The Ability of GAP1IP4BP To Function as a Rap1 GTPase-Activating Protein (GAP) Requires Its Ras GAP-Related Domain and an Arginine Finger Rather than an Asparagine Thumb

GAP1^IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) that includes GAP1^m, CAPRI, and RASAL. Composed of a central Ras GAP-related domain (RasGRD), surrounded by amino-terminal C2 domains and a carboxy-terminal PH/Btk domain, these proteins, with the notable exception of GAP1^m, possess an unexpected arginine finger-dependent GAP activity on the Ras-related protein Rap1 (S. Kupzig, D. Deaconescu, D. Bouyoucef, S. A. Walker, Q. Liu, C. L. Polte, O. Daumke, T. Ishizaki, P. J. Lockyer, A. Wittinghofer, and P. J. Cullen, J. Biol. Chem. 281:9891-9900, 2006). Here, we have examined the mechanism through which GAP1^IP4BP can function as a Rap1 GAP. We show that deletion of domains on either side of the RasGRD, while not affecting Ras GAP activity, do dramatically perturb Rap1 GAP activity. By utilizing GAP1^IP4BP/GAP1^m chimeras, we establish that although the C2 and PH/Btk domains are required to stabilize the RasGRD, it is this domain which contains the catalytic machinery required for Rap1 GAP activity. Finally, a key residue in Rap1-specific GAPs is a catalytic asparagine, the so-called asparagine thumb. By generating a molecular model describing the predicted Rap1-binding site in the RasGRD of GAP1^IP4BP, we show that mutagenesis of individual asparagine or glutamine residues that lie in close proximity to the predicted binding site has no detectable effect on the in vivo Rap1 GAP activity of GAP1^IP4BP. In contrast, we present evidence consistent with a model in which the RasGRD of GAP1^IP4BP functions to stabilize the switch II region of Rap1, allowing stabilization of the transition state during GTP hydrolysis initiated by the arginine finger.The Ras-like family of small GTPases are ubiquitously expressed, evolutionarily conserved proteins that, by undergoing conformational changes in response to the alternate binding of GDP and GTP, function as binary switches (28, 31, 35). The GDP-bound “off” state and the GTP-bound “on” state recognize distinct effector proteins, thereby allowing the regulation of a variety of downstream signaling events (28, 31, 35). While Ras is the best-known and best-studied Ras-like GTPase, Rap1 has recently attracted considerable attention (reviewed in reference 20).Rap1 was originally identified through its ability, when overexpressed, to reverse the phenotype of K-Ras-transformed NIH 3T3 cells (19). As Ras and Rap1 have very similar effector regions, the ability of Rap1 to reverse the transformed phenotype appeared to arise through an ability to compete with K-Ras effectors. For example, Rap1 binds the Ras effector Raf1 but this does not lead to its activation (11). This is consistent with a simple model in which Rap1 functions as a Ras antagonist (6, 37). However, recent work has challenged this view. Increasing evidence points to Rap1 interacting with its own panel of effectors through which it controls cell-cell adhesion and cell-matrix interactions (reviewed in reference 20).Like that of other GTPases, the activation of Ras and Rap1 is regulated through guanine nucleotide exchange factors, which control activation by stimulating the exchange of GDP for GTP. Inactivation is driven by GTPase-activating proteins (GAPs). These enhance the intrinsic GTPase activity of Ras and Rap1, thereby leading to GTP hydrolysis. A wide variety of guanine nucleotide exchange factors and GAPs specific for these GTPases have been identified (14). Through the arrangement of different modular domains, these proteins are regulated following the activation of cell surface receptors. This occurs either through direct association with the activated receptor or indirectly through second messengers (4, 5, 14, 41).Mammalian proteins capable of functioning as Ras GAPs include NF1 (3, 27, 40), p120^GAP (38), the semaphorin 4D receptor plexin-B1 (29), and members of the GAP1 (reviewed in reference 41) and SynGAP (DAB2IP, nGAP, and SynGAP) families (10, 18, 39). These function as Ras GAPs by supplying a catalytic arginine residue—the arginine finger—into the active site of Ras. This stabilizes the transition state of the GTPase reaction, increasing the reaction rate by more than 1,000-fold (1, 33, 34).Rap1 GAPs include Rap GAPs I and II, the SPA-1 family (SPA-1, SPAR, SPAL, and E6TP1), and tuberin (16, 17, 26, 32). Unlike Ras, Rap1 does not possess the catalytic glutamine residue that is critical for GTP hydrolysis in Ras. This fundamental difference means that the mechanisms by which Ras and Rap1 GAPs function are distinct. Rap1 GAPs do not employ a catalytic arginine residue (8, 9); instead, they provide a catalytic asparagine—the asparagine thumb—to stimulate GTP hydrolysis (15). Here the asparagine carboxamide side chain has a function similar to that of the glutamine residue in Ras, stabilizing the position of the nucleophilic water and γ-phosphate in the transition complex (15, 36).Given such distinct catalytic mechanisms, surprisingly, some Ras GAPs, while having no detectable sequence homology with any Rap1 GAPs, are capable of stimulating the GTPase activity of Rap1. The first protein found to display such dual activities was GAP1^IP4BP (13) (also known as RASA3, GAPIII, and R-Ras GAP). This is a member of the GAP1 family, which also comprises GAP1^m, CAPRI, and RASAL (2, 23-25). These proteins are characterized by a domain architecture comprising amino-terminal tandem C2 domains, a highly conserved central Ras GAP-related domain (RasGRD), and a carboxy-terminal pleckstrin homology (PH) domain that is associated with a Bruton''s tyrosine kinase (Btk) motif (41). Consistent with the presence of the RasGRD, all proteins display Ras GAP activity, although each is differentially regulated following receptor stimulation (41). With the notable exception of GAP1^m, all GAP1 proteins also possess efficient Rap1 GAP activity (22). Such dual specificity is not restricted solely to GAP1 proteins. Recently, C2 domain-containing SynGAP—a neuronal Ras GAP—has also been shown to display Rap1 GAP activity (21), an activity that appears to require, alongside the RasGRD, the presence of a single C2 domain (30).Here we have examined the mechanism behind the dual Ras and Rap1 GAP activities of GAP1^IP4BP. Through the generation of a series of GAP1^IP4BP/GAP1^m chimeras, we have established that while the C2 domains of GAP1^IP4BP are required to stabilize the RasGRD, these domains do not supply catalytic residues required for Rap1 GAP activity. Rather, the Rap1 GAP catalytic machinery appears to reside solely within the RasGRD. By the site-directed mutagenesis of selected asparagine and glutamine residues within this domain—selected following the generation of a predicted molecular model of the GAP1^IP4BP RasGRD-Ras(Rap1) complex—we establish that the ability of GAP1^IP4BP to function as a Rap1 GAP does not occur via a mechanism that utilizes a classic asparagine thumb. Rather, we suggest that the GAP1^IP4BP RasGRD functions to stabilize the switch II region of Rap1 in a manner that allows a catalytic arginine finger from GAP1^IP4BP to drive the hydrolysis of GTP.     

Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMP- and Rap-mediated signaling to regulate adhesion to the extracellular matrix.Cyclic AMP (cAMP) is a second messenger that relays a wide range of hormone responses. The discovery of Epac as a direct effector of cAMP (15, 29) has triggered the elucidation of many cAMP-regulated processes that could not be explained by the previously known effectors protein kinase A (PKA) and cyclic nucleotide-regulated ion channels (21). Both Epac family members, Epac1 and Epac2, act as guanine nucleotide exchange factors (GEFs) for the small G proteins Rap1 and Rap2. Thereby, Epac functions in processes such as exocytosis (28, 48, 59), cell-cell junction formation (13, 20, 30, 53, 64), and cell-extracellular matrix (ECM) adhesion (55). Adhesion to the ECM induced by Epac1 and Rap is mediated by actin-linked integrin molecules and is implicated in diverse biological processes such as homing of endothelial progenitor cells to ischemic tissue (9), remodeling of the vasculature (10, 36), and transendothelial migration of leukocytes (37, 60).Epac1 and Epac2 are multidomain proteins containing a C-terminal catalytic region, which consists of a CDC25 homology domain responsible for GEF activity, a Ras exchange motif (REM), which stabilizes the CDC25 homology domain, and a Ras association (RA) domain. In the autoinhibited state, the catalytic site is sterically covered by the N-terminal regulatory region, which harbors a DEP (Dishevelled, Egl-10, and pleckstrin) domain and one or two cyclic nucleotide-binding domains in Epac1 and Epac2, respectively. As demonstrated by the crystal structures of both active and inactive Epac2, autoinhibition is released by a conformational change induced by the binding of cAMP (56, 57).After its production at the plasma membrane (PM) by adenylate cylases, cAMP becomes compartmentalized due to local degradation by spatially restricted phosphodiesterases (1). Further compartmentalization of cAMP signaling is established by the confined targeting of the cAMP effector proteins. Numerous adaptor proteins that target PKA to distinct subcellular locations and mediate the assembly of large signaling complexes have been identified (3). Similarly, cAMP-Epac signaling appears to be spatially regulated by diverse anchoring mechanisms, which may reflect the many different functions assigned to Epac. For instance, the DNA damage-responsive kinase DNA-PK is regulated by nuclear Epac1 (26), whereas membrane recruitment by activated Ras is essential for the role of Epac2 in neurite outgrowth (34, 35). Recently, we reported that Epac1 translocates to the PM upon the binding of cAMP and that this translocation contributes to Rap-mediated cell-ECM adhesion (51). Although the anchor at the PM remains elusive, it has become clear that the cAMP-dependent translocation of Epac1 involves its DEP domain (amino acids 50 to 148) and requires the cAMP-induced conformation.In this study, we reveal an additional targeting mechanism of Epac1 by showing that its N terminus interacts with members of the ezrin-radixin-moesin (ERM) family. ERM proteins show high sequence similarity and function as scaffolding proteins that link the actin cytoskeleton to the PM (18, 42, 47). Inactive ERM proteins reside in the cytoplasm in an autoinhibited state maintained by an intramolecular interaction between the N-terminal FERM (4.1 protein, ezrin, radixin, moesin) domain and the C-terminal actin binding domain (ABD). This autoinhibition is released by binding to phosphatidylinositol-4,5-bisphosphate (PIP₂) and threonine phosphorylation of the ABD, which induce the open conformation of the protein (reviewed in reference 8). Several kinases have been implicated in phosphorylation of this threonine in the ABD, including protein kinase C α (PKC α), PKC θ, NIK, Mst4, and the Rho effector ROCK (2, 40, 46, 50, 61). Active ERM proteins directly link the actin cytoskeleton to the PM and allow the recruitment of multiple signaling proteins. In this manner, ERM proteins function in numerous processes, such as the formation of microvilli, adherens junction stabilization, and leukocyte polarization (12, 18, 42, 47). Here, we demonstrate that ERM proteins also function as PM anchors for Epac1. The underlying interaction is mediated by the N terminus (residues 1 to 49) of Epac1 and is independent of its conformational state. Instead, the interaction is regulated at the level of the ERM proteins, which bind Epac1 when they are in their active, open conformation. G protein-coupled receptor (GPCR)-mediated signaling that results in activation of ERM proteins increases binding of Epac1 and results in a clustered localization of Epac1 at the PM. Together with DEP domain-mediated PM translocation, ERM proteins control cell adhesion mediated by Epac1. In conclusion, our data show that ERM proteins mediate PM recruitment of Epac1 and couple Epac1 activity to integrin-mediated cell adhesion.     

The Adenovirus E4orf4 Protein Induces G2/M Arrest and Cell Death by Blocking Protein Phosphatase 2A Activity Regulated by the B55 Subunit

Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the Bα subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. Bα is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that Bα-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70^S6K, were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I₁^PP2A enhanced E4orf4-induced cell killing and G₂/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with Bα-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.Our research group and others have shown that the 114-residue product of early region E4 of human adenoviruses, termed E4orf4, induces p53-independent cell death in human tumor cells (24, 25, 34-36, 55) and in Saccharomyces cerevisiae (23, 53). E4orf4 protein, which shares no obvious homology with other viral or cellular products, kills a wide range of human cancer cells but is believed to have reduced activity against normal human primary cells (6, 55, 56). Although in some cases E4orf4-expressing cells exhibit characteristics typical of apoptosis, including the presence of irregularly shaped and shrunken nuclei, cytoplasmic vacuolization, and membrane blebbing (24, 25, 50, 55), cell death may more typically be independent of caspase activation (24, 25, 30, 32, 50). With H1299 human non-small-cell lung carcinoma cells, death is characterized by rapid cell rounding, enlargement, release from the surface of culture plates, cell cycle arrest in G₂/M and possibly G₁, and eventually, after an extended period, loss of membrane integrity (30). Both cytoplasmic and nuclear pathways have been observed, the former involving interactions with c-Src family kinases, activation of calpain, and remodeling of the actin cytoskeleton (7, 24, 50, 51, 58). Little is known about the nuclear pathway, which may represent the predominant death-inducing process. Our current evidence suggests that H1299 cells die following prolonged irreversible cell cycle arrest leading to mitotic catastrophe and death by a necrosis-like process (30).E4orf4 is known to associate with the Bα regulatory subunit of protein phosphatase 2A (PP2A) (22, 34), and this interaction appears to be necessary for the majority of E4orf4 toxicity in both yeast (23, 53) and human tumor cells (34, 56). PP2A is an abundant serine-threonine phosphatase involved in regulation of metabolism, splicing, translation, morphogenesis, development, and cell cycle progression (15, 19, 27, 43, 59). PP2A holoenzymes exist as multiple heterotrimeric complexes composed of a catalytic C subunit, an A subunit that functions as a scaffold, and a B-type regulatory subunit. Two forms each of the A and C subunits exist in mammalian cells; however, more than 20 B-type subunits have been identified in three unique classes (B/B55, B′/B56, B″/PR72), plus striatin/SG2NA (sometimes called B‴) (10, 19, 26). Although one group has suggested that E4orf4 protein interacts with one or more members of the B′/B56 class (57), it is generally accepted that interaction with the Bα/B55 subunit (Cdc55 in yeast) is important for induction of cell death in both human tumor cells and yeast (53, 57). Interestingly, a recent report has also suggested that in yeast, growth suppression induced by E4orf4 is mediated only in part by the catalytic C subunit of PP2A (31).In the present report, we show that E4orf4 protein interacts uniquely with members of the B55 class of PP2A B-type subunits, and at sufficient concentrations, it appears to become toxic by reducing dephosphorylation of substrates of B55-containing PP2A holoenzymes. As cell death is preceded by cell cycle arrest, we believe that key substrates may include proteins required for cell cycle progression.     

Serine Protease HtrA1 Associates with Microtubules and Inhibits Cell Migration

HtrA1 belongs to a family of serine proteases found in organisms ranging from bacteria to humans. Bacterial HtrA1 (DegP) is a heat shock-induced protein that behaves as a chaperone at low temperature and as a protease at high temperature to help remove unfolded proteins during heat shock. In contrast to bacterial HtrA1, little is known about the function of human HtrA1. Here, we report the first evidence that human HtrA1 is a microtubule-associated protein and modulates microtubule stability and cell motility. Intracellular HtrA1 is localized to microtubules in a PDZ (PSD95, Dlg, ZO1) domain-dependent, nocodazole-sensitive manner. During microtubule assembly, intracellular HtrA associates with centrosomes and newly polymerized microtubules. In vitro, purified HtrA1 promotes microtubule assembly. Moreover, HtrA1 cosediments and copurifies with microtubules. Purified HtrA1 associates with purified α- and β-tubulins, and immunoprecipitation of endogenous HtrA1 results in coprecipitation of α-, β-, and γ-tubulins. Finally, downregulation of HtrA1 promotes cell motility, whereas enhanced expression of HtrA1 attenuates cell motility. These results offer an original identification of HtrA1 as a microtubule-associated protein and provide initial mechanistic insights into the role of HtrA1 in theregulation of cell motility by modulating microtubule stability.HtrA1 (for high temperature requirement) belongs to a family of serine proteases and is so named because of its essential role in thermal tolerance in Escherichia coli, which requires HtrA (also known as DegP) for survival at elevated temperatures (14). This survival is attributed to the ability of HtrA proteins to switch from chaperones to proteases that reduce the amount of unfolded and aggregated protein upon heat stress (46). Human, as well as bacterial, HtrA proteins contain trypsin and PDZ (PSD95, Dlg, ZO1) domains that display a high degree of sequence conservation from bacteria to human (14). Of the four human HtrA proteins, HtrA1, HtrA3, and HtrA4 also contain a signal peptide, insulin-like growth factor binding protein (IGFBP), and Kazal-type trypsin inhibitor domains, while HtrA2 lacks these domains. Although HtrA1 contains signal peptide, an intracellular form of HtrA1 has been reported as well (15, 17). The mitochondrial protein HtrA2 is well characterized and has been shown to be involved in apoptosis (27, 37, 39, 47, 52, 53) and neurodegenerative disease (35). However, HtrA1 is the first in the family to be implicated as a tumor suppressor in ovarian cancer and melanoma (3, 5, 13). In addition, HtrA1 is implicated in various pathogenic and developmental processes, including osteoarthritis, Alzheimer''s disease, neuronal maturation and development, age-related macular degeneration, and tumor progression (11, 23, 24, 33, 36, 50, 56). Specific to its role in tumor progression, HtrA1 is downregulated in various cancers, and its downregulation is associated with resistance to chemotherapy and a metastatic phenotype (4, 11, 19). Recently, we developed a mixture-based peptide library to determine the specificities of cleavage site motifs for HtrA1 serine protease. The results identified tubulins as potential substrates of HtrA1. Furthermore, we showed that exogenously expressed HtrA1 disrupts microtubules (MTs) and targets tubulins for degradation (data not shown). These results suggest a potential role for HtrA1 as an MT-associated protein (MAP) and its potential to regulate MT and tubulin stability and MT-associated cellular functions.MTs are highly dynamic noncovalent polymers of α- and β-tubulins that undergo cyclical shrinking (catastrophe) and growing (rescue) (18, 31, 43). The dynamic instability of MTs is central to their diverse biological functions, including the coordination of cell division (40, 55), morphogenesis (25), cell polarity (42), and motility (48). MT instability is, in part, modulated by MAPs (2, 29). Many tumor suppressors, such as adenomatous polyposis coli (APC) (20), RASSF1A (45), and Dlg (6), associate with MTs and impose tumor suppressor activities by regulating their functions related to cell division, polarity, and motility. Deregulation of these processes, as a consequence of loss of function of these tumor suppressors, contributes to unchecked proliferation; cytoarchitecture disruption; and the ability to migrate, invade, and metastasize distant organs (6, 7, 26). Therefore, the regulation of MT stability and dynamics or the lack of it has dire consequences for normal cell functions.Given the fact that HtrA1 is downregulated in various cancers, particularly in metastatic cancer, it is possible that HtrA1 may regulate certain aspects of cancer, namely, the motility of cancer cells, by modulating MT stability and dynamics. Therefore, to better characterize the interaction between HtrA1 and MTs and to gain mechanistic insights into the functional consequences of HtrA1 downregulation in cancer, we investigated the biochemical interaction between HtrA1 and tubulin, the domain within HtrA1 required for localization to MTs, and the effect on cell migration. Here, we describe the identification of HtrA1 as an MT-associated serine protease and a novel role of HtrA1 in the regulation of cell motility.     

Direct Spatial Control of Epac1 by Cyclic AMP

Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane. Combining high-resolution confocal fluorescence microscopy with total internal reflection fluorescence and fluorescent resonance energy transfer assays, we observed that Epac1 translocation is a rapid and reversible process. This dynamic redistribution of Epac1 requires both the cAMP-induced conformational change as well as the DEP domain. In line with its translocation, Epac1 activation induces Rap activation predominantly at the plasma membrane. We further show that the translocation of Epac1 enhances its ability to induce Rap-mediated cell adhesion. Thus, the regulation of Epac1-Rap signaling by cAMP includes both the release of Epac1 from autoinhibition and its recruitment to the plasma membrane.Cyclic AMP (cAMP) is an important second messenger that mediates many cellular hormone responses. It has become more and more appreciated that, along with the cAMP effector protein kinase A (PKA), Epac proteins also play pivotal roles in many cAMP-controlled processes, including insulin secretion (23, 39), cell adhesion (9, 17, 25, 49, 60), neurotransmitter release (22, 53, 63), heart function (13, 35, 54), and circadian rhythm (38). Epac1 and Epac2 are cAMP-dependent guanine nucleotide exchange factors (GEFs) for the small G proteins Rap1 and Rap2 (12, 24). They contain a regulatory region with one (Epac1) or two (Epac2) cAMP-binding domains, a Dishevelled, Egl-10, Pleckstrin (DEP) domain, and a catalytic region for GEF activity (11). The binding of cAMP is a prerequisite for catalytic activity in vitro and in vivo (11). Recently, the structures of both the inactive and active conformations of Epac2 were solved (51, 52). This revealed that in the inactive conformation, the regulatory region occludes the Rap binding site, which is relieved by a conformational change induced by cAMP binding.Like all G proteins of the Ras superfamily, Rap cycles between an inactive GDP-bound and active GTP-bound state in an equilibrium that is tightly regulated by specific GEFs and GTPase-activating proteins (GAPs). The GEF-induced dissociation of GDP results in the binding of the cellularly abundant GTP, whereas GAPs enhance the intrinsic GTPase activity of the G protein, thereby inducing the inactive GDP-bound state. Besides Epac, several other GEFs for Rap have been identified, including C3G, PDZ-GEF, and RasGRP, and these act downstream of different signaling pathways (7). Since Rap localizes to several membrane compartments, including the Golgi network, vesicular membranes, and the plasma membrane (PM) (2-4, 37, 42, 48), the spatial regulation of its activity is expected to be established by the differential distributions of its upstream GEFs, each activating distinct pools of Rap on specific intracellular locations.Similarly to Rap, Epac1 also is observed at many locations in the cell, including the cytosol, the nucleus, the nuclear envelope, endomembranes, and the PM (5, 11, 14, 21, 29, 47). These various locations may reflect the many different functions assigned to Epac1, such as the regulation of cell adhesion, cell junction formation, secretion, the regulation of DNA-dependent protein kinase by nuclear Epac1, and the regulation of the Na⁺/H⁺ exchanger NHE3 at the brush borders of kidney epithelium (19, 21, 26). Apparently, specific anchors are responsible for this spatial regulation of Epac1. Indeed, Epac1 was found to associate with phosphodiesterase 4 (PDE4) in a complex with mAKAP in cardiomyocytes (13), with MAP-LC bound to microtubules (62), and with Ezrin at the brush borders of polarized cells (M. Gloerich, J. Zhao, and J. L. Bos, unpublished data).In this study, we report the unexpected observation that, in addition to the temporal control of Epac1 activity, cAMP also induces the translocation of Epac1 toward the plasma membrane. Using confocal fluorescence microscopy, total internal reflection fluorescence (TIRF) microscopy, and fluorescence resonance energy transfer (FRET)-based assays for high spatial and temporal resolution, we observed that the translocation of Epac1 is immediate and that Epac1 approaches the PM to within ∼7 nm. In line with this, Epac1-induced Rap activation was registered predominantly on this compartment. Epac1 translocation results directly from the cAMP-induced conformational change and depends on the integrity of its DEP domain. We further show that Epac1 translocation is a prerequisite for cAMP-induced Rap activation at the PM and enhances Rap-mediated cell adhesion. Thus, cAMP exerts dual regulation on Epac1 for the activation of Rap, controlling both its GEF activity and targeting to the PM.     

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

In most eukaryotic cells, tubulin is subjected to posttranslational glutamylation, a conserved modification of unclear function. The glutamyl side chains form as branches of the primary sequence glutamic acids in two biochemically distinct steps: initiation and elongation. The length of the glutamyl side chain is spatially controlled and microtubule type specific. Here, we probe the significance of the glutamyl side chain length regulation in vivo by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena. Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic microtubules and destabilized axonemal microtubules. Our observations suggest that the cellular outcomes of glutamylation are mediated by spatially restricted tubulin interactors of diverse nature.Microtubules are dynamic elements of the cytoskeleton that are assembled from heterodimers of α- and β-tubulin. Once assembled, tubulin subunits undergo several conserved posttranslational modifications (PTMs) that diversify the external and luminal surfaces of microtubules (51). Two tubulin PTMs, glycylation and glutamylation, collectively known as polymodifications, form peptide side chains that are attached to the γ-carboxyl groups of glutamic acids in the primary sequence of the C-terminal tails (CTTs) of α- and β-tubulin (14, 36). Glutamylated microtubules are abundant in projections of neurons (14), axonemes (8, 15, 17), and centrioles/basal bodies (5, 31) and are detectable in the mitotic spindle and on a subset of cytoplasmic network microtubules (1, 5). The modifying enzymes, tubulin glutamic acid ligases (tubulin E-ligases), belong to the family of proteins related to the tubulin tyrosine ligase (TTL), known as TTL-like (TTLL) proteins (22, 50, 53). Tubulin glutamylation appears to be important in vivo. A knockdown of the TTLL7 E-ligase mRNA in cultured neurons inhibits the outgrowth of neurites (20). A loss of PGs1, a protein associated with TTLL1 E-ligase (22, 37), disorganizes sperm axonemes in the mouse (11), and a morpholino knockdown of TTLL6 E-ligase expression in zebrafish inhibits the assembly of olfactory cilia (33). The biochemical consequences of tubulin glutamylation in vivo are poorly understood, but the emerging model is that this PTM regulates interactions between microtubules and microtubule-associated proteins (MAPs) (6, 7, 19, 27).The ciliate Tetrahymena thermophila has 18 types of diverse microtubules that are all assembled in a single cell. Although most, if not all, of these microtubules are glutamylated, the length of glutamyl side chains is spatially regulated (8, 53). Minimal side chains composed of a single glutamic acid (monoglutamylation) are present on the cytoplasmic and nuclear microtubules, whereas elongated side chains are present on the basal bodies and axonemes (53). In Tetrahymena, Ttll6Ap is a β-tubulin-preferring E-ligase (22), with a strong if not exclusive, side chain elongating activity (50). Here, by overproducing Ttll6Ap in vivo, we explore the consequences of glutamyl side chain hyper-elongation. Unexpectedly, we show that in the same cells, hyperelongation of glutamyl side chains stabilizes cell body and destabilizes axonemal microtubules. The simplest explanation of these data is that, in vivo, the cellular outcomes of tubulin glutamylation are mediated by diverse microtubule type-specific MAPs. To our knowledge, we are first to report that excessive tubulin glutamylation can either stabilize or destabilize microtubules in the same cell.     

Borna Disease Virus Requires Cholesterol in both Cellular Membrane and Viral Envelope for Efficient Cell Entry

Borna disease virus (BDV), the prototypic member of the family Bornaviridae within the order Mononegavirales, provides an important model for the investigation of viral persistence within the central nervous system (CNS) and of associated brain disorders. BDV is highly neurotropic and enters its target cell via receptor-mediated endocytosis, a process mediated by the virus surface glycoprotein (G), but the cellular factors and pathways determining BDV cell tropism within the CNS remain mostly unknown. Cholesterol has been shown to influence viral infections via its effects on different viral processes, including replication, budding, and cell entry. In this work, we show that cell entry, but not replication and gene expression, of BDV was drastically inhibited by depletion of cellular cholesterol levels. BDV G-mediated attachment to BDV-susceptible cells was cholesterol independent, but G localized to lipid rafts (LR) at the plasma membrane. LR structure and function critically depend on cholesterol, and hence, compromised structural integrity and function of LR caused by cholesterol depletion likely inhibited the initial stages of BDV cell internalization. Furthermore, we also show that viral-envelope cholesterol is required for BDV infectivity.Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization (3′-N-p10/P-M-G-L-5′) is characteristic of mononegaviruses (6, 28, 46, 48). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales (8, 28, 46, 49).BDV can infect a variety of cell types in cell culture but in vivo exhibits exquisite neurotropism and causes central nervous system (CNS) disease in different vertebrate species, which is frequently manifested in behavioral abnormalities (19, 33, 44, 53). Both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (14, 16, 29, 42, 44). BDV provides an important model for the investigation of both immune-mediated pathological events associated with virus-induced neurological disease and mechanisms whereby noncytolytic viruses induce neurodevelopmental and behavioral disturbances in the absence of inflammation (15, 18, 41). Moreover, serological data and molecular epidemiological studies suggest that BDV, or a BDV-like virus, can infect humans and that it might be associated with certain neuropsychiatric disorders (17, 24), which further underscores the interest in understanding the mechanisms underlying BDV persistence in the CNS and its effect on brain cell functions. The achievement of these goals will require the elucidation of the determinants of BDV cell tropism within the CNS.BDV enters its target cell via receptor-mediated endocytosis, a process in which the BDV G protein plays a central role (1, 5, 13, 14, 39). Cleavage of BDV G by the cellular protease furin generates two functional subunits: GP1 (GP_N), involved in virus interaction with a yet-unidentified cell surface receptor (1, 39), and GP2 (GP_C), which mediates a pH-dependent fusion event between viral and cellular membranes (13). However, a detailed characterization of cellular factors and pathways involved in BDV cell entry remains to be done.Besides cell surface molecules that serve as viral receptors, many other cell factors, including nonproteinaceous molecules, can influence cell entry by virus (52). In this regard, cholesterol, which plays a critical role in cellular homeostasis (55), has also been identified as a key factor required for productive infection by different viruses. Accordingly, cholesterol participates in a variety of processes in virus-infected cells, including fusion events between viral and cellular membranes (3), viral replication (23), and budding (35, 37), as well as maintenance of lipid rafts (LR) (12) as scaffold structures where the viral receptor and coreceptor associate (11, 26, 32, 36). LR are specialized microdomains within cellular membranes constituted principally of proteins, sphingolipids, and cholesterol. LR facilitate the close proximity and interaction of specific sets of proteins and contribute to different processes associated with virus multiplication (38). Cholesterol can also influence virus infection by contributing to the maintenance of the properties of the viral envelope required for virus particle infectivity (21, 54). Here, we show for the first time that cholesterol plays a critical role in BDV infection. Depletion of cellular cholesterol prior to, but not after, BDV cell entry prevented productive BDV infection, likely due to disruption of plasma membrane LR that appear to be the cell entry point for BDV. In addition, we document that cholesterol also plays an essential role in the properties of the BDV envelope required for virus particle infectivity.     

Poxvirus Complement Control Proteins Are Expressed on the Cell Surface through an Intermolecular Disulfide Bridge with the Viral A56 Protein

The vaccinia virus (VACV) complement control protein (VCP) is an immunomodulatory protein that is both secreted from and expressed on the surface of infected cells. Surface expression of VCP occurs though an interaction with the viral transmembrane protein A56 and is dependent on a free N-terminal cysteine of VCP. Although A56 and VCP have been shown to interact in infected cells, the mechanism remains unclear. To investigate if A56 is sufficient for surface expression, we transiently expressed VCP and A56 in eukaryotic cell lines and found that they interact on the cell surface in the absence of other viral proteins. Since A56 contains three extracellular cysteines, we hypothesized that one of the cysteines may be unpaired and could therefore form a disulfide bridge with VCP. To test this, we generated a series of A56 mutants in which each cysteine was mutated to a serine, and we found that mutation of cysteine 162 abrogated VCP cell surface expression. We also tested the ability of other poxvirus complement control proteins to bind to VACV A56. While the smallpox homolog of VCP is able to bind VACV A56, the ectromelia virus (ECTV) VCP homolog is only able to bind the ECTV homolog of A56, indicating that these proteins may have coevolved. Surface expression of poxvirus complement control proteins may have important implications in viral pathogenesis, as a virus that does not express cell surface VCP is attenuated in vivo. This suggests that surface expression of VCP may contribute to poxvirus pathogenesis.Poxviruses, including vaccinia virus (VACV), encode large numbers of immunomodulatory proteins that help them establish an infection and combat the host''s immune response (10, 32). One of these is the vaccinia virus complement control protein (VCP), which is both secreted from and expressed on the surface of infected cells (9, 14, 16, 17). VCP acts against the complement system, a series of soluble proteins that is an important early component of the innate immune system and also shapes adaptive immune responses (15, 42, 43). In response to viral infection, complement can opsonize or inactivate virions and can lyse enveloped virus or infected cells (1, 3, 7, 12). Because of these pressures, a number of viruses, including herpes simplex virus, flaviviruses, and poxviruses, encode novel or host-derived regulators of complement, while others, including HIV and poxviruses, incorporate host complement regulatory proteins into virus particles (7, 11, 31, 39). Many orthopoxviruses encode a complement regulator (8, 20, 23, 29), and the most studied of these is VCP. Structurally, VCP is made up of four short consensus repeats (SCR) that are the basic units of mammalian complement regulators (17, 25), and VCP has been shown to interfere with the complement cascade at multiple steps (2, 16, 20-22, 25, 28-30, 33). Additionally, a VCP knockout virus generates smaller lesions in animal models (14, 16). While some host complement control proteins (CCPs) are secreted, many contain transmembrane domains (or a glycophosphatidylinositol anchor) and are thus expressed on the cell surface (42, 43). Thus, when we found that VCP is also expressed on the infected cell surface and protects infected cells from complement-mediated lysis in vitro (9), we believed this to be an important interaction that required further investigation. We previously found that the N-terminal cysteine on VCP was needed for surface expression and that the VACV transmembrane protein A56 was also required (9). The vaccinia virus A56 protein is a type 1 transmembrane glycoprotein that is found on the surface of infected cells and on extracellular virus particles (4, 18, 26, 27, 36). It interacts with another viral protein, K2 (19, 37, 45), which lacks a transmembrane domain and binds to A56 noncovalently (36). The A56/K2 complex prevents syncytium formation between infected cells and superinfection by interacting with the vaccinia virus entry/fusion complex on virions (24, 38, 40, 41). Here we provide evidence that the N-terminal cysteine on VCP forms an intermolecular disulfide bond with cysteine 162 on the ectodomain of A56. We also demonstrate that similar interactions can occur with other poxvirus CCPs, as the smallpox virus and ectromelia virus homologs of VCP also exhibit A56-dependent surface expression.     

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC₉₀] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5, 33, 45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12, 23, 64, 65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21, 37, 44, 54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17, 43, 44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1, 18, 21, 36, 62, 63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15, 43, 55, 61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22, 28, 48, 63).In immunocompetent individuals T-cell and antibody responses efficiently control HCMV infection and reduce pathological consequences of maternal-fetal transmission (13, 67), although this is usually not sufficient to eradicate the virus. Albeit with controversial results, HCMV immunoglobulins (Igs) have been administered to transplant patients in association with immunosuppressive treatments for prophylaxis of HCMV disease (56, 57), and a recent report suggests that they may be effective in controlling congenital infection and preventing disease in newborns (32). These products are plasma derivatives with relatively low potency in vitro (46) and have to be administered by intravenous infusion at very high doses in order to deliver sufficient amounts of neutralizing antibodies (4, 9, 32, 56, 57, 66).The whole spectrum of antigens targeted by HCMV-neutralizing antibodies remains poorly characterized. Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6, 14, 26, 29, 34, 41, 52, 60). Remarkably, we have recently shown that human sera exhibit a more-than-100-fold-higher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20). Similarly, CMV hyperimmunoglobulins have on average 48-fold-higher neutralizing activities against epithelial cell entry than against fibroblast entry (10). However, epitopes that are targeted by the antibodies that comprise epithelial or endothelial cell-specific neutralizing activity of human immune sera remain unknown.In this study we report the isolation of a large panel of human monoclonal antibodies with extraordinarily high potency in neutralizing HCMV infection of endothelial and epithelial cells and myeloid cells. With the exception of a single antibody that recognized a conserved epitope of UL128, all other antibodies recognized conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex.     

Site-Specific mTOR Phosphorylation Promotes mTORC1-Mediated Signaling and Cell Growth

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) functions as a rapamycin-sensitive environmental sensor that promotes cellular biosynthetic processes in response to growth factors and nutrients. While diverse physiological stimuli modulate mTORC1 signaling, the direct biochemical mechanisms underlying mTORC1 regulation remain poorly defined. Indeed, while three mTOR phosphorylation sites have been reported, a functional role for site-specific mTOR phosphorylation has not been demonstrated. Here we identify a new site of mTOR phosphorylation (S1261) by tandem mass spectrometry and demonstrate that insulin-phosphatidylinositol 3-kinase signaling promotes mTOR S1261 phosphorylation in both mTORC1 and mTORC2. Here we focus on mTORC1 and show that TSC/Rheb signaling promotes mTOR S1261 phosphorylation in an amino acid-dependent, rapamycin-insensitive, and autophosphorylation-independent manner. Our data reveal a functional role for mTOR S1261 phosphorylation in mTORC1 action, as S1261 phosphorylation promotes mTORC1-mediated substrate phosphorylation (e.g., p70 ribosomal protein S6 kinase 1 [S6K1] and eukaryotic initiation factor 4E binding protein 1) and cell growth to increased cell size. Moreover, Rheb-driven mTOR S2481 autophosphorylation and S6K1 phosphorylation require S1261 phosphorylation. These data provide the first evidence that site-specific mTOR phosphorylation regulates mTORC1 function and suggest a model whereby insulin-stimulated mTOR S1261 phosphorylation promotes mTORC1 autokinase activity, substrate phosphorylation, and cell growth.The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine/threonine protein kinase, senses and integrates signals from diverse environmental cues (14, 31, 50, 74). mTOR associates with different partner proteins to form functionally distinct signaling complexes (4). The immunosuppressive drug rapamycin acutely inhibits signaling by mTOR complex 1 (mTORC1) (22), which contains mTOR, mLST8/GβL, raptor, and PRAS40 (24, 33, 34, 54, 67). Rapamycin fails to acutely inhibit signaling by mTORC2, which contains mTOR, mLST8/GβL, rictor, mSin1, and PRR5/Protor (18, 32, 47, 55, 73, 76). mTORC1 promotes various biosynthetic processes, including protein synthesis, cell growth (an increase in cell mass and size), and cell proliferation (an increase in cell number) (14, 40, 74). During growth factor (e.g., insulin) and nutrient (e.g., amino acids and glucose) sufficiency, mTORC1 phosphorylates the translational regulators p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) to coordinately upregulate protein biosynthesis (40). Both S6K1 and 4EBP1 contain a TOR signaling motif, which mediates their interaction with raptor and thus facilitates their recruitment to the mTOR kinase (10, 44, 57, 58). In addition to regulating protein synthesis, mTORC1-mediated phosphorylation of S6K1 and 4EBP also promotes cell growth and cell cycle progression (15, 16). While more recently identified and thus less well characterized than mTORC1, mTORC2 mediates the phosphorylation of AGC kinase family members (e.g., Akt [also known as protein kinase B, PKB], PKCα, and SGK1) on their hydrophobic motifs and modulates the organization of the actin cytoskeleton (20, 26, 32, 55, 56).The insulin pathway represents the best-characterized activator of mTORC1 signaling to date, and thus many signaling intermediates that link insulin receptor activation to mTORC1 have been identified (12, 31). Complementary work using Drosophila melanogaster genetics and mammalian cell culture identified TSC1 (hamartin) and TSC2 (tuberin) as upstream negative regulators of mTORC1 (27). Inactivation of either the TSC1 or TSC2 genes, whose protein products heterodimerize to form a tumor suppressor complex, causes the development of benign tumors in diverse organs in both humans and rodents, a disease known as tuberous sclerosis complex (TSC) (36). TSC2 contains a GTPase-activating protein domain that acts on Rheb, a Ras-like GTP binding protein that activates mTORC1 (27). Thus, in TSC-deficient cells, constitutive Rheb-GTP leads to chronically high mTORC1 signaling. While the mechanism by which Rheb-GTP activates mTORC1 remains incompletely understood, Rheb coimmunoprecipitates with mTOR and directly activates mTORC1 kinase activity in vivo and in vitro when GTP bound (2, 38, 54). Rheb has been reported to augment the activity of PLD1, an enzyme that catalyzes the production of the lipid second messenger phosphatidic acid, which contributes to the mitogenic activation of mTORC1 signaling (13, 62). Additionally, Rheb-GTP was reported to induce the dissociation of the endogenous mTOR inhibitor FKBP38 (3), although aspects of this model have been questioned (72). Insulin/phosphatidylinositol 3-kinase (PI3K) signaling reduces the inhibitory effect of TSC on mTORC1 via Akt-mediated phosphorylation of TSC2 (29, 42, 64). Additionally, Ras-regulated signaling via mitogen-activated protein kinase (MAPK) and RSK also inhibits TSC via PI3K/Akt-independent phosphorylation of TSC2 (39, 51, 63). In contrast, glucose deprivation enhances TSC''s inhibitory effect on mTORC1 signaling via AMP-activated protein kinase (AMPK)-mediated phosphorylation of TSC2 (on different sites) (30). Thus, TSC functions as a central nexus of diverse physiological signals to fine-tune mTORC1 signaling depending on environmental conditions (27). While the mechanism by which amino acids promote mTORC1 signaling has remained elusive, compelling new data reveal that the Rag GTPases link amino acid sensing to mTORC1 activation (35, 52, 53). During amino acid sufficiency, GTP-bound Rag heterodimers bind raptor and recruit mTORC1 to an endomembrane compartment that contains the mTORC1 activator Rheb; thus, amino acid sufficiency may function to prime mTORC1 for subsequent growth factor-mediated activation via a dynamic subcellular redistribution mechanism (52).Despite the well-characterized regulation of mTORC1 signaling by growth factors (e.g., insulin), nutrients (e.g., amino acids and glucose), and cellular stress (e.g., hypoxia) and the identification of numerous signaling mediators of these pathways, the direct molecular mechanisms by which cellular signals modulate mTORC1 action remain obscure (31). While three phosphorylation sites (P-sites) on mTOR have been reported to date (T2446, S2448, and S2481), no function has yet been ascribed to any site (7, 43, 49, 59). Here we identify S1261 as a novel mTOR phosphorylation site in vivo in cultured mammalian cells and provide the first evidence that site-specific mTOR phosphorylation regulates mTORC1 function. We show that insulin signals via the PI3K/TSC/Rheb pathway in an amino acid-dependent and rapamycin-insensitive manner to promote mTOR S1261 phosphorylation, which regulates mTORC1 autokinase activity, biochemical signaling to downstream substrates, and cell growth to increased cell size, a major cellular function of mTORC1. Elucidation of the molecular mechanisms underlying mTORC1 regulation will enable us to better understand how mTORC1 senses environmental stimuli to control cellular physiology. As aberrantly upregulated mTORC1 signaling likely contributes to cancer, insulin-resistant diabetes, and cardiovascular diseases, understanding mTORC1 regulation may aid in the development of novel therapeutics for these prevalent human diseases (11, 21, 28).     

gp340 Promotes Transcytosis of Human Immunodeficiency Virus Type 1 in Genital Tract-Derived Cell Lines and Primary Endocervical Tissue

The human scavenger receptor gp340 has been identified as a binding protein for the human immunodeficiency virus type 1 (HIV-1) envelope that is expressed on the cell surface of female genital tract epithelial cells. This interaction allows such epithelial cells to efficiently transmit infective virus to susceptible targets and maintain viral infectivity for several days. Within the context of vaginal transmission, HIV must first traverse a normally protective mucosa containing a cell barrier to reach the underlying T cells and dendritic cells, which propagate and spread the infection. The mechanism by which HIV-1 can bypass an otherwise healthy cellular barrier remains an important area of study. Here, we demonstrate that genital tract-derived cell lines and primary human endocervical tissue can support direct transcytosis of cell-free virus from the apical to basolateral surfaces. Further, this transport of virus can be blocked through the addition of antibodies or peptides that directly block the interaction of gp340 with the HIV-1 envelope, if added prior to viral pulsing on the apical side of the cell or tissue barrier. Our data support a role for the previously described heparan sulfate moieties in mediating this transcytosis but add gp340 as an important facilitator of HIV-1 transcytosis across genital tract tissue. This study demonstrates that HIV-1 actively traverses the protective barriers of the human genital tract and presents a second mechanism whereby gp340 can promote heterosexual transmission.Through correlative studies with macaques challenged with simian immunodeficiency virus (SIV), the initial targets of infection in nontraumatic vaginal exposure to human immunodeficiency virus type 1 (HIV-1) have been identified as subepithelial T cells and dendritic cells (DCs) (18, 23, 31, 36-38). While human transmission may differ from macaque transmission, the existing models of human transmission remain controversial. For the virus to successfully reach its CD4⁺ targets, HIV must first traverse the columnar mucosal epithelial cell barrier of the endocervix or uterus or the stratified squamous barrier of the vagina or ectocervix, whose normal functions include protection of underlying tissue from pathogens. This portion of the human innate immune defense system represents a significant impediment to transmission. Studies have placed the natural transmission rate of HIV per sexual act between 0.005 and 0.3% (17, 45). Breaks in the epithelial barrier caused by secondary infection with other sexual transmitted diseases or the normal physical trauma often associated with vaginal intercourse represent one potential means for viral exposure to submucosal cells and have been shown to significantly increase transmission (reviewed in reference 11). However, studies of nontraumatic exposure to SIV in macaques demonstrate that these disruptions are not necessary for successful transmission to healthy females. This disparity indicates that multiple mechanisms by which HIV-1 can pass through mucosal epithelium might exist in vivo. Identifying these mechanisms represents an important obstacle to understanding and ultimately preventing HIV transmission.Several host cellular receptors, including DC-specific intercellular adhesion molecule-grabbing integrin, galactosyl ceramide, mannose receptor, langerin, heparan sulfate proteoglycans (HSPGs), and chondroitin sulfate proteoglycans, have been identified that facilitate disease progression through binding of HIV virions without being required for fusion and infection (2, 3, 12, 14, 16, 25, 29, 30, 43, 46, 50). These host accessory proteins act predominately through glycosylation-based interactions between HIV envelope (Env) and the host cellular receptors. These different host accessory factors can lead to increased infectivity in cis and trans or can serve to concentrate and expose virus at sites relevant to furthering its spread within the body. The direct transcytosis of cell-free virus through primary genital epithelial cells and the human endometrial carcinoma cell line HEC1A has been described (7, 9); this is, in part, mediated by HSPGs (7). Within the HSPG family, the syndecans have been previously shown to facilitate trans infection of HIV in vitro through binding of a specific region of Env that is moderately conserved (7, 8). This report also demonstrates that while HSPGs mediate a portion of the viral transcytosis that occurs in these two cell types, a significant portion of the observed transport occurs through an HSPG-independent mechanism. Other host cell factors likely provide alternatives to HSPGs for HIV-1 to use in subverting the mucosal epithelial barrier.gp340 is a member of the scavenger receptor cysteine-rich (SRCR) family of innate immune receptors. Its numerous splice variants can be found as a secreted component of human saliva (34, 41, 42) and as a membrane-associated receptor in a large number of epithelial cell lineages (22, 32, 40). Its normal cellular function includes immune surveillance of bacteria (4-6, 44), interaction with influenza A virus (19, 20, 32, 51) and surfactant proteins in the lung (20, 22, 33), and facilitating epithelial cell regeneration at sites of cellular inflammation and damage (27, 32). The secreted form of gp340, salivary agglutinin (SAG), was identified as a component of saliva that inhibits HIV-1 transmission in the oral pharynx through a specific interaction with the viral envelope protein that serves to agglutinate the virus and target it for degradation (34, 35, 41). Interestingly, SAG was demonstrated to form a direct protein-protein interaction with HIV Env (53, 54). Later, a cell surface-associated variant of SAG called gp340 was characterized as a binding partner for HIV-1 in the female genital tract that could facilitate virus transmission to susceptible targets of infection (47) and as a macrophage-expressed enhancer of infection (10).     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.