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Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ~930 Å2 surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.
Author Summary
A novel coronavirus emerged in 2003 and was identified as the etiological agent of the deadly disease called Severe Acute Respiratory Syndrome. This coronavirus replicates and transcribes its giant genome using sixteen non-structural proteins (nsp1-16). Viral RNAs are capped to ensure stability, efficient translation, and evading the innate immunity system of the host cell. The nsp16 protein is a RNA cap modifying enzyme only active in the presence of its activating partner nsp10. We have crystallized the nsp10/16 complex and report its crystal structure at atomic resolution. Nsp10 binds to nsp16 through a ~930 Å2 activation surface area in nsp10, and the resulting complex exhibits RNA cap (nucleoside-2′-O)-methyltransferase activity. We have performed mutational and functional assays to identify key residues involved in catalysis and/or in RNA binding, and in the association of nsp10 to nsp16. We present two additional crystal structures, that of the known inhibitor Sinefungin bound in the SAM binding pocket, and that of a tighter complex made of the mutant nsp10(Y96F) bound to nsp16. Our study provides a basis for antiviral drug design as well as the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.
Citation: Decroly E, Debarnot C, Ferron F, Bouvet M, Coutard B, et al. (2011) Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex. PLoS Pathog 7(5): e1002059. doi:10.1371/journal.ppat.1002059
Editor: Félix A. Rey, Institut Pasteur, France
Received: December 7, 2010; Accepted: March 23, 2011; Published: May 26, 2011
Copyright: © 2011 Decroly et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported, in its initial phase, by the Euro-Asian SARS-DTV Network (SP22-CT-2004-511064) from the European Commission specific research and technological development Programme "Integrating and strengthening the European Research area." It was subsequently supported by the VIZIER integrated project (LSHG-CT-2004-511960) of the European Union 6th Framework and the French National Research agency, under reference "ANR-08-MIEN-032," and subsequently the Fondation pour la Recherche Médicale (Programme Équipe FRM), and the Direction Générale de l'Armement (contrat n°07co404). MB has a fellowship from the Direction Générale de l'Armement. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Most eukaryotic cellular and viral mRNAs are modified by the addition of a polyadenine tail at the 3′- terminal and a cap structure at the 5′-terminal. The RNA cap protects mRNA from degradation by 5′ exoribonucleases, ensures efficient mRNA translation, and prevents recognition of viral RNA via innate immunity mechanisms[1], [2], [3], [4]. The RNA cap is made of an N7-methylated guanine nucleotide connected through a 5′-5′ triphosphate bridge to the first transcribed nucleotide, generally an adenine. Through 2′-O methylation of the latter, this cap-0 structure (7MeGpppA…) may be converted into a cap-1 structure (7MeGpppA2′-O-Me…). In the eukaryotic cell, the cap is added co-transcriptionally in the nucleus by three sequential enzymatic reactions[1], [5]: (i) an RNA triphosphatase (RTPase) removes the 5′ γ-phosphate group of the nascent mRNA; (ii) a guanylyltransferase (GTase), dubbed capping enzyme, catalyses the attachment of GMP to the 5′-diphosphate mRNA; and (iii) an S-adenosylmethionine (SAM)-dependent (N7-guanine)-methyltransferase (N7MTase) methylates the cap onto the N7-guanine, releasing S-adenosylhomocysteine (SAH). In general, a SAM-dependent (nucleoside-2′-O-)-methyltransferase (2′-O-MTase) further intervenes, in higher eukaryotes, to yield a cap-1 structure.
The viral RNA capping machinery is structurally and mechanistically diverse, and RNA viruses often deviate from the paradigmic eukaryotic mRNA capping scheme. For example, alphaviruses methylate GTP onto the N7-guanine before the presumed attachment of 7MeGMP to the nascent viral 5′-diphosphate mRNA[6]. In the case of single-stranded negative-sense (-)RNA viruses, such as the vesicular stomatitis virus, the L polymerase attaches GDP rather than GMP to a nascent viral 5′-monophosphate mRNA, covalently linked to the viral capping enzyme[7]. Other viruses, such as influenza virus capture a short capped RNA oligonucleotide from host cell mRNAs and use it as an RNA synthesis primer. This process is known as « cap snatching »[8].
In 2003, a novel coronavirus named Severe Acute Respiratory Syndrome coronavirus (SARS-CoV[9]) was responsible for the first viral pandemic of the new millennium with ~8000 cases globally and a 10 % case-fatality rate. Coronaviruses encode an unusually large membrane-associated RNA replication/transcription machinery comprising at least sixteen proteins (nsp1-to-16)[10]. For SARS-CoV, the RNA cap structure likely corresponds to a cap-1 type[11], [12], [13]. As in many other (+)RNA viruses, the RTPase activity is presumably embedded in the RNA helicase nsp13, whereas the GTase remains elusive. RNA cap 2′-O-MTase activity was first discovered in the feline coronavirus (FCoV) nsp16[14]. Shortly after, SARS-CoV nsp14 was shown to methylate RNA caps in their N7-guanine position[15]. Curiously, although closely homologous to that of FCoV, recombinant SARS-CoV nsp16 alone was devoid of enzymatic activity. It was demonstrated[16], [17], [18], [19] that nsp10 interacts with nsp16, conferring 2′-O-MTase activity to nsp16 on N7-methyl guanine RNA caps selectively[16]. The latter selectivity implies that RNA cap methylation obeys an ordered sequence of events during which nsp14-mediated N7-guanine methylation precedes nsp10/nsp16 RNA 2′-O methylation. Nsp10 is a double zinc finger protein of 148 residues whose crystal structure is known[20], [21]. Together with nsp4, nsp5, nsp12, nsp14, and nsp16, nsp10 has been found to be essential in the assembly of a functional replication/transcription complex[22]. Drawing on these observations, nsp10 has been proposed to play pleiotropic roles in viral RNA synthesis[23] and polyprotein processing through interaction with the main protease nsp5[24].
SAM-dependent MTases belong to a large class of enzymes present in all life forms. These enzymes catalyze the transfer of the SAM methyl group to a wide spectrum of methyl acceptors, indicating that a common chemical reaction is used on a variable active-site environment able to activate the methyl acceptor atom. Although SAM-dependent MTases share little sequence identity, 2′O-MTases exhibit a KDKE catalytic tetrad and a very conserved folding made of a seven-stranded β-sheet surrounded by one to three helices on each side[25], always similar to the paradigmatic catechol-O-MTase[26]. The SAM binding site general location is conserved, suggesting that evolutionary pressure on the MTase fold has maintained the same SAM-binding region whilst accommodating the versatile chemistry of the methyltransfer reaction.
Structural and functional studies of viral MTases involved in RNA capping is an expanding research area, since these enzymes show unexpected diversity relative to their cellular counterparts, and thus constitute attractive antiviral targets. Crystal structures of viral RNA cap MTases exist for only three viral families, namely Poxviridae, Reoviridae, and Flaviviridae. The Vaccinia virus VP39 crystal structure was the first to be elucidated in 1996[27]. The structure of this DNA virus RNA 2′-O-MTase revealed a conserved MTase fold similar to that of RrmJ (also named FtsJ), the canonical reference folding for RNA cap MTases[26]. More recently, the crystal structure of a second Vaccinia virus N7-guanine RNA cap MTase domain (D1) was determined in complex with its activator protein D12[28]. The study revealed that D12 also bears an MTase fold, but has lost catalytic capability due to truncation of its SAM binding site. In turn, Reoviridae provided the first RNA cap MTase structures at 3.6 Å resolution as forming part of the reovirus core[29]. Another RNA cap machinery was more recently described for the non-turreted orbivirus Bluetongue virus VP4 protein at 2.5 Å resolution[30], which revealed a three-domain protein, with a "head" guanylyltransferase domain, a central N7-guanine MTase, and a "bottom" 2′-O-MTase domain. This architecture illustrates the sequence of three out of the four chemical reactions involved in RNA capping described above.
Regarding (+)RNA viruses, MTase structural information at the atomic level is only available for a single genus. The flavivirus N-terminus domain (residues 1–265) of the NS5 RNA-dependent RNA polymerase harbors an RrmJ fold with an N-terminus extension able to accommodate RNA cap structures[31], [32]. This enzyme carries both N7-guanine MTase and 2′-O-MTase activities on a single domain with one shared active site[33]. Homologous domains have been crystallized for a number of flaviviruses, revealing a conserved fold and activity[34], suggesting that MTases might represent interesting targets for drug design. No other (+)RNA virus RNA cap MTase crystal structures have as yet been defined.
In 2003, the identification of the 2′-O-MTase signature sequence in the SARS-CoV genome added nsp16 to the list of putative targets for antiviral drugs[35]. Several compounds have been shown to inhibit viral MTases, such as the co-product of the MTase reaction SAH, Sinefungin, and aurintricarboxylic acid (ATA)[14], [36], [37], [38], [39]. In this paper, we report the crystal structure of the SARS-CoV 2′-O-MTase nsp16 in complex with its activator, the zinc finger protein nsp10, at 2.0 Å resolution, in conjunction with mutagenesis experiments, binding and activity assays. These results lay down the structural basis for the nsp10 function as an activator of nsp16-mediated 2′-O-MTase. We identify residues playing key roles in the nsp10/nsp16 interaction, as well as other residues involved in 2′-O-MTase catalysis and RNA binding. We also report the crystal structure of the nsp10/nsp16 complex bound to the inhibitor Sinefungin. Comparison with known cellular SAM binding sites points to the nsp16 nucleobase binding pocket as a possible target for the design of selective antiviral molecules.
Crystallization and Structure Determination of an Active nsp16 2′-O-MTase
We observed that purified nsp16 was unstable in solution, impeding crystallogenesis. Yeast double-hybrid and co-immunoprecipitation experiments on purified SARS-CoV nsp10 and nsp16 have uncovered the reciprocal interaction of these two proteins[16], [17], [18]. Indeed, SARS-CoV nsp16 exhibits 2′-O-MTase activity only when complemented with SARS-CoV nsp10, raising the interesting possibility that nsp10 acted as a scaffold for nsp16. Co-expression of nsp10 and nsp16 using a bi-cistronic prokaryotic expression vector facilitated affinity chromatography purification and crystallization of the complex[16], [40]. Crystals diffracted to ~1.9 Å. The position of the nsp10 protein was determined using molecular replacement with the SARS-CoV nsp10 protein structure[20] as a search model. Strong peaks in both the residual and anomalous Fourier maps confirmed the presence of two zinc ions. Nsp16 was well defined by its electron density except for two flexible loops (residues 19–35 and 135–137) with high B factors and weak or missing electron density. These loops are solvent-exposed at each side of the putative RNA-binding groove (see below). Structure determination data and refinement statistics are reported in Table 1.
Structure of the nsp10/16 Heterodimer
The heterodimer can be conveniently viewed as nsp16 sitting on top of a nsp10 monomer (Fig. 1A). The nsp10 overall structure in the complex remains essentially unchanged relative to published structures of nsp10 alone, with its N-terminus comprising two α-helices, a central β-sheet domain, and a C-terminus domain containing various loops and helices (see[20], [21], Fig. 1B). Comparison with existing crystal structures of nsp10 using DaliLite[41] rendered nsp10 atomic coordinates very similar to those of nsp10 in our nsp10/nsp16 complex. The average RMSD is about 0.77 Å in 118 residues (PDB codes 2FYG, 2G9T and 2GA6[20], [21]). This indicates that neither significant conformational change nor surface modification occurs in nsp10 when binding to nsp16. The nsp10 structural Zn2+ ions are not directly involved in the nsp10/nsp16 interface (Fig. 1A).
A) The nsp16 protein (green) is bound to nsp10 (yellow) through an interface which does not involve Zn ions (grey spheres) present in nsp10. One metal ion (blue sphere) is found in nsp16 on the opposite face from the active site to which a SAH molecule is found (red sticks). B) Ribbon representation of nsp16, rainbow colors from N- to C-terminus. Top: Each secondary structure element is labeled according to[25] (see also Fig. S3). The SAH molecule shown in sticks colored following atom type. Bottom: View of interface involving nsp16 (green surface) and nsp10 (yellow ribbons) showing the nsp10 secondary structure elements involved in the interface.
Nsp16 adopts a canonical SAM-MT fold (Figs. 1B, 2A and B), as defined initially for the catechol O-MTase[25]. The seven-stranded β-sheet MTase fold has been described as having a secondary structure topology defining two binding domains, one for SAM and the other for the methyl acceptor substrate (Fig. 2A). The nsp16 topology matches those of dengue virus NS5 N-terminal domain and of vaccinia virus VP39 MTases[27], [31]. Nsp16 lacks several elements of the canonical MTase fold, such as helices B and C (Fig. 2B).
A) The consensus topology diagram is shown with a two domain organization for methyl acceptor substrate and SAM, as defined[25] with the catechol O-MTase and its canonical seven strand beta sheet. B) Topology diagram of nsp16 colored according to rainbow colors from N-to C-terminus as in Fig. 1B. The missing helix B and helix C are indicated by dashed ovals. The approximate general binding site of nsp10 is shown (yellow oval).
S-adenosylhomocysteine- and Sinefungin-Binding
Electron density corresponding to one molecule of S-adenosylhomocysteine (SAH), the co-product of the methylation reaction, was identified in the putative SAM-binding site (Figs. 1A and 3A). Neither SAM nor SAH was added to the purification or crystallization buffers, therefore it must have been captured from the medium by nsp16 during bacterial growth. The SAH molecule is found with its adenine in an anti conformation and the ribose pucker in a southern (2′-endo/3′-exo) conformation. All the residues involved in SAM/SAH binding are absolutely conserved in coronavirus np16s (Fig. S1). Binding specificity for SAM/SAH is achieved by holding distal SAM/SAH carboxylic and amino groups through five hydrogen bonds (G81, N43, Y47, G71, and D130) (Fig. 3A and Fig. S2A). The ribose moiety is held by three hydrogen bonds involving Y132, G73, and D99. As in the case of other MTases[25], the SAH binding cleft is globally positively charged. However, an aspartic acid (D99) acts as the ribose-sensing residue with its side chain carboxyl making strong hydrogen bonds with both ribose hydroxyls (Fig. S2A). Binding of the adenine base involves few contacts. The nucleobase occupies a loose hydrophobic pocket engaging two hydrogen bonds of moderate strength with side chain and main chain atoms of conserved residues D114 and C115, respectively.
A) SAH modeled in a simulated annealing Omit map contoured at 1σ. Carbons, oxygens, sulfur, and nitrogens are in grey, red, yellow, and blue sticks, respectively. Water molecules are shown as red spheres. B) Sinefungin bound in the SAM/SAH binding site, with main catalytic residues and the water molecule indicative of a catalytic mechanism. Colors as in A). C) SAH modeled in a 2Fo-Fc map (green) of Sinefungin contoured at 1σ with a Fo-Fc difference map (red) contoured at 3σ. A peak of negative density appears clearly on the sulfur atom of the SAH molecule showing that SAH was indeed replaced by Sinefungin. Colors as in A).
Soaking the crystals into a Sinefungin-containing buffer captured this MTase inhibitor in the SAH binding site almost perfectly superimposable on SAH (Fig. 3B and Fig. S2B). Binding involved the same residues and contacts as SAH. Inhibition of the MTase reaction by Sinefungin therefore probably occurs competitively. The Sinefungin amino group quasi-isosteric to the donated SAM methyl group indicates a cavity where the 2′-hydroxyl of the capped RNA is expected to bind. Lining this empty substrate cavity are the residues proposed to be involved in the catalytic reaction: K46, D130, K170, and E203[16]. Alanine substitutions in the catalytic tetrad (K46, D130, K170, or E203) almost completely block 2′-O-MTase activity without jeopardizing binding to nsp10 (Table 2, and [16]). Several SAM-binding residues (N43, G73, D99 and Y132, Fig. S2A) were substituted by alanine. Although they conserve their specific nsp10 binding properties, indicating that they are correctly folded, they all show a drastically reduced MTase activity (Table 2), validating the structural description of the nsp10/nsp16/SAH ternary complex.
A Mg2+ Cation is Present in nsp16, Outside the Active Site
We recently reported[16] that nsp10/nsp16 MTase activity requires Mg2+. Although the crystallization buffer contains Mg2+, we were unable to locate any such cation in the nsp16 active site. In enzyme activity assays, the Mg2+ ion can be substituted by Mn2+ or Ca2+, but not Zn2+ (data not shown, see also[16]). A peak of electron density presumably corresponding to Mg2+ is localized onto nsp16, distant from the SAH-binding cavity. The Mg2+ coordination mode is through six first-shell water molecules in an octahedral geometry (Fig. 4). Binding via water molecules, involves T58 and S188 side chain hydroxyls and the main chain carbonyl of E276. Since there are no carboxylic acids involved in binding this cation, it was suspected that its presence resulted from the crystallization procedure[42], with no biological relevance. However, the T58A, T58N, T58E and S188A substitutions show 43, 70, 99 and 72% loss of activity, respectively (Table 2), with no significant effect on the stability of the nsp10/nsp16 complex except for T58E whose association was 54 % that of wild-type. These residues are located on three distinct structural elements at the C-terminus of helix Z (T58), the N-terminus of β6 (S188), and in the central part of helix A3 (E276), respectively (Fig. 4 and Fig. S3). The cation may thus hold these elements together.
Left, the putative Mg2+ ion (blue sphere) is shown solvated in its first atomic shell by six water molecules (red spheres). The corresponding 2Fo-Fc electron density map (contoured 1σ) with a cross shape is shown in purple. Residues of Nsp16 involved in coordination of Mg2+ ion via water molecules are labeled. Right, a global view of the bound metal ion in its electron density on the opposite side from the SAH molecule (red sticks). Nsp16 is shown as green ribbons, nsp10 as yellow ribbons with its two bound Zn2+ as grey spheres as in Fig. 1.
The Putative RNA Binding Site: Mutagenesis and Effect on Activity
The nsp10/nsp16 complex absolutely requires an N7-methyl guanine capped RNA substrate to exhibit MTase activity[16]. The structural basis for the preferential binding to methylated N7-guanine versus non-methylated caps has been elucidated in four cases, those of VP39[27], eIF4E[43], CBC[44], and PB2[45] proteins (PDB codes 1AV6, 1EJ1, 1H2T, and 2VQZ, respectively) bound to cap analogues or capped RNAs. In these cases, the methylated base specificity is achieved through increased binding energy resulting from the stacking of the N7-methyl guanine between parallel aromatic residues of the cap binding protein. The presence of the methyl group greatly enhances π-π stacking, providing a dominant effect over unmethylated guanine[46]. Despite numerous attempts, cap analogues (m7GpppA, GpppA, m7GpppG, GpppG) and short capped RNA substrates (m7GpppA(C)n) could neither be co-crystallized with nsp10/nsp16 nor soaked and bound onto preformed nsp10/nsp16 crystals. However, the atomic coordinates of the N7-methyl guanine RNA oligomer in complex with VP39[47] provided data from which a model of RNA binding to the nsp16 protein was derived. SAM molecules identified in both structures were superimposed, and the VP39-bound RNA was positioned onto the nsp16 structure. After minimal manual adjustments not exceeding 5 Å, the VP39 RNA was a reasonably good fit into an nsp16 hydrophobic groove radiating from the catalytic site (Fig. 5), establishing very few contacts with nsp10. We note that the protein side diametrically opposite to the proposed hydrophobic RNA binding groove is highly positively charged (not shown), an observation that may account for the difficulty of achieving experimental RNA binding in the proposed RNA binding site. In the absence of robust data to guide docking of the guanine cap, the m7Gpp cap structure was not positioned in the structure but two possible N7-methylated cap guanine binding areas are indicated by arrows (Fig. 5). The first transcribed nucleotide together with its ribose receiving the methyl group fit well in the active site (Fig. 5, panel B) as predicted in the proposed mechanism. The same holds for the immediately preceding three nucleotides. The base of the first transcribed nucleotide may be held by contact with P134 and Y132, bending the extending RNA cap structure. Accordingly, the substitution of Y132 greatly depresses MTase activity (Table 2). We also note that Y132 is located in the vicinity of a highly mobile loop (residues 135-138) not always visible in our crystal structures suggesting that this loop may move in order to wrap the triphosphate moiety of the RNA cap and/or the RNA cap itself. The solvent exposed side chain of Y30 may also participate in RNA binding. In the model, the highly mobile side chain of Y30 was flipped out in an alternative conformation in order to open the groove. In that position, Y30 should specifically contact the third transcribed nucleotide. Our mutagenesis data confirms the importance of Y30 since its replacement with either Ala or Phe severely impairs MTase activity without affecting the interaction with nsp10 (Table 2).
A) In this representation, Sinefungin was preferred over SAH because one of its NH2 groups approximates the direction of a transferred CH3 group from the SAM substrate. Carbon is white, oxygen is red, nitrogen is blue and phosphorous is orange. Nsp16 and nsp10 are rendered as a solvent-accessible surface colored grey and wheat respectively. The Sinefungin molecule defines the methyltransferase active site. Missing residues in the 135-137 loop (see text) are indicated by a shaded blue dotted box. Position of Y30 and Y132 are indicated. Y30 generated poor electron density (see « Methods ») and its aromatic ring position has been manually adjusted before generation of this image. B) Close caption of the methyltransferase active site showing distance between the NH2 of Sinefungin to the 2′-O of the ribose of the first base, thus mimicking the position of the methyl of the S-adenosylmethionine.
Interface of the Heterodimer
All nsp10 secondary structure elements but helices 2 and 5 contact nsp16 (Fig. 1). The nsp10 contact points can be viewed as 5 small patches A to E (residues 40–47, 57–59, 69–72, 77–80, and 93–96, respectively, Fig. 6A). In turn, these five patches contact most of the nsp16 SAM-binding structural elements in 4 areas, I to IV (Fig. 6B, Fig. S3)), mainly involving β2, β3, αA, αZ, and for area IV, the loop connecting helices A2 and A3 at the C-terminus (Figs. 1 and 6). In total, the interface of the heterodimer involves 53 residues, 23 and 30 from nsp10 and nsp16, respectively. In nsp10, a single residue (Asn10, at the edge of the interaction surface) is not conserved out of 23 (4.3 %), whereas in nsp16 there are 8 non-conserved residues out of 30 (26.7 %) (Fig. S1). The interface has a buried surface area of 1820 Å2, with nsp10 contributing to 930 Å2 and nsp16 to 890 Å2.
A) nsp16 (left) and nsp10 (right) are rendered as a solvent-accessible surface colored green and yellow, respectively. Residues involved in the interface are rendered as sticks with the same color code, in transparency into respective proteins. B) Separate representation of the nsp16 (left) and nsp10 (right) interface. Nsp16 residues defining patch I, II, III, and IV are rendered as sticks as above in transparency into nsp16. Nsp10 residues defining patch A, B, C, D, and E are rendered as sticks as above in transparency into nsp10. C) A plot of MTase activity as a function of nsp10/nsp16 complex formation using the data reported in Table 2.
Four nsp10 patches included in the 5 interaction patches identified here were recently mapped using reverse yeast two-hybrid methods coupled to bioluminescence resonance energy transfer and in vitro pull-down assays (see[18] and below). To probe the observed crystal structure of the interface further, we engineered 5 new nsp10 alanine mutants (N40A, L45A, T58A, G69A, and H80A, see Table S1) sitting in patches A, B, C and D. Whereas T58A, G69A and H80A showed limited effect on nsp16 binding, N40A reduced it to 64% of wild type affinity, and L45A almost abrogated it. The crystal structure indicates that the nsp10 interface proposed by Lugari et al.[18], is a correct and conservative estimation, as the interface also includes L45 belonging to patch A. We also confirm the positive co-relation of the detected nsp10/nsp16 interaction with MTase activity. In no instance can nsp16 be active in the absence of nsp10/16 complex formation.
The Y96 position is of particular interest. Alanine substitution (Y96A) abrogates interaction whereas a phenylalanine (Y96F) increases both interaction and MTase activity[18]. In order to understand how residue 96 plays such a pivotal role, we determined at 2.0 Å resolution the crystal structure of this nsp10(Y96F)/nsp16 complex (Table 1). Strikingly, the absence of the hydroxyl group does not alter the topology of the interface. Wild-type and Y96F residues superimpose without significant difference at all atomic positions (not shown). Either Y96 or F96 is in direct contact with nsp16 helix αZ, which carries the catalytic residue K46. Detailed surface analysis using PISA indicates that the position of K46 in Y96F nsp10 is identical to that of K46 in wild-type nsp10, ruling out a better alignment of catalytic residues of the Y96F mutant. The nsp10(Y96F)/nsp16 differs from wild-type nsp10/nsp16 in the SAH binding site, though. When compared to that of wild-type, the SAH occupancy is much lower (~0.3 versus ~1), leading to poor density definition. SAH is known to be a fairly good inhibitor of the methylation reaction. Therefore, a lower binding affinity might translate into less end-product inhibition, and account for the observed increased activity. We measured the affinity of nsp16 for SAH using fluorescence spectroscopy, but no significant differences were found (not shown). Likewise, the MTase inhibition pattern by SAH was identical for wild-type and nsp10(Y96F)/nsp16 (not shown). We therefore infer that the previously observed ~10-fold increased stability of the heterodimer[18] may be responsible for the increased activity. A more hydrophobic character of the interaction may appear upon the loss of the tyrosine hydroxyl which, in the wild-type protein, was not engaged in any polar contact. We thus attribute the increased activity of the nsp10(Y96F)/nsp16 complex relative to wild-type to a stronger equilibrium association of nsp10(Y96F) with nsp16 than that of wild-type nsp10 with nsp16.
Mutation analysis was also conducted on nsp16 residues presumably involved in the interface and interfacial activation (Tables 2 and S2). Several mutants (V78A, V104A, L244A, M247A) in patches II, III and IV completely disrupt the nsp10/nsp16 complex and annihilate nsp16 MTase activity. Interestingly, we also identified nsp16 mutants still interacting with nsp10, but with a strongly reduced 2′-O-MTase activity (I40A, M41A, V44A, T48A, Q87A, D106A) suggesting, that these mutations in the nsp10/nsp16 interface may alter the fine positioning of catalytic residues without any significant effect on nsp10 binding. Accordingly, most of these mutants are localized in αZ helix of patch I which contains the K46 catalytic residue. On the other hand, patch II and III mutants tend to have more mitigated phenotypes, yielding to full-blown interaction with only about half of the expected activity. Finally, patch IV mutants were totally inactive. Using all mutants reported in Table 2, a plot (Fig. 6C) of interaction versus activity shows that the nsp10/nsp16 interaction is strictly required to obtain significant nsp16 MTase activity.
Mechanism of 2′-O-Methyltransfer
The SARS-CoV RNA cap 2′-O-MTase is a heterodimer comprising SARS-CoV nsp10 and nsp16. When bound to nsp10, nsp16 is active as a type-0 RNA cap-dependent 2′-O-MTase, ie., active only when the cap guanine is methylated at its N7 position[16]. The nsp10/nsp16 crystal structure shows that nsp16 adopts a typical fold of the S-adenosylmethionine-dependent methyltransferase family as defined initially for the catechol O-MTase[25]. A good alignment (170°) is found between the SAH sulfur atom, a water molecule present in both SAH- and sinefungin-bound nsp16 structures, and the K46 ε-amino group (Fig. 3A and B). This geometry provides interesting hints for a catalytic mechanism, as the positions of the catalytic residues (K46, D130, K170, E203) match spatially those of the vaccinia virus VP39 2′-O-MTase[47]. At the initial stage of the reaction the 2′-hydroxyl of the capped RNA substrate would occupy the position of the water molecule. In turn, E203 and K170 decrease the pKa of the K46 ε-amino group that becomes a deprotonated general base (-NH2) able to activate the RNA 2′-hydroxyl at neutral pH. In VP39, K175 has been identified as the general base catalyst[47] with a pKa depressed by ~ 2 pH units by the neighbouring D138 and R209 residues[48]. These findings indeed suggest a related mechanism: once K46 has activated the 2′-hydroxyl group, the 2′-oxygen would produce an in line attack through a SN2-like mechanism onto the electrophilic SAM methyl group. The methyl group would pass through a pentavalent intermediate with the 2′-O and sulfur at apical positions. D130 is positioned to stabilize the transient positive charge on the donated methyl atom of SAM before the sulfur recovers a neutral electric charge during SAH generation (Fig. 3B).
Role of Mg2+ on MTase Activity
Unlike most SAM-dependent MTases, the SARS-CoV nsp10/nsp16 enzyme requires a divalent cation, either magnesium, manganese or calcium[25]. We have found that this cation does not reside in the active site. Instead, the cation is coordinated through water molecules by three residues located on three distinct structural elements. It is thus possible that one divalent cation, presumably Mg2+, present in the host cell at millimolar levels, plays a structural role in holding these three nsp16 structural elements together and so regulate the enzyme activity. It is intriguing that T58A is more active than T58N or T58E that can still bind the water that chelates to the metal. Alternatively, it is possible that divalent cations such as Mg2+ or Ca2+ act as a phosphodiester charge shield to allow RNA binding in the hydrophobic binding groove[49].
MTase Activity is Regulated via Protein-Protein Interactions
The main regulation mechanism of nsp16 is through its physical association with nsp10. Nsp16 is unstable in solution, and nsp10 acts as a scaffold for nsp16, yielding a stable dimer active as an RNA cap-dependent (nucleoside-2′-O)-MTase. The complex is assembled through a ~890 Å2 contact surface in nsp16, an area typically in the intermediate zone differentiating strongly from weakly associated dimers[50]. This finding is consistent with a Kd estimated at ~0.8 µM[18] that qualifies the nsp10/nsp16 complex as a rather weak heterodimer. The nsp10 interaction surface identified in the crystal structure was confirmed by site-directed mutagenesis and overlaps that previously identified by indirect methods[18]. Remarkably, the nsp10 surface in the nsp10/nsp16 complex is essentially identical to that of uncomplexed nsp10 crystallized alone by others[20], [21](Fig. S4). It is therefore reasonable to see this heterodimer as a non-permanent species which would tolerate nsp10 or nsp16 engaging in interactions with other partners. This notion is actually in line with the involvement of nsp10 in a network of protein-protein interactions that we and others have proposed[17], [19]. Donaldson et al.[23] have engineered mutations in nsp10 using reverse genetics. Out of eight mutations that turned out to be in the nsp10/nsp16 interface (this work), five, two and one rendered lethal, debilitated, and viable phenotypes, respectively[23]. Interestingly, the nsp10(Q65E) mutant providing a temperature-sensitive phenotype[22], [23] does not map in the nsp10/nsp16 interface, confirming that nsp10 has a pleïotropic role.
Our mutagenesis analysis shows that the formation of an nsp10/nsp16 complex is a pre-requisite for MTase activity (Fig. 6C) indicating that physical association of nsp10 and nsp16 is essential to activate nsp16 2′-O-MTase activity and foster efficient virus replication. We note that most interface mutants exhibit a severe loss of their 2′-O-MTase activity, whereas the apparent association affinity is often only modestly affected. That minor changes in the interface translate into potent effects is also dramatically illustrated by the Y96F mutation, where the loss of a single hydroxyl provokes a significant change in affinity[18]. Remarkably, it is not the most active complex that was selected in nature, since the nsp10(Y96F)/nsp16 complex is both more stable and more active than the wild-type heterodimer (this work and[18]). This is yet another observation hinting at the involvement of nsp10 in protein-protein interaction networks including other partners than nsp16, such as nsp5 and nsp14[17], [19]. In most other coronaviruses, the nsp10 residue at position 96 is a phenylalanine. It would be interesting to determine whether this polymorphism is relevant to the SARS-CoV pathogenicity at any (direct or indirect) level, or if compensating polymorphisms in other coronaviral nsp10 (or nsp16) restore a weaker nsp10/nsp16 association equivalent to that of the SARS-CoV pair. Since a bona fide viral RNA cap is key in evading the host cell innate immunity[4], [51], a minimal level of 2′-O-MTase activity would be expected to be critical to virus survival.
MTase activation through dimerisation of two viral protein partners has already been reported in the case of the vaccinia virus D1/D12 N7-guanine MTase[28]. However, the activating D12 subunit does not contact the D1 subunit through a homologous surface mainly defined by canonical αA and αZ helices. Rather, the D1/D12 activation surface would be located at a 90° clockwise rotation relative to the nsp10/nsp16 interface depicted in Fig. 1A. In the case of dengue virus, the bi-functional N7-guanine and 2′-O-MTase is part of the N-terminus of the dengue NS5 protein. Based on reverse genetic data and modeling[52], the MTase domain would be associated with the Pol domain through an interface topologically similar to that of nsp10/nsp16, i.e., involving mainly helices αA, αZ and strands β2 and β3 as depicted in Fig. 1A.
We have previously shown that the nsp10/nsp16 is only active as N7-guanine methylated capped RNA, implying that RNA cap methylation obeys to an ordered sequence of events where nsp14-mediated N7-guanine methylation precedes nsp10/nsp16 RNA 2′-O methylation[16]. In the absence of data regarding the RNA substrate, we built a model of RNA binding based on that of the vaccinia virus VP39 ternary complex structure. Interestingly, our model proposes that the RNA interacts only with nsp16 residues, in keeping with what was recently suggested based on RNA binding assays[14]. Although the position of the cap structure on the nsp16 surface remains to be determined, our model suggests a well-defined position for the ribose of the first transcribed nucleotide in the active site. In agreement with mutagenesis analysis, the model also suggests that the transcribed RNA 5′-end stacks between Y132 and Y30. Furthermore, this model is consistent with the observation that coronavirus MTase requires RNA substrates of at least 3 transcribed nucleotides in length[14]. It is also worth to know that a comparison of nsp16 and VP39 electrostatic surfaces reveals that the putative RNA-binding groove of nsp16 is mostly hydrophobic, whereas the VP39 RNA-binding groove is positively charged. This variation would imply a change in the nature of the RNA/protein interaction.
Binding of Sinefungin to nsp16/nsp10 Suggests Feasibility of a Drug Design Approach to Inhibit MTase Activity
Viral MTases are increasingly evaluated as potential drug design targets[34], [37], [53]. We have crystallized the inhibitor Sinefungin with the nsp10/nsp16 complex. Sinefungin exhibits an IC50 of 0.74 µM, 16-fold lower than that of SAH as reported by Bouvet et al.[16] using purified nsp10/nsp16. Analysis of the structure suggests a likely mechanism of action that also accounts for the observed inhibitory effect of this drug. We note that the adenine nucleobase does not fit snugly into its binding pocket, raising interest regarding structure-based drug design. Preliminary examination of eukaryotic non-viral MTase structures from main classes as defined in Martin and McMillan[25] indicates that the SAH adenine is bound tighter in any of the latter enzymes than in the nsp16 SAM-binding site, indicating a possible breach to achieve anti-coronavirus selectivity with a small molecule inhibitor of nsp16.
In conclusion, the crystal structures presented here extend our general understanding of the mechanism and regulation of viral RNA cap MTases in (+)RNA viruses, and point to both the nsp10/nsp16 interface and the substrate binding sites as putative antiviral targets.
Crystallization and Data Collection
Both nsp10 and nsp16 were expressed from the same dual expression vector pmCOX [16]. Nsp10 had a N-terminal strep-tag (WSHPQFEK), and nsp16 a N-terminal hexa-histidine tag. The purification and crystallogenesis of the nsp10/nsp16 complex was performed as described in [40]. Typical crystals of the wild-type nsp10/nsp16 appear in hanging drops after 24 h at 20°C in 0.1 M CHES pH 9, 1.52 M MgCl2 hexahydrate. Crystals (a = 68.53 Å, b = 184.74 Å, c = 129.01 Å, C2221) contain one nsp10/nsp16 complex per asymmetric unit, with a solvent content of 70 % and Vm of 4.17 Å3/Da. Crystals of nsp10(Y96F)/nsp16 were grown in 67 mM CHES pH 8.5, 0.99 M MgCl2 hexahydrate, 33 mM Tris-HCl, 8.3 % PEG 8000. Both crystallization conditions yielded crystals diffracting to 1.9 Å when exposed to synchroton radiation at the ID14-1 beamline of the European Synchrotron Radiation Facility, Grenoble, France. Crystals were cryo-cooled in the same buffer supplemented with 15 % glycerol. Crystal soaking was performed in the same buffer supplemented with 5 mM SAH or Sinefungin during 24 h.
Structure Determination and Refinement
The position of the nsp10 protein was unambiguously determined by molecular replacement using the program PHASER[54] with the nsp10 protein (2FYG), as search probe[20]. Strong peaks in both the residual and anomalous Fourier maps confirmed the presence of two Zinc ions at the expected positions within the nsp10 protein, thus giving confidence in the validity of the MR solution. Phases calculated from this partial model were combined with SAD phases from the Zn atoms using PHASER. To ameliorate the resulting low quality density map, phases were improved with PARROT[55]. An initial model, comprising both nsp10 and nsp16, was automatic built by successive use of BUCCANEER[56] and ARP/wARP[57]. The resulting model was subject to several cycles of manual rebuilding using COOT [58] and refinement with REFMAC [59]. The protein structure model could be built, except the strep and hexahistidine tags. In nsp16, density was too weak for the mobile, solvent exposed nsp16 loop 136–139, Y30 (see "Results"), and 2 and 6 residues in N- and C-terminus, respectively. Likewise, nsp10 solvent exposed 9 and 8 residues in N- and C-terminus were missing, respectively.
Overall, the chain traces are unambiguous, with clear electron density including for a single SAH residue bound to the nsp16 protein. Solvent accessible surfaces were calculated using program AREAIMOL [60] with a 1.7 Å radius sphere as the probe (Table 1) and values rounded to the nearest 5 Å2. Conformational differences were analyzed using the DynDom server (http://www.cmp.uea.ac.uk/dyndom/main.jsp). Figures were created using PYMOL (http://www.pymol.org). The coordinates of the wild-type/SAH, mutant, and wild-type/Sinefungin structures have been deposited at the Protein Data bank under PDB codes 2XYQ, 2XYV, and 2XYR, respectively.
RNA Cap Structure Modeling
The modeling of the RNA cap structure in the nsp10/nsp16 complex structure is derived from the analysis of the structure of the vaccinia virus methyltransferase VP39 crystallized in complex with a capped RNA and a S-Adenosylhomocysteine[47] (SAH) (pdb code: 1AV6). The two structures are manually aligned using COOT[58] based on the position of SAH binding sites, as well as SAH, and Sinefungin (SFG) molecules. The RNA binding site of VP39 is only partly overlapping that of nsp16 whilst the shape of the cavity is similar; thus local adjustments necessary to accommodate the RNA molecule in its binding groove were done manually using COOT. The side chain of tyrosine 30 of nsp16initially pointed to the putative RNA binding site, preventing any bona fide modeling. In order to fit the RNA molecule in the cavity, an alternative conformation was sought for this side chain. The second most common conformation for the tyrosine side chain was selected. Due to the biochemistry data and surface electrostatic analysis, it is not possible to describe with certainty the final position of the cap, thus the cap was removed and replaced by arrows symbolizing possible positions. No other modification was performed on the RNA, the Sinefungin molecule or the nsp16 structure.
The SARS-CoV nsp10 and nsp16-coding sequences were amplified by RT-PCR from the genome of SARS-CoV Frankfurt-1 (accession number AY291315) as previously described[16]. The nsp10 and nsp16 genes (encoding residues 4231–4369, 5903–6429, and 6776–7073 of replicase pp1ab) were cloned into a Gateway modified dual-promotor expression plasmid and in the gateway pDest 14 expression vector. In this backbone, SARS CoV nsp10 can be expressed under a tet promoter and encodes a protein in fusion with a N-terminal strep tag, whereas nsp16 is expressed under a T7 promoter and encodes a protein in fusion with a N-terminal hexahistidine tag. The mutants were generated by PCR using the Quickchange site–directed mutagenesis kit (Stratagene), according to the manufacturer's instructions.
AdoMet and cap analogs GpppA and 7MeGpppA were purchased from New England BioLabs, the[3H]-AdoMet was purchased from Perkin Elmer and Sinefungin (adenosylornithine) from Sigma-Aldrich.
Expression and Purification of SARS-CoV nsp10, nsp16 and nsp10/nsp16 Complex
E. coli C41 (DE3) cells (Avidis SA, France), containing the pLysS plasmid (Novagen), were transformed with nsp10 or nsp16 cloned in pDest14, or nsp10/nsp16 cloned in pmCox, and grown in 2YT medium supplemented with appropriate antibiotics. The expression of strep-tagged nsp10 or 6His-tagged nsp16 mutants was induced (DO600 = 0.6) by adding 50 µM IPTG, and the expression of the nsp10/nsp16 complex by adding 50 µM IPTG and 200 µg/L of anhydrotetracycline. After an incubation for a 16 h at 24°C, the cell were pellets, frozen and resuspended in lysis buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM MgSO4, 5 mM β-mercaptoethanol (only for nsp10) supplemented with 1 mM PMSF, 40 mM imidazole, 10 µg/ml DNase I, and 0.5% Triton X-100. After sonication and clarification, proteins were purified either by IMAC (HisPurTM Cobalt Resin; Thermo Scientific) chromatography[16] (nsp10 mutants and nsp16 mutants), and the nsp10/nsp16 complex was purified by using Strep-Tactin sepharose (IBA Biotagnology) as previously described[16]. All purified proteins were analyzed by SDS-PAGE. The binding of wild-type nsp10 to mutant nsp16, and that of mutant nsp10 to wild-type nsp16 was quantified using ImageJ as described[16].
Radioactive Methyltransferase Assay
MTase activity assays were performed in 40 mM Tris-HCl, pH 8.0, 5 mM DTT, 1 mM MgCl2, 2 µM 7MeGpppAC5 or GpppAC5, 10 µM AdoMet, and 0.03 µCi/µl [3H]AdoMet (GE Healthcare). Short capped RNAs (7MeGpppAC5, GpppAC5, were synthesized in vitro using bacteriophage T7 DNA primase and were purified by high-performance liquid chromatography (HPLC) as previously described[61]. In the standard assay, nsp10 and nsp16 were added at final concentrations of 600 nM, and 200 nM, respectively, and the amount of 3H-CH3 transferred onto 7MeGpppAC5 substrates was determined by filter binding assay as previously described[16].
Supporting Information
Figure S1.
Sequence alignment and amino acid conservation in coronavirus nsp16. The alignment of coronavirus nsp16 sequences was generated with Muscle program (http://www.ebi.ac.uk/Tools/msa/muscle/), and the resulting alignment converted using the ESPript program, (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Residues that are conserved in all or >70% sequences are boxed in red and yellow, respectively. National Center for Biotechnology Information (NCBI) accession numbers for replicase polyprotein sequences that include nsp16 are as follows: Severe acute respiratory syndrome virus SARS-CoV Frankfurt isolate (SARF), Severe acute respiratory syndrome virus SARS-CoV Tor2 isolate (SART), NP_828873.2; Turkey coronavirus (Turk), YP_001941189; Infectious Bronchitis Virus (IBV), NP_066134; Feline Coronavirus (Feli), YP_239426;Porcine Transmissible Gastroenteritis Coronavirus (PTGC), P18457; Transmissible Gastroenteritis coronavirus (TGEV), NP_840013; Porcine epidemic diarrhea virus CV777 (PEDV), NP_839969; Bat coronavirus 512/2005 (BATC), YP_001351683; Human coronavirus NL63 (NL63), AAS58176; Human coronavirus 229E (229E), NP_073549; Murine hepatitis virus strain JHM (MurJ), YP_209243; Mouse Hepatitis virus strain A59 (MA59), NP_740613; Human coronavirus HKU1 genotype A (HKU1), YP_460023; Human enteric coronavirus 4408 (4408), ACJ35483; Bovine coronavirus (BCoV), NP_742142; Human coronavirus OC43 (OC43), AAT84359. Numbering was made using SARF as a reference.
Figure S2.
Binding determinants in the SAM/SAH/Sinfungin binding site. A) LIGPLOT diagram (www.ebi.ac.uk/thornton-srv/software/LIGPLOT/) of the SAH ligand molecule interacting with the nsp16 binding site. Ligand bonds are in purple, neighbor residue (non-ligand) bonds are in light brown, hydrogen bonds are green dashed lines. Ligand atoms surrounded by a yellow circle are highly accessible. Non-ligand residues in hydrophobic contacts with the ligand are presented by red semi-circles with radiating spokes. B) Superimposition of SAH and Sinefungin molecules. The nsp16 residues are green sticks, the SAH is represented in red sticks, and Sinefungin and water molecules are in sticks and spheres as in Fig. 3, respectively.
Figure S3.
Primary and secondary structure elements of nsp16. Helices and sheets are colored according to rainbow colors from N- to C-terminus as in Fig. 1. Patches I to IV are boxed in grey, labeled above the sequences, and correspond to residues represented in Figs. 6A and B.
Figure S4.
Nsp10 interface comparison between the nsp10/nsp16 complex and nsp10 homo multimer. A) For the corresponding nsp10/nsp16 interface nsp10 (cyan) interacts with 2 monomers (green and orange). Left: Cartoon representation of 3 nsp10 monomer part of the dodecamer structure (PDB: 2G9T). Right: Cartoon representation of the nsp10(cyan)-nsp16(dark red) complex. Nsp10 molecules in cyan are in the same orientation. B) Nsp10 sequence is presented with above the corresponding secondary structure elements. Below dark red colored dots indicate residues involved in the interaction between nsp10 and nsp16 while green and orange dots indicate residues involved in the homomultimer in the dodecamer complex.
Table S1.
Effect of alanine mutations in nsp10 interface residues. Residues were identified using PISA (http://www.ebi.ac.uk/msd-srv/prot_int/pi_link.html). Bold on grey background: strictly conserved residues amongst coronaviruses; Bold on white background: conserved a.a. (>70%) amongst coronaviruses (see Fig. S1). The % of Bioluminescence Resonance energy Transfer (BRET) signal was previously reported[18]. The interaction of each nsp10 or nsp16 mutant was determined using strep-tactin pull-down experiments of strep-tagged nsp10 co-expressed with nsp16 followed by SDS-PAGE analysis, and quantitation (see Methods). The interaction of wild-type nsp10 with wild-type nsp16 was normalized to 100%. The % of MTase activity was determined using filter binding assays (see Methods) relative to wild-type.
Table S2.
Effect of alanine mutations in nsp16 interface residues. Residues were identified using PISA (http://www.ebi.ac.uk/msd-srv/prot_int/pi_link.html). Same legend as Table S1 except that no BRET experiments were performed.
We thank Barbara Selisko for helpful discussion, comments and support along this work, and Joëlle Boretto-Soler for help in mutagenesis and protein purification. We are indebted to Karen Dalle, Violaine Lantez for excellent technical assistance and General Hugues Toulou for his support.
Author Contributions
Conceived and designed the experiments: E. Decroly, B. Canard. Performed the experiments: E. Decroly, C. Debarnot, F. Ferron, M. Bouvet, B. Coutard, I. Imbert, L. Gluais, N. Papageorgiou, B. Canard. Analyzed the data: E. Decroly, F. Ferron, I. Imbert, N. Papageorgiou, A. Sharff, G. Bricogne, M. Ortiz-Lombardia, J. Lescar, B. Canard. Wrote the paper: E. Decroly, B. Canard.
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- 58. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta crystallographica Section D, Biological crystallography 60: 2126–2132.
- 59. Collaborative Computational Project N (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50: 760–763.
- 60. Saff EB, Kuijlaars ABJ (1997) Distributing Many Points on a Sphere. Math Intell 19: 5–11.
- 61. Peyrane F, Selisko B, Decroly E, Vasseur JJ, Benarroch D, et al. (2007) High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2′O positions. Nucleic Acids Res 35: e26.


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The market for potable or drinking quality water is large, growing and global. As the world population expands, people are using more fresh water, but natural systems are not producing any more of it. In addition, the demand for water has been increasing at a rate that is double the rate of population growth. Higher per capita income levels lead to higher water usage by individuals. But the biggest impact is from agricultural use as the agricultural sector accounts for 71 percent of global water usage.[1] As societies become richer, the proportion of meat in the diet increases. This, in turn, leads to an increase in water usage since the amount of water required to produce a pound of meat is ten times the amount required to produce a pound of rice.[2]
The growing demand for water is increased further by several factors:
- Higher-than-average rates of population growth and economic growth in areas with limited natural water resources;
- Greater demand by agriculture to produce not only more meat but also crops for energy (e.g., corn to produce ethanol); and
- Extreme weather events such as droughts that create water shortages in specific regions.
But, in stark contrast to oil and other natural resources, the problem of water scarcity is not caused by a declining supply of water. Water is a fully renewable resource, renewed constantly through the water cycle. And, in fact, we use only a small percentage of the water available on the planet. Water scarcity is really a result of the mismatch between the demand for water and the supply of water, either in location, in time or in the quality of the water required. One forecast estimates that by 2025 water scarcity will affect 14% of the world's population, or 1 billion people.[3]
There are many ways to address this mismatch of potable water supply and demand, including transporting water using canals or pipelines, building more water storage capacity such as dams and reservoirs and increasing the efficiency of water usage. The other way is to produce drinking water from lower quality but available water sources, such as desalinating seawater or cleaning up brackish water. This is the area which is the focus of Trevi's efforts. It is also a very strong growth area since it is drought-proof and frequently easier to implement than other options.
The Trevi FO process can be used in a variety of applications, from new desalination plants, improving existing desalination plants, brackish water treatment, water and waste water treatment facilities, brine concentration, and mining and fracking. Our near-term strategic focus is on desalination applications since the demand for them is worldwide, large and growing, and our product development efforts have yielded a working seawater desalination system.

subset: commoncrawl


text:
Tina Fey wants Ashley Dupre. We had no idea she was so kinky! Kidding aside, Tina actually wants Ashley for a different kind of work: acting! As producer of the show, Tina has offered Spitzer's girl a role on 30 Rock.
The only problem is that Tina says she has no idea how to get a hold of her. We'll give her a hint. It's starts with www. Wonder what the role is! This season of 30 Rock is going to rock! Britney, Oprah, AND Ashley Dupre? Beat that, How I Met Your Mother!


text:
Recorded with Zoom H1 inbuilt microphones
Typing on a keyboard
Writing on a piece of paper then signing a signature at the end.
Hard patch cable clicks in Phonos studio.
Screwing a screw down on a thingy.
Baseball Bat slam, hit close Ext.
Creaking some tree. Recorded with Zoom H4.
This is me in the garden tearing away rotten wood from my fence before fitting in new wood pieces. Recorded ...
Sound of a person pressing mobile phone keystrokes (blackberry style phone) 24/96 Stereo
My female cat, 1 year 9 months old. Can be very greedy with food and very noisy eating - specially ...
sound of typing on a notebook keyboard (noise in a background)
soft sound produced by book pages moving fast. recorded with Rode NT4 mic->Fel preamplifier->IRiver IHP120 (line-in) /sonido muy suave producido ...
move book pages. Zoom H1 recorder, unprocessed
Recorded with Zoom H1 inbuilt microphones
Flipping the pages of a book. Recorded at the second floor of the library at Communication Campus.


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Reflecting on Reflections - June 2014
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Measurement of Small Time Constants of Thermal Impedance of Bipolar Transistors with an Audio Analyzer
For the estimation and the simulation of linear and nonlinear distortions caused by thermally effected power-dependent internal feedback (TPF), the thermal impedance has to be measured. Conventional measurements refer to the time domain; they analyze the response to switched changes of power dissipation. This paper proposes measurements in the frequency domain with the advantage that an audio analyzer can be employed.
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Comfort Inn & Suites Airport
6701 Buckley Road
Syracuse, NY 13212
Phone: (315) 457-4000
Fax: (315) 453-7877
Syracuse Funny Bone
10301 Destiny USA Drive
Syracuse, NY 13290
+1 315 423 8669 (Box Office)
Break away from your daily stressful routine and book the seats for a performance at the Syracuse Funny Bone to get your dose of laughter. A part of the renowned chain of comedy clubs, the Syracuse Funny Bone has entertained the people of Syracuse by hosting the performances of many established as well as upcoming stand-up comedians. And while you are here, you can also enjoy a full-fledged dinner, which comprises tasty burgers, sandwiches, salads and desserts.
Earn valuable points toward free nights.


text:
Civilian Personnel Law Manual: Title III -- Travel
OGC-89-8: Jan 1, 1989
Additional Materials:
- Full Report:
(202) 512-4800
This is Title III of the Third Edition of the Civilian Personnel Law Manual relating to travel issues. The Manual is prepared by the Office of General Counsel. The purpose of the Manual is to present the legal entitlements of federal employees, including an overview of the statutes and regulations which give rise to those entitlements, in the following areas: Title I--Compensation, Title II--Leave, Title III--Travel, and Title IV--Relocation. The Manual generally reflects decisions of this Office issued through September 30, 1988. The material in the Manual is, of course, subject to revision by statute or through the decisionmaking process. Accordingly, this Manual should be considered as a general guide only and should not be cited as an independent source of legal authority. This Manual supersedes the Second Edition of the Civilian Personnel Law Manual which was published in June 1983 and the supplements published in 1984, 1985, and 1986.
Mar 31, 2015
- Advanced Communication Cabling, Inc.B-410898.2
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- Sigmatech, Inc.B-410933
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- ExecuTech Strategic Consulting, LLC; TRI-COR Industries, Inc.B-410893,B-410893.2,B-410893.3
Looking for more? Browse all our products here


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- Shopping Bag ( 0 items )
Janet MaslinBy focusing a book entirely on the tactical maneuvers that got Lincoln through 1864, the historian Charles Bracelen Flood makes a smart tactical choice of his own…Mr. Flood's 1864 compresses the multiple demands upon Lincoln into a tight time frame and thus captures a dizzying, visceral sense of why this single year took such a heavy toll.
—The New York Times


text:
Resolution: standard / high
Figure 1.
Network models for the heat shock system. The network from [2] is represented in black, from [3] in red and from [4] in green. HSP:HSF denotes HSP bound to HSF and similar for all other complexes. The solid lines represent chemical reactions and regulatory interactions.
Cates et al. BMC Biophysics 2011 4:16 doi:10.1186/2046-1682-4-16


text:
(Updated, Aug. 4, 3:45 p.m.) GateHouse Media will not honor the collective bargaining agreements negotiated between the Providence Journal and A.H. Belo Corp. and may lay off as many as 40 Journal employees upon its purchase of the paper who will be covered by Belo's severance policy, according to an asset purchase agreement filing with the U.S. Securities and Exchange Commission. more
By Kaylen Auer
Women & Infants Hospital laid off 41 employees, and also reduced hours for six additional employees, as part of an effort to "address inefficiencies" and "bring its cost structure in line with other organizations," it said in a press release Monday. more
By PBN Staff
(Updated, Feb. 23, 6:10 p.m.) All has not been well between labor and management at Rhode Island's two largest hospitals in recent weeks, with unions publicly criticizing administration for decisions they say will jeopardize not … more
By Mary MacDonald
The phrase "public school teacher" conjures myriad conflicting images and memories, depending on our personal experiences. more
Hotel workers and their supporters said Monday afternoon that three or more of them will begin a hunger strike if the R.I. Senate passes a state budget that bans municipalities from individually voting to raise the minimum wage. more
By Patricia Daddona
Superior Court Judge Sarah Taft-Carter on Wednesday denied a motion by the state of Rhode Island to dismiss the pension lawsuit scheduled to go to trial in September, ruling that "a valid contract exists" between retirees and the state regarding promised pension benefits. more
By Kaylen Auer
Court-ordered settlement talks between the state and Rhode Island public employees and retirees over cuts in pension benefits have broken down, sending the dispute to trial, both parties in the case said Friday. more
By Patrick Anderson
The Region 1 director of the National Labor Relations Board has approved a settlement regarding workers' complaints involving the Renaissance Providence Hotel. more
The pending sale of The Providence Journal to a sprawling national newspaper chain whose past acquisition frenzy drove it into bankruptcy caused many local observers to scratch their heads. more
By Patrick Anderson
The union representing Providence Journal newsroom employees says it will fight plans by new owners Gatehouse Media to outsource editing jobs. more
By Patrick Anderson


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text:
Home » Comics » Revolutionary Girl Utena » Souji Mikage
Mikage is the pink-haired head of the popular Mikage Seminar at Ohtori Academy. Originally he was Nemuro, a professor at Ohtori Academy working a project to "obtain eternity". Emotionless and uncaring, he learned to feel when he fell in love with Tokiko, and threw himself into his research in order to save her ailing brother Mamiya and preserve their family forever. His dreams were dashed when Akio seduced Tokiko and Mamiya died, causing him to burn down the dormitory because he believed it to be the last step to opening the gate to "revolutionize the world", killing the 100 boys inside its walls.
Source from Wikipeida
Souji Mikage Costumes
Souji Cosplay (Jacket) from Revolutionary Girl Utena


text:
I could make a list of a bunch of guns i have, or "have", or wish I had. Why would one feel compelled to provide that infornation freely? Do you feel the need to show off? Is it some immature game of oneupsmanship?
Why do you feel it necessary to list your guns?
Maybe so. But whenever they say the caller told them something they say "RP states that blah blah blah". I could tell cops my neighbor has a SCUD but they would not say that so and so has a SCUD registered if Joe Blow just tells them that.
My pistols, however, I always kept by me.
Jesse James
Supposedly the Feds are restricted from maintaining a registry of firearms owned by the same law that gave us background checks. When a FFL goes out of business the records they have are turned over to the Feds.
Recently, talking with a dealer ,he stated that there is a push by the Feds to have all gun purchases "on line" right along with the background checks. Watching how well our government is a nation that follows "the rule of law" is anyone going to be surprised?
Around here you can contact the local Sheriff and register your guns. From there, any name search, driver's license query, or license plate with your info attached will highlight a tab on the officers' and dispatchers' computers. But like I said, you can (not must) register your firearms, and it is very few and far between that are registered in this fashion. Here, when dispatchers advise on firearms, it is almost always third party information; "Johnny is known to carry a gun/knife/pointy rock" or "Johnny owns firearms, unsure if he is armed at this time."
Texan By Birth & Choice
In my state you can report longarm transfers at the capital.


text:
Posted March 27, 2015
As we begin planning for September, it is important for us to know of any upcoming changes to our students population prior to the start of the new school year. This will enable us to work with accurate information when setting up classrooms for the fall. If you are moving and changing schools, please contact [...]
Read more about Planning for September »
Posted March 23, 2015
The Waterloo Region District school board has partnered with My Class Needs, a not-for-profit foundation associated with Curriculum Services Canada, to offer a unique opportunity to support classroom teachers in securing additional resources to support student learning, well-being and achievement. The program provides grants for materials/services up to a maximum of $750 per class. To qualify, [...]
Read more about New Pilot Funding Initiative for Schools »
Categories: Broadcast
Posted March 13, 2015
Registration is now open for the PIC Family Event 'Building Skills to Navigate the School Years', April 18, 2015, Bluevale Collegiate Institute. This FREE event is open to parents, guardians and the community. Keynote speaker, Lesley Andrews, will share with us her inspiring story of 'overcoming the odds' to follow her passion in life and [...]
Read more about Register Now for the PIC Family Event! »
Categories: Broadcast
Posted March 13, 2015
Saturday, April 18th, 2015 Bluevale Collegiate Institute, Waterloo The conference is FREE and we are also providing FREE childcare for children ages 2 to 11. We have a FANTASTIC collection of speakers and topics for our conference this year! Our keynote, Ms. Lesley Andrew is a Canadian singer, actress and motivational speaker "Overcoming Obstacles and [...]
Read more about FREE Parent Conference (pssst….. Free Childcare is available) »
Posted March 13, 2015
We are currently going through our yearly process of snow melting, and our tarmac being very icy and wet. If you are on our tarmac, please be cautious as we do get a lot of water pooling and freezing. Yesterday and today we have some people working on our drain to remove the frozen ice [...]
Read more about Tarmac excitement at EZ »
Posted March 5, 2015
Bravo to all our dancers – students and staff alike! We had a great day of moving to the music provided by DJ Professor Jamz. Thanks to students and families for your enthusiastic support and participation!
Posted March 5, 2015
Click here to keep up to date with the EZ School Council.
Read more about School Council Winter Newsletter Now Available »
Posted February 26, 2015
Yesterday was Pink Shirt Day, and EZ students and staff were encouraged to wear pink to show support for anti-bullying.
Check out this awesome picture that appeared in the Waterloo Region Record today!!
Read more about Elizabeth Ziegler – in the news! »
Posted February 26, 2015
Our dance-a-thon has been rescheduled for Thursday, March 5th. We look forward to this great event and some excellent movement from our students! Thanks to all for supporting this fundraising activity! m4s0n501
Posted February 26, 2015
Here is some information regarding Canada's Anti Spam Legislation (CASL) and parent communication. Click this letter to read more.


text:
Library Branch
Book Clubs & Writers' Groups
Mount Pleasant
Join in on diverse and thought-provoking discussions of non-fiction books selected by the group's members. Copies of the books are available at Mount Pleasant Branch. New Members Welcome....
Thu Apr 02
Thu Jun 04
Join us for a discussion on new books and old favourites....
Tue Apr 21
Tue May 26
Parliament Street
Library staff will treat you to some new reading suggestions and a lovely cup of tea! For more information visit the branch in person or call 416-393-7663.
Wed Apr 08
Wed May 13
Wed Jun 10
Discuss selected books in Spanish. Meetings are once a month on a Monday night at Palmerston Branch....
Mon Apr 13
Mon May 04
Mon Jun 01
Join us for an exciting afternoon of great books and conversation. Please register and pick up your copy at the Information Desk....
Tue Apr 21
Tue May 19
Tue Jun 16
All are welcome who are interested in learning about, sharing and discussing diverse collections of poetry. Drop-in.
Wed Apr 01
Wed May 06
Wed Jun 03
Wed Jul 01
Discuss new books and old favourites with library staff. Light refreshments provided. Call the library for more information.
Fri Apr 10
Fri May 01
Fri Jun 05
Mount Pleasant
Do you love to read? Do you enjoy discussing the adventure, romance, or mystery unveiled by a good book? Join the discussion of books selected by the group. Copies of the books are available at Mount Pleasant Branch. New mem...
Tue Apr 07
Tue May 05
Tue Jun 02
Tue Jul 07
Toronto WordSmiths is a writing group for youth ages 16-29 to come together to share and learn from one another....
Mon Apr 06
Mon Apr 13
Mon Apr 20
Mon Apr 27
Mon May 04
Eglinton Square
Members discuss a book by a woman author or about women read by the group. Call 416-396-8920 to register.
Mon Apr 13
Mon May 11
Mon Jun 08
Mon Jul 13


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About Us
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Thursday 02 April 2015
Book Mark
PDF newspaper
Local News
World News
UN says at least 62 children killed in Yemen violence
37 dead in bombing of dairy in west Yemen: Governor
Former Yemeni President Saleh 'in hometown hideout'
Al-Azhar: Houthis have crushed Yemen security
Pakistani delegation in Riyadh for talks on Yemen
Yemenis flee capital to escape airstrikes
News Analysis: Ground military intervention in Yemen likely tough task
UN says Yemen 'on verge of total collapse'
Will Yemen kick-off the 'War of the two Blocs?'
Saudi Arabia's Ominous Reach Into Yemen
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Arab in Persian Medis
by Fouad Zubairy
IRAN Mullahs expose their fear of PMOI by attributing lies to the dead
Ahwazi workers protest in front of Water Department over unpaid salaries
For 3rd month Iraqi forces block gasoline entry into Camp Liberty
GCC chief condemns Iranian regime official 'defamatory' remarks
Iranian opposition PMOI official passes away
Iran Ahvaz residents protest regime's inaction on dust pollution
Iranian opposition is opposed to any alliance with Tehran
Tehran youth destroyed motorcycles belong to the Basij paramilitaries
Tehran Bazar Shop keepers clash with police forces
political prisoner Reyhaneh Haj-Ibrahim
Saleh attempts to drag Yemen into a civil war
by Fouad Zubairy
Published Since: 3 Years and 5 Months and 4 Days
Wednesday 26 October 2011 11:54 pm
president Ali Abdullah Saleh- during a meeting with his party in Sana'a
Observers believe that the UN resolution about Yemen which called on President Ali Abdullah Saleh to step down looks weak and unable to change the existing balance in the absence of external real pressure against Selah's regime.
The lack of pressures against Saleh makes him dragging Yemen to a war, Persian News Agency Reported.
Observers in Yemeni affairs consider the UN resolution which calls on President Saleh to step down as weak and unable to change the existing balance in the absence of external pressure against Yemeni regime, which push for blowing the situation militarily.
The war could be a way for Saleh to impose more conditions for his departure and a way for the armed opposition to resolve constant conflict, since in nine months. Analysts stated.
Saleh would not sign the initiative of the Cooperation Council, likely to take a neroni decision to burn everything before leaving power. The Director of future Studies Center , "Fares Saqqaf" said.
Al-saqqaf said that UN resolution is a success for the path of political and diplomatic revolution, but noted that the recent Security Council resolution, which calls for the signing of the Cooperation Council initiative, will be most successful if it came under the Chapter VII sanctions against President Saleh, "Saqqaf" said.
The government expressed in an official statement Saturday willingness to deal positively with the UN Security Council Resolution 2014, but observers do not believe much on the Declaration of Intent by Sana'a, the president - according to their opinion – Saleh claimed repeatedly that he agreed on the initiative of the GCC, but he has backed three times from sign it.
Saleh is likely to announce his agreement to the international resolution, at the same time try to drag the country to the fighting, which will - according to his opinion – push for military escalation in coming days without a declaration of war officially. According Saqqaf.
The president will not be able to win the war in case it ignited, and eventually it will be channeled in the interest of the opposition. He added.
_BACKTO Arab in Persian Medis
Arab in Persian Medis
S. Arabia fears from Yemeni's Revolution to extend to Riyadh
by Fouad Zubairy
Egyptian Authorities Use Israeli Weapons against Protestors
by Fouad Zubairy
Analyst Raps US, S. Arabia, Qatar for Sowing Discord among Egyptians
by Fouad Zubairy
Iran: Bahraini people is killed by Saudi Arabia military
by Fouad Zubairy
Arab countries Spending Huge Sums to Derail Egyptian Revolution
by Fouad Zubairy
Saleh Regime Supporters Wound 40 Women in Yemen
by Fouad Zubairy
About Us
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All rights reserved © 2010-2015 Yemen Fox


text:
23 Avalon Way
Commute to Downtown Forest Park
Almost all errands require a car.
Score Details
23 Avalon Way has a Walk Score of 9 out of 100. This location is a Car-Dependent neighborhood so almost all errands require a car.
Nearby schools include Kendrick Middle School, Callaway Elementary School and Pointe South Elementary School.
The closest grocery store is ALDI Jonesboro, GA.
Nearby restaurants include Rival Group, Wings R Us and Wings R Us.
Explore how far you can travel by car, bus, bike and foot from 23 Avalon Way.


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London hit by 50mph gusts as winds cause damage and disruption across the capital
London has been battered by 50mph winds that have felled trees and caused travel chaos. Powerful gusts swept across the capital as the Met Office issued a yellow "be aware" weather alert for most of the country.
Driver held over 'drunk crash' into tree in south London which left passengers seriously injured
A driver suspected of drinking or taking drugs while behind the wheel was seriously injured after her car smashed into a tree in south London.
Alps air disaster: Families may have to wait months for remains to be identified
Families of the Germanwings crash victims may have to wait up to four months for their remains to be identified.
Related Articles
Published: 15 November 2012
When you write regularly about sexism, an odd thing starts happening. Emails pop into your inbox (all penned by men) demanding you stop holding forth on the globe's ovary-owners. "Aren't there bigger worries in the world?" they demand. Which is strange, because when I wrote only about the stock market, that didn't seem to trouble anyone, nor have I ever heard of a football correspondent being routinely scolded for focusing on the young men who destroy lawns for a living.
God forbid you touch on a lighter subject, though. For you can't bemoan the sexism of advertising (which — incidentally — often patronises men too), without one of these chaps asking why you aren't devoting your energies to ending female genital mutilation. For women can only care about one issue at a time, apparently.
But what stops me, when I read one of these rants, from unhooking my bra and searching for some lighter fluid is that there has never been a less lonely time to be making the case for equality.
Currently, the Everyday Sexism Project is documenting the daily jabs to the female spirit — from catcalling to unequal pay — while the No More Page 3 Campaign is asking The Sun to end the newspaper industry's version of a pervy uncle, whose sole purpose for staying in the paper in the age of internet porn seems to be to annoy feminists.
This Sunday, the two campaigns are teaming up for a comedy night — Stand up to Sexism — at the Harold Pinter Theatre, thus addressing another issue: the silly stereotype of the women's libber as a humourless harridan. Meanwhile, at the Royal Court, feminism underpins Lucy Kirkwood's darkly funny play NSFW, which suggests that the 'sleb magazines that trade in circles of shame (A spot! A bunion! Six fingers like Anne Boleyn!) can be just as cruel to women as lads' mags are.
Then there's the list Woman's Hour is currently compiling of the 100 most powerful women in the UK, which draws attention to the male domination of influential roles. In mixed-sex accolades, on average only 15 per cent of those who make the cut are women, and often some of those will owe their appearance to the man they have married. So perhaps the judges should let 15 men on to the list to ram that point home (Prince Philip, perhaps, as well as Theresa May's hubbie). There's a second, even more important list that is currently being put together, too. The Women's Room, an index of female experts, is intended to help address the lack of female voices on the radio and female faces on TV.
So to those behind the email invective, I'm afraid women's voices will only be getting louder and more numerous. We're not going to quieten down until equality has been won.
Let Oscar mind games begin
Having recently dubbed the Oscar race "total, utter bullshit", Joaquin Phoenix tried a corrective this week. In an interview with the Sydney Morning Herald, the actor — tipped for his role in The Master — said his nomination for Gladiator had clearly boosted his standing. "It's not like I f**king hate the Oscars," he added. "What I was reacting to was sometimes the reverence that we have about these things."
I'd guess the original outburst was part of an ingenious plan to ensure that the statuette is his this time. For how better to silence the only actor in Hollywood who is speaking sense than by dragging him over to the dark side? Just like those who rail against the honours system and yet jump joyfully to the Palace when they get the nod, I'll bet Phoenix attends the ceremony. But boy, he had better not cry now.
Super VAT: it's a no-brainer
In the fight against tax avoiders, the attention has now turned to those naughty foreign firms who consider paying corporation tax the preserve of their weaker competitors. But we mustn't forget those naughty deep-pocketed folk who consider paying tax to be the preserve of us plebs.
Since the dark arts of the bean-counters are finally getting the coverage they deserve, I propose a tax they will struggle to help their rich clients avoid: a super-VAT rate on the most luxurious goods. Sales taxes are, of course, usually regressive but this would hit the 0.01 per cent: those who consider the Financial Times' supplement, How to Spend It, a shopping list and who feature in Chrystia Freeland's brilliant book, Plutocrats. So when they buy a Bugatti to keep the Lamborghini company, or a Patek Philippe to "look after for the next generation", they'll pay this rate. Since these are arguably both "Veblen goods" — where they become more desirable as the price climbs — I suspect it won't even hurt sales.
Old dogs can teach us tricks
The Today programme yesterday promised to reveal the secret of happiness. After a 72-year study by the Harvard Medical School — no doubt costing some insane sum — its current director shared what I could have imparted for free: if you want to be more content (and — before the RSPCA calls in — you have the spare time and space), get a puppy.
This was oddly well-timed for me, as Saturday will see a much-wanted addition to my family: Wilbur, a former racing dog we are adopting from the charity Greyhound Rescue West of England. At 11, Wilbur clearly can't be classed as a puppy, but old dogs can equally teach humans the tricks of happiness: affection, an extreme appreciation of food and the habit of howling at the men and women from Royal Fail.
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Here is a great Postcard that I bought in Maryland - I was not looking for anything like this - but I realized that I had to have it. I will try a better scan. It was published by F. W. Brown Book Company of Ann Arbor. Very cool - especially as I love taking photos of squirrels on campus.
_Abi_ and cindy.hall added this photo to their favorites.


text: Buffalo Talk [VIDEO] By Joe August 18, 2014 10:18 AM Share on Facebook Share on Twitter Just like everywhere you go… people are going to say you have an accent… or they are just going to think you are silly and not talk to you in the first place. Lets just thank our lucky stars that we aren't Canadian, aye. Filed Under: accent, Buffalo Category: Jack Around Town, Jacktube Share on Facebook Share on Twitter More Leave a comment Send to a friend! Print this page Pin it! Reddit This! Share on Tumblr! Best of 92.9 Jack FM Recommended For You Around the Web


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"And the way they approach it, in this very refined but scary manner, I think is what sets them apart."
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