This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G0/G1 phase and caspase-3 activation.A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS) initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01) with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G Caspase-3 then cleaves cellular targets to promote apoptosis Apoptosis plays a crucial role in the normal development and pathology of a wide variety of tissues  23 mg to 1 g Citrus paradisi), oranges (Citrus sinensis) and in tomato (Solanum lycopersicum) Over 4000 different flavonoids have been identified in the human diet and the daily intake ranges fromOxidative stress induced DNA damage or deficient DNA-repair is reported to have an etiological or prognostic role in cancer via estrogen receptor viz. human epidermoid carcinoma A431 cell.Several mechanisms have been proposed for naringenin induced anti-proliferative effects i.e. antioxidant activities, kinase and glucose uptake inhibition, and to hamper cell proliferation o's modified eagle medium (DMEM) and HaCaT cell was cultured in DMEM:F12 (1∶1) medium supplemented with 10% (v/v) fetal calf serum (Himedia), 2.0 mM L-glutamine,1.5 g/l NaHCO3, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate and 1% antibiotic solution. Cells were maintained at 37°C, 5% CO2 in a humidified air.Normal skin cell (HaCaT) and Human epidermoid carcinoma (A431) cell line were obtained from Cell Repository, National Centre for Cell Sciences (NCCS), Pune, India. A431 was cultured in Dulbeccviz. 50 µM, 100 µM, 200 µM, 300 µM, 400 µM, 500 µM and 750 µM of naringenin were prepared in dimethyl sulfoxide (DMSO) in final volume of 100 µl media. After 21 h exposure, 10 µl of MTT solution (5 mg/ml stock solution) was added in each well re-incubated for 3 h at 37°C until formazan blue crystal developed. Media was discarded from each well and 100 µl of DMSO was added to dissolve formazan crystals for 10 min at 37°C. The absorbance was recorded at 540 nm by microplate reader (BIORAD-680) and relative percentage cell viability was evaluated.This assay is based on the enzymatic reduction phenomenon of 3--2,5-diphenyltetrazolium bromide (MTT) dye and provides a direct relationship between the viable cells and absorbance. The effect of naringenin on cell viability was evaluated by MTT reduction assay as performed earlier 41×10 cells/well in 96-well culture plate. After overnight incubation, the cells were treated with different concentrations of naringenin for 24 h. The cellular morphology was observed under inverted phase contrast microscope .The effect of naringenin was analyzed for morphological changes in the cultured cells 4 per well) were seeded as described above for the MTT assay. Cells were then exposed to 50 µM, 100 µM, 200 µM, 300 µM, 400 µM, 500 µM and 750 µM concentrations of naringenin for 12 h. Cells were incubated with 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) (10 mM) for 30 min at 37°C. The reaction mixture was aspirated and replaced by 200 µl of phosphate-buffered saline (PBS) in each well. The plate was kept on a shaker for 10 min at room temperature in the dark. An inverted fluorescent microscope  was used to visualize intracellular fluorescence of cells and to capture images. For quantitative ROS analysis, cells (1×104 per well) were re-seeded in 96-well black bottom culture plate and allowed to adhere for 24 h in a CO2 incubator at 37°C. A431 cells were treated with different concentrations of naringenin for 12 h. After exposure, cells were incubated with DCFH-DA (10 mM) for 30 min at 37°C. Fluorescence intensity was measured by multiwell micro-plate reader  at excitation wavelength of 485 nm and emission wavelength of 528 nm. Values were expressed as the percentage of fluorescence intensity relative to the control wells.Microscopic fluorescence imaging was used to study ROS generation in A431cells after exposure to different concentrations of naringenin  10 min. Subsequently the cells were permealized with permealizing buffer  and stained with DAPI dye. After staining, the images were captured and numbers of cells were quantified using a fluorescent microscope .The apoptotic effect of compounds was analyzed by using florescent nuclear dye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1) is a cationic, lipophilic dye and therefore has been extensively used to study the loss of the mitochondrial membrane potential which occurs during apoptosis  24 h exposure, the treated cells were washed with PBS and stained with 2 µg/ml of JC-1 dye in DMEM media without phenol red at 37°C in dark for 30 min. The photographs were then taken by inverted fluorescent phase contrast microscope and the mitochondrial depolarization patterns of cells for cells quantification were examined by using imaging software NIS-Elements F 4.00.00.Flouroprobe 5, 10 mM Tris-HCl pH 8.0, 0.2% Triton X-100 and 100 µg/ml Proteinase K) for 1.5 h at 37°C. The samples were centrifuged at 6000×g for 5 min to collect the supernatant containing DNA. The supernatant was added with equal volume of isopropanol and 25 µl 4M NaCl to make 100 mM final concentration and incubated overnight at −20°C. Sample was then centrifuged at 6000×g for 25 min at room temperature. The pellet was dissolved in 50 µl ddH2O and 2 µl RNase A (10 mg/ml) and reincubated at 37°C for 1 h. The extracted DNA was subjected to electrophoresis on 1.5% agarose gel containing ethidium bromide. The DNA bands were read under ultraviolet illumination gel-doc system .Assay was done by the method of 61×10 cells/ml and treated with 100 µM, 300 µM and 500 µM of naringenin for 24 h in 5% CO2 incubator and at 37°C temperature  24 h incubation, the cultured cells were harvested, washed with cold PBS, fixed in 70% ethanol and treated with RNase A (10 mg/ml). Fixed cells were stained with propidium iodide (PI) dye followed by incubation for 30 min at room temperature in dark. The PI fluorescence of individual nuclei was measured using flow cytometer . Data were analyzed with the Cell Quest Pro V 3.2.1 software .Cell cycle phase distribution with cellular DNA contents were carried out using flow cytometry. A431 cells were seeded into 6-well plate at density 63×10 (treated and untreated) of cells were resuspended with chilled lysis buffer. The cell lysate was incubated on ice for 10 min prior to centrifugation . The reaction buffer, with 10 mM DTT was added to the supernatant of cell lysate and incubated further for 30 min on ice. The cell lysate (50 µl) was aliquoted into wells of 96-well microplate and 50 µl of reaction buffer containing 10 mM DTT was then added to the lysate. About 5 µl of 4 mM DEVD-pNA substrate was added in each wells and incubated at 37°C for 2 h. Absorbance at 405 nm was then read in a microplate reader. The absorbance of treated cells was compared with untreated control to find the change in caspase-3 activity.The caspase-3 activity was assayed using Caspase-3 Colorimetric Assay Kit . Approximately Data were expressed as the mean ± SD from three independent experiments. One-way ANOVA and Dunnett’s multiple comparison test were performed using Graph Pad prism (Version 5.01) software for significance test, using a p value≤0.05.To find out the experimental doses of naringenin, cytotoxic tests were performed on normal skin cells HaCaT using MTT assay and cell morphology assessment. Morphological study suggests that higher concentration 750 µM of naringenin changes cell morphology and induces cell death. However, doses 50 µM, 100 µM, 200 µM, 300 µM, 400 µM and 500 µM of naringenin did not affect significantly on cell morphology as revealed in  24 h treatment of naringenin. The effect of different concentrations of naringenin on cell viability was shown in The cell morphology was assessed using inverted phase contrast microscopy under 20× objective . PhotogrNuclear apoptosis were observed by using florescent DAPI dye. Condensed and fragmented nuclei were regarded as apoptotic cells. As observed from photomicrograph, naringenin induces the nuclear condensation in a dose dependent manner as indicated by arrow . Maximum 12 h after naringenin treatment. Florescent micrograph of stained A431 cells (ROS generation was assessed at31 cells  depicts 31 cells . Maximum31 cells . IncreasΔΨm). Result indicated that cells treated with 100 µM, 300 µM and 500 µM of naringenin induced a strong green fluorescence in a dose dependent manner , an important biochemical feature in apoptotic signaling, was further investigated to determine whether the apoptosis was induced by naringenin. Caspase-3 is the main downstream effecter caspase in apoptotic pathway and hence we compared its activity in treated and untreated control cells. The result showed that caspase-3 was significantly increased to 29.57% and 85.10% (***p<0.001) at concentrations 100 and 300 µM of naringenin. However, caspase-3 activity was dramatically increased to 114.02% (***p<0.001) at 500 µM naringenin concentration .Phyto-chemicals have been demonstrated significant potential as anticancer therapeutic agents due to their ability to inhibit tumor growth, angiogenesis, and metastasis without many side effects Here we have endeavored to work out the effects of the naringenin on epidermoid carcinoma A431 cell line. Cell viability analysis suggests that naringenin halts proliferation of cells . Furtherviz. glutathione (GSH) and glutathione peroxidase (GPX) under the influence of anti-oxidant agents which perturb the anti-oxidant system and lead apoptosis ΔΨm) is as an early event in apoptotic process as shown by earlier workers The accumulation of free radical mediated damage may be a possible mechanism of cancer development DNA fragmentation is generally considered to be the hallmark of apoptosis. Our DNA fragmentation data support earlier observation that naringenin induced the DNA fragmentation and subsequent cellular damage Fig. 5. 1 peak) of the cells were analyzed by flow cytometry using PI staining. G1 and G2 phases of the cell cycle are the major check points and have an important role in cell cycle progression 0/G1 phase of cell cycle and consequently lead to apoptosis (The results support the findings that key changes in cell death can occur without DNA fragmentation and it alone should not be considered as a criterion for assessing apoptotic cell death poptosis . Caspasepoptosis .0/G1 phase and caspase-3 activity. Our data confirm the potential of naringenin as an agent of chemotherapeutic and cytostatic activity in human skin carcinoma cancer and therefore, may be potentially valuable for application in drug developments. For further confirmation, molecular mechanism will be carried out to elucidate the molecular pathways.In conclusion, the present study provides a novel insight into the mechanism of action of naringenin-induced apoptosis in human skin carcinoma cells without/little affecting normal skin cells and shows a link between antiproliferative and apoptotic induction, and the cell death was due to the induction of ROS mediated mitochondrial membrane depolarization, nuclear condensation and DNA fragmentation. We also observed that naringenin induced cell cycle arrest at G


Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA.Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit   •X-ray structures of major yeast eIF3 components and subcomplexes•Crosslinking coupled to mass-spectrometry analysis of 40S⋅eIF1⋅eIF3 complex•Integrative modeling reveals architecture of 40S⋅eIF1⋅eIF3 complex A hybrid approach drawing on X-ray structures, crosslinking coupled to mass spectrometry, electron microscopy, and integrative modeling yields mechanistic insights into how eIF3 coordinates translation initiation. Protein synthesis is catalyzed by the ribosome in a process that consists of initiation, elongation, and termination. In bacteria, three initiation factors  and the Shine-Dalgarno sequence are sufficient to accurately pair the AUG start codon in messenger RNA (mRNA) with the anticodon loop of methionyl initiator transfer RNA (Met-tRNAi). Eukaryotes, in contrast, require at least 25 different polypeptides assembled into eight eukaryotic initiation factors (eIFs) to initiate protein synthesis and employ a complex scanning mechanism to probe the 5′ leader sequences of mRNA for the correct start site . Initiati ternary complex (TC), eIF1, eIF1A, eIF5, and eIF3 on the 40S ribosomal subunit. In canonical eukaryotic translation, the 43S PIC recruits mRNAs by engaging the eIF4F cap-binding complex to form the 48S PIC. Within the 48S PIC, eIF1, eIF1A, eIF3, and eIF4G promote the accurate scanning of the mRNA leader region (i.Eukaryotic translation initiation begins with the cooperative assembly of the 43S preinitiation complex (PIC), composed of the eIF2/GTP/Met-tRNAr region  and ensuproteosome, COP9/signalosome, eIF3) modules, and two (eIF3f and eIF3h) contain MPN  domains. The PCI⋅MPN core is structurally conserved in the 26S proteasome lid, the COP9 signalosome, and eIF3, forming a distinctive, multilobed structure   with a molecular weight of 600–800 kDa . Six of tructure . PCI modtructure , which mtructure . In the tructure , an orgatructure . S. cere3j/Hcr1) A. The co3j/Hcr1) .Figure 1subunits and the dynamic nature of the 40S⋅eIF3 interaction. In this study, we present X-ray structures of all major structured domains of S. cerevisiae eIF3 and utilize a combination of negative-stain electron microscopy (EM), chemical crosslinking followed by mass spectrometry (CX-MS) and integrative structural modeling to derive a detailed molecular architecture of the yeast 40S⋅eIF1⋅eIF3 complex.Recent EM reconstructions as well as in vivo and in vitro experiments indicate that the N-terminal ends of the helical subdomains of the eIF3a/eIF3c PCI modules form two intermolecular bridges with the 40S subunit in the vicinity of ribosomal proteins rpS13/uS15 and rpS27/eS27 as well as rpS26/eS26, rpS1/eS1, and rpS0/uS2 . In addiS. cer. residues 1–496) was solved by single-wavelength anomalous dispersion (SAD) with selenomethionine (Se-Met)-labeled protein to a resolution of 3.3 Å  and residues 228–496 of eIF3a. The structure of the dimeric eIF3a/eIF3c complex was solved at 3.5 Å resolution using Se-Met SAD -containing RNA elements require the presence of eIF3. The recent structure of the 40S-bound CSFV IRES in complex with eIF3 revealed the structural basis of this interaction, with the IRES RNA occupying the eIF3 binding site at the 40S platform and creating a secondary binding site for the PCI⋅MPN core . BecauseBased on the convincing fit of the eIF3a/eIF3c structure into the EM density, we attempted to dock the recently solved paralogs of eIF3e (CSN1)  and the S. cer. residues 132–626) and solved its structure using Se-Met SAD to a resolution of 2.2 Å , full-length eIF3i, and the eIF3g-NTD  (S. cer. residues 1–135). The heterotrimeric structure was solved to a resolution of 2 Å using molecular replacement with the eIF3b-CTD/eIF3i complex as the search model  sample preparation as described for both yeast and mammalian 40S⋅eIF1 complexes . Because complex B reveals complex A. SimilaS. cerevisiae eIF3 samples to form dimers due to the blade swapping within the eIF3b subunit  . SpecifiS. cerevisiae 40S subunit  and eIF3c (S. cer. residues 1–251) provide critical additional restraints  to regulate their access to the ribosome. In rganisms . Our detA full list of constructs used in this study is given in All proteins (15–20 mg/ml) were crystallized by sitting-drop vapor diffusion. Native and Se-Met SAD data were collected at beamline PX of the Swiss Light Source and processed in XDS . StructuEIF3 samples were assembled from purified, recombinant subunits and were subsequently mixed with 40S subunits as described in the Lachancea kluyveri 40S⋅eIF1⋅eIF3 complex was prepared and crosslinked as described for CX-MS. The sample was then subjected to gradient fixation using GraFix  phosphine hydrochloride (TCEP) and 10% Glycerol. S. cer. or L. klu. His-MBP-tagged eIF3c and His-tagged eIF3a were independently purified over Ni-affinity columns, then combined and purified over a Heparin column. D. han. His-eIF3b/eIF3i/eIF3g was expressed from a polycistronic construct and purified over Ni-Sepharose and Heparin columns. Bacterial cells expressing S. cer. or L. klu. eIF3i were mixed with cells expressing S. cer. or L. klu His-MBP-tagged eIF3g before lysis and purification over Ni-Sepharose and HiTrap Q. L. klu. His-eIF3b, His-MBP-eIF3j and His-eIF1 were expressed separately and purified over Ni-Sepharose and Heparin columns. Samples for crystallography were assembled and digested with TEV protease overnight to remove all tags before final purification over a Superdex 200 gel-filtration column. The 6-subunit eIF3 core complex was assembled from purified L. klu. eIF3a/eIF3c, eIF3b, eIF3i/eIF3g (or eIF3b/eIF3i/eIF3g in the case of D. han.) and eIF3j before overnight TEV cleavage to remove all tags. After orthogonal purification to remove uncleaved proteins, tags and TEV protease, the sample was applied onto a Superdex 200 column to yield the full eIF3 complex. Se-Met-derivatized proteins were expressed in minimal media supplemented with an amino acid cocktail including Se-Met (Proteins were cloned into pET-derived vectors using ligation-independent-cloning and expressed in g Se-Met .Lachancea kluyveri (DSMZ strain 70517). Cells were grown to an OD of 10 in a 50-l fermenter (INFORS) at 24°C, harvested by centrifugation, washed with 1x PBS, resuspended in lysis buffer and snap frozen in liquid nitrogen. Yeast cells were lysed using a cell cracker at 40 kpsi, the lysate cleared by centrifugation  and then applied onto a high-density sucrose cushion (60% w/v) in low salt for a 24 hr ultracentrifugation at 50k rpm in a TI-70 rotor. Pelleted ribosomal particles were resuspended in high-salt buffer and the subunits separated on 10%–47% w/v sucrose gradients by ultracentrifugation. 40S bands were carefully removed and pooled, then buffer exchanged into 50 mM HEPES pH 7.5, 10 mM MgCl2, 75 mM KCl and 0.5 mM TCEP.40S ribosomal subunits were purified from S. cer. eIF3b to form dimers, we focused on two related budding yeasts for our EM and CX-MS experiments. The full 6-component eIF3 complex, lacking only the disordered C terminus of eIF3a  was assembled from individual components. While we were able to assemble the full eIF3 complex from Lachancea kluyveri, we were unable to purify eIF3a and eIF3c from Debaromyces hansenii. Because our polycistronic construct for D. han. eIF3b/eIF3i/eIF3g expressed large quantities of the trimeric complex, and because of the high degree of conservation among these related budding yeasts, we decided to test whether we could obtain a hybrid complex of L. klu. and D. han. to increase the variability of the samples for CX-MS. Hybrid eIF3 complexes behaved similarly to the nonhybrid L. klu. complex and showed the same areas of extra density in negative-stain single particle reconstructions (data not shown). 40S⋅eIF1⋅eIF3 (10 μM) was assembled with a 1.2x molar excess of initiation factors in 25 mM HEPES, 15 mM MgCl2, 125 mM KCl and 2 mM TCEP prior to crosslinking. For each round of experiments, several different salt concentrations (40 mM to 175 mM KCl) as well as sample concentrations (2 μM to 10 μM) were explored to find optimal conditions for stable complex formation prior to crosslinking.Because of the propensity of 221 appeared overnight and achieved maximum size after 3 days. Crystals were stabilized and dehydrated over a 2 hr period by the addition of 25% ethylene glycol and 12% PEG 20K over 4 equal concentration steps. This dehydration treatment was essential for diffraction beyond 4.5Å and to promote isomorphicity along the crystallographic c-axis between native and Se-Met crystals. The eIF3b β propeller (20 mg/ml) was crystallized in space group P21 after 2-5 days in 10%–12% PEG MME 5k, 100-200 mM KSCN or KH2PO4 and pH 8 – 9.5 using Tris-Acetate or CHES as buffers. Crystals were cryo-stabilized with the addition of 25% Glycerol and flash frozen in liquid nitrogen. The trimeric eIF3b-CTD/eIF3i/eIF3g-NTD complex (15 mg/ml) was crystallized in 5%–7% PEG 20K, 25%–28% PEG MME 550 at pH 6.5 with Imidazole or MES buffers and belonged to space group C2221. The structures of eIF3a, eIF3a/eIF3c and eIF3b were solved by single-wavelength anomalous dispersion techniques using Se-Met substituted proteins. eIF3b-CTD/eIF3i/eIF3g-NTD was solved using the yeast eIF3b/eIF3i structure  prior to crystallization. The eIF3a-PCI domain (18 mg/ml) was crystallized by sitting-drop vapor diffusion against a solution of 14%–18% PEG 3350, 400-600 mM KSCN in a broad pH range (6.8-8.8) using bis-tris-propane, HEPES or bicine as buffers. Crystals belonging to space group C2 grew over 2-5 days and were cryo-stabilized with the addition of 25% ethylene glycol. The eIF3a/eIF3c dimer (20 mg/ml) was crystallized by sitting-drop vapor diffusion against a solution of bis-tris-propane pH 6.0-6.3, 9%–11% PEG 6000 and 400-500 mM KSCN. Crystals belonging to space group P3tructure  as a mol2; Average refined B factor = 175 Å2). Initial models were built into the 3.7 Å experimental SAD maps obtained from AutoSol  at room temperature (RT) for 30 min and quenched for 10 min at RT with ammonium bicarbonate . Samples assembled in high salt, where no significant ribosome oligomerization occurred, were processed immediately, while low salt samples, which showed a significant level of ribosome oligomerization, were applied to 10%–47% sucrose gradients to separate monomeric 40S fractions from oligomeric assemblies and unbound eIF3 before further processing. There are strength and weaknesses in each of these approaches, as the extra purification steps lower the final peptide yields, while the nonpurified samples retain a background of eIF3 interlinks from the unbound eIF3 fraction of the sample. To ensure a large and unbiased set of identified crosslinked peptides, we included crosslinks from both data types in the final data set, although we did exclude eIF3⋅eIF3 interlinks from our nonpurified samples, as there is no unbiased way to determine whether the crosslink was generated when eIF3 was bound to 40S or free in solution.EIF3 samples from After 1 hr treatment with 10 μg/ml RNase A (Roche), the samples were reduced with 2.5 mM TCEP in 8 M urea at 37°C for 30 min and alkylated with 5 mM iodoacetamide (Sigma-Aldrich) for 30 min at RT in the dark. Before digestion, the samples were diluted with ammonium bicarbonate to a 1 M final concentration of urea. 2% w/w trypsin (Promega) was added overnight at 37°C and inactivated by acidification with 2% (w/v) trifluoroacetic acid (TFA). Peptides were purified with Sep-Pak C18 MicroSpin columns , according to the manufacturer’s protocol, followed by enrichment of crosslinked peptides using size exclusion chromatography . LC-MS/MS. cerevisiae 40S⋅eIF1⋅eIF3 complex architecture proceeds through four stages (S. cerevisiae sequence.The integrative approach to determine the r stages : (1) gatr stages . All resS. cerevisiae eIF1⋅40S interface was modeled using the Tetrahymena thermophila 40S⋅eIF1 complex structure  as well as the eIF3 subunit domains described in the main text . We removed chain h (the Stm1 protein) from the 40S structure, and rigid-body adjusted the position of helix ES6A to better fit the 43S EM density . We incrtructure . Sequenctructure . The 965tructure  were idetructure  and FDR tructure  to defintructure .The domains of the 40S⋅eIF1⋅eIF3 subunits were represented by beads, arranged into either a rigid body or a flexible string, based on the available crystallographic structures and comparative models . To balaM ≡  includes the structure coordinates X and additional parameters {αi} (below). Using Bayes’ theorem, the posterior probability p, given data D and prior knowledge I, isp is the probability of observing data D, given I and M; and the prior is the probability of model M, given I. To define the likelihood function, one needs a forward model that predicts the data point  given the structure, and a noise model that specifies the distribution of the deviation between the observed and predicted data points. The Bayesian scoring function is the negative logarithm of pp(M|I), which ranks the models identically to the posterior probability. All information gathered in Stage 1 is encoded into the Bayesian scoring function as a likelihood function (crosslinking data), as well as priors .The Bayesian approach  estimatedn between two lysines i and j, the forward model predicts the presence of the crosslink given the structural coordinates X. Because of the multi-scale representation, residues are mapped into coarse-grained beads with variable radius, which represents the uncertainty of their positions. Furthermore, the rigid-body representation of crystallographic structures does not account for the flexibility of residues placed in loops. Therefore, we introduced the parameters {σk}, one for each crosslinked lysine, that define the unknown radii of the spheres, centered on the beads representing the lysines, within which the position of the lysine Cα is uniformly distributed. Given ri and rj (the coordinates of the Cα of the two lysines) and the maximum length for the crosslinker lXL, including the lysine side chains, we defined the forward model fn as the probability of randomly picking two points ri and rj, with radii σi and σj, such that the distance between them lXL. The forward model value is one  when the spheres are closer than lXL and the radii are sufficiently small. It has a value of zero  if the distance between the spheres exceeds lXL. To reduce the number of parameters in the model, we utilized a single structural uncertainty σ, whose value estimates the average of {σk} for all crosslinked residues. We estimated lXL = 21 Å from potential of mean force calculations of a system comprising of the DSS crosslinker covalently attached to the two lysine sidechains .For a given experimentally identified crosslink dn is defined by the following data noise model:ψn ∈ is the uncertainty associated with the crosslink identification. When the value of ψn is 0.5, the uncertainty is maximal and the likelihood does not depend on the forward model fn or on the distance between the lysines. When ψn is 0.0, the uncertainty is minimal, and the likelihood is identical to the forward model, i.e., it reaches a maximum when the distance between the spheres rij is below the crosslink length lXL. The joint likelihood function p for a data set D = {dn} of NXL of independently observed crosslinks is the product of likelihood functions for each data point. Crosslinks observed multiple times are considered as independent observations, and therefore considered multiple times in the joint likelihood function. Analysis of the intra-40S crosslink distances , ψM (263 crosslinks) and ψL (344 crosslinks) for the three classes.The likelihood function for an identified crosslink istances C allowedistances . We redup(M|I) is defined as a product of the individual priors p(X), p(σ), p(ψH), p(ψM), and p(ψL) on the structural coordinates, structural uncertainties, and the three data point uncertainties, respectively. The prior p(X) included the excluded volume and terms to maintain sequence connectivity. The excluded volume restraint was implemented as a pairwise hard-sphere repulsive potential, where the bead radii were determined using the statistical relationship between the volume and the number of residues (−1. p(σ), p(ψH), p(ψM), and p(ψL) are uniform distributions, that bound σ to interval , and the ψ parameters to the interval .The model prior residues . The seqStructural models of the 40S⋅eIF1⋅eIF3 complex were computed by Replica Exchange Gibbs sampling, based on Metropolis Monte Carlo sampling . This saFor each ensemble, the solutions  were grouped using k-means clustering using the root-mean-square deviation (rmsd) of all eIF3 subunits as a structural similarity criterion when the models were superposed on the 40S subunit structure. The rmsd distance matrix identified two dominant clusters of similar structures A, here nJ.P.E., F.S., R.P., and N.B. conceived the study and experimental approach; J.P.E., S.Z., and T.S. cloned, purified, and crystallized eIF3 samples, collected and processed X-ray diffraction data, solved the structures, and built the atomic models; J.P.E., T.S., F.S., and C.H.S.A. prepared samples for EM and CX-MS experiments. C.H.S.A. and D.B. collected EM data and calculated the EM reconstructions; F.S. performed CX-MS experiments; R.P. carried out the integrative modeling with the help of P.C.; J.P.E., F.S., and R.P. analyzed the integrative modeling results, and J.P.E., F.S., R.P., A.S., R.A., and N.B. wrote the paper with input from all authors.


The magnetization trajectory was simulated to identify sources of T1ρ error. To limit motion artifacts, an optical flow-based image registration method was used to align T1ρ images. The reproducibility and accuracy of these methods was assessed in phantoms and 10 healthy subjects. Results are shown in 1 patient with pre-ventricular contractions (PVCs), 1 patient with chronic myocardial infarction (MI) and 2 patients with hypertrophic cardiomyopathy (HCM).We developed a breath-held T1ρ images in subjects, as determined by the increase in Dice Score Coefficient (DSC) from 0.76 to 0.88. T1ρ reproducibility was high  = 0.85–0.97). Mean myocardial T1ρ value in healthy subjects was 63.5 ± 4.6 msec. There was good correspondence between late-gadolinium enhanced (LGE) MRI and increased T1ρ relaxation times in patients.In phantoms, the mean bias was 1.0 ± 2.7 msec (100 msec phantom) and 0.9 ± 0.9 msec (60 msec phantom) at 60 bpm and 2.2 ± 3.2 msec (100 msec) and 1.4 ± 0.9 msec (60 msec) at 80 bpm. The coefficient of variation (COV) was 2.2 (100 msec) and 1.3 (60 msec) at 60 bpm and 2.6 (100 msec) and 1.4 (60 msec) at 80 bpm. Motion correction improved the alignment of T1ρ mapping in a breath-hold with good accuracy and precision.Single-shot, motion corrected, spin echo, spin lock MRI permits 2D T In vivo myocardial T1ρ mapping has detected relaxation time changes that correlate with histologically-confirmed scar in animal models of ischemic heart disease T. Simulations were performed in Matlab .Simulations were performed to test the magnetization response to pulse sequence parameters. The time-dependent magnetization was solved using piecewise, time-independent matrix solution to the Bloch equations M(t)=eLt1ρ MRI pulse sequences were determined in phantoms and human subjects. A phantom was prepared with multiple known nuclear relaxation properties, as determined by the reference T1ρ sequence, and differences between measured and true T1ρ relaxation times were quantified. The phantom contained 8 cylindrical samples prepared with solutions of H2O and MnCl2 in concentrations of 0.002, 0.004, 0.006, 0.008, 0.010, 0.012, 0.015, 0.017% solutions, providing a range of T1ρ relaxation times that would be expected in vivo. Mean bias was analyzed for two phantoms with relaxation times similar to normal myocardium  and scar tissue . Scan parameters were the same as in the previous section in a single slice intersecting all phantoms and with TSL = 50, 42, 34, 26, 18, 10, 2 msec.The accuracy and precision of different T1ρ images were acquired in 10 normal subjects  without a previous history of cardiovascular events, 1 patient with chronic MI, 1 patient with PVCs, and 2 patients with HCM on a 1.5 T whole-body MRI system  at the Hospital of the University of Pennsylvania. The University of Pennsylvania Institutional Review Board approved this study and all subjects gave written informed consent to participate. 5 of the subjects underwent 3 MRI scans to assess the reproducibility of T1ρ maps.T1ρ-weighted images were acquired, during end-systole and end-expiration, to reduce the effects of cardiac and respiratory motion. The end-systolic cardiac phase was determined by first examining the cardiac motion on cine-SSFP short-axis images and then adjusting the beginning of spatial encoding with the period of maximum myocardial wall thickness. To further reduce respiratory motion artifacts, all T1ρ-weighted images were acquired within a single end-expiratory breath-hold using the same acquisition parameters as in the phantom experiments, and with flip angle = 70°, Nshots = 1, TSL = 50, 42, 34, 26, 18, 10, 2 msec, and number of slices = 7–10.In humans, 2D multislice ECG-gated T1ρ MRI were performed 2 weeks apart in different imaging sessions.Late gadolinium enhanced (LGE) MRI was performed in patients using a 2D multislice, short-axis, phase-sensitive inversion recovery pulse sequence . Approximately 15 minutes prior to LGE MRI, 0.15 mmol/kg gadolinium-based contrast agent  was administered intravenously. In the patient with chronic scar, LGE and T1ρ images were acquired at the same end-systolic cardiac phase and during an end-expiratory breathhold. Even though this strategy reduced the influence of motion, residual myocardial motion was observed in images due to respiration and small variations in sinus rhythm. This issue was more problematic when the subjects were unable to hold their breath or were uncooperative.To mitigate cardiac motion, T1ρ and R2 mapping were implemented into the online reconstruction software utilizing the Image Calculation Environment (ICE) framework of the clinical MRI System . Each T1ρ image series consisted of 8 images. Frame 4 (TSL = 34 msec) was chosen as the reference frame as it showed moderate T1ρ contrast and, in most cases, less motion compared to other frames. Two-frame image registration was performed between each of the 7 moving frames and the reference frame (frame 4).To further reduce the effects of residual motion, we investigated image-based optical flow (OF) motion correction. The OF algorithm ,19 was iTo quantify the accuracy of motion correction, we measured the Dice similarity coefficient (DSC) between image pairs with large, moderate and no motion before and after motion correction. DSC was defined as:1ρ relaxation to assess their accuracy and precision (We tested three models of Trecision . The mai1ρ MRI accuracy was quantified as the percent error N = 3 measurements and σ(Tρ1) the standard deviation. The intra-class correlation coefficient (ICC) was used to assess the T1ρ ROI measurement reproducibility between different scans (R software package). ICC values > 0.75 represent a good agreement . In both previous reports, motion and heart rate correction were not discussed, but could reduce T1ρ reproducibility. As was observed in Previous CMR T studies ,27 were mans. In , relaxatThere was moderate qualitative correspondence between T1ρ and LGE lesions. Specifically, for the patient with ischemic heart disease, T1ρ MRI was consistent with non-transmural scar at a mid-ventricular imaging slice. The reduced wall thickness indicated a very large apical aneurysm and this was supported by LGE hyperenhancement. The spatial distribution of the hyperintense region was consistent with scar, since the lesion appeared predominantly in the mid-myocardium. For HCM patients, there was asymmetric septal wall thickening of the anterosuperior right ventricular insertion zone and this corresponded to elevated T1ρ and LGE lesions. In a fourth patient with non-ischemic cardiomyopathy, there was elevated midwall T1ρ corresponding to a similar LGE lesion pattern. There were several features that were inconsistent, such as the apparent LGE scar size in the ischemic patient or relative lesion position in HCM patients. Myocardial or patient motion may have contributed to incomplete overlap. In this study, LGE scans were performed 10–20 minutes after pre-contrast T1ρ scans and LGE was performed in diastole and T1ρ in systole. Other studies have also reported moderate correspondence between LGE and T1ρ. In van Oorschot et al., double-blinded overlap of LGE and T1ρ in 21 ischemic patients was 72% and unblinded was 74% . Inconsi1ρ MRI using similar parameters [1ρ measurements, it was desirable to change the field-of-view instead of parameters that would affect the number segments and readout duration. The bias for all flip angles and segment sizes was low. The range of T1ρ relaxation times was 10 msec in the normal subject group, but does not account for potential effects of age or gender in these populations and may impact the detectability of diffuse disease.We observed that single-shot bSSFP imaging at high flip angle (70°) had high accuracy compared to the standard, partially because bSSFP has lower perturbation of the transverse relaxation compared to spoiled gradient echo sequences, even at very high flip angles . The accrameters , primari1ρ MRI arising from pulse sequence parameter selection. In particular, Bloch equation simulations showed that proper choice of pulse sequence parameters is essential to reduce bias and reproducibly measure T1ρ relaxation times in the heart. Motion was a major contributing factor to error in cardiovascular T1ρ mapping. Our initial experience using breath-held multi-shot T1ρ imaging was highly unsatisfactory due to the presence of image artifacts, which caused inconsistency in phase encoded k-space data. Single-shot imaging and OF motion correction was found to improve the quality of the T1ρ map  fraction, upstream coronary perfusion or tracer kinetics may alter contrast agent distribution in tissue and LGE signal intensity. T1ρ is sensitive to interactions between tissue water 1H and the macromolecular environment. Increased myocardial fibrosis associated with extracellular matrix expansion may reduce water 1H rotational correlation times and increase T1ρ. In addition, T1ρ imaging has shown increased signal intensity in areas of edema [1ρ MRI. A study by Wang et al. [1ρ maps and LGE cardiac magnetic resonance using quantitative thresholding in HCM patients. In this group, myocardial disarray and replacement fibrosis may contribute to increased T1ρ relaxation.There is considerable interest to characterize myocardial edema and fibrosis in ischemic patients and those with non-ischemic cardiomyopathy. Qualitative visual comparison of the injured myocardium in of edema ,5 and thg et al.  showed s1ρ to T2 mapping, is that T1ρ limits low frequency contributions to the measured relaxation rate, increasing the dynamic range of transverse relaxation times in normal and diseased myocardial tissue. This property was observed in ex vivo myocardial tissue and scar at several B1 amplitudes [1ρ patterns, which may depend on reperfusion time, ischemia-reperfusion injury, extent of coronary ischemia, and hemorrhage. Animal models and prospective studies of T1ρ in humans will be crucial to address these issues.The putative advantage of Tplitudes  and woulplitudes  have non1ρ imaging methods are necessary for parametric mapping of myocardial disease with high accuracy and reproducibility in humans. The methods shown here significantly reduce measurement error of T1ρ relaxation times associated with pulse sequence parameters, magnetic field heterogeneity, and myocardial and respiratory motion and will be essential in the assessment of myocardial disease.In conclusion, highly robust TS1 Dataset(DOCX)Click here for additional data file.S2 Dataset(DOCX)Click here for additional data file.


B-factors. These corrections significantly improved phasing by molecular replacement and subsequent refinement.The refined structures of all four extended NS6 proteases are very similar in structure to the mature MNV NS6—and in one case reveal additional details of a surface loop. Although the packing arrangement observed showed some similarities to those observed in the adventitious protease-product crystals reported previously, in no case were specific protease–substrate interactions observed.Noroviruses are positive-sense single-stranded RNA viruses. They encode an NS6 protease that cleaves a viral polyprotein at specific sites to produce mature viral proteins. In an earlier study we obtained crystals of murine norovirus (MNV) NS6 protease in which crystal contacts were mediated by specific insertion of the C-terminus of one protein (which contains residues P5-P1 of the NS6-7 cleavage junction) into the peptide binding site of an adjacent molecule, forming an adventitious protease-product complex. We sought to reproduce this crystal form to investigate protease–substrate complexes by extending the C-terminus of NS6 construct to include residues on the C-terminal (P′) side of the cleavage junction. We report the crystallization and crystal structure determination of inactive mutants of murine norovirus NS6 protease with C-terminal extensions of one, two and four residues from the N-terminus of the adjacent NS7 protein . We also determined the structure of a chimeric extended NS6 protease in which the P4-P4′ sequence of the NS6-7 cleavage site was replaced with the corresponding sequence from the NS2-3 cleavage junction (NS6 4′ 2|3).The constructs NS6 1′ and NS6 2′ yielded crystals that diffracted anisotropically. We found that, although the uncorrected data could be phased by molecular replacement, refinement of the structures stalled unless the data were ellipsoidally truncated and corrected with anisotropic  Caliciviridae, a family of positive-sense, single-stranded RNA viruses with a ∼7.5 kb genome that generally contains three open reading frames (ORF)  ; a noveles (ORF) . Whereases (ORF) . The vires (ORF) .β-barrel domains separated by a cleft that contains a Cys-His-Asp/Glu catalytic triad similar in arrangement to the Ser-His-Asp triad characteristic of serine proteases  . The solroteases . Calicivroteases .trans cleavage by NS6 at the NS6–NS7 junction. An almost identical packing arrangement was obtained in a different space-group with crystals of Norwalk virus NS6 protease (Previously our lab determined the structure of full-length Murine norovirus NS6 (residues 1-183) . Strikinprotease . These sprotease  revealedprotease ; in partprotease , and theprotease . HoweverWe reasoned that catalytically inactive MNV NS6 constructs extended at the C-terminus might be able to crystallise with the same packing contacts as we had observed for the full-length protein, and that this could therefore give us a convenient way to investigate the structures of NS6-substrate complexes. To this end we extended an inactive MNV NS6 construct (which incorporates a C139A mutation to knock out the active site nucleophile) by adding residues from NS7, which is immediately downstream in the polyprotein. We made three constructs, NS6 1′, NS6 2′ and NS6 4′, which were extended by 1, 2 and 4 residues respectively, generating proteases that contain substrates that correspond to P4–P1′, P4-P2′ and P4-P4′ of the NS6–NS7 cleavage junction. To investigate the structural variation in substrate recognition we also made an NS6 chimera which interchanged the residues P4-P4′ of the NS6-7 cleavage junction with the sequence of the NS2-3 junction from MNV (NS6 4′ 2|3).R-factors. However, this was overcome by the successful application of an anisotropic data correction procedure , inactivated by mutation of the active site Cys 139 to Ala (C139A) were generated by the polymerase chain reaction using the inactive full length MNV NS6 as a template . NS6 1′ and NS6 2′ constructs were transformed into E. coli BL21-CodonPlus (Stratagene) and grown in lysogeny broth (LB) supplemented with 25 µg/mL kanamycin and 35 µg/mL chloramphenicol, while NS6 4′ and NS6 4′ 2|3 constructs were transformed into E. coli BL21(DE3) pLysS (Promega) and grown in LB supplemented with 25 µg/mL kanmycin and 35 µg/mL chloramphenicol. For large-scale protein expression, 1 L of LB was inoculated with overnight cultures. The cultures were incubated at 37 °C with shaking at 220 RPM and protein expression was induced for 3 to 4 h by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) once the cultures had attained an OD at 600 nm of 1.0. Cells were harvested by centrifugation at 4,000 g for 10 min, and pellets frozen at −80 °C.C-terminally extended constructs of the murine norovirus NS6 protease ) supplemented with 0.5 mM PMSF, 2 mg/mL lysozyme, and 0.1% (v/v) Triton X-100 and the cells disrupted by sonication. Cell debris was removed by centrifugation of the bacteria lysate at 29,000 g for 1 h. Clarified lysates were incubated with 1 mg/mL protamine sulphate for 20 min at 4 °C to precipitate nucleic acid contaminants, which were removed by re-centrifugation at 29,000 g for 20 min. The lysate was loaded onto TALON metal affinity resin  in a gravity flow column. After washing with 25 volumes of lysis buffer, 25 volumes of lysis buffer containing 5 mM imidazole, and finally a further 25 volumes of lysis buffer containing 10 mM imidazole, the MNV NS6 proteins were eluted with a step gradient to 100 mM imidazole. The His6 tag was removed during overnight dialysis in lysis buffer in the presence 2 mM CaCl2, and 10 units of thrombin  per mg of MNV NS6 protein. Following dialysis, uncleaved material and cleaved tags were removed in a second round of TALON purification. As a final polishing step, the unbound fractions containing cleaved MNV NS6 variants were pooled, concentrated and loaded onto a Superdex-75 size-exclusion column  equilibrated with 50 mM Tris pH 8.0, 100 mM NaCl, 1 mM DTT (SEC buffer).In the case of NS6 4′ and NS6 4′ 2|3 constructs the above protocol (and additives) were used but the lysis buffer was composed instead of 50 mM HEPES pH 6.5, 300 mM NaCl, 1 mM DTT. Size-exclusion chromatography was performed in 25 mM Tris pH 8.0, 200 mM NaCl, 5 mM DTT.All crystallisation experiments were performed with a protein concentrations of 10–14.5 mg/mL in the SEC buffer used for the final purification step. Efforts to use the original crystallisation conditions to reproduce the MNV NS6 protein crystals with the packing arrangement that would place the C-terminus of one molecule in the active site of a neighbour  failed fWe therefore widened the search for crystallisation conditions, carrying out screening on a sub-microlitre scale by sitting-drop vapour-diffusion. A 100 nL amount of protein solution at 14 mg/mL was mixed with 100 nL reservoir solution and equilibrated against 90 µL of reservoir solution using a Mosquito automated pipetting system  and the following commercial crystallisation screens: Wizard 1 & 2 , PACT Premier, JCSG+, Morpheus and PGAScreen . Larger crystals were grown from initial hits by sitting-drop vapour diffusion at 18 °C in CompactClover Plates EBS-XPT .Useable crystals of MNV NS6 1′ were obtained by mixing 1 µL of protein with 1 µL mother liquor consisting of 10% (v/v) poly-ethylene glycol (PEG) 10000, 20% (v/v) ethylene glycol, 0.1 M MES/Imidazole pH 6.3. Crystals with cuboid shape appeared between 2 and 5 days and grew to full size (80 × 50 × 50 µm) in about 15 days and were flash-cooled in liquid nitrogen prior to data collection without any additional cryo-protectant.Crystals of MNV NS6 2′ were obtained in 15% (v/v) PEG 3350, 0.1 M glycine, 0.1 M Na-citrate pH 7.0. They grew as long rods in 1–2 days and achieved full size (400 × 30 × 30 µm) in 5–10 days. The crystals were cryo-protected in mother liquor supplemented with 15% (v/v) PEG 200 and flash-cooled in liquid nitrogen.Crystals of NS6 4′ and NS6 4′ 2|3 grew from 0.2 M KSCN, 0.1 M Bis-Tris propane pH 6.5–7.5, 20% w/v PEG 3350. They were cryo-protected adjusting the mother liquor solution to a final concentration of 30% (v/v) PEG 3350.X-ray diffraction data from crystals of NS6 1′ and NS6 2′ were collected on a Pilatus 6M-F detector at the I03 beamline at the Diamond Light Source . For the MNV NS6 1′ crystals a 2.3 Å data set of 200 frames was collected with an oscillation width of 1° per frame. Diffraction images were integrated and scaled using the CCP4 program suite . Data-coFor the MNV NS6 2′ crystals, 720 frames with a 0.5° oscillation were collected. A 3.1 Å dataset was integrated and scaled as described above; see MNV NS6 4′ and NS6 4′ 2|3 crystals data was collected in-house using a Rigaku MicroMax-007 HF-M X-ray generator and a Saturn 944 + CCD detector. Data sets of 316 and 344 0.5° oscillation frames were collected for NS6 4′ and NS6 4′ 2|3 crystals at 2.47 and 2.42 Å respectively. The data were processed and scaled as described above.http://services.mbi.ucla.edu/anisoscale) for anisotropic analysis. Following the server indication of severe anisotropy diffraction of the MNV NS6 1′ and NS6 2′ crystals both data sets were truncated/scaled using the server default values, in particular using a 3.0 cut-off for F/sigma ] for the highest-resolution shells of data  and Rfree (∼39%). A similar problem was encountered with initial attempts to refine the MNV NS6 2′ model.Initial structural factors  were used for molecular replacement, and in the case of MNV NS6 1′ produced four possible solutions with a highest LLG of 2,723 and TFZ of 19.7. However, initial refinement of the model obtained by molecular replacement stalled at relatively high values of c∗ axis as stronger diffracting than the a∗ and b∗ directions in each case, and detected anisotropic ΔB values of 68.6 Å2 and 51.0 Å2 for MNV NS6 1′ and NS6 2′, respectively. Anisotropic ΔB reports the directionality dependence of the intensity falloff with resolution . Following the F/sigma analysis, the MNV NS6 1′ data were truncated to 3.1 Å, 2.3 Å, and 2.1 Å along a∗, b∗ and c∗, respectively. To create a nominally isotropic data set, B-factor corrections of 39.2, −9.8 and −29.4 Å2 were applied to the observed structure factors along the a∗, b∗ and c∗ directions respectively. The reduction in the anisotropy of the corrected data in the a∗c∗(h0l) plane can been seen by comparing At this stage we re-visited the processed data to determine if the anisotropy was at the root of the refinement problems. Plots of F/sigma against resolution for each of the 3 principal axes revealed the severity of the anisotropy of the diffraction from both crystals , 2B; whea∗, b∗ and c∗ directions. To generate the nominally isotropic data set, B-factor correction of 17.0, 17.0 and −34.0 Å2 along the same axes were applied to the observed structure factors. The anisotropic truncation of the data, with the new limit of 2.7 Å along the c∗ axis, resulted in a dramatic increase in the number of unique reflections from 8,555 in the “pre-treated” data to 12,786.Following anisotropy analysis the MNV NS6 2′ data were truncated to 3.2 Å, 3.2 Å, and 2.7 Å along B values of 13.3 Å2 and 15.4 Å2 for MNV NS6 4′ and NS6 4′ 2|3, respectively). In these cases, no directional-dependent truncation of the data was applied.MNV NS6 4′ or NS6 4′ 2|3 data sets plots of F/sigma against resolution for each of the 3 principal axes revealed only mild anisotropy of the diffraction from both crystals  and yielded electron density maps that were much more interpretable . Nevertheless, this resulted in immediate improvements in refinement: R factors (Rwork/Rfree) of 20.9/25.2 and 21.0/26.2%, respectively.The translation Z-scores and LLG’s for the Phaser molecular replacement solutions for MNV NS6 4′ are 13.8 and 1,042, respectively, and for MNV NS6 4′ 2|3 27.1 and 1,394, respectively. The crystals structures of NS6 4′ and NS6 4′ 2|3 were refined to β-strand aII and bII  or residue 174 (chain C). The electron density for the last four residues modelled in chain D (Ala 170 to Gly 173) is poor, presumably due to disorder. Nevertheless, we included these residues in the final model because removal increased the Although the full C-termini of chains A, C and D of the NS6 1′ could not be modelled, the electron density map revealed additional features that could be built as short stretches of polypeptide. These correspond to portions of the missing C-termini but, because of discontinuities in the electron density, it is not possible to unambiguously ascribe which monomer they belong to. These short segments were present in the asymmetric unit, and were modelled as (i) a stretch of five residues (Chain E) that could be assigned confidently as corresponding to residues Leu 180 to Gly 184 that lies near to the active site of a neighbouring chain A; and (ii) an extended portion of electron density that was not of sufficient quality to identify the amino acid side chains and was built as a seven-residue poly-Ala peptide (chain F), that lies between chains A and D and may be part of a symmetry-related molecule of chain A. However, in neither case were specific interactions with the putative active sites of neighbour monomers observed.In contrast to chains A, C and D, the electron density for the B chain in the asymmetric unit was sufficiently clear to permit inclusion of all 183 residues of NS6 and the extra first residue of NS7 (here labelled Gly 184) in the refined model . The C-tP6122 with two molecules in the asymmetric unit. The electron density indicates the positions of residues 3-181 in chain A and 4-181 in chain B, but is not good enough to allow complete modelling of the N- or C-termini. The two monomers found in the asymmetric unit are related by a quasi-2-fold symmetry axis and form an apparent homodimer with an interface area of 1,097 Å2 . This may explain why trans interactions were not observed in our crystals but in turn raises a further question: why was this conformational state not captured in the crystal form? One possibility is that the concentration of proteins inevitably involved in crystallisation somehow destabilizes the cis interaction but does not necessarily capture a catalytically competent trans interaction between the cleavage junction within the extended C-terminus and a protease neighbour. Why this would be the case, particularly when a protease–substrate complex would be expected to be a relatively stable state, remains a mystery.Why did we not get the same crystal form as for the full-length protein? At present it is difficult to give a definitive answer to this question. Although we screened extensively for crystallisation conditions, we cannot claim to have searched exhaustively and it may be that further efforts might yet succeed. Moreover, it is well known that even very modest changes to protein constructs may alter substantially the way that they crystallise, though it is worth noting that a similar strategy to explore the specific interactions of different protease-product complexes was applied successfully to Norwalk virus NS6 . In prel


NS2 from influenza A virus mediates Crm1-dependent vRNP nuclear export through interaction with Crm1. However, even though the nuclear export signal 1 (NES1) of NS2 does not play a requisite role in NS2–Crm1 interaction, there is no doubt that NES1 is crucial for vRNP nuclear export. While the mechanism of the NES1 is still unclear, it is speculated that certain host partners might mediate the NES1 function through their interaction with NES1. In the present study, chromodomain-helicase-DNA-binding protein 3 (CHD3) was identified as a novel host nuclear protein for locating NS2 and Crm1 on dense chromatin for NS2 and Crm1-dependent vRNP nuclear export. CHD3 was confirmed to interact with NES1 in NS2, and a disruption to this interaction by mutation in NES1 significantly delayed viral vRNPs export and viral propagation. Further, the knockdown of CHD3 would affect the propagation of the wild-type virus but not the mutant with the weakened NS2–CHD3 interaction. Therefore, this study demonstrates that NES1 is required for maximal binding of NS2 to CHD3, and that the NS2–CHD3 interaction on the dense chromatin contributed to the NS2-mediated vRNP nuclear export.The online version of this article (doi:10.1007/s00018-014-1726-9) contains supplementary material, which is available to authorized users. Influenza A virus (IAV) is the causative agent of seasonal influenza  and of tThe nuclear export is a significant event in the viral life cycle. Both cellular and viral factors participate in the process. An important cellular factor for vRNP export is Crm1, a nuclear export receptor for proteins containing a characteristic leucine-rich nuclear export signal (NES) , 10. In In the present study, CHD3 was identified as a novel host protein involved in the NS2-associated nuclear export of vRNP. Our data suggest that the interaction would assist in tethering NS2 and Crm1 to vRNP facilitating the export. In addition, since only after CHD3 interacted with NES1, would the vRNPs be exported, it suggests that the interaction would expose the otherwise hidden NES1 for nuclear export.COS-1 cells were cultured in RPMI 1640 complete medium  supplemented with 10 % heat-inactivated fetal bovine serum . MDCK cells were cultured in Dulbecco’s Modified Eagle complete medium (with 10 % FBS) (DMEM) .The WD virus (named as “rNS1-wt” in reference ) was conaggacata 3′) were introduced (5′ttcccgggcata 3′) to prevent splicing at this site without changing NS1 protein. A new splice acceptor site corresponding to nucleotides 459-527 of the NS segment, a 2× Flag sequence, the entire NS2 ORF and 3′ noncoding region were added successively after NS1 ORF . All other pHW2000 plasmids used in this study had been generated previously  and mutants  were constructed by site mutation based on pBIND-NS2. The C-terminus of human CHD3 (cCHD3) was cloned into the pCMVtag2B (Flag-cCHD3) for producing Flag-tagged cCHD3 and pACT (pACT-cCHD3) for M2H assay. A nuclear localization signal   containing the 1 mM phenylmethyl sulfonylfluoride . Cell debris and high molecular weight DNA were removed by centrifugation at 10,000g for 30 min. For Co-IP, the supernatant of lysate was diluted in the binding buffer , and incubated with 1 μg of an anti-Flag tag antibody and A/G PLUS-agarose (Santa Cruz Biotechnology) for 3 h at 4 °C. The beads were washed and then resuspended in sodium dodecyl sulfate (SDS) loading buffer. The bound proteins were resolved via SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to a nitrocellulose membrane for western blot assay with ECL illumination . For GST pull-down, the GST–NS2 and GST proteins were expressed in Escherichia coli BL21 (DE3) and bound to glutathione-Sepharose 4B  for 2 h at 4 °C, respectively. The same procedure as Co-ip was performed except that cell lysate was incubated with the beads for 6 h at 4 °C.At 36 h after expression of Flag-cCHD3, cells were lysed using lysis buffer [50 mM Tris (pH 8.0), 100 mM NaCl, 20 mM NaF, 50 mM KHDuplex small interfering RNAs (siRNAs) targeting CHD3 (5′ gcgugacagugaggaggaa 3′) (si-CHD3)  and a vaThe cells were fixed with 4 % paraformaldehyde, permeabilized using 0.2 % Triton X-100, and incubated with primary antibody for 30 min, then second antibody for 40 min. Nuclei were stained with DAPI (Invitrogen). Samples were examined by confocal microscopy with the LSM510 system . Colocalization was analyzed using the software Image J (NIH).7 COS-1 cells were used for cell fractionation. The cytoplasmic fraction (cyt), nucleoplasmic fraction (nuc), low-salt chromatin fraction (ch150) and high-salt chromatin fraction (ch500) were resolved in 100 μl of sucrose buffer, 70 μl of nucleoplasm extraction, 70 µl of nuclease incubation buffer and 70 µl of chromatin extraction buffer, respectively. After collecting the ch500, the remaining pellet was resuspended in 70 µl of sample buffer without bromophenol blue. The supernatant was saved as the >ch500 fraction. The relative quantity of the proteins in each fraction was analyzed using the software Image J (NIH).Subcellular fractionation was performed as described previously  with modp values were obtained according to Student’s t test for paired data. Statistical significance was defined as p < 0.05.The mean values ± standard deviation (SD) were calculated, and A human fetal brain cDNA library was screened by Y2H with NS2 as the bait to identify host proteins that interact with NS2. The C-terminus of human CHD3 (cCHD3)  was identified and its interaction with NS2 was confirmed in the M2H assay . CHD3 could be immunoprecipited with mouse anti-Flag monoclonal antibody with WD-Flag-NS2 infection  (with amino acid residues 54–121 deleted), NS2(54–121aa), NS2(6–121aa), NS2(9–121aa), NS2(16–121aa), NS2(20–121aa), and NS2(26–121aa), were prepared for analysis in M2H assay. The expression of the mutant NS2 proteins was similar to the wild-type levels  in NS2 , 19, it It was shown that, during IAV infection, vRNPs export complexes along with NS2 and Crm1 were tethered to the host dense chromatin with very high affinity, which was essential for the NS2 and Crm1-dependent vRNP nuclear export . It is pa), the amount of NS2 in >ch500 fraction was reduced at 4 h p.i. , which was confirmed by a statistical test showing significant difference of NS2 content between ch150 and >ch500 in si-NC but not in si-CHD3 treatment . In the experiment, it also showed that the distribution of Crm1 also changed with more Crm1 in nuc and less in >ch500 , in which the significant difference of Crm1 between nuc and >ch500 was observed in si-NC but not in si-CHD3 sample . The distribution of NP/vRNP was not altered with the knockdown of CHD3 . The observation were further supported by statistical analysis which showed significant difference of Crm1 between nuc and >ch500  in WD but not WD–NS2(L19S) infection, confirming the role of NS2–CHD3 interaction in locating NS2 and Crm1 on dense chromation. Further, IFA showed that vRNP/NP was mostly distributed in the nuclei at 4 h p.i. in cells infected by either WD–NS2(L19S) or WD , which encoded the mutant NS2 weakening the NS2–CHD3 interaction, propagated poorly at all time points Fig. a. The pea) significantly decreased the propagation of the wild-type WD virus. The titers of virus were 4.3 and 18.8 times lower than the control at 24 and 36 h p.i., respectively . In comparison, knockdown of CHD3  had less effect on the propagation of WD–NS2(L19S) virus . Additionally, though NS2 has been shown to regulate polymerase activity [Similarly, the knockdown of CHD3 Fig. b a signiactivity , the NS2Selected strains of IAV were also tested for their NS2 interaction with CHD3. The NS2 of pandemic A/Mexico City/001/2009 (H1N1) virus, A/WSN/1933 (H1N1) virus, and avian influenza virus A/duck/Hubei/W1/2004 (H9N2) were employed in the M2H assay. The assay showed that all these NS2 could strongly interact with CHD3 Fig. b, regardIAV vRNPs display a biphasic pattern of intracellular localization during infection. They translocate in the nucleus to allow transcription to take place early in the life cycle of the virus, and are exported to the cytoplasm for package into progeny virions later in the life cycle. The current ‘‘daisy-chain’’ model indicates that during translocation of vRNPs, interaction between the IAV NS2 and Crm1  is impora).The interaction would increase the export factors (NS2 and Crm1) around the vRNP, facilitating the export. It has been demonstrated that vRNPs were assembled on the chromatin before being exported to the cytoplasm . Our datThe interaction would expose the NES1 for performing its function in vRNP export. During infection, it can be demonstrated that NS2 locates in the nucleus at an early stage 3.5 p.i.)  and in the cytoplasm (5 p.i.) or cell membrane (6 p.i.) at later stages of the infection . NS2 can also be shown to colocalize with NP/vRNP throughout the infection . This implies that NES1 in NS2 could be hidden in the early stages of the viral life cycle and exposed to execute its function in the later stages. The data in the present study demonstrated that the vRNP export was executed only after the CHD3–NES1 interaction occurred. Thus, our data raised the possibility that the interaction between NES1 in NS2 and CHD3 on chromatin kept the NES1 exposed for nuclear export after vRNP assembly. Supporting this, when the NS2 was fused with the RFP, the NS2–RFP fusion protein instead completely located in the cytoplasm Supplementary material 2 (TIFF 497 kb)Supplementary material 3 (TIFF 918 kb)Supplementary material 4 (DOC 27 kb)Below is the link to the electronic supplementary material.


Histone acetyltransferases can be classified into two distinct groups (type A and B) on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4.The acetylation of the core histone NHTo better understand the role of histone acetylation in the assembly of chromatin structure, we have identified additional type B histone acetyltransferase activities specific for histone H3. One such activity, termed HatB3.1, acetylated histone H3 with a strong preference for free histones relative to chromatin substrates. Deletion of the GCN5 and ADA3 genes resulted in the loss of HatB3.1 activity while deletion of ADA2 had no effect. In addition, Gcn5p and Ada3p co-fractionated with partially purified HatB3.1 activity while Ada2p did not.Yeast extracts contain several histone acetyltransferase activities that show a strong preference for free histone H3. One such activity, termed HatB3.1, appears to be a novel Gcn5p-containing complex which does not depend on the presence of Ada2p. Histones H3 and H4 are among the most evolutionarily conserved proteins (>90% identity from yeast→humans) . Octamer2-terminal tails of the histones. These domains, which protrude from the core of the nucleosome, are free to interact with, and be acted upon by, the nuclear environment. The past several years has seen the identification of numerous enzymes that are capable of modifying the histones. These enzymes are generally found in large, multi-subunit complexes and have activities that are not only specific for a given histone but are specific for particular amino acid residues within the histone ). Supernatant from a 1500 × g, 15' spin at 4°C was retained as a cytosolic extract. Pelleted material was washed once with Buffer A then resuspended in DN(1000) . Supernatant from another 1500 × g spin as above yielded the nuclear extract. This extract was dialyzed O/N at 4°C into DN(0) to a conductivity similar to that of the cytosolic extract. Extracts were cleared by high speed centrifugation  prior to their chromatographic fractionation.Cells were grown to mid-log phase in 1% yeast extract, 2% peptone, 2% glucose and 50 μg/mL ampicillin at 30°C. Cells were harvested at 4000 × g, 10', 4°C and total grams of cells recorded. All buffers contain 1.0 mM PMSF. Spheroplasts were prepared essentially as described previously using 0.25 mg of Zymolyase  per gram of cells for spheroplasting . Spheroppurifier – Pharmacia) was employed for all column runs.All columns were equilibrated with and run using DN Buffers. HPLC (ÄKTA DEAE – Cleared extracts were loaded onto a HiPrep 16/10 DEAE FF column (Pharmacia). Following a 5 C.V. wash with DN(50), proteins were eluted with a linear, 20 C.V., salt gradient from 50 mM to 1.0 M NaCl. A flow rate of 1.0 mL/min. was used and 3.0 mL fractions were collected.CM – Either pooled peak fractions, dialyzed into DN(0) until at similar conductivity as DN(50) start buffer, or Flowthrough from the DEAE were loaded onto a HiPrep 16/10 CM FF column (Pharmacia). The column was washed and proteins eluted as described above.Mono Q – The flowthrough fraction from the CM column was loaded onto a Mono Q HR 5/5 column (Pharmacia). Following a 5 C.V. wash with DN(50), a 20 C.V., linear, salt gradient was employed as above and 0.5 mL fractions were collected.4)2SO4 (0.516 g/mL) over 30' at 4°C. Following an additional 30' equilibration period at 4°C, precipitated protein was pelleted  and resuspended in 300 μL cold, DN(0).Peak fractions of HatB3.1 activity from the Mono Q column were pooled and brought to 75% . The column was equilibrated with and run in DN(350) at a flow rate of 0.3 mL/min. and 0.25 mL fractions were collected. Molecular weight standards  were run using the same parameters and 24 μL aliquots of every other fraction run on a 10% SDS-polyacrylamide gel. The elution profile of the MW standards was determined by protein visualization via Coomassie blue staining.3H-Acetyl Coenzyme A  and ~1.0 mg/mL core histones or chromatin in a final volume of 100 μL at 1X [DN(75)]. 50 μL of each reaction was analyzed for HAT activity via liquid scintillation counting. The remaining assay mixture was brought to 1X [SDS Load Dye] to stop the reaction. In general, aliquots (24 μL) of these remaining assay mixtures were run on 18% SDS-polyacrylamide gels to resolve the histones. Gels were incubated in Autofluor , dried down and acetylated histone species visualized via fluorography.Chicken erythrocyte core histones and chromatin were isolated as previously described ,76. TypiSuperose 6 fractions exhibiting HAT B3 activity, as determined above, were run on 10% SDS-polyacrylamide gels and proteins were either visualized by silver staining or transferred to nitrocellulose using a semi-dry transfer apparatus (Biorad). Blots were processed following standard procedures. Goat, polyclonal antibodies against Gcn5p, Ada2p and Ada3p  were used at 1:100 dilutions in 5% Milk/TBS-T. Donkey, HRP-labeled Anti-Goat IgG secondary antibody  was used at 1:2500 dilution followed by detection with ECL+Plus (Pharmacia) and visualization via phosphoimager .A.R.S. performed all of the experiments presented here and drafted the manuscript. M.R.P. directed the project and edited the manuscript.


Fast, cheap and reliable methods are needed to identify large populations, which may be at risk in relation to environmental exposure. Polymorphisms in NAT1 (N-acetyl transferase) may be suitable markers to identify individuals at risk.A strategy allowing to address simultaneously 24 various genetic variants in the NAT1 gene using the single sequencing reaction method on the same PCR product is described. A modified automated DNA sequencing using only one of the sequence terminators was used to genotype PCR products in single-track sequencing reactions of NAT1 and was shown to be universal for both DNA sequencing using labeled primers and labeled nucleotides. By this method we detected known SNPs at site T640G, which confers the NAT1*11 allele with frequency of 0.036, further T1088A and C1095A with frequency of 0.172 and 0.188, respectively and a deletion of TAATAATAA in the poly A signal area with a frequency 0.031. All observed frequencies were in Hardy Weinberg equilibrium and comparable to those in Caucasian population. The single-track signatures of the variant genotypes were verified on samples previously genotyped by RLFP.The method could be of great help to scientists in the field of molecular epidemiology of screening of large populations for known informative biomarkers of susceptibility, such as NAT1. CYP19 (aromatase) [NAT1 gene. The increasing number of detected mutations in the NAT1, makes genotyping using conventional restriction fragment length polymorphism (RFLP) or allele-specific amplification complicated. We developed a rapid and universal strategy based on single-track DNA sequencing analysis of a unique PCR product encompassing the entire NAT1 coding region (contains no introns) along with the flanking 5' and 3' untranslated regions. It allows a rapid and economic characterization of NAT1 alleles. Our method brings reproducible results on both Alf Express™ (Pharmacia) and ABI310 PRISM sequencing instruments and may be adopted for majority of epidemiological studies with relevance of NAT1 in environmentally related diseases.We have previously described the single sequencing reaction (SSR) protocol for assessment of a known polymorphism in the 3'utr region of omatase) . Here weNAT1 gene is highly polymorphic . Expression of NAT1*16 but not NAT1*10 and NAT1*11 caused a 2-fold decrease in the amount and catalytic activity of NAT1 in COS-1 cell cytosol [NAT1 allelic variants that encode reduced expression of NAT1 and/or less-stable NAT1 protein [N-acetyltransferases  are implicated in the biotransformation of primary arylamines (e.g. 2-naphthylamine and aminobiphenyls), heterocyclic amines, hydrazines, and their N-hydroxylated metabolites present in tobacco smoke and food . An incr cytosol -14. All  protein .NAT1 and NAT2 acetylation polymorphisms modify the risk of developing urinary bladder, colorectal, breast, head and neck, lung, and possibly prostate cancers [NAT2*4 and NAT1*10 were suggested by the increased frequency of the NAT2*4/NAT1*10 haplotype [NAT1 genotypes in the population, the attributable cancer risk may be high. Large-scale molecular epidemiological studies that investigate the role of NAT1 genotypes and/or phenotypes together with other genetic susceptibility gene polymorphisms and biomarkers of carcinogen exposure are necessary to expand our current understanding of the role of NAT1 acetylation polymorphisms in cancer risk [Epidemiological studies suggest that the  cancers -18. Inteaplotype -21. The cer risk .NAT1 with two sequencing tracks: A and G and 11 variants in the 3' flanking area with two tracks: A and T . Despite the different chemistry, Figures A-track sequencing of the 3'-untranslated region of e et al. . This chNAT1 gene by single-track sequencing reactions in 192 control individuals. Previously, we have shown that single-track sequencing reactions performed on PCR products, with subsequent analysis on an Alf Express™ DNA Sequencer, can be as informative, sensitive, and accurate as complete sequencing reactions but more economical option for the genotyping of known polymorphisms in the human aromatase gene [NAT1.A modified automated DNA sequencing with a fluorescent label was used to genotype PCR products spanning through the whole This paper shows that single-track sequencing reactions can be used as a tool for screening for all types of known polymorphisms in field laboratories with limited or overloaded sequence capacity. Compared to standard sequencing, this single track sequencing may have better signal to noise ratio. This approach is time and cost effective and can be accommodated for high-throughput analyses in epidemiology studies.Chemicals for PCR and sequencing were purchased from ABI  and Amersham Biosciences .The study included 192 DNA samples from Norwegian healthy individuals obtained through Norwegian Population Registry as a population-based series of residents in the Oslo area. Test samples from healthy Norwegian controls with known genotypes (previously assessed by RFLP) were available.NAT1 (923 bp) using forward primer: 5'-tactgggctctgaccactat-3' and reverse primer: 5'-tgctttctagcataaatcacc-3' was performed. PCR mix contained 16.9 μl dH2O, 10 × PCR buffer (2.5 μl), 1.25 mM MgCl2 (4 μl), 2.0 mM dNTP (1.0 μl mixture of each), 10 μM oligonucleotide primer, 0.2 μl Taq DNA polymerase  and 1.0 μl of genomic DNA (100 ng/μl) in a final volume of 25 μl. Thermal cycling  included initial denaturation 2 min at 94°C, 10 cycles of 30 sec at 94°C, 30 sec at decreasing annealing temperature 58 to 48°C, 1 min at 72°C, and 25 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C. Quality of PCR products was checked on 6% acrylamide gels.A single PCR amplification of the entire coding region and the 3' flanking area of NAT1, respectively. The single sequencing reactions (7 μl) contained: 1.25 μl of dH2O, 0.5 μl of 10-times concentrated FS polymerase buffer 2SO4, 150 mM MgCl2), 2 μl of ddNTP termination mix (containing 10 mM dNTPs and either ddATP or ddTTP (10 μM) for A-track or T-track sequencing respectively, 1 μl of 2 μM primer, 0.25 μl of FS polymerase (5 U/μl), and 2 μl of NAT1 PCR product. The following conditions were used for thermal cycling: initial denaturation for 5 min at 95°C, 35 cycles of 30 sec at 95°C, 30 sec at 50°C, and 1 min at 68°C. The single-tracks were evaluated using conventional software AlfWin, Fragment Analyzer, v. 1.02 supplied with the sequencer (Alf Express™). For the single-track sequencing by ABI310 PRISM we used the same sequencing primers as for Alf Express™ system however labeled with the fluorophor 6-FAM. Sequencing reactions (10 μl) contained: 5.0 μl dH2O, 1.0 μl of 10-times concentrated Thermo Sequenase buffer , 2.0 μl of ddNTP termination mix (containing dNTPs and either ddATP or ddGTP for A-track or G-track sequencing respectively – for composition see Table 3), 0.5 μl of 1 μM primer, 0.5 μl of Thermo Sequenase DNA polymerase (with pyrophosphatase) 3.2 U/μl and 1.0 μl NAT1 PCR product (described above). Thermal cycling conditions were as follows: initial denaturation 20 sec at 95°C, 25 cycles of 20 sec at 95°C, 20 sec at 55°C and 1 min at 72°C, and then 10 cycles of 20 sec at 95°C, 1 min at 72°C. Results were evaluated using GeneScan Analysis software, v. 3.7.The SNPs studied by this assay are summarized in Table ABPs – aminobiphenyls, NAT1 – N-acetyltransferase 1, SSR (Single Sequencing Reaction)PS performed the SSR analysis on an Alf Express™ and prepared the first draft of the paper, CFS and MS optimized the SSR analysis for an ABI system in the lab of EHK, TK designed primers and contributed with reagents, materials, and advice, VNK brought the idea, organized the study and was responsible for the revisions of the paper.


Isoquercitrin (IQC) is a component abundantly present in many plants and is known to have an anti-viral effect against various viruses. In this study, we demonstrate that IQC exhibits strong anti-influenza A virus infection, and its effect is closely related to the suppression of hemagglutinin (HA) and neuraminidase (NA) activities. We used green fluorescent protein-tagged Influenza A/PR/8/34 (H1N1), A/PR/8/34 (H1N1), and HBPV-VR-32 (H3N2) to evaluate the anti-IAV effect of IQC. The fluorescence microscopy and fluorescence-activated cell sorting analysis showed that IQC significantly decreases the levels of GFP expressed by IAV infection, dose-dependently. Consistent with that, IQC inhibited cytopathic effects by H1N1 or H3N2 IAV infection. Immunofluorescence analysis confirmed that IQC represses the IAV protein expression. Time-of-addition assay showed that IQC inhibits viral attachment and entry and exerts a strong virucidal effect during IAV infection. Hemagglutination assay confirmed that IQC affects IAV HA. Further, IQC potently reduced the NA activities of H1N1 and H3N2 IAV. Collectively, IQC prevents IAV infection at multi-stages via virucidal effects, inhibiting attachment, entry and viral release. Our results indicate that IQC could be developed as a potent antiviral drug to protect against influenza viral infection. Influenza viruses, a family of orthomyxoviridae with segmented RNA genome , produceMangifera indica, Rheum nobile, Annona squamosa, Camellia sinensis, and Vestia foetida. It is also called isoquercetin, isotrifoliin, or quercetin 3-glucoside. IQC has been reported to have efficacy against various diseases and has anti-cancer [Dianthus superbus L inhibits IAV infection through the suppression of ROS, acidic vesicular organelle formation, and PB2 protein. In the present study, we first demonstrate that the antiviral effect of IQC against IAV infection is related to the suppression of viral binding and entry and a virucidal effect via the blockage of hemagglutination in the early stages of IAV infection. Further, IQC exhibited inhibitory effects on the neuraminidase of H1N1 and H3N2 IAV.Isoquercitrin (IQC) is a flavonoid, which is rich in various plants such as i-cancer , anti-fai-cancer , anti-dii-cancer ,14,15, ai-cancer ,17,18. Ii-cancer ,20, herpi-cancer ,22, Eboli-cancer , and Mayi-cancer . In addii-cancer  showed ti-cancer  reportedThe cytotoxicity of IQC to RAW 264.7 cells was determined by a CCK-8 assay. When the cell viability was checked at 24 h post-treatment with IQC at indicated concentrations, IQC did not show a toxic effect on the cell viability up to 25 µM B.We used the GFP-tagged Influenza A/PR8/34 (PR8-GFP IAV) virus to assess the anti-influenza viral effect. IQC  and PR8-GFP IAV were mixed and placed for 1 h at 4 °C, and were treated to the RAW 264.7 cells for 2 h at 37 °C. The cells were washed with PBS and further incubated for 24 h at 37 °C to express GFP. As shown in Next, we examined whether IQC could affect IAV protein expression using immunofluorescence analysis. IQC (2.5 μM or 25 μM) and PR8-GFP IAV were mixed for 1 h at 4 °C and cotreated to the RAW 264.7 cells for 2 h at 37 °C. The cells were further incubated for 24 h at 37 °C after washing with PBS and were fixed with absolute methanol and 4% paraformaldehyde. The cells were stained with Alexa 594-tagged antibodies against M2, NP, HA, PA, PB1, and NA viral proteins. The nuclei in the cells were detected by staining with Hoechst 33342. Since IQC significantly inhibited IAV infection, we examined whether IQC affects IAV infection at the early stages. First of all, the RAW 264.7 cells were treated with medium, PR8-GFP IAV or a mixture of PR8-GFP IAV and IQC for 30 min at 4 °C before incubation for 24 h at 37 °C to investigate the effect of IQC on viral binding to the cells. To examine the effect of IQC on the viral entry into the cells, the RAW 264.7 cells were infected with medium or PR8-GFP for 30 min at 4 °C before adding IQC. After washing the cells to remove unbound virus, the cells were incubated with medium or IQC for 30 min at 37 °C, followed by further incubation for 24 h at 37 °C after the removal of IQC. We also checked whether IQC could kill the virus before viral infection into the cells. To evaluate the virucidal effect of IQC, medium, PR8-GFP IAV only, or a mixture of PR8-GFP IAV and IQC were incubated for 30 min at 4 °C. Each mixture was treated to the RAW 264.7 cells for 30 min at 37 °C. After washing, the cells were further incubated for 24 h at 37 °C. The HA protein of the influenza virus is crucial for viral attachment to the cells and inducing hemagglutination in red blood cells. Since IQC has a strong inhibitory effect on viral attachment and entry during IAV infection, we examined whether IQC could influence hemagglutination by IAV infection. To address this, H1N1 IAV and IQC at various concentrations were cotreated to the RAW 264.7 cells, as described in Neuraminidase activity of the influenza virus is essential for the release of IAV progeny from the cells. Neuraminidase inhibitors, such as oseltamivir and zanamivir, are well-known anti-influenza viral drugs. We investigated whether IQC could inhibit the NA activity of IAV. Nelumbo nucifera Gaertn.) leaves extract exhibits antiviral efficacy by directly inhibiting IAV infection [Camellia sinensis [Eupatorium perfoliatum [Jatropha curcas [Isatis indigotica [Epimedium koreanum Nakai [Rhus verniciflua [In the previous study, we reported that lotus -tagged Influenza A/PR8/34 (PR8-GFP) and A/PR8/34 (H1N1) viruses were kindly received from Dr. Jong-Soo Lee  and HBPV-VR-32 (H3N2) was purchased from the Korea Bank for Pathogenic Viruses (KBPV). The viruses were amplified in the allantoic fluid of a 10-day-old chicken embryo as described previously [Isoquercitrin with 98% purity was purchased from Wuhan ChemFaces Biochemical Co., Ltd. , and dissolved in DMSO. RAW 264.7 cells  were grown in Le Roswell Park Memorial Institute medium  containing 10% fetal bovine serum and 100 U/mL of Penicillin and Streptomycin at 37 °C with 5% COeviously ,49,50. A5 cells/well) were incubated with IQC at concentrations from 0.25 to 100 µM. At 24 h post-treatment, 10 μL of reagent was added to the plate and incubated for 1 h, and then the absorbance at 450 nm was measured using a spectrophotometer .To evaluate the toxicity of IQC to the cell, the CCK-8 assay was used, according to the manufacturer’s recommendation . Briefly, RAW 264.7 cells seeded in 96 wells (1 × 10The influenza A/PR8-GFP virus (PR8-GFP IAV) at 10 multiplicity of infection (MOI) and IQC at indicated concentrations were mixed for 1 h at 4 °C and the mixtures were added to RAW 264.7 cells for 2 h at 37 °C. After washing with PBS, the cells were further incubated for 24 h. Fluorescent microscopy or fluorescence-activated cell sorting (FACS) was used to detect GFP expression. To evaluate the inhibitory effect of IQC on the cytopathic effect (CPE) of H1N1 or H3N2 IAV, IQC with H1N1 or H3N2 at 50 MOI were placed for 1 h at 4 °C and were cotreated to RAW 264.7 cells for 2 h at 37 °C. After washing with PBS, the cells were further incubated until CPE formation. The cell viability was determined by CCK-8 assay, according to the manufacturer’s recommendation.5 cells/well) were incubated with a mixture of PR8-GFP IAV and 2.5 or 25 µM of IQC for 2 h at 37 °C. At 24 h post-incubation, the cells were fixed with cold absolute methanol for 10 min and 4% paraformaldehyde for 10 min. The cells were blocked with 1% BSA-PBS for 30 min and incubated with antibodies  specific for influenza viral proteins for 1 h at room temperature. After washing with PBST (PBS with 0.05% Tween 20), the cells were incubated with Alexa Fluor 594-tagged secondary antibody  in PBST for 1 h in the dark. The cells were incubated with Hoechst 33342  for 5 min. The images of red viral proteins and blue nuclei were captured using fluorescent microscopy.RAW 264.7 cells seeded in 12 wells (5 × 105 cells/well) for 24 h at 37 °C. The cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. The cells resuspended in PBS were analyzed using the CytoPLEX flow cell counter  to detect the levels of GFP expression.IQC at 0, 1, 5, 10, or 25 µM was mixed with PR8-GFP IAV for 1 h at 4 °C and the mixtures were added to the RAW 264.7 cells in 12 wells (5 × 105 cells/well) were infected with 10 MOIPR8-GFP IAV and 25 µM IQC for 30 min at 4 °C. After washing, the cells were incubated for 24 h at 37 °C. To check the effect on the entry step, the cells were infected with 10 MOI PR8-GFP IAV for 30 min at 4 °C followed by the addition of 25 µM IQC for 30 min at 37 °C. After washing, the cells were incubated for 24 h at 37 °C. For checking the effect on the virucidal step, 10 MOI PR8-GFP IAV and 25 µM IQC were co-incubated for 30 min at 4 °C. The mixtures were added to the cells and incubated at 37 °C for 30 min. After washing, the cells were incubated for 24 h at 37 °C. The brightfield and fluorescence images of cells were obtained using fluorescence microscopy with 200× magnification. The cells were fixed with 4% paraformaldehyde for 10 min and were analyzed by FACS to compare GFP expression.The time-of-addition assay was performed on binding, entry and virucidal stages upon IAV infection. Briefly, to examine the effect of IQC on the attachment step, RAW 264.7 cells in 12 wells (5 × 105 cells/well) for 2 h at 37 °C. After washing with PBS, the cells were further incubated for 24 h at 37 °C. The supernatant was harvested for the hemagglutination assay. Briefly, each supernatant was serially two-fold diluted and added to a round-bottomed 96-well plate. The blank medium was used as a negative control. Each sample (100 µL) was mixed with an equal volume of 1% chicken red blood cells (RBCs)  in PBS, and incubated for 1 h at room temperature. RBCs in negative control exhibited agglutination and RBCs in the virus-infected well were lysed without agglutination. HA titers were calculated as HA unit/100 µL in comparison with values of the virus control treatment. HA units were determined as the point at which hemolysis occurs.H1N1 IAV at 10 MOI and IQC  were mixed for 1 h at 4 °C and the mixtures were added to RAW 264.7 cells in 12 wells (5 × 106 PFU) IAV was added to each well and incubated for 30 min at 37 °C. The mixtures were incubated with 200 µM NA-Fluor Substrate for 1 h for 37 °C and terminated using NA-Fluor stop solution. The reaction was monitored by a fluorescence spectrometer  with an excitation wavelength of 365 nm and an emission wavelength of 445 nm.A neuraminidase (NA) inhibition assay was performed using the NA-Fluor influenza Neuraminidase Assay Kit  according to the manufacturer’s instructions. IQC was serially 2-fold diluted from 100 μM to 1.56 μM in 96 well black plates. As a positive control, oseltamivir carboxylate , a specific NA inhibitor, was used from 10 μM to 0.0001 μM. H1N1 or H3N2 (2 × 10Isoquercitrin, an ingredient abundantly found in natural products, prevented IAV binding and entry into the cells and exhibited a strong virucidal effect at an early stage upon IAV infection. Additionally, isoquercitrin inhibited viral progeny release via blocking neuraminidase activity. Our results elucidated the underlying mechanism of the antiviral efficacy of isoquercitrin against IAV infection and suggested the potential for development as an antiviral agent.