Functional Analysis of the Putative Integrin Recognition Motif on Adeno-associated Virus 9

Adeno-associated viruses (AAVs) display a highly conserved NGR motif on the capsid surface. Earlier studies have established this tripeptide motif as being essential for integrin-mediated uptake of recombinant AAV serotype 2 (AAV2) in cultured cells. However, functional attributes of this putative integrin recognition motif in other recombinant AAV serotypes displaying systemic transduction in vivo remain unknown. In this study, we dissect the biology of an integrin domain capsid mutant derived from the human isolate AAV9 in mice. The AAV9/NGA mutant shows decreased systemic transduction in mice. This defective phenotype was accompanied by rapid clearance of mutant virions from the blood circulation and nonspecific sequestration by the spleen. Transient vascular hyperpermeability, induced by histamine coinjection, exacerbated AAV9/NGA uptake by the spleen but not the liver. However, such treatment did not affect AAV9 virions, suggesting a potential entry/post-entry defect for the mutant in different tissues. Further characterization revealed modestly decreased cell surface binding but a more pronounced defect in the cellular entry of mutant virions. These findings were corroborated by the observation that blocking multiple integrins adversely affected recombinant AAV9 transduction in different cell types, albeit with variable efficiencies. From a structural perspective, we observed that the integrin recognition motif is located in close proximity to the galactose binding footprint on AAV9 capsids and postulate that this feature could influence cell surface attachment, cellular uptake at the tissue level, and systemic clearance by the reticuloendothelial system.     

Mutations in the Putative Dimer-Dimer Interfaces of the Measles Virus Hemagglutinin Head Domain Affect Membrane Fusion Triggering

Measles virus (MV), an enveloped RNA virus belonging to the Paramyxoviridae family, enters the cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin (H) and a fusion (F) protein. The crystal structure of the receptor-binding head domain of MV-H bound to its cellular receptor revealed that the MV-H head domain forms a tetrameric assembly (dimer of dimers), which occurs in two forms (forms I and II). In this study, we show that mutations in the putative dimer-dimer interface of the head domain in either form inhibit the ability of MV-H to support membrane fusion, without greatly affecting its cell surface expression, receptor binding, and interaction with the F protein. Notably, some anti-MV-H neutralizing monoclonal antibodies are directed to the region around the dimer-dimer interface in form I rather than receptor-binding sites. These observations suggest that the dimer-dimer interactions of the MV-H head domain, especially that in form I, contribute to triggering membrane fusion, and that conformational shift of head domain tetramers plays a role in the process. Furthermore, our results indicate that although the stalk and transmembrane regions may be mainly responsible for the tetramer formation of MV-H, the head domain alone can form tetramers, albeit at a low efficiency.     

Terminal N-linked galactose is the primary receptor for adeno-associated virus 9

Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3-15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general.     

Identification of interactions in the E1E2 heterodimer of hepatitis C virus important for cell entry

Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry.     

The Collagen-like Protein gp12 Is a Temperature-dependent Reversible Binder of SPP1 Viral Capsids

Icosahedral capsids of viruses are lattices of defined geometry and homogeneous size. The (quasi-)equivalent organization of their protein building blocks provides, in numerous systems, the binding sites to assemble arrays of viral polypeptides organized with nanometer precision that protrude from the capsid surface. The capsid of bacterial virus (bacteriophage) SPP1 exposes, at its surface, the 6.6-kDa viral polypeptide gp12 that binds to the center of hexamers of the major capsid protein. Gp12 forms an elongated trimer with collagen-like properties. This is consistent with the fold of eight internal GXY repeats of gp12 to build a stable intersubunit triple helix in a prokaryotic setting. The trimer dissociates and unfolds at near physiological temperatures, as reported for eukaryotic collagen. Its structural organization is reacquired within seconds upon cooling. Interaction with the SPP1 capsid hexamers strongly stabilizes gp12, increasing its T_m to 54 °C. Above this temperature, gp12 dissociates from its binding sites and unfolds reversibly. Multivalent binding of gp12 trimers to the capsid is highly cooperative. The capsid lattice also provides a platform to assist folding and association of unfolded gp12 polypeptides. The original physicochemical properties of gp12 offer a thermoswitchable system for multivalent binding of the polypeptide to the SPP1 capsid surface.     

Three Different Functional Microdomains in the Hepatitis C Virus Hypervariable Region 1 (HVR1) Mediate Entry and Immune Evasion

High genetic heterogeneity is an important characteristic of hepatitis C virus (HCV) that contributes to its ability to establish persistent infection. The hypervariable region 1 (HVR1) that includes the first 27 amino acid residues of the E2 envelope glycoprotein is the most variable region within the HCV polyprotein. HVR1 plays a major role in both HCV cell entry and immune evasion, but the respective contribution of specific amino acid residues is still unclear. Our mutagenesis analyses of HCV pseudoparticles and cell culture-derived HCV using the H77 isolate indicate that five residues at positions 14, 15, and 25–27 mediate binding of the E2 protein to the scavenger receptor class B, type I receptor, and any residue herein is indispensable for HCV cell entry. The region spanning positions 16–24 contains the sole neutralizing epitope and is dispensable for HCV entry, but it is involved in heparan binding. More importantly, this region is necessary for the enhancement of HCV entry by high density lipoprotein and interferes with virus neutralization by E2-neutralizing antibodies. Residues at positions 1–13 are also dispensable for HCV entry, but they can affect HCV infectivity by modulating binding of the envelope protein to scavenger receptor class B, type I. Mutations occurring at this site may confer resistance to HVR1 antibodies. These findings further our understanding about the mechanisms of HCV cell entry and the significance of HVR1 variation in HCV immune evasion. They have major implications for the development of HCV entry inhibitors and prophylactic vaccines.     

The final conformation of the complete ectodomain of the HA2 subunit of influenza hemagglutinin can by itself drive low pH-dependent fusion

One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1–185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.     

Directed Evolution of an Angiopoietin-2 Ligand Trap by Somatic Hypermutation and Cell Surface Display

Tie2 is a receptor tyrosine kinase that is essential for the development and maintenance of blood vessels through binding the soluble ligands angiopoietin 1 (Ang1) and 2 (Ang2). Ang1 is constitutively produced by perivascular cells and is protective of the adult vasculature. Ang2 plays an important role in blood vessel formation and is normally expressed during development. However, its re-expression in disease states, including cancer and sepsis, results in destabilization of blood vessels contributing to the pathology of these conditions. Ang2 is thus an attractive therapeutic target. Here we report the directed evolution of a ligand trap for Ang2 by harnessing the B cell somatic hypermutation machinery and coupling this to selectable cell surface display of a Tie2 ectodomain. Directed evolution produced an unexpected combination of mutations resulting in loss of Ang1 binding but maintenance of Ang2 binding. A soluble form of the evolved ectodomain binds Ang2 but not Ang1. Furthermore, the soluble evolved ectodomain blocks Ang2 effects on endothelial cells without interfering with Ang1 activity. Our study has created a novel Ang2 ligand trap and provided proof of concept for combining surface display and exogenous gene diversification in B cells for evolution of a non-immunoglobulin target.     

Physical Interaction of Jab1 with Human Serotonin 6 G-protein-coupled Receptor and Their Possible Roles in Cell Survival

The 5-HT₆ receptor (5-HT₆R) is one of the most recently cloned serotonin receptors, and it plays important roles in Alzheimer disease, depression, and learning and memory disorders. However, unlike the other serotonin receptors, the cellular mechanisms of 5-HT₆R are poorly elucidated relative to its significance in human brain diseases. Here, using a yeast two-hybrid assay, we found that the human 5-HT₆R interacts with Jun activation domain-binding protein-1 (Jab1). We also confirmed a physical interaction between 5-HT₆R and Jab1 using glutathione S-transferase pulldown, fluorescence resonance energy transfer, co-immunoprecipitation, and immunocyto(histo)chemistry assays. The manipulation of Jab1 expression using Jab1 small interference RNA decreased 5-HT₆R-mediated activity and cell membrane expression of 5-HT₆R, whereas overexpression of Jab1 produced no significant effect. In addition, we demonstrated that the activation of 5-HT₆R induced the translocation of Jab1 into the nucleus and increased c-Jun phosphorylation and the interaction between Jab1 and c-Jun. Furthermore, we found that 5-HT₆R and Jab1 were up-regulated in middle cerebral artery occlusion-induced focal cerebral ischemic rats and in cultured cells exposed to hypoxic insults, suggesting possible protective roles for 5-HT₆R and Jab1. These findings suggest that Jab1 provides a novel signal transduction pathway for 5-HT₆R and may play an important role in 5-HT₆R-mediated behavior changes in the brain.     

Solution structure of the monovalent lectin microvirin in complex with Man(alpha)(1-2)Man provides a basis for anti-HIV activity with low toxicity

Lectins that bind surface envelope glycoprotein gp120 of HIV with high avidity can potently inhibit viral entry. Yet properties such as multivalency that facilitate strong interactions can also cause nonspecific binding and toxicity. The cyanobacterial lectin microvirin (MVN) is unusual as it potently inhibits HIV-1 with negligible toxicity compared with cyanovirin-N (CVN), its well studied antiviral homolog. To understand the structural and mechanistic basis for these differences, we solved the solution structure of MVN free and in complex with its ligand Manα(1-2)Man, and we compared specificity and time windows of inhibition with CVN and Manα(1-2)Man-specific mAb 2G12. We show by NMR and analytical ultracentrifugation that MVN is monomeric in solution, and we demonstrate by NMR that Manα(1-2)Man-terminating carbohydrates interact with a single carbohydrate-binding site. Synchronized infectivity assays show that 2G12, MVN, and CVN inhibit entry with distinct kinetics. Despite shared specificity for Manα(1-2)Man termini, combinations of the inhibitors are synergistic suggesting they recognize discrete glycans and/or dynamic glycan conformations on gp120. Entry assays employing amphotropic viruses show that MVN is inactive, whereas CVN potently inhibits both. In addition to demonstrating that HIV-1 can be inhibited through monovalent interactions, given the similarity of the carbohydrate-binding site common to MVN and CVN, these data suggest that gp120 behaves as a clustered glycan epitope and that multivalent-protein interactions achievable with CVN but not MVN are required for inhibition of some viruses.     

Rapid Fusion and Syncytium Formation of Heterologous Cells upon Expression of the FGFRL1 Receptor

The fusion of mammalian cells into syncytia is a developmental process that is tightly restricted to a limited subset of cells. Besides gamete and placental trophoblast fusion, only macrophages and myogenic stem cells fuse into multinucleated syncytia. In contrast to viral cell fusion, which is mediated by fusogenic glycoproteins that actively merge membranes, mammalian cell fusion is poorly understood at the molecular level. A variety of mammalian transmembrane proteins, among them many of the immunoglobulin superfamily, have been implicated in cell-cell fusion, but none has been shown to actively fuse cells in vitro. Here we report that the FGFRL1 receptor, which is up-regulated during the differentiation of myoblasts into myotubes, fuses cultured cells into large, multinucleated syncytia. We used luciferase and GFP-based reporter assays to confirm cytoplasmic mixing and to identify the fusion inducing domain of FGFRL1. These assays revealed that Ig-like domain III and the transmembrane domain are both necessary and sufficient to rapidly fuse CHO cells into multinucleated syncytia comprising several hundred nuclei. Moreover, FGFRL1 also fused HEK293 and HeLa cells with untransfected CHO cells. Our data show that FGFRL1 is the first mammalian protein that is capable of inducing syncytium formation of heterologous cells in vitro.     

Determinants of the specificity of rotavirus interactions with the alpha2beta1 integrin

The human α2β1 integrin binds collagen and acts as a cellular receptor for rotaviruses and human echovirus 1. These ligands require the inserted (I) domain within the α2 subunit of α2β1 for binding. Previous studies have identified the binding sites for collagen and echovirus 1 in the α2 I domain. We used CHO cells expressing mutated α2β1 to identify amino acids involved in binding to human and animal rotaviruses. Residues where mutation affected rotavirus binding were located in several exposed loops and adjacent regions of the α2 I domain. Binding by all rotaviruses was eliminated by mutations in the activation-responsive αC-α6 and αF helices. This is a novel feature that distinguishes rotavirus from other α2β1 ligands. Mutation of residues that co-ordinate the metal ion (Ser-153, Thr-221, and Glu-256 in α2 and Asp-130 in β1) and nearby amino acids (Ser-154, Gln-215, and Asp-219) also inhibited rotavirus binding. The importance of most of these residues was greatest for binding by human rotaviruses. These mutations inhibit collagen binding to α2β1 (apart from Glu-256) but do not affect echovirus binding. Overall, residues where mutation affected both rotavirus and collagen recognition are located at one side of the metal ion-dependent adhesion site, whereas those important for collagen alone cluster nearby. Mutations eliminating rotavirus and echovirus binding are distinct, consistent with the respective preference of these viruses for activated or inactive α2β1. In contrast, rotavirus and collagen utilize activated α2β1 and show an overlap in α2β1 residues important for binding.     

Early murine T-lymphocyte activation is accompanied by a switch from N-Glycolyl- to N-acetyl-neuraminic acid and generation of ligands for siglec-E

It is well established that murine T-lymphocyte activation is accompanied by major changes in cell-surface sialylation, potentially influencing interactions with sialic acid-binding immunoglobulin-like lectins (siglecs). In the present study, we analyzed early activation of murine CD4+ and CD8+ T-lymphocytes at 24 h. We observed a striking and selective up-regulation in the binding of a recombinant soluble form of siglec-E, an inhibitory siglec, which is expressed on several myeloid cell types including antigen-presenting dendritic cells. In contrast, much lower levels of T cell binding were observed with other siglecs, including sialoadhesin, CD22, and siglec-F and the plant lectins Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin. By mass spectrometry, the sialic acid content of 24-h-activated CD4+ and CD8+ T-lymphocytes exhibited an increased proportion of N-acetyl-neuraminic acid (NeuAc) to N-glycolyl-neuraminic acid (NeuGc) in N-glycans. Reduced levels of NeuGc on the surface of activated T cells were demonstrated using an antibody specific for NeuGc and the expression levels of the gene encoding NeuAc- to NeuGc-converting enzyme, CMP-NeuAc hydroxylase, were also reduced. Siglec-E bound a wide range of sialylated structures in glycan arrays, had a preference for NeuAc versus NeuGc-terminated sequences and could recognize a set of sialoglycoproteins that included CD45, in lysates from activated T-lymphocytes. Collectively, these results show that early in T cell activation, glycan remodelling involves a switch from NeuGc- to NeuAc-terminating oligosaccharides on cell surface glycoproteins. This is associated with a strong up-regulation of siglec-E ligands, which may be important in promoting cellular interactions between early activated T-lymphocytes and myeloid cells expressing this inhibitory receptor.     

NMR Structure of a Viral Peptide Inserted in Artificial Membranes: A VIEW ON THE EARLY STEPS OF THE BIRNAVIRUS ENTRY PROCESS*

Nonenveloped virus must penetrate the cellular membrane to access the cytoplasm without the benefit of membrane fusion. For birnavirus, one of the peptides present in the virus capsid, pep46 for infectious bursal disease virus, is able to induce pores into membranes as an intermediate step of the birnavirus-penetration pathway. Using osmotic protection experiments, we demonstrate here that pep46 and its pore-forming N-terminal moiety (pep22) form pores of different diameters, 5–8 and 2–4 nm, respectively, showing that both pep46 moieties participate to pore formation. The solution structures of pep46, pep22, and pep24 (the pep46 C-terminal moiety) in different hydrophobic environments and micelles determined by ¹H NMR studies provide structural insights of the pep46 domain interaction. In CDCl₃/CD₃OH mixture and in dodecylphosphocholine micelles, the N-terminal domain of pep46 is structured in a long kinked helix, although the C terminus is structured in one or two helices depending upon the solvents used. We also show that the folding and the proline isomerization status of pep46 depend on the type of hydrophobic environment. NMR spectroscopy with labeled phospholipid micelles, differential scanning calorimetry, and plasmon waveguide resonance studies show the peptides lie parallel to the lipid-water interface, perturbing the fatty acid chain packing. All these data lead to a model in which the two domains of pep46 interact with the membrane to form pores.     

Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain

The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp⁶³, Arg¹¹⁶, and Lys⁹⁸ are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp⁶³ and Arg¹¹⁶ form a salt bridge, whereas Lys⁹⁸ is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.     

High mannose-binding lectin with preference for the cluster of alpha1-2-mannose from the green alga Boodlea coacta is a potent entry inhibitor of HIV-1 and influenza viruses

The complete amino acid sequence of a lectin from the green alga Boodlea coacta (BCA), which was determined by a combination of Edman degradation of its peptide fragments and cDNA cloning, revealed the following: 1) B. coacta used a noncanonical genetic code (where TAA and TAG codons encode glutamine rather than a translation termination), and 2) BCA consisted of three internal tandem-repeated domains, each of which contains the sequence motif similar to the carbohydrate-binding site of Galanthus nivalis agglutinin-related lectins. Carbohydrate binding specificity of BCA was examined by a centrifugal ultrafiltration-HPLC assay using 42 pyridylaminated oligosaccharides. BCA bound to high mannose-type N-glycans but not to the complex-type, hybrid-type core structure of N-glycans or oligosaccharides from glycolipids. This lectin had exclusive specificity for α1-2-linked mannose at the nonreducing terminus. The binding activity was enhanced as the number of terminal α1-2-linked mannose substitutions increased. Mannobiose, mannotriose, and mannopentaose were incapable of binding to BCA. Thus, BCA preferentially recognized the nonreducing terminal α1-2-mannose cluster as a primary target. As predicted from carbohydrate-binding propensity, this lectin inhibited the HIV-1 entry into the host cells at a half-maximal effective concentration of 8.2 nm. A high association constant (3.71 × 10(8) M(-1)) of BCA with the HIV envelope glycoprotein gp120 was demonstrated by surface plasmon resonance analysis. Moreover, BCA showed the potent anti-influenza activity by directly binding to viral envelope hemagglutinin against various strains, including a clinical isolate of pandemic H1N1-2009 virus, revealing its potential as an antiviral reagent.     

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

HIV-1 Nef Binds a Subpopulation of MHC-I throughout Its Trafficking Itinerary and Down-regulates MHC-I by Perturbing Both Anterograde and Retrograde Trafficking

The HIV protein Nef is thought to mediate immune evasion and promote viral persistence in part by down-regulating major histocompatibility complex class I protein (MHC-I or HLA-I) from the cell surface. Two different models have been proposed to explain this phenomenon as follows: 1) stimulation of MHC-I retrograde trafficking from and aberrant recycling to the plasma membrane, and 2) inhibition of anterograde trafficking of newly synthesized HLA-I from the endoplasmic reticulum to the plasma membrane. We show here that Nef simultaneously uses both mechanisms to down-regulate HLA-I in peripheral blood mononuclear cells or HeLa cells. Consistent with this, we found by using fluorescence correlation spectroscopy that a third of diffusing HLA-I at the endoplasmic reticulum, Golgi/trans-Golgi network, and the plasma membrane (PM) was associated with Nef. The binding of Nef was similarly avid for native HLA-I and recombinant HLA-I A2 at the PM. Nef binding to HLA-I at the PM was sensitive to specific inhibition of endocytosis. It was also attenuated by cyclodextrin disruption of PM lipid micro-domain architecture, a change that also retarded lateral diffusion and induced large clusters of HLA-I. In all, our data support a model for Nef down-regulation of HLA-I that involves both major trafficking itineraries and persistent protein-protein interactions throughout the cell.     

Features of a Spatially Constrained Cystine Loop in the p10 FAST Protein Ectodomain Define a New Class of Viral Fusion Peptides

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only ∼20–40 residues, it is unclear how such diminutive fusion proteins can mediate cell-cell fusion and syncytium formation. Contained within the 40-residue ectodomain of the p10 FAST protein resides an 11-residue sequence of moderately apolar residues, termed the hydrophobic patch (HP). Previous studies indicate the p10 HP shares operational features with the fusion peptide motifs found within the enveloped virus membrane fusion proteins. Using biotinylation assays, we now report that two highly conserved cysteine residues flanking the p10 HP form an essential intramolecular disulfide bond to create a cystine loop. Mutagenic analyses revealed that both formation of the cystine loop and p10 membrane fusion activity are highly sensitive to changes in the size and spatial arrangement of amino acids within the loop. The p10 cystine loop may therefore function as a cystine noose, where fusion peptide activity is dependent on structural constraints within the noose that force solvent exposure of key hydrophobic residues. Moreover, inhibitors of cell surface thioreductase activity indicate that disruption of the disulfide bridge is important for p10-mediated membrane fusion. This is the first example of a viral fusion peptide composed of a small, spatially constrained cystine loop whose function is dependent on altered loop formation, and it suggests the p10 cystine loop represents a new class of viral fusion peptides.