The VP3 Protein of Bluetongue Virus Associates with the MAVS Complex and Interferes with the RIG-I-Signaling Pathway

Bluetongue virus (BTV), an arbovirus transmitted by Culicoides biting midges, is a major concern of wild and domestic ruminants. While BTV induces type I interferon (alpha/beta interferon [IFN-α/β]) production in infected cells, several reports have described evasion strategies elaborated by this virus to dampen this intrinsic, innate response. In the present study, we suggest that BTV VP3 is a new viral antagonist of the IFN-β synthesis. Indeed, using split luciferase and coprecipitation assays, we report an interaction between VP3 and both the mitochondrial adapter protein MAVS and the IRF3-kinase IKKε. Overall, this study describes a putative role for the BTV structural protein VP3 in the control of the antiviral response.     

Molecular Docking and Virtual Screening of an Influenza Virus Inhibitor That Disrupts Protein–Protein Interactions

Influenza is an acute respiratory infection caused by the influenza virus, but few drugs are available for its treatment. Consequently, researchers have been engaged in efforts to discover new antiviral mechanisms that can lay the foundation for novel anti-influenza drugs. The viral RNA-dependent RNA polymerase (RdRp) is an enzyme that plays an indispensable role in the viral infection process, which is directly linked to the survival of the virus. Methods of inhibiting PB1–PB2 (basic polymerase 1–basic polymerase 2) interactions, which are a key part of RdRp enzyme activity, are integral in the design of novel antiviral drugs, a specific PB1–PB2 interactions inhibitor has not been reported. We have screened Enamine’s database and conducted a parallel screening of multiple docking schemes, followed by simulations of molecular dynamics to determine the structure of a stable ligand—PB1 complex. We also calculated the free energy of binding between the screened compounds and PB1 protein. Ultimately, we screened and identified a potential PB1–PB2 inhibitor using the ADMET prediction model.     

Cellular PSMB4 Protein Suppresses Influenza A Virus Replication through Targeting NS1 Protein

The nonstructural protein 1 (NS1) of influenza A virus (IAV) possesses multiple functions, such as the inhibition of the host antiviral immune responses, to facilitate viral infection. To search for cellular proteins interacting with the IAV NS1 protein, the yeast two-hybrid system was adopted. Proteasome family member PSMB4 (proteasome subunit beta type 4) was found to interact with the NS1 protein in this screening experiment. The binding domains of these two proteins were also determined using this system. The physical interactions between the NS1 and cellular PSMB4 proteins were further confirmed by co-immunoprecipitation assay and confocal microscopy in mammalian cells. Neither transiently nor stably expressed NS1 protein affected the PSMB4 expression in cells. In contrast, PSMB4 reduced the NS1 protein expression level, especially in the presence of MG132. As expected, the functions of the NS1 protein, such as inhibition of interferon activity and enhancement of transient gene expression, were suppressed by PSMB4. PSMB4 knockdown enhances IAV replication, while its overexpression attenuates IAV replication. Thus, the results of this study suggest that the cellular PSMB4 protein interacts with and possibly facilitates the degradation of the NS1 protein, which in turn suppresses IAV replication.     

Identification of RSV Fusion Protein Interaction Domains on the Virus Receptor,Nucleolin

Nucleolin is an essential cellular receptor to human respiratory syncytial virus (RSV). Pharmacological targeting of the nucleolin RNA binding domain RBD1,2 can inhibit RSV infections in vitro and in vivo; however, the site(s) on RBD1,2 which interact with RSV are not known. We undertook a series of experiments designed to: document RSV-nucleolin co-localization on the surface of polarized MDCK cells using immunogold electron microscopy, to identify domains on nucleolin that physically interact with RSV using biochemical methods and determine their biological effects on RSV infection in vitro, and to carry out structural analysis toward informing future RSV drug development. Results of immunogold transmission and scanning electron microscopy showed RSV-nucleolin co-localization on the cell surface, as would be expected for a viral receptor. RSV, through its fusion protein (RSV-F), physically interacts with RBD1,2 and these interactions can be competitively inhibited by treatment with Palivizumab or recombinant RBD1,2. Treatment with synthetic peptides derived from two 12-mer domains of RBD1,2 inhibited RSV infection in vitro, with structural analysis suggesting these domains are potentially feasible for targeting in drug development. In conclusion, the identification and characterization of domains of nucleolin that interact with RSV provide the essential groundwork toward informing design of novel nucleolin-targeting compounds in RSV drug development.     

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T₃₇/T₄₆, followed by T₇₀ and S₆₅. Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, ⁵⁴YXXXXLΦ⁶⁰ (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m⁷GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP1_50–84). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S₆₅ and T₇₀). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1–mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.Translation control of gene expression allows cells to respond quickly to external cues. In eukaryotic cells, this regulation occurs mainly at the translation initiation step (reviewed in ref. 1). Cellular eukaryotic mRNAs have a cap structure at their 5′ terminus, which is a modified nucleotide (7-methylguanosine triphosphate, m⁷GpppN, where N is any nucleotide) (2). The translational preinitiation complex assembles at the m⁷GpppN cap via the translation initiation complex 4F (eIF4F) (3), which comprises a cap-binding protein, eIF4E, a DEAD-Box RNA helicase, eIF4A, and a large scaffold protein, eIF4G. The scaffold protein eIF4G interacts with eIF4E through a consensus motif, YXXXXLΦ, where X is any amino acid and Φ is a hydrophobic residue. This motif is also shared by eIF4E binding proteins (4E-BPs). The interaction between eIF4E and 4E-BP is phosphorylation-dependent (4–8). When hypophosphorylated, 4E-BP binds tightly to eIF4E. Hyperphosphorylation of 4E-BP, however, decreases its affinity for eIF4E, enabling eIF4G to interact with eIF4E.Altered regulation of translation initiation has been linked to prion formation (9) and to several human diseases, including autism (10) and cancer (11). eIF4E is overexpressed in a variety of tumor cells (12, 13). This overexpression has been implicated in oncogenic transformation (14, 15), a process that 4E-BPs can effectively revert (14–16). Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin and its analogs, exert antitumor activity by suppressing 4E-BP1’s phosphorylation, thus enabling its interaction with eIF4E (17). The ability of 4E-BPs to compete with eIF4G for eIF4E binding is explained by the shared YXXXXLΦ binding motif (18, 19). Crystal structures of mouse eIF4E complexed with either 4E-BP1_51–64, eIF4G-I_569–580, or eIF4G-II_621–637, all short fragments containing the consensus-binding motif, are virtually identical (19–22). NMR spectroscopy titration experiments (23) and small angle X-ray scattering of full-length 4E-BP1 bound to eIF4E (24) suggested that 4E-BP1 has a larger binding interface on eIF4E than eIF4G. In agreement with this observation, mutagenesis analysis and affinity binding measurements showed that the C-terminal segment of 4E-BPs is auxiliary for binding to eIF4E (25, 26). More recently, a conserved ⁷⁹PGVTS/T⁸³ motif found in the C terminus of 4E-BPs was shown to enhance its binding affinity to eIF4E from micromolar to nanomolar range (27, 28), revealing that 4E-BP1 has, in fact, a bipartite binding interface with eIF4E.Our group has identified a small-molecule inhibitor, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), which specifically disrupts association of eIF4G-derived peptides with eIF4E but stabilizes the eIF4E/4E-BP1 interaction (29). 4EGI-1 is of particular interest because it inhibits cap-dependent translation, is active against numerous cancer cell lines, and reduces growth of human cancer xenografts in vivo (29–31). Its effect is partially explained by the recent crystal structure of an eIF4E/4EGI-1 complex, in which the inhibitor binds to a site located remotely from the YXXXXLΦ binding interface, suggesting that it allosterically represses translation initiation (32). However, the mechanism by which 4EGI-1 stabilized 4E-BP1 binding remains unclear.In this study, we describe a crystal structure of eIF4E bound to a 35-residue fragment of 4E-BP1. This fragment comprises the consensus-binding motif and also a proline-turn-helix segment containing two phosphorylation sites (S₆₅ and T₇₀) followed by a loop of irregular structure. We find that the C-terminal loop of 4E-BP1 partially overlaps with the binding site of 4EGI-1, which enables us to understand the molecular mechanism through which 4EGI-1 inhibits translation initiation: by dissociating eIF4G from eIF4E but also stabilizing the interaction between eIF4E and the unphosphorylated form of 4E-BP1. We further find that the C-terminal loop of 4E-BP1 is required to inhibit cap-dependent translation and mediates cell cycle arrest in mammalian cells.     

Discovery of New Small Molecule Hits as Hepatitis B Virus Capsid Assembly Modulators: Structure and Pharmacophore-Based Approaches

Hepatitis B virus (HBV) capsid assembly modulators (CpAMs) have shown promise as potent anti-HBV agents in both preclinical and clinical studies. Herein, we report our efforts in identifying novel CpAM hits via a structure-based virtual screening against a small molecule protein-protein interaction (PPI) library, and pharmacophore-guided compound design and synthesis. Curated compounds were first assessed in a thermal shift assay (TSA), and the TSA hits were further evaluated in an antiviral assay. These efforts led to the discovery of two structurally distinct scaffolds, ZW-1841 and ZW-1847, as novel HBV CpAM hits, both inhibiting HBV in single-digit µM concentrations without cytotoxicity at 100 µM. In ADME assays, both hits displayed extraordinary plasma and microsomal stability. Molecular modeling suggests that these hits bind to the Cp dimer interfaces in a mode well aligned with known CpAMs.     

Amino Terminal Region of Dengue Virus NS4A Cytosolic Domain Binds to Highly Curved Liposomes

Dengue virus (DENV) is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. Non-structural protein 4A (NS4A) is a vital component of the viral replication complex (RC) and plays a major role in the formation of host cell membrane-derived structures that provide a scaffold for replication. The N-terminal cytoplasmic region of NS4A(1–48) is known to preferentially interact with highly curved membranes. Here, we provide experimental evidence for the stable binding of NS4A(1–48) to small liposomes using a liposome floatation assay and identify the lipid binding sequence by NMR spectroscopy. Mutations L6E;M10E were previously shown to inhibit DENV replication and to interfere with the binding of NS4A(1–48) to small liposomes. Our results provide new details on the interaction of the N-terminal region of NS4A with membranes and will prompt studies of the functional relevance of the curvature sensitive membrane anchor at the N-terminus of NS4A.     

Foot-and-Mouth Disease Virus: Molecular Interplays with IFN Response and the Importance of the Model

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals with a significant socioeconomic impact. One of the issues related to this disease is the ability of its etiological agent, foot-and-mouth disease virus (FMDV), to persist in the organism of its hosts via underlying mechanisms that remain to be elucidated. The establishment of a virus–host equilibrium via protein–protein interactions could contribute to explaining these phenomena. FMDV has indeed developed numerous strategies to evade the immune response, especially the type I interferon response. Viral proteins target this innate antiviral response at different levels, ranging from blocking the detection of viral RNAs to inhibiting the expression of ISGs. The large diversity of impacts of these interactions must be considered in the light of the in vitro models that have been used to demonstrate them, some being sometimes far from biological systems. In this review, we have therefore listed the interactions between FMDV and the interferon response as exhaustively as possible, focusing on both their biological effect and the study models used.     

Na,K-ATPase α3 is a death target of Alzheimer patient amyloid-β assembly

Neurodegeneration correlates with Alzheimer’s disease (AD) symptoms, but the molecular identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na⁺/K⁺-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aβ–derived “thorns” responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn⁸⁷⁹ and Trp⁸⁸⁰ is essential for ASPD–NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD–NAKα3 interaction.Alzheimer’s disease (AD) brains characteristically display fibrillar and nonfibrillar (oligomeric) protein assemblies composed of the amyloid β-protein (Aβ) (1–6). Aβ has been shown to bind to postsynaptic receptors, such as α7-nicotinic acetylcholine receptor (α7nAChR) (7), receptor for advanced glycation end products (RAGE) (8), receptor tyrosine kinase EPHB2 (9), and cellular prion protein PrP^C (10). These “Aβ receptors,” except for RAGE, have been reported to mediate toxicity of Aβ oligomers through modulating NMDA receptors (NMDAR) (11). Aβ oligomers, including dimers from AD brains (12, 13), dodecamers (Aβ*56) from AD model mice (14), and in vitro-generated Aβ-derived diffusible ligands (ADDLs) (15, 16), induce synaptic impairment by affecting NMDAR (11). Thus, NMDAR are a common target for synaptic impairment in AD. However, these oligomers do not cause neuronal death (12, 14). The atomic resolution structures of neurotoxic Aβ oligomers and their in vivo targets leading to neuronal death in AD remain unclear (6), even though neuronal death is the central mechanism responsible for symptomatic onset in AD (17).We previously isolated neurotoxic Aβ oligomers, termed amylospheroids (ASPD), from the brains of AD patient (18–20). ASPD appear in transmission electron microscopic (TEM) images as spheres of diameter ∼11.9 ± 1.7 nm (19). ASPD appear to be unique Aβ assemblies, as determined immunochemically. These structures are recognized strongly by ASPD-specific antibodies (K_d ∼ pM range), but not with the oligomer-specific polyclonal antiserum A11 (19). ASPD are distinct from Aβ dimers, ADDLs, dodecamers, and other A11-reactive entities (19).ASPD cause severe degeneration of mature human neurons (19). ASPD levels in the cortices of AD patients correlate well with disease severity (19). In contrast, ASPD-like oligomers were minimally detectable in the brains of transgenic mice expressing human amyloid precursor protein (APP), in which no significant neuronal loss is observed (19). These findings suggest that ASPD are an important effector of neuronal death in AD patients. We sought to elucidate mechanisms of ASPD-induced neurotoxicity. We report here that ASPD interact with the α-subunit of neuron-specific Na⁺/K⁺-ATPase (NAKα3), resulting in presynaptic calcium overload and neuronal death.     

Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis

Although the pimeloyl moiety was long known to be a biotin precursor, the mechanism of assembly of this C7 α,ω-dicarboxylic acid was only recently elucidated. In Escherichia coli, pimelate is made by bypassing the strict specificity of the fatty acid synthetic pathway. BioC methylates the free carboxyl of a malonyl thioester, which replaces the usual acetyl thioester primer. This atypical primer is transformed to pimeloyl-acyl carrier protein (ACP) methyl ester by two cycles of fatty acid synthesis. The question is, what stops this product from undergoing further elongation? Although BioH readily cleaves this product in vitro, the enzyme is nonspecific, which made assignment of its physiological substrate problematical, especially because another enzyme, BioF, could also perform this gatekeeping function. We report the 2.05-Å resolution cocrystal structure of a complex of BioH with pimeloyl-ACP methyl ester and use the structure to demonstrate that BioH is the gatekeeper and its physiological substrate is pimeloyl-ACP methyl ester.     

Pepducin targeting the C-X-C chemokine receptor type 4 acts as a biased agonist favoring activation of the inhibitory G protein

Short lipidated peptide sequences derived from various intracellular loop regions of G protein-coupled receptors (GPCRs) are named pepducins and act as allosteric modulators of a number of GPCRs. Recently, a pepducin selectively targeting the C-X-C chemokine receptor type 4 (CXCR4) was found to be an allosteric agonist, active in both cell-based assays and in vivo. However, the precise mechanism of action of this class of ligands remains poorly understood. In particular, given the diversity of signaling effectors that can be engaged by a given receptor, it is not clear whether pepducins can show biased signaling leading to functional selectivity. To explore the ligand-biased potential of pepducins, we assessed the effect of the CXCR4 selective pepducin, ATI-2341, on the ability of the receptor to engage the inhibitory G proteins (Gi1, Gi2 and Gi3), G13, and β-arrestins. Using bioluminescence resonance energy transfer-based biosensors, we found that, in contrast to the natural CXCR4 ligand, stromal cell-derived factor-1α, which promotes the engagement of the three Gi subtypes, G13 and the two β-arrestins, ATI-2341 leads to the engagement of the Gi subtypes but not G13 or the β-arrestins. Calculation of the transduction ratio for each pathway revealed a strong negative bias of ATI-2341 toward G13 and β-arrestins, revealing functional selectivity for the Gi pathways. The negative bias toward β-arrestins results from the reduced ability of the pepducin to promote GPCR kinase-mediated phosphorylation of the receptor. In addition to revealing ligand-biased signaling of pepducins, these findings shed some light on the mechanism of action of a unique class of allosteric regulators.Pepducins represent a class of molecules that regulate the activity of G protein-coupled receptors (GPCRs). These lipid-modified peptides are derived from the amino acid sequences of one of the four intracellular loops of a target GPCR (1). Although the precise mode of action is not completely understood, it is believed that pepducins bind to their target receptors and allosterically modulate their signaling activity (2, 3). Pepducins have been identified for several GPCRs, including the protease-activated receptors PAR1 (1, 4–10), PAR2 (1, 11–13), and PAR4 (6, 9), the formyl peptide receptor-2 (14), the melanocortin type 4 receptor (1), the sphingosine 1-phosphate receptor 3 (15), and the C-X-C chemokine receptor type 1, 2 (CXCR1, CXCR2) (16), and 4 (CXCR4) (2, 17, 18). They have been found to act as allosteric agonists as well as negative or positive allosteric modulators. However, in most cases, their activity was assessed for only one or a few signaling pathways engaged by the receptors.One of the receptors for which pepducins were developed is CXCR4. This receptor, expressed in many tissues including hematopoietic and circulating cells, is a coreceptor for the entry of HIV (19, 20) and controls many physiological functions, including cell migration, mobilization, and retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem cells, as well as progenitor cells (HSPCs) in the bone marrow niche (21–23). It has also been found to play an important role in tumor progression, angiogenesis, and metastasis of a variety of cancers (24–26). A CXCR4 antagonist, AMD-3100 (Mozobil), is used to mobilize HSPCs from the bone marrow for transplantation of leukemic patients (27).A pepducin derived from the first intracellular loop of CXCR4 (ATI-2341) was found to be an allosteric agonist on the chemotactic response elicited on a human T-lymphoblastic leukemia cell line (CCRF-CEM cells) that endogenously expresses CXCR4 (18). This activity of ATI-2341 was confirmed in fresh human PMNs as well as in vivo for their ability to promote the mobilization of PMNs and HSPCs in the peripheral circulation of both mice and monkeys (18). Of note, unlike the clinically used CXCR4 antagonist AMD-3100 and stromal cell-derived factor-1α (SDF-1; also known as CXCL12) that promote the mobilization of lymphocytes in addition to PMNs and HSPCs (18, 28–30), ATI-2341 was without effect on the mobilization of lymphocytes (18), indicating that ATI-2341 may display functional selectivity.When assessing the effect of ATI-2341 on the signaling activity of CXCR4, the pepducin was found to be an allosteric agonist, activating the inhibitory heterotrimeric G protein (Gi) to promote inhibition of cAMP production and induce calcium mobilization (18, 31). In recent years, many GPCRs have been shown to engage in promiscuous signaling activities involving more than one G protein subtype as well as G protein-independent signaling. More importantly, it was found that different ligands can selectively couple to a subset of the signaling pathways that can be engaged by a receptor. In some cases, a given ligand can even have opposite efficacies on two different signaling pathways: a concept known as “ligand-biased signaling” or “functional selectivity” (32–36). In addition to its coupling to Gi, CXCR4 has also been found to signal through the engagement and activation of G13 (37, 38) and β-arrestin2 (39, 40), both pathways being proposed to contribute to the chemotactic responses. These observations raise the question of whether the CXCR4-selective pepducin, ATI-2341, is an allosteric agonist on all of the signaling pathways identified for the chemokine receptor or whether it could show bias toward selective signaling pathways and thus be functionally selective.To determine whether pepducins can display functional selectivity on CXCR4 signaling at the molecular level, we took advantage of bioluminescence resonance energy transfer (BRET)-based assays that allow the direct monitoring of the engagement and activation of proximal signaling effectors. More specifically, we compared the ability of ATI-2341 and the natural agonist of the receptor, SDF-1, to promote the engagement/activation of three Gi family members (Gi₁, Gi₂, and Gi₃), G13, β-arrestin1, and β-arrestin2. We found that, whereas SDF-1 promotes the engagement of all of the signaling effectors, ATI-2341 selectively led to the functional engagement of the Gi family members and had no effect on G13 or the two β-arrestins. The lack of recruitment of β-arrestins results from the poor recruitment of G protein-coupled receptor kinases (GRKs) to the receptor because, in contrast to SDF-1 that stimulates the phosphorylation of CXCR4 by protein kinase C (PKC), GRK2/3, and GRK6, ATI-2341 promotes effective PKC-dependent phosphorylation of the receptor but minimal GRK2/3 recruitment, as well as minimal GRK6-dependent phosphorylation.Taken together, our results demonstrate that the pepducin ATI-2341 is a functionally selective allosteric regulator of CXCR4 that activates Gi-dependent pathways without modulating G13 and β-arrestin pathways. These data indicate that ATI-2341 could have physiological actions that may differ from the natural ligand SDF-1 and the clinically used AMD-3100, raising the intriguing possibility that ATI-2341 may have distinct clinical properties. These findings also shed some light on the mechanism of action of pepducins and indicates that, similar to orthosteric ligands, these allosteric regulators can be functionally selective.     

Construction of a Recombinant Japanese Encephalitis Virus with a Hemagglutinin-Tagged NS2A: A Model for an Analysis of Biological Characteristics and Functions of NS2A during Viral Infection

Nonstructural protein 2A (NS2A) of the Japanese encephalitis virus (JEV) contributes to viral replication and pathogenesis; however, a lack of NS2A-specific antibodies restricts studies on the underlying mechanisms. In this study, we constructed a recombinant JEV with a hemagglutinin (HA)-tagged NS2A (JEV-HA/NS2A/∆NS1’) to overcome this challenge. An HA-tag was fused to the N-terminus of NS2A (HA-NS2A) at the intergenic junction between NS1 and NS2A. A peptide linker, “FNG”, was added to the N-terminus of HA-tag to ensure correct cleavage between the C-terminus of NS1 and the N-terminus of HA-NS2A. To avoid the side effects of an unwanted NS1’ tagged with HA (HA-NS1’), an alanine-to-proline (A30P) substitution was introduced at residue 30 of NS2A to abolish HA-NS1’ production. The HA-tag insertion and A30P substitution were stably present in JEV-HA/NS2A/∆NS1’ after six passages and did not exhibit any significant effects on viral replication and plaque morphology. Taking advantage of HA-NS2A, we examined the activities of NS2A during JEV infection in vitro using anti-HA antibodies. NS2A was observed to be localized to the endoplasmic reticulum and interact with viral NS2B and NS3 during virus infection. These data suggest that JEV-HA/NS2A/∆NS1’ can serve as a model for the analysis of the biological characteristics and functions of NS2A in vitro during JEV infection.     

Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3

Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease (PD); however, the molecular mechanism and mode of action of this protein remain elusive. cAMP-dependent protein kinase (PKA), along with other kinases, has been suggested to be an upstream kinase regulating LRRK2 function. Using MS, we detected several sites phosphorylated by PKA, including phosphorylation sites within the Ras of complex proteins (ROC) GTPase domain as well as some previously described sites (S910 and S935). We systematically mapped those sites within LRRK2 and investigated their functional consequences. S1444 in the ROC domain was confirmed as a target for PKA phosphorylation using ROC single-domain constructs and through site-directed mutagenesis. Phosphorylation at S1444 is strikingly reduced in the major PD-related LRRK2 mutations R1441C/G/H, which are part of a consensus PKA recognition site (¹⁴⁴¹RASpS¹⁴⁴⁴). Furthermore, our work establishes S1444 as a PKA-regulated 14-3-3 docking site. Experiments of direct binding to the three 14-3-3 isotypes gamma, theta, and zeta with phosphopeptides encompassing pS910, pS935, or pS1444 demonstrated the highest affinities to phospho-S1444. Strikingly, 14-3-3 binding to phospho-S1444 decreased LRRK2 kinase activity in vitro. Moreover, substitution of S1444 by alanine or by introducing the mutations R1441C/G/H, abrogating PKA phosphorylation and 14-3-3 binding, resulted in increased LRRK2 kinase activity. In conclusion, these data clearly demonstrate that LRRK2 kinase activity is modulated by PKA-mediated binding of 14-3-3 to S1444 and suggest that 14-3-3 interaction with LRRK2 is hampered in R1441C/G/H-mediated PD pathogenesis.Parkinson disease (PD), one of the most prevalent neurodegenerative afflictions, is characterized pathologically by the selective loss of dopaminergic neurons in the midbrain and by the presence of intracellular inclusions in the remaining cells, termed Lewy bodies (1). However, the molecular mechanisms underlying the complex pathological process are poorly understood. Many genetic and environmental factors contribute to the disease, and mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial PD. LRRK2 is a large protein of 285 kDa and encodes several structural motifs, such as armadillo, ankyrin, and the namesake leucine-rich repeats, a Ras of complex proteins (ROC) GTPase, a C-terminal of ROC (COR), a kinase domain [with sequence homology to MAP kinase kinase kinase (MAPKKKs)], and a C-terminal WD40 domain (2). Notably, mutations known to cause PD are located within the catalytically active GTPase (ROC) and kinase domains of LRRK2 (see Fig. 3A) (3). Particularly for a single residue located within the ROC domain, three independent PD-associated mutations (R1441C, R1441G, and R1441H) have been found (4), whereas the kinase domain may harbor the most frequent pathogenic mutation, G2019S. Mutations at both these sites have been associated with enhanced kinase activity compared with wild type (5, 6), suggesting that dysregulation of these enzymatic activities may contribute to PD pathogenesis.Open in a separate windowFig. 3.S1444 on LRRK2 is a PKA-induced 14-3-3 binding site. (A) Multidomain structure of LRRK2. ANK, ankyrin repeat region; LRR, leucine-rich repeat domain; ROC, Ras of complex (GTPase); COR, C-terminal of ROC. The potential 14-3-3 interaction motif in LRRK2 is shown and aligned to a mode I 14-3-3 binding consensus sequence. Known pathogenic mutations R1441C/G/H and G2019S are indicated. (B) LRRK2 pull-down with recombinant GST–14-3-3 gamma. LRRK2 WT and LRRK2 WT ∆967 were expressed in Sf9 cells. Lysates were incubated with GST–14-3-3 gamma for 4 h. GST–14-3-3 gamma pulled down both LRRK2 WT full length and LRRK2 WT ∆967. Samples were separated on a 4–12% gradient SDS gel, and the membranes were probed with Strep-Tactin HRP or GST antibody. (C) LRRK2 WT ∆967 was precipitated with GST–14-3-3-agarose in the absence or in the presence of increasing concentrations of chemically synthesized LRRK2 peptides (LFNIKARASSSPVILVGT) phosphorylated or nonphosphorylated at S1444. Sample separation and Western blotting were carried out as described under B. (D) Two micrograms of His-ROC WT, S1443A, S1444A, or S1443A-S1444A mutant proteins was incubated in the presence or absence of PKA for 1 h at 30 °C. Far western blotting was performed using GST–14-3-3 protein as a probe. PKA-induced ROC–GST–14-3-3 interaction could be observed only when S1444 was present. Equal loading was demonstrated using anti-His antibody.LRRK2 is a cytosolic phosphoprotein (7) phosphorylated in vitro by a variety of serine/threonine kinases, including PKC zeta (8), serine protein kinase ataxia telangiectasia mutated (9), the IκB kinase family (10), and cAMP-dependent protein kinase (PKA) (11, 12). PKA is a key regulator of a vast number of signaling molecules and is critical for neuronal functions such as synaptic plasticity, protein trafficking, protein degradation, neuronal excitability, and regulation of dopamine physiology (13–17). In LRKK2, the conserved residue (S935) was shown to be phosphorylated by PKA (12), and recently this site was proposed as a biomarker for LRRK2 activity (10, 18, 19). Phosphorylation of S910 and S935 within LRRK2 promotes binding of 14-3-3 proteins (19), a family of small 29–30 kDa acidic regulatory proteins, highly conserved and ubiquitously expressed in various tissues. The binding of 14-3-3 proteins results in various downstream effects, such as changes in structural conformations, kinase activity, and subcellular localization of the target proteins (20, 21). Nichols et al. (19) proposed a role for 14-3-3 proteins in regulating the cytoplasmic localization of LRRK2, whereas Li et al. (12) observed protection from dephosphorylation of S935 after 14-3-3 binding. Furthermore, recent data from Fraser et al. (22) suggest a regulatory function of 14-3-3 binding in controlling extracellular release of LRRK2. Dysregulation of 14-3-3/client protein interaction has been shown to facilitate the development of several human disorders (23, 24), and an influence of the pathogenic mutation R1441G on LRRK2/14-3-3 interaction has been demonstrated (12, 19). Although these data support an involvement of PKA and 14-3-3 proteins in regulating LRRK2 function, LRRK2 phosphorylation by PKA, as well as 14-3-3 binding and possible (patho)physiological consequences of this interplay, have not yet been addressed in detail.Here, we systematically mapped PKA phosphorylation sites in LRRK2 and further investigated the impact of this phosphorylation in terms of 14-3-3 binding and LRRK2 function. Our results predict an essential function for PKA phosphorylation and subsequent 14-3-3 interaction in the negative regulation of LRRK2 kinase activity.     

The Tn7 transposition regulator TnsC interacts with the transposase subunit TnsB and target selector TnsD

The excision of transposon Tn7 from a donor site and its insertion into its preferred target site, attachment site attTn7, is mediated by four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC, and TnsD. Transposition requires the assembly of a nucleoprotein complex containing all four Tns proteins and the DNA substrates, the donor site containing Tn7, and the preferred target site attTn7. TnsA and TnsB together form the heteromeric Tn7 transposase, and TnsD is a target-selecting protein that binds specifically to attTn7. TnsC is the key regulator of transposition, interacting with both the TnsAB transposase and TnsD-attTn7. We show here that TnsC interacts directly with TnsB, and identify the specific region of TnsC involved in the TnsB–TnsC interaction during transposition. We also show that a TnsC mutant defective in interaction with TnsB is defective for Tn7 transposition both in vitro and in vivo. Tn7 displays cis-acting target immunity, which blocks Tn7 insertion into a target DNA that already contains Tn7. We provide evidence that the direct TnsB–TnsC interaction that we have identified also mediates cis-acting Tn7 target immunity. We also show that TnsC interacts directly with the target selector protein TnsD.In DNA cut-and-paste transposition, the transposon is excised from the donor site and then integrated into a new insertion site. These reactions are mediated by nucleoprotein complexes called transpososomes (1, 2). Understanding the mechanism and regulation of transposition requires identification of the protein–DNA and protein–protein interactions that underlie transpososome assembly and activity.The transpososomes that promote transposition of the bacterial transposon Tn7 are particularly elaborate. Tn7 encodes five transposition proteins—TnsA, TnsB, TnsC, TnsD, and TnsE—that mediate transposition to two classes of target sites (3–5). TnsABC+D promote target site-specific insertion of Tn7 into its preferred chromosomal target site attTn7 (3), whereas TnsABC+E promote Tn7 insertion into non-attTn7 sites on conjugal plasmids (6, 7). Thus, TnsABC form the core of the transposition machinery. This viewpoint is reinforced by the finding that although WT TnsABC alone do not promote transposition, transposition with TnsABC can occur when TnsC is activated by gain-of-function mutations that allow TnsC target binding and transposase activation in the absence of TnsD or TnsE (8, 9). In contrast, other characterized transposition systems involve only one or two transposition proteins (10).Tn7 transposition also requires ATP (11), given that TnsC is an ATP-dependent DNA-binding protein (12) and an ATPase (9). It should be noted that ATP is not required for the chemical steps of transposition (13); rather, it regulates assembly of the Tn7 transpososomes (14). TnsC contains AAA+ ATPase motifs (15). Bacteriophage Mu, whose breakage and joining reactions are performed by the transposase MuA, also uses the ATP-dependent target-binding protein MuB to regulate transposition (16, 17).Our work is focused on the TnsABC+D system. All of these Tns proteins, as well as the DNA substrates for transposition (i.e., the Tn7 ends and the attTn7 target DNA), must assemble into the elaborate transpososome in which transposition actually occurs (11, 18). The targeting protein TnsD binds to a specific sequence in attTn7 (11, 19). Subsequent TnsC recruitment involves both TnsC–DNA interactions that depend on TnsD-induced distortions in attTn7 (20, 21) and likely TnsC–TnsD interactions that we previously detected using yeast two-hybrid assays (19). Although we have shown that a TnsCD-attTn7 complex can form (20), TnsC also may be recruited to attTn7 as part of a TnsA-TnsC complex, because TnsA and TnsC can copurify as a TnsA₂C₂ heterotetramer (22, 23). The stoichiometery of the Tns proteins in the target complex remains to be directly established, but we have observed an TnsACD-attTn7 complex (23). Analysis of the stoichiometery of the Tns proteins in a posttransposition complex has revealed the likely presence of multiple TnsA₂C₂s in this complex (23).TnsA and TnsB together form the Tn7 transposase (13). TnsB is a sequence-specific DNA-binding protein that binds to multiple sites at both transposon ends (24, 25). TnsB also mediates DNA breakage and joining at the 3′ ends of the transposon and is a member of the RNase H transposase-retroviral integrase superfamily (26). TnsA is a nuclease that mediates cleavage at the 5′ ends of the transposon (27, 28). Although TnsA lacks specific DNA-binding activity, it is positioned at the transposon ends by interaction with TnsB (29). Thus, TnsA and TnsB collaborate to excise Tn7 from the donor site and insert it into a target site. Notably, TnsA and TnsB are interdependent, with breakage and joining occurring only in the presence of both proteins, even when their catalytic activity has been abolished by mutations in their active sites (26, 27).TnsA and TnsB do not promote breakage and joining in the absence of TnsC, however. How does TnsC activate the TnsAB transposase? As noted above, TnsA and TnsC can interact directly, forming a TnsA₂C₂ heterotetramer. The structure of a cocrystal of TnsA/TnsC_504–555 has been solved (22), locating the TnsC region that interacts with TnsA to the very C-terminal region of TnsC. The resulting model for TnsA–TnsC_504–555 interaction also led to the proposal that the TnsC_495–501 region, which is lysine-rich, may play a significant role in interacting with the donor DNA near the transposon end. Such an interaction could be the basis for the stabilization of transpososomes by the presence of TnsA and in its role in stabilizing the TnsACD-target complex (18, 23, 30).We have previously suggested that the transposition regulator TnsC and the transposase subunit TnsB interact (31), but a direct TnsB–TnsC interaction has not yet been demonstrated. Support for the view that TnsB and TnsC interact is that TnsB bound to a Tn7 end-containing DNA and a TnsD- and TnsE-independent gain-of-function TnsC mutant, TnsC^A225V, bound to a target DNA can form a donor-target complex in the presence of the crosslinker glutaraldehyde (18).Other observations suggesting a TnsB–TnsC interaction come from the study of Tn7 cis-acting target immunity, i.e., the process that inhibits the insertion of Tn7 into a target DNA that already contains Tn7 ends (31, 32). A target DNA already containing Tn7 ends is immune to Tn7 insertion because TnsB can bind to the Tn7 ends, leading to an increase in the local concentration of TnsB. This increase in TnsB concentration on the target DNA results in ATP hydrolysis by TnsC that attempts to bind to the target DNA, thereby clearing TnsC from that potential target DNA (30, 31). Thus, the key step in Tn7 target immunity is TnsB-induced inhibition of the binding of TnsC to the Tn7-containing target DNA, blocking the formation of a stable TnsC-target complex (30, 31). We have visualized this TnsB-induced dissociation of TnsC from attTn7 by analysis of Tns-attTn7 complexes by EMSAs (30). Such target immunity established by transposase-induced ATP-dependent dissociation of the target-binding protein from a target DNA containing the Mu ends occurs in the Mu system as well (33).Thus, several lines of evidence support the view that TnsB interacts with TnsC; however, direct demonstration of this point has not yet been reported.What regions of TnsB and TnsC might interact? We previously suggested that the region of TnsB that interacts with TnsC lies at the C terminus of TnsB. We isolated mutants of TnsB that have reduced effectiveness of transposition immunity (30). These “immunity bypass” mutants are located in the C-terminal region of TnsB at TnsB^P686S, TnsB^V689M, and TnsB^P690L, TnsB being 702-aa long. We demonstrated that these TnsB mutants have reduced ability to promote dissociation of the TnsCD-attTn7 target complex in vitro, consistent with the view that the region of TnsB in which the immunity bypass mutations are located interacts with TnsC (30). Moreover, whereas short C-terminal TnsB WT peptides can promote TnsCD-attTn7 complex dissociation, such C-terminal TnsB peptides from immunity bypass mutants do not promote target complex dissociation (30). Here we identify a region of TnsC that is critical to TnsB–TnsC interaction using a photocrosslinking assay and analysis of the effects of TnsC mutants on TnsB-dependent transposition activities.TnsC also plays a key role in target site selection, being recruited to the TnsD-attTn7 complex to form a TnsCD-attTn7 complex (11, 20). TnsD binds specifically to attTn7, thereby identifying this preferred target site (11, 19). A key step in TnsC recruitment is the interaction of TnsC with TnsD-induced distortions in attTn7 (20, 21). We previously detected TnsC–TnsD interactions using a yeast two-hybrid assay (19). In the present work, we used affinity chromatography to show that TnsC also interacts directly with the attTn7-binding protein TnsD. Thus, TnsC is central to transposition and participates in multiple protein–protein and protein–DNA interactions.     

NSvc4 Encoded by Rice Stripe Virus Targets Host Chloroplasts to Suppress Chloroplast-Mediated Defense

Our previous research found that NSvc4, the movement protein of rice stripe virus (RSV), could localize to the actin filaments, endoplasmic reticulum, plasmodesmata, and chloroplast, but the roles of NSvc4 played in the chloroplast were opaque. Here, we confirm the accumulation of NSvc4 in the chloroplasts and the N-terminal 1–73 amino acids of NSvc4 are sufficient to localize to chloroplasts. We provide evidence to show that chloroplast-localized NSvc4 can impair the chloroplast-mediated immunity. Expressing NSvc4 in Nicotiana benthamiana leaves results in the decreased expression of defense-related genes NbPR1, NbPR2, and NbWRKY12 and the inhibition of chloroplast-derived ROS production. In addition, generation of an infectious clone of potato virus X (PVX) carrying NSvc4 facilitates PVX infection in N. benthamiana plants. Moreover, we identify two chloroplast-related host factors, named NbGAPDH-A and NbPsbQ1, both of which can interact with NSvc4. Knockdown of NbGAPDH-A or NbPsbQ1 can both promote RSV infection. Our results decipher a detailed function of NSvc4 in the chloroplast.