides to interact with each other in solution may be correlated with antiviral activity, whereas their ability to interact with the membrane, together with their location near the fusion peptide and the transmembrane domain, suggests a revision to the currently accepted model for viral-induced membrane fusion. In the revised model, in the sequence of events associated with viral entry, the two heptad-repeat sequences may assist in bringing the viral and cellular membranes closer, thus facilitating membrane fusion.     

A leucine zipper motif in the ectodomain of Sendai virus fusion protein assembles in solution and in membranes and specifically binds biologically-active peptides and the virus

We have detected a leucine zipper-like motif in the ectodomain of the Sendai virus fusion protein (aa 269-307) which is extremely conserved in the family of Sendai viruses. To find a possible role for this motif, we synthesized SV-269, a 39 amino acid peptide corresponding to this domain, and a mutant peptide, MuSV-269, with an amino acid pair interchanged their positions. The peptides were labeled with fluorescent probes at their N-terminal amino acid and functionally and structurally characterized. The data show that SV-269, but not MuSV-269, specifically binds Sendai virus. Expectedly, SV-269 is more active than the mutant MuSV-269 in inhibiting Sendai virus-mediated hemolysis. Fluorescence studies reveal that SV-269 assembles in aqueous solution, binds to zwitterionic PC and negatively-charged PS/PC vesicles, and assembles therein. Although MuSV-269 similarly binds to both types of vesicles, it only slightly assembles in solution and not at all in membranes. Moreover, SV-269, but not MuSV-269, coassembles with the biologically-active heptad repeats SV-150 and SV-473 (Rapaport et al. , 1995) in solution as revealed by fluorescence and circular dichroism (CD) spectroscopy, and with SV-150 within negatively-charged PS/PC and zwitterionic PC vesicles. Despite these differences, both SV-269 and MuSV-269 adopt similar secondary structures in 40% TFE and 1% SDS as revealed by CD spectroscopy, and disrupt the packing of the lipid bilayers to the same extent, as shown by the dissipation of diffusion potential. The role of this leucine zipper motif is discussed in terms of the assembly of the Sendai virus fusion protein in solution and within membranes. Since most of the heptadic leucines are also conserved in the corresponding domains of other paramyxoviruses such as rinderpest, measles, SV5, and parainfluenza, it may indicate a similar role of this domain in these viruses as well.     

The perceptual span and oculomotor activity during the reading of Chinese sentences

The paramyxovirus fusion (F) protein mediates membrane fusion. The biologically active F protein consists of a membrane distal subunit, F2, and a membrane-anchored subunit, F1. We have identified a highly stable structure composed of peptides derived from the F1 heptad repeat A, which abuts the hydrophobic fusion peptide (peptide N-1), and the F1 heptad repeat B, located 270 residues downstream and adjacent to the transmembrane domain (peptides C-1 and C-2). In isolation, peptide N-1 is 47% alpha-helical and peptide C-1 and C-2 are unfolded. When mixed together, peptides N1 + C1 form a thermostable (Tm >90 degreesC), 82% alpha-helical, discrete trimer of heterodimers (mass 31,300 Mr) that is resistant to denaturation by 2% SDS at 40 degreesC. We suggest that this alpha-helical trimeric complex represents the core most stable form of the F protein that either is fusion competent or forms after fusion has occurred. Peptide C-1 is a potent inhibitor of both the lipid mixing and the aqueous content mixing fusion activity of the SV5 F protein. In contrast, peptides N-1 and N-2 inhibit cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Thus, these peptides define functionally different steps in the fusion process. The parallels among both the fusion processes and the protein structures of paramyxovirus F proteins, HIV gp41, and influenza virus hemagglutinin are discussed, as the analogies are indicative of a conserved paradigm for fusion promotion among fusion proteins from widely disparate viruses.     

Cross-reactive and group-specific immune responses to a neutralizing epitope of the human respiratory syncytial virus fusion protein

Immune responses to a synthetic peptide corresponding to amino-acids 205-225 of the fusion protein from group B respiratory syncytial (RS) virus, were studied in mice and rabbits, and compared to a similar peptide from group A RS virus. Peptide 205-225 (B) was recognized by monoclonal antibody RS-348, and was immunogenic in both mice and rabbits, as was peptide 205-225 from the fusion protein of a group A strain. Peptide 205-225 (B) induced a proliferative T-cell response, demonstrating the existence of a T-cell epitope in this region of the fusion protein of group B viruses. Both peptides were able to induce a T-cell cross-reactive proliferation when mice were primed with either the homologous or the heterologous peptide. ELISA were performed using synthetic peptides or whole virus (from group A and B) as antigens. Mice anti-peptide sera recognized both homologous and heterologous peptides. A similar pattern was observed with RS virus strains. In indirect immunofluorescence assays, both anti-peptide rabbit sera recognized human nasal epithelial cells infected with A or B strains of RS virus. In contrast, while anti-peptide 205-225 rabbit serum from group A neutralized group A and B strains of RS virus, anti-peptide 205-225 rabbit serum from group B was unable to neutralize a group A virus, although it neutralized a group B strain. These results are similar to the immune response observed in children following primary RS virus infection.     

Paramyxovirus fusion protein: characterization of the core trimer, a rod-shaped complex with helices in anti-parallel orientation

The fusion (F) protein of the paramyxovirus SV5 contains two heptad repeat regions, HRA adjacent to the fusion peptide and HRB proximal to the transmembrane domain. Peptides, N-1 and C-1, respectively, corresponding to these heptad repeat regions form a thermostable, alpha-helical trimer of heterodimers (S. B. Joshi, R. E. Dutch, and R. A. Lamb (1998). Virology 248, 20-34). Further characterization of the N-1/C-1 complex indicated that the C-1 peptides, which are predicted to residue on the outside of the complex, are resistant to digestion by several proteases when present in the complex. Only proteinase K digested most of the C-1 peptide, though the small remaining protease protected fragment of C-1 confers extreme thermostability on the proteinase-K-resistant N-1 trimeric coiled-coil. Carboxypeptidase Y digestion of the N-1/C-1 complex indicates that the C-1 peptides associate in an antiparallel orientation relative to the N-1 peptides. Electron microscopy of the N-1/C-1 complex showed a rod-shaped complex with an average length of 9.7 nm, consistent with all of N-1 existing as an alpha helix. Mutations at heptad repeat a and d residues of N-1, positions that are predicted to point inward to the center of the N-1 trimeric coiled-coil, were found to have varying effects as analyzed by circular dichroism measurements. The mutation I137M did not affect the helical structure of the isolated N-1 peptide but did affect the thermostability of the N-1/C-1 complex. Mutations L140M and L161M perturbed the helical structure formed by N-1 in isolation but did not affect formation of a thermostable N-1/C-1 complex. Finally, a peptide, SV5 F 255-293, corresponding to a proposed leucine zipper region, was analyzed for effects on N-1, C-1, or the N-1/C-1 complex. Circular dichroism analysis demonstrated that while the presence of peptide 255-293 increased the helical signal from either N-1 or the N-1/C-1 complex, no change in thermostability was observed, indicating that this region is not a component of the final, most stable core of the F protein.     

Identification of a novel domain of HIV tat involved in monocyte chemotaxis

Human immunodeficiency virus (HIV) Tat is chemotactic for monocytes and dendritic cells, an activity that could play a key role in the expansion of HIV infection of accessory cells. To date, domains of Tat previously found to interact with cell surface molecules have shown only partial chemotactic activity toward monocytes. Using overlapping Tat peptides, we identify a novel region of Tat with a potent chemotactic activity for monocytes, reaching levels equal to Tat itself. This peptide also provokes monocyte polarization similar to Tat and is able to compete with Tat for induction of monocyte migration. Specific high affinity (kd = 3 x 10(-9) M) cell surface binding sites on monocyte cell surfaces for this region of Tat are demonstrated. These data indicate that the majority of Tat effects on monocytes are mediated by a novel region in the cysteine-rich and core domains. These domains are highly conserved among different HIV isolates, suggesting an important role in the establishment of HIV infection.     

Analysis of the neutralizing antibody response to the measles virus using synthetic peptides of the haemagglutinin protein

Infection or immunization with measles virus induces a protective immune reaction including neutralizing antibodies against the haemagglutinin and fusion protein. The reactivity of the polyclonal IgG response of sera obtained from late convalescent donors was studied, using overlapping 15mer peptides covering the complete sequence of the measles virus haemagglutinin. Most sera reacted with a similar set of peptides generating a characteristic binding pattern. The reactive peptides correspond to a region mediating cell hemolysis (aa310-325), to regions which serve as targets to neutralizing antibodies and to a putative transmembrane region (aa35-58). The latter region contains also a human T-cell epitope providing evidence of a non-random association of T- and B-cell epitopes. We also immunized different strains of mice and rabbits with measles virus. In contrast to the human sera, animal sera with strong neutralizing activities did not react with any of the H-protein peptides. The mostly weak reactivities with the linear sequences contrast with the strong neutralizing activities of the human or animal antibodies, suggesting that these primarily recognize the fusion protein or conformational epitopes of the haemagglutinin protein.     

Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates

The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage sites in the nucleocapsid protein were also found to be substrates of the EIAV proteinase. However, these peptides were not hydrolyzed by the HIV proteinases. While peptides representing the corresponding sequences in the first cysteine arrays of the nucleocapsid proteins of HIV-1 and HIV-2 were substrates of the proteinases, peptides representing the homologous sequences in the second Cys arrays were resistant against the proteolytic attack. A three-dimensional model of the EIAV proteinase built on the basis of homology with HIV-1 proteinase was used to interpret the differences. In addition to the oligopeptides representing cleavage sites in the Gag and Gag-Pol polyproteins, the EIAV proteinase was also able to cleave an oligopeptide mimicking a cleavage site in the transmembrane protein. Our results suggest that the specificity of lentiviral proteinases share common characteristics, although substantial differences may exist in hydrolysis of some peptides.     

Inhibition of natural killer cell activities from normal donors and AIDS patients by envelope peptides from human immunodeficiency virus type I

Recombinant and synthetic peptides derived from the human immunodeficiency virus type I (HIV-1) genome corresponding to portions of the envelope (env) and internal core protein (gag) were examined for their immunoregulatory effects on the natural killer (NK) cell activity of lymphocytes from healthy donors and from patients with the acquired immunodeficiency syndrome (AIDS). Two recombinant peptides (env-gag and Env 80-DHFR) and three chemically synthesized peptides (env 487-511, env 578-608 and env 647-659) were used. Normal lymphocytes precultured for 24 to 72 hrs. with either env-gag, env 487-511, or env 647-659 at 5 and 50 ng/ml concentrations which significantly stimulated lymphocyte proliferation, produced significant suppression of NK activities. Two control peptides, one derived from the E. coli vector used to clone the HIV env-gag fusion peptide and another, a non-HIV-1 viral antigen (rubeola virus) did not produce any observable effect on NK activity of normal lymphocytes demonstrating the specificity of the reaction. Env-gag peptide also inhibited the NK activities of Percoll-separated, NK-enriched large granular lymphocytes. In target binding assays, lymphocytes precultured with env-gag significantly suppressed the target binding capacity of effector cells and produced significantly lower levels of natural killer cytotoxic factor (NKCF). In kinetic studies, lymphocytes from normal donors preincubated with env-gag for 24 to 72 hrs. produced significant inhibition of their NK activity and an even greater inhibitory effect on NK activities was observed when lymphocytes from AIDS patients were preincubated with HIV peptides. Thus HIV-1 peptides, which we previously demonstrated could regulate B- and T-lymphocyte activities, are also capable of regulating the NK activities of lymphocytes from HIV-1-infected and normal individuals.     

Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathione S-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.     

Pseudopeptide TASP inhibitors of HIV entry bind specifically to a 95-kDa cell surface protein

The template assembled synthetic peptide constructs (TASP), pentavalently presenting the tripeptide KPR or RPK, are potent and specific inhibitors of human immunodeficiency virus (HIV) infection by preventing viral entry into permissive cells. Here the 5[KPsi(CH2N)PR]-TASP construct, Psi(CH2N) for reduced peptide bond, was used in studies to demonstrate its specific binding to a 95-kDa cell surface protein ligand. Compared to its nonreduced 5[KPR]-TASP counterpart, the pseudopeptide 5[KPsi(CH2N)PR]-TASP manifested higher affinity to bind to its cell surface ligand, increased activity to inhibit HIV infection, and resistance to degradation when incubated in serum from an HIV-1 seropositive individual. In ligand blotting experiments, the biotin-labeled 5[KPsi(CH2N)PR]-TASP identified a single 95-kDa protein in crude cell extracts. This 95-kDa protein (p95) is expressed on the cell surface since surface iodination of cells resulted in its labeling, and moreover, following incubation of cells with the biotin-labeled 5[KPsi(CH2N)PR]-TASP, the p95.TASP complex was recovered by affinity chromatography using avidin-agarose. All anti-HIV TASP constructs but not their control derivatives affected the binding of biotin-labeled 5[KPsi(CH2N)PR]-TASP to p95, thus emphasizing the specific nature of this binding. Since 5[KPsi(CH2N)PR]-TASP does not interact with HIV-envelope glycoproteins, our results suggest that TASP inhibitors mediate directly or indirectly a block in HIV-mediated membrane fusion process by binding to the cell surface expressed p95.     

Spectral analysis of a thalamus-to-cortex seizure pathway

CD4 ligation of HIV envelope gp120 results in conformational changes in gp120 that lead to exposure of the gp41 fusogenic domain and fusion with the host cell membrane. One determinant at or near the CD4-binding site exposed on gp120 subsequent to CD4 binding is defined by two human MAbs termed 17b and 48d. These MAbs do not block CD4 binding to gp120; rather, their binding to gp120 is upregulated following CD4 binding. To determine if synthetic peptide mimetopes could be found that reflect conformational determinants on the surface of gp120, synthetic gp120 peptides from 10 divergent HIV isolates were screened for their ability to bind to 17b and 48d in ELISAs. Although MAb 48d binds to HIV IIIB recombinant gp120 protein, in our studies 48d selectively bound only to the HIV Can0A V3 peptide and not to HIV IIIB V3 peptide, whereas MAb 17b bound none of the peptides tested. Monoclonal antibody 48d bound to the HIV Can0A V3 peptide both in solid-phase ELISA and in solution in a competitive ELISA, but could not bind to HIV Can0A V3 peptide bound to human T cells. The HIV Can0A V3 peptide induced anti-HIV antibodies in rhesus monkeys that neutralized the laboratory-adapted HIV MN strain but did not induce antibodies that neutralized HIV IIIB/LAI, HIV SF-2, or HIV RF isolates, or that neutralized HIV primary isolates. These data suggested that the primary sequence of the HIV Can0A V3 loop exists in a conformer that mimicks a non-V3 determinant of native gp120 exposed subsequent to CD4 binding on the surface of gp120 of laboratory-adapted HIV strains. Structural studies of the Can0A V3 peptide and/or the 48d MAb may provide important information regarding the nature of gp120 conformational changes that occur following gp120 ligation by CD4.     

Measles virus fusion protein is palmitoylated on transmembrane-intracytoplasmic cysteine residues which participate in cell fusion

[3H]palmitic acid was metabolically incorporated into the viral fusion protein (F) of Edmonston or freshly isolated measles virus (MV) during infection of human lymphoid or Vero cells. The uncleaved precursor F0 and the F1 subunit from infected cells and extracellular virus were both labeled, indicating that palmitoylation can take place prior to F0 cleavage and that palmitoylated F protein was incorporated into virus particles. [3H]palmitic acid was released from F protein upon hydroxylamine or dithiothreitol treatment, indicating a thioester linkage. In cells transfected with the cloned MV F gene, in which the cysteines located in the intracytoplasmic and transmembrane domains (Cys 506, 518, 519, 520, and 524) were replaced by serine, a major reduction of [3H]palmitic acid incorporation was observed for F mutated at Cys 506 and, to a lesser extent, at Cys 518 and Cys 524. We also observed incorporation of [3H]palmitic acid in the F1 subunit of canine distemper virus F protein. Cell fusion induced by cotransfection of cells with MV F and H (hemagglutinin) genes was significantly reduced after replacement of Cys 506 or Cys 519 with serine in the MV F gene. Transfection with the F gene with a mutation for Cys 518 abolished cell fusion, although less mutant protein was detected on the cell surface. These results suggest that the F protein transmembrane domain cysteines 506 and 518 participate in structures involved in cell fusion, possibly mediated by palmitoylation.     

The vaccinia virus 14-kilodalton (A27L) fusion protein forms a triple coiled-coil structure and interacts with the 21-kilodalton (A17L) virus membrane protein through a C-terminal alpha-helix

The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.     

Mutational analysis of residues in the coiled-coil domain of human immunodeficiency virus type 1 transmembrane protein gp41

The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263-273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426-430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.     

Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX1-2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.     

Identification and characterization of golgin-84, a novel Golgi integral membrane protein with a cytoplasmic coiled-coil domain

The cytoplasmic face of the Golgi contains a variety of proteins with coiled-coil domains. We identified one such protein in a yeast two-hybrid screen, using as bait the peripheral Golgi phosphatidylinositol(4,5)P2 5-phosphatase OCRL1 that is implicated in a human disease, the oculocerebrorenal syndrome. The approximately 2.8-kilobase mRNA is ubiquitously expressed and abundant in testis; it encodes a 731-amino acid protein with a predicted mass of 83 kDa. Antibodies against the sequence detect a novel approximately 84-kDa Golgi protein we termed golgin-84. Golgin-84 is an integral membrane protein with a single transmembrane domain close to its C terminus. In vitro, the protein inserts post-translationally into microsomal membranes with an N-cytoplasmic and C-lumen orientation. Cross-linking indicates that golgin-84 forms dimers, consistent with the prediction of an approximately 400-residue dimerizing coiled-coil domain in its N terminus. The dimerization potential is supported by a data base search that showed that the N-terminal 497 residues of golgin-84 contain a coiled-coil domain that when fused to the RET tyrosine kinase domain had the ability to activate it, forming the RET-II oncogene. Data base searching also indicates golgin-84 is similar in structure and sequence to giantin, a membrane protein that tethers coatamer complex I vesicles to the Golgi.     

Phosphatidylinositol-dependent membrane fusion induced by a putative fusogenic sequence of Ebola virus

The membrane-interacting abilities of three sequences representing the putative fusogenic subdomain of the Ebola virus transmembrane protein have been investigated. In the presence of calcium, the sequence EBO(GE) (GAAIGLAWIPYFGPAAE) efficiently fused unilamellar vesicles composed of phosphatidylcholine, phosphatidylethanolamine, cholesterol, and phosphatidylinositol (molar ratio, 2:1:1:0.5), a mixture that roughly resembles the lipid composition of the hepatocyte plasma membrane. Analysis of the lipid dependence of the process demonstrated that the fusion activity of EBO(GE) was promoted by phosphatidylinositol but not by other acidic phospholipids. In comparison, EBO(EA) (EGAAIGLAWIPYFGPAA) and EBO(EE) (EGAAIGLAWIPYFGPAAE) sequences, which are similar to EBO(GE) except that they bear the negatively charged glutamate residue at the N terminus and at both the N and C termini, respectively, induced fusion to a lesser extent. As revealed by binding experiments, the glutamate residue at the N terminus severely impaired peptide-vesicle interaction. In addition, the fusion-competent EBO(GE) sequence did not associate significantly with vesicles lacking phosphatidylinositol. Tryptophan fluorescence quenching by vesicles containing brominated phospholipids indicated that the EBO(GE) peptide penetrated to the acyl chain level only when the membranes contained phosphatidylinositol. We conclude that binding and further penetration of the Ebola virus putative fusion peptide into membranes might be governed by the nature of the N-terminal residue and by the presence of phosphatidylinositol in the target membrane. Moreover, since insertion of such a peptide leads to membrane destabilization and fusion, the present data would be compatible with the involvement of this sequence in Ebola virus fusion.     

Roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein

The spike (S) protein of coronavirus mouse hepatitis virus (MHV), mediates attachment and fusion during viral entry and cell-to-cell fusion later in infection. By analogy with other viral proteins that induce cell fusion the MHV S protein would be expected to have a hydrophobic stretch of amino acids that serves as a fusion peptide. Sequence analysis suggests that the S protein falls within the group of fusion proteins having internal rather than N-terminal fusion peptides. Based on the features of known viral fusion peptides, we identified two regions (PEP1 and PEP2) of MHV-A59 S2 as possible fusion peptides. Site-directed mutagenesis and an in viro cell-to-cell fusion assay were used to evaluate the roles of PEP1 and PEP2, as well as a third previously identified putative fusion domain (PEP3) in membrane fusion. Substitution of bulky hydrophobic residues with charged residues within PEP1 affects the fusion activity of the S protein without affecting processing and surface expression. Similar substitutions within PEP2 result in a fusion-negative phenotype; however, these mutant S proteins also exhibit defects in protein processing and surface expression which likely explain the loss of the ability to induce fusion. Thus PEP1 remains a candidate fusion peptide, while PEP2 may play a significant role in the overall structure or oligomerization of the S protein. PEP3 is an unlikely putative fusion peptide since it is not conserved among coronaviruses and nonconservative amino acid substitutions in PEP3 have minimal effects on cell-to-cell fusion.