The improvement in cycling stability of S/EDA-RGO composites was further verified at different current rates. These findings demonstrated that proper surface modification of RGO by EDA reducing agent might improve the electrochemical performances of Li–S batteries.Sulfur/ethylenediamine-functionalized reduced graphene oxide (S/EDA-RGO) nanocomposites were synthesized using a simple process. Ethylenediamine (EDA) was employed as both the reducing agent and the modification component. The morphologies, microstructures, and compositions of S/EDA-RGO composites were characterized by various detection techniques. The data indicated that EDA-RGO used as scaffolds for sulfur cathodes could enhance the electronic conductivity of the composites and strengthen the adsorbability of polysulfides. Meanwhile, the electrochemical properties of both S/EDA-RGO and S/RGO composites that were used as cathodes for lithium-sulfur (Li-S) batteries were investigated. The initial discharge capacity of S/EDA-RGO composites reached 1240 mAh g Ethylenediamine (EDA) acted as both the reducing agent and the modification component. The electrochemical performances of S/EDA-RGO nanocomposites cathodes showed obvious improvements when compared to sulfur/reduced graphene oxide (S/RGO) cathodes.−1) were fabricated by the modified Hummers’ method [Graphene oxides  measurements were analyzed with a PHI 5000 Versa Probe system . The surface morphologies and the microstructure were examined by scanning electron microscopy  and high-resolution transmission electron microscopy .The crystalline phases of the samples were conducted by X-ray diffraction  using Cu Kα radiation (λ = 0.1542 nm). The chemical transformations of the composites were detected by Fourier Transform Infrared Spectroscopy  using KBr pellet method. The Raman spectra were obtained by a Raman microscope , from 100 cm+) at room temperature. The cell consisted of S/EDA-RGO composite cathode and lithium metal anode sandwiched by microporous polypropylene membrane as the separator soaked in 75 μL electrolyte, which is a solution of 1M lithium bis (trifluoromethane) sulfonamide (LiTFSI) mixed with 1, 2-dioxolane, and dimethoxymethane (1:1 by volume). The cathode comprised of S/EDA-RGO composite, Super P conducting agent, and polyvinylidene fluoride (PVDF) as a binder (8:1:1 by weight), dissolved in N-methylpyrrolidone. The resultant slurry was uniformly spread on an aluminum foil current collector, dried at 60 °C for 12 h, and then cut into circular disks with 1 cm in diameter. The sulfur loading amount of each electrode sheet is approximately 3 mg cm-2.CR2025 coin-type cells were assembled in high purity argon-filled MBraun glove box. All of the electrochemical measurements were carried out using a Neware battery testing system  between 1.5 V and 3 V , and X-ray photoelectron spectroscopy (XPS) were used for characterization. The XRD patterns of EDA-RGO showed broad and weak peaks that were centered at around 23.6° , corresp−1. The two sharp peaks below 600 cm−1 can be assigned to the S-S bond in the S/EDA-RGO composite. The three typical peaks of graphene-based materials were indexed to D band, G band, and very weak two-dimensional (2D) band at around 2700 cm−1 [−1 and G band around 1597 cm−1, corresponding to defect-induced band and crystalline graphite band, respectively [DI/GI) reflects the disorder degree of carbon materials. The DI/GI value of GO was less than that of the EDA-RGO composite (0.82 vs. 1.03). This partially confirmed disordered structures after the removal of oxygen-containing functional groups and attachments of amino functional groups after the oil bath process doped into the graphene layers [DI/GI value of the S/EDA-RGO composite dropped to 0.96, demonstrating that the reaction of sulfur with amino functional groups may decrease the disorder degree of the S/EDA-RGO composite [Raman spectroscopy is a powerful non-destructive tool that is suitable for distinguishing ordered from disordered crystalline structures of carbon-based materials . The Ram700 cm−1 . The Figectively . In genee layers ,33. Afteomposite .−1 was assigned to O-H stretching vibration, the adsorption band at 1720 cm−1 was attributed to C=O stretching vibration of carboxyl or carbonyl groups, the band at 1670 cm−1 was associated with the overlapping absorption signals from C=C stretching vibration, and the band at 1050 cm−1 was linked to C-O stretching vibration [−1) of primary amines and aliphatic C–N (1200 cm−1) stretching vibrations were observed, demonstrating the successful functionalization of EDA on GO [−1, corresponding to C-H stretching and suggesting the presence of amine functional groups on the GO surface [The FT-IR spectra of S/EDA-RGO and EDA-RGO composites are depicted in ibration . For theDA on GO ,38,39. T surface .2-NH2) appeared at 285.6 eV. These features confirmed the successful reduction of GO to EDA-RGO [2–NH2, respectively [The XPS spectra of S/EDA-RGO composite are depicted in  EDA-RGO . On the ectively . These dectively . The chaThe morphology and microstructure of the S/EDA-RGO composite were carried out by SEM in −1. Distinct oxidation and reduction peaks were visible. The cathodic peaks of S/EDA-RGO that were located at around 2.3 V and 2.1 V were assigned to reduction of sulfur (S8) to soluble high-order lithium polysulfides , and then further to insoluble lower-order lithium polysulfides Li2S2 (Li2S), respectively [2S2 (Li2S) to sulfur [To gain a better understanding of the influence of EDA functionalized RGO in trapping polysulfides, various electrochemical measurements of Li–S battery cathodes that were based on S/RGO and S/EDA-RGO composites were carried out. ectively . The onlo sulfur . As can -1 and 1240 mAh g−1 for the first cycle, respectively. The initial coulombic efficiency of S/EDA-RGO reached 100%, whereas that of S/RGO composite decreased to 95%. The S/EDA-RGO composite cathode also showed better cycling performance than unfunctionalized S/RGO. After 100 cycles, and when compared to S/RGO, the S/EDA-RGO composite revealed exceptionally stable discharge capacity of 714 mAh g−1 with a corresponding coulombic efficiency of 98%. Therefore, EDA functionalization effectively improved the cycling stability of the S/RGO composite cathode.The cycling performance of S/RGO and S/EDA-RGO composites for Li–S batteries was tested between 1.5 V and 3.0 V at 0.1 C, and the results are represented in −1, 1106 mAh g−1, 1052 mAh g−1, 985 mAh g−1, and 932 mAh g−1 at 0.1 C, 0.2 C, 0.5 C, 1 C, and 2 C, respectively. After switching again to 0.1 C, the obtained discharge capacity of S/EDA-RGO composite cathode reached 1013 mAh g−1.The rate capability of S/RGO and S/EDA-RGO composites were performed at various C rates . The S/E1 (resistance of electrolyte and electrode), R2 (charge transfer resistance), CPE (constant phase capacitance), and ZW (Warburg impedance) [The electrochemical impedance spectroscopy (EIS) measurements of S/RGO and S/EDA-RGO cathodes were carried out, and the data are summarized in pedance) . The semTo confirm the enhanced electrochemical performances of EDA functionalization-modified RGO, the further comparison among S/GO, S/MWNT, S/RGO, and S/EDA-RGO cathodes of Li-S batteries were carried out and the results are compiled in −1 at 2 C, which was higher than that of the S/RGO cathode. When the current rate returned back to 0.1 C, the capacity of S/EDA-RGO composite cathode recovered to 1013 mAh g−1, indicating the stable structure of S/EDA-RGO and its promise as a cathode material for Li-S batteries.S/EDA-RGO composite was successfully obtained by an EDA functional-modified RGO. The EDA functionalization did not only improve the electronic conductivity of carbon framework, but also suppressed lithium polysulfides diffusion in the electrolyte. As the C rate gradually increased, the discharge capacities of S/RGO and S/EDA-RGO cathodes decreased. It will be noted that the S/EDA-RGO composite presented a steady reversible discharge capacity of 932 mAh g


Posttranslational modifications of core histones are correlated with changes in transcriptional status, chromatin fiber folding, and nucleosome dynamics. However, within the centromere-specific histone H3 variant CENP-A, few modifications have been reported, and their functions remain largely unexplored. In this multidisciplinary report, we utilize in silico computational and in vivo approaches to dissect lysine 124 of human CENP-A, which was previously reported to be acetylated in advance of replication.Computational modeling demonstrates that acetylation of K124 causes tightening of the histone core and hinders accessibility to its C-terminus, which in turn diminishes CENP-C binding. Additionally, CENP-A K124ac/H4 K79ac containing nucleosomes are prone to DNA sliding. In vivo experiments using a CENP-A acetyl or unacetylatable mimic  reveal alterations in CENP-C levels and a modest increase in mitotic errors. Furthermore, mutation of K124 results in alterations in centromeric replication timing. Purification of native CENP-A proteins followed by mass spectrometry analysis reveals that while CENP-A K124 is acetylated at G1/S, it switches to monomethylation during early S and mid-S phases. Finally, we provide evidence implicating the histone acetyltransferase (HAT) p300 in this cycle.Taken together, our data suggest that cyclical modifications within the CENP-A nucleosome contribute to the binding of key kinetochore proteins, the integrity of mitosis, and centromeric replication. These data support the paradigm that modifications in histone variants can influence key biological processes.The online version of this article (doi:10.1186/s13072-017-0124-6) contains supplementary material, which is available to authorized users. Posttranslational modifications in histones play an important role in chromosome biology. The majority of such modifications discovered exist on the N-terminal tails of histones H3, H2A, H3.3, and H4 –3. N-terA plethora of CENP-A modifications have been discovered in recent years . Of thesIn this report, we dissect the role of CENP-A K124ac in silico and in vivo. First, using all-atom computational modeling, we simulate the presence of K124ac and H4K79ac in the octameric CENP-A nucleosome. We find that these modifications result in a loosening of DNA at the pseudo-dyad, followed by asymmetric site exposure of the terminal ends of the DNA. These DNA dynamics are driven, in part, by an unexpected compaction of the CENP-A protein, accompanied by a locking of the CENP-A C-terminus. Consistent with this finding, further computational analysis of this acetylated CENP-A nucleosome shows dramatically reduced contacts with the key kinetochore protein CENP-C. To examine the function of CENP-A K124ac in vivo, we generated mutants of CENP-A K124, which mimic either the constitutively acetylated (K124Q), or the unacetylated state (K124A) and observe three aspects of centromere dynamics are impacted. First, gain or loss of CENP-A K124ac results in a quantifiable decrease in association of modified CENP-A nucleosomes with native CENP-C. Second, there is a modest accumulation of downstream mitotic errors. Third, relative to wild-type CENP-A, in cells expressing the unacetylatable CENP-A K124A, centromeric foci are delayed in replication timing, whereas the acetyl-mimic K124Q loses mid–late S phase replication bias. Using gel-coupled mass spectrometry analysis of native CENP-A at distinct points spanning G1/S to late S phase, we uncover a switch in modifications of chromatin-bound native CENP-A, from acetylation of K124 at G1/S to monomethylation at S phase. Finally, using two custom antibodies against CENP-A K124ac, our data suggest p300 as a putative HAT, which promotes acetylation of chromatin-bound native CENP-A.Together, these results suggest a working model wherein cyclical changes in CENP-A K124 modifications facilitate accurate timing of centromeric replication and contribute to mitotic integrity.In recent work, using all-atom molecular dynamics (MD), we showed that the 4-helix bundle in the CENP-A nucleosome core particle (CENP-A NCP) is highly dynamic , such thDue to the loss of positive charge on CENP-A K124ac and H4K79ac at the interface of DNA and histones  of dimers. In results above, CENP-A/H4 and CENP-A′/H4′ dimers are closer together in the acetyl NCP Fig. c. In othcore) where high-frequency vibrational motions are filtered out within the first few PC modes, thereby revealing large global distortions. In this analysis, the most significant mode of motion, PC1core, revealed a surprising “freezing” or lack of motion at histone interfaces at the pseudo-dyad, adjacent to the K124ac modifications  of Cα atoms . Visual analysis of the most significant mode, PC1NUC, demonstrated a pronounced untwisting motion of DNA ends in the acetyl NCP  or a constitutively acetylated state . These mutants were expressed in the endogenous CENP-A background and used for the series of experimental investigations discussed below . WCE for total protein revealed that exogenously expressed HA-tagged proteins, the CENP-A chaperone HJURP, and the inner kinetochore protein CENP-C levels remained relatively equal , 2 days after transfection, when mutant protein expression was at its highest and most detectable in WCE. Western blots were performed to detect the presence and levels of CENP-C relative to levels of CpA/K124A/K124Q Fig. a. Data fTo verify this result using an alternative approach, we turned to co-immunofluorescence (co-IF) on chromatin fibers extracted from cells expressing either CpA, K124A, or K124Q. Next, we used anti-HA and anti-CENP-C antibodies to examine the association of CENP-C on CpA/K124A/K124Q positive chromatin fibers Fig. c. Our inThese chromatin fiber co-IF analyses support the biochemical results above, namely that relative to wild-type CpA, K124A has enhanced CENP-C associations, whereas K124Q has reduced associations with CENP-C.In previous experiments, over-expression of CENP-C in chicken DT40 cells led to higher rates of chromosomal segregation errors . Since oCells expressing CpA/K124A/K124Q were first synchronized with a double thymidine block. FACS analysis suggested that there were no appreciable defects or delays in cell cycle progression , a critical junction before the onset of replication . This leWe synchronized cells to S phase and staged cells to early, mid- and late S phase time-points  diagram. These dConversely, we observed that K124Q displayed a different phenotype. Whereas CpA shows a bias toward late S replication, K124Q mutants appear to have lost this bias, so that replicating centromeric origins fire equally throughout S phase Fig. b. An altThe replication defects noted above show that centromeric foci are modestly delayed in replication timing in K124A, and lack of a late S phase centromeric replication bias in K124Q cells. These phenotypes suggest that K124ac may be removed by early S phase, in order for centromeric replication to progress correctly. To address whether acetylation at K124ac is removed, or replaced by another modification at S phase, we utilized a native CENP-A gel-purification strategy coupled to a MS/MS approach , 39, 40,Mass spectra of CENP-A containing bands from these DLτ gels Fig. b confirmunpublished observations), which co-migrates with CENP-A on standard SDS-PAGE gels. To avoid this problem, we couple TAU gels to WBs for studying CENP-A modifications, where H3 and CENP-A run far apart, avoiding potential cross-reactivity. We developed a custom rabbit polyclonal antibody against CENP-A K124ac. Using recombinant histone proteins including CENP-A, H4, synthetic CENP-A K124ac, H4K79ac, H2A and H2B, we first tested this polyclonal antibody , trichostatin A (TSA) is a potent pan HDAC inhibitor (HDACiPAN), and quisinostat 2HCl is a pan inhibitor that targets predominantly HDAC1, 2, 4 and 10 . P300 was predicted to be a HAT target for CENP-A, because of a previous study, in which co-localization of p300 with CENP-A was observed at centromeres  or [Q] mutation  and a reverse primer that includes the XmaI site and STOP codon. The final fusion PCR product was excised, gel purified and finally ligated downstream of plasmid that had either GFP or HA-tags, driven by a constitutive CMV promoter.HeLa cells were grown to ~75% confluency and transfected using Lonza’s Amaxa Cell Line Nucleofector Kit R (Cat #VCA-1001) using Amaxa Biosystems Nucleofector II electroporation system according to the manufacturer’s guidelines using program O-005. After transfection, cells were plated with fresh media and grown for 48 h before harvesting for experiments.HeLa cells were grown in DMEM (Invitrogen/ThermoFisher Cat #11965) supplemented with 10% FBS and 1X Pen/Strep. nChIP experiments were performed without fixation, and with or without a double thymidine block to synchronize cells. For the complete double thymidine block protocol, please refer to Bui et al. (2012).2) with 0.5% Nonidet P 40 Substitute (Sigma Cat #74385). Afterward, nuclei were washed with TM2 and chromatin was either digested for 4 min for nChIP or 8 min for ChIP-seq with 1.0 U MNase (Sigma Cat #N3755-500UN) in nuclei solubilized with 2 mL of 0.1 M TE  and supplemented with 1.5 mM CaCl2. MNase reactions were quenched with 10 mM EGTA and centrifuged at 1000 rpm at 4 °C. Supernatant was removed, and nuclei extracted overnight at 4 °C in 0.5X PBS supplemented with a protease inhibitor cocktail (Roche Cat #05056489001). ChIP was performed with anti-HA antibody (Santa Cruz Cat #sc-805). nChIP’ed chromatin bound to Protein G Sepharose beads  were washed 3X with cold 0.5X PBS. Westerns were done using LiCor’s Odyssey CLx scanner and Image Studio Ver 2.0. Antibodies used for Westerns include: CENP-A (AbCam Cat #ab13939), CENP-B , HA-tag (GenScript Cat #A01244), FLAG-tag (AbCam Cat #ab1162), and H2B (AbCam Cat#52484).After cells were harvested, they were washed with PBS and PBS containing 0.1% Tween 20. Nuclei were released with TM2 . GFP tags were fused in-frame and upstream of wild-type CpA, K124A or K124Q, and may be co-transfected with mCh-CENP-A for co-localization assays. IF was performed using CENP-B (AbCam Cat #ab25734) and CENP-C (MBL Cat #PD030) antibodies. Cells were pulsed with EdU 30 min prior to the desired time point using the Click-iT EdU Alexa Fluor 594 kit (Life Technologies Cat #C10639) and imaged using a DeltaVision RT system fitted with a CoolSnap charged-coupled device camera and mounted on an Olympus IX70. Deconvolved IF images were processed using Image J and to assess co-localization with its ‘Colocalization Finder’ plug-in.Nature Protocols1, 2856–2860, 2007). Each band was split in two, and separate trypsin and chymotrypsin in-gel digestions were performed. The samples then underwent shotgun proteomic analysis on a nano-HPLC system  coupled to a hybrid mass spectrometer . Samples were injected using an auto-sampler and loaded onto a self-packed trap column , and the samples were then analyzed on a self-packed C18  column with a laser-pulled tip  using a flow rate of 200nL/min. The column was heated to 50 °C using column heater . Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid. The analytical gradient was a 90-min linear gradient from 5 to 35% buffer B. Eluting peptides were electrosprayed at 2.3 kV, and the ion transfer capillary was heated to 250 °C. The Orbitrap was operated in data-dependent mode with different settings depending on the cleavage enzyme used: Trypsin-cleaved samples were analyzed with a CID top 18 method, and chymotrypsin-cleaved samples were analyzed with a CID and ETD decision tree top 12 method. Precursor resolution was set to 60,000, CID collision energy was 35%, and ETD time reaction time was 100 ms with supplemental activation.Tau gel bands were processed using an in-gel digestion protocol from Shevchenko et al. (Protein identification was performed against the UniProt database entry for CENP-A using Proteome Discoverer 2.1 (Thermo Fisher Scientific) equipped with SEQUEST HT and Mascot . Search settings included tryptic or chymotryptic digest with up to two missed cleavages or nonspecific cleavage. Carbamidomethylation of cysteine was set as a static modification, while dynamic modifications included Met oxidation, Asp, Glu deamidation, Ser, Thr, Tyr phosphorylation, Lys acetylation, Lys ubiquitination, Arg and Lys methylation. Only matches with XCorrs greater than 2.0 or ion scores greater than 20 were considered. All the spectra matches were manually validated.Additional file 1: Movie S1. Histone dimers CENP-A/H4 is shown in red, CENP-A′/H4′ in blue, H2A/H2B in light blue, and H2A′/H2B′ in white. Movies presented here are animations of the most significant mode of motion, PC1core, of the principal component analysis of histones (PCAcore). The first clip shows the histones in both systems rocking with a “freezing” of motion in the acetyl CENP-A histones on the right. The second clip shows the 4-helix bundle in isolation to highlight the interface formed between CENP-A and CENP-A′. Next, the histones are rotated to focus on the described scissoring motion between helices α2 and α3 in the acetyl NCP. Here, the two helices move apart and then together, modulating the widths of the major and minor grooves in the acetyl NCP. Next, flipped to the other side of the nucleosome core, observe the rigidification of the H2A/H2B to H2A′/H2B′ interface in the acetylated nucleosome. To clarify this further, we then show the H2A to H2A′ interface in isolation.Additional file 2: Fig. S1. RMSF of proteins. A) This decrease in RMSF of Cα residues upon acetylation is more pronounced on the histone heterotetramer adjacent to the entry DNA. Of particular interest, the RMSF of the acetylated H2A acidic patch was suppressed with acetylation by −1 Å, and suppression is shown in the CENP-A C-terminus. The greater similarity shown in the RMSF of the reciprocal histones—CENP-A′, H4′. H2A′, and H2B′—could potentially be explained by the observed asymmetric unwrapping of DNA where the exit end in both systems dissociates to a similar amount . B) The RMSF of whole base pairs is shown for each DNA strand. Regions marked by I are DNA wrapped near the entry or near CENP-A and II are near the exit end of CENP-A′. The pseudo-dyad is marked by the vertical dotted line.Additional file 3: Movie S2. Histone dimers CENP-A/H4 is shown in red, CENP-A′/H4′ in blue, H2A/H2B in light blue, and H2A′/H2B′ in white. Presented are animations of the most significant mode of motion of the whole nucleosome, PC1nuc, of the principal component analysis (PCAnuc). The pseudo-dyad is labeled PD, and the modified lysine side chains are shown in green with or without acetylation dependent on the system. It is worth noting that our PCAnuc calculations are based on DNA phosphate positions and protein Cα’s—therefore, side chains are stagnant relative to the protein backbone. In the first two clips, two unique features of the acetyl NCP are shown: the modulation in the width of the major and minor DNA grooves and the inter-helical DNA bubble formed adjacent to H4 and H4′ K79ac. The final clip then shows the NCP on the side to highlight DNA end untwisting in the acetyl NCP with the last ten base pairs were truncated from the analysis.Additional file 4: Fig. S2. Experimental scheme for experiments and cell synchronization. A) Computation and biological experimental scheme for this publication. B) Cell cycle synchronization with a 30-min EdU pulse prior to preparing slides for EdU and immunofluorescence.Additional file 5: Fig. S3. Co-CENP-B/CENP-C staining with HA-tagged K124A/Q proteins. A) Unsynchronized cells stained with CENP-B (CpB) and B) percentage of CENP-B co-localizing with GFP-CENP-A (CpA) or K124A/Q exogenously expressed proteins after a double thymidine block and released for 1 or 8 h. C) Co-localization of the GFP-tagged CpA/K124A/K124Q with CpB during metaphase.Additional file 6: Fig. S4. K124A/Q have altered affinity for CENP-C. A) Whole cell extracts (WCE) from CpA/K124A/K124Q mutants reveal no noticeable differences in CpC/HJURP levels. B) Levels of the mutants are tracked across 8 days, revealing 2d post-transfection accrued the peak level of mutant proteins, allowing us to ChIP at 2 days against the HA-tag to observe any CENP-C binding defects. C) Percent distribution of CENP-C/HA-tagged mutant for the fiber IF experiments. D) Medium-sized arrays from 4-min MNase-digested chromatin were used in our ChIP assays, and overall comparison between ChIP’ed HA-tagged H3 versus CpA mutant chromatin. kD mark = 20kD, I = Input, U = Unbound. E) CENP-C interacts with endogenous CENP-A after ACA ChIP.Additional file 7: Fig. S5. K124A/Q mutants have normal cell ploidy and progress through the cell cycle with no aberrant defects. A) Overlaid FACS analysis of GFP-tagged CpA/K124A/Q mutants. B) Separated FACS profiles for each mutant and time point. X-axis is the propidium iodide level and the Y-axis is the cell count.Additional file 8: Fig. S6. Identifying CENP-A on double long TAU (DLτ) and long TAU (Lτ) gels for mass spectrometry and HAT/HDAC inhibitor drug treatments. A) dLT gels remove excess canonical histone components, leaving behind predominantly CENP-A and histone H2A. Numbers on the DLτ represent bands that were sent for mass spec, and duplicate gel was used for Western and probed against CENP-A. B) Whole cell extracts from cells that were untreated, treated with a HAT inhibitor or HDAC inhibitor, were probed against CpA K124ac, CpA, H2A, H4K5ac and H4. The probes were also used against recombinant CpA/H4 (rCpA + rH4) or recombinant chemically ligated K124ac (rK124ac) to determine antibody specificity. C) Untreated cells with chromatin-bound, hydroxyapatite-purified histones were ran on a long TAU (LT) gel, and duplicate gel transferred to a membrane was probed for K124ac, H2A and H4.Additional file 9: Fig. S7. A model depicting how cyclical switching in CENP-A K124 acetylation and methylation might affect centromeric replication dynamics. A) Model of CENP-A K124me-containing nucleosome. B) Cell cycle progression of K124ac during G1/S to K124me at S phase. C) Current model encompassing the dynamics of how K124 modifications affect replication and kinetochore protein CENP-C binding, before and during replication.


Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (mT of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation.The Sso7d protein from the hyperthermophilic archaeon  Antibodies are broadly used as research reagents and therapeutics because of their ability to recognize a wide range of target molecules with high affinity and specificity. However, applications requiring cytoplasmic expression, small size, complex modular fusion protein topologies, or high stability motivate exploration of alternative protein scaffolds for molecular recognition.The ideal general binder scaffold would exhibit properties such as small size, lack of glycosylation sites and disulfide bonds, high stability, high solubility, negligible aggregation, and absence of polyspecificity. A number of alternative scaffolds have been developed by diversifying the amino acid composition at the surface of a stable structure . Example2–9–Sulfolobus solfataricus and Sulfolobus acidocaldarius, respectively. Several advantages make these proteins particularly promising as binder scaffolds. (i) They are highly stable with mT values of 98 and 91 °C for Sso7d and Sac7d, respectively (A)  (–A), which could strongly inhibit interaction with positively charged antigens or epitopes  , 17. Apar Sac7d) –21. More Sac7d) –A, which epitopes .Therefore, we constructed a charge-neutralized Sso7d mutant that largely maintains high thermal stability. We demonstrate that this charge neutralization reduces nonspecific interactions with mammalian cells. Based on the neutralized scaffold, high affinity binders against mouse serum albumin (MSA) and human epidermal growth factor receptor (hEGFR) were generated. Surprisingly, the distribution of amino acids on the rigid binding surfaces of Sso7d-based binders is very different from that of the flexible complementarity-determining region (CDR) loops of antibodies but closely matches that of antibody binding energetic hot spots. In particular, the aromatic residues Trp, Tyr, and Phe were observed at high frequencies. Remarkably, binders tolerated up to four of these aromatic residues in their nine-residue binding sites and maintained high thermal stability and monomeric behavior without any observed aggregation. Together, these data demonstrate that the optimal distribution of amino acids in small, rigid binding surfaces is distinct from the composition of loop-based paratopes and that the hyperstability of the scaffold promotes an unusually high tolerance for aromatic residues.mT of the resulting mutant Sso7d-Short was not decreased (B). Next, lysines at positions Lys-6, Lys-8, Lys-27, and Lys-39 were mutated to Leu, Ile, Asn, Gln, Ser, or Thr in silico. These residues were chosen based on both their net charge and size. The most stable in silico mutation at each position was subsequently incorporated into Sso7d-Short and analyzed by DSC (B). Compared with wild-type Sso7d , the mT value of the mutant Short-K27Q was unchanged (98.9 °C), whereas the mutants Short-K6T and Short-K8Q were slightly destabilized by 1.6 and 0.6 °C, respectively (B). In silico calculations predicted that mutating both residues (K6T and K8Q) simultaneously would result in improved stability, whereas individual mutations would lead to destabilization. Consistent with in silico results, combining these mutations completely restored thermal stability, resulting in a mT of 99.1 °C. Thus, we identified the neutralizing mutations K6T, K8Q, and K27Q, which do not affect thermal stability.First, we aimed at neutralizing the charge of Sso7d without significantly impacting its thermal stability. We started by deleting the two C-terminal lysines (Lys-62 and Lys-63). Differential scanning calorimetry (DSC) analysis demonstrated that the ecreased B. Next, d by DSC B. Comparectively B. In silin silico experiments identified a favorable mutation to either Gln or Thr. Because this residue protrudes from the anticipated binding surface , potentially sterically clashing with bound antigen, we tested mutations not only to Gln and Thr but also to the small side chains of Ala, Gly, and Ser. These mutations at position 39 were combined with the above mentioned mutations , resulting in five clones termed Short-TQQX with X being either Gln, Ala, Gly, Ser, or Thr at position 39. Short-TQQQ was the most stable version followed by Short-TQQA . Given the small side chain of alanine at position 39, which reduces the risk of clashing with the antigen, we chose Short-TQQA as our final scaffold. This mutant was termed reduced charge Sso7d (rcSso7d). Importantly, the net formal charge of rcSso7d was only +1 as opposed to +7 for Sso7d-WT, and the protein remained highly stable (mT of 95.5 °C).At position Lys-39, C) with the final scaffold rcSso7d showing no binding.To test whether this charge neutralization had an effect on nonspecific cell binding, we analyzed the binding of all mutants to HeLa cells. Remarkably, nonspecific binding strongly correlated with the net formal charge of the proteins C with thA, magenta). Because only every other position is randomized, all nine residues are solvent-exposed. To increase the frequency of potential binders in the library, we mimicked the amino acid diversity found in protein-protein interactions (PPIs) using two previously published data sets: (i) the -fold enrichment of amino acids in PPI hot spots  over the entire database of alanine mutations that was analyzed in the study by Bogan and Thorn . In addition, proline was excluded to avoid disruption of the β-strands. This resulted in the library rcSso7d-11, which was designed to contain a mixture of 11 different amino acids  at equal frequencies (9.1% each) at all nine randomly mutated positions. As a control, we constructed the library rcSso7d-18 containing all amino acids at a frequency of 5.6% each except for proline and cysteine. To control the distribution of amino acids in a precise manner, the randomized oligonucleotides were constructed by trinucleotide synthesis. Finally, both rcSso7d-11 and rcSso7d-18 were constructed as yeast display libraries with 1.4 × 109 transformants each.Using the charge-neutralized mutant rcSso7d as a scaffold, we constructed yeast display libraries that were randomly mutated at nine positions within three adjacent β-strands nd Thorn . We exclB). Importantly, both libraries were expressed on the surface of yeast at high levels (C and D). This indicates that the vast majority of the mutant proteins in the libraries are well folded and thermally stable as multiple studies have demonstrated that expression levels correlate with thermal stability (–E), again confirming the high tolerance to mutation. Only a minor fraction of the libraries displayed truncated proteins , likely due to the introduction of frameshifts and stop codons (data not shown). This high fraction of expressed and screenable diversity is an unusual feature by comparison with single chain Fv . Yeast surface titration of the MSA binders revealed affinities in the high nanomolar range , with M11.1 having the highest affinity (226 nm). Next, these four binders were subjected to two rounds of affinity maturation with each round consisting of error-prone PCR (epPCR) and two to three FACS sorts. For each of the two library designs (rcSso7d-11 and rcSso7d-18), the two lead clones were affinity-matured in the same tube, resulting in direct competition between mutants derived from either of the two parental clones. The majority of the resulting clones were variants of M11.1 and M18.2, respectively. The affinity-matured binders are referred to here according to their parental clone. For example, M11.1.2 is a variant of M11.1. MSA titrations of yeast-displayed binders showed 10–30-fold improvements in affinity over the respective parental clones  with M11.1.2 being the strongest binder with a dK of 8 nm. Finally, the results from yeast surface titrations were confirmed by biolayer interferometry (BLI) where solubly expressed rcSso7d clones were incubated with MSA-loaded tips. Representative measurements are shown for the parental binder M11.1 and its affinity-matured version M11.1.2, clearly demonstrating an increase in affinity during affinity maturation . Affinities obtained from BLI experiments were severalfold higher compared with yeast surface titrations, but the relative ranking of affinities was consistent between the two methods. According to BLI, the highest affinities were 4 nm for M11.1.2 and M11.1.3 .MSA selections were started with two rounds of enrichment for binding to MSA-loaded magnetic beads . To reduFig. 3A).For hEGFR selections, a fusion protein consisting of the extracellular domain of hEGFR and human IgG1-Fc (hEGFR-Fc) was used. Similar to MSA selections, both libraries were subjected to two rounds of positive and three rounds of negative magnetic bead selections using hEGFR-Fc-loaded and unloaded beads, respectively, followed by FACS. To avoid enrichment of Fc binders, one FACS round of negative selection against hIgG1-Fc was included. 18–36 clones were sequenced from both libraries after the third, fourth, and fifth FACS rounds. Sequence analysis showed that, in contrast to MSA, several distinct sequence families had emerged A.A). Instead of choosing only the binders that showed the highest signal for hEGFR-Fc binding, we also included binders from different clone families, such as the non-cetuximab-competitive binders E11.8 and E18.6. Similar to MSA affinity maturations, for each of the two library designs, one affinity maturation library was constructed starting with a mixture of the parental clones. After two rounds of affinity maturation, binding was significantly improved compared with parental clones (data not shown). To determine monovalent dK values, the binders were expressed solubly and titrated on human EGFR-positive A431 cells (A and B). Binders isolated from the naïve library without affinity maturation already showed monovalent affinities of up to 20 nm. In agreement with yeast display experiments, the binding signal of affinity-matured binders was increased . However, the dK values were only slightly improved compared with the parental clones , which may be due to increased repulsion between anionic cell surfaces and the negative charges that accumulated on the binders during affinity maturation.To narrow down the number of mutants for further analysis, individual mutants were displayed on the surface of yeast and tested for binding to hEGFR-Fc (data not shown). In addition, these clones were also analyzed for competition with the clinical anti-EGFR antibody cetuximab. Remarkably, all tested mutants except for two (E11.8 and E18.6) were blocked by preincubation of hEGFR-Fc with cetuximab, indicating that the majority of the isolated rcSso7d mutants binds to an epitope overlapping with that of cetuximab (data not shown). Based on single clone analysis, we chose a set of mutants for affinity maturation . In agreement with yeast display competition experiments, all binders except for E11.8 and E18.6 competed with cetuximab. Moreover, all of these cetuximab-competitive binders also competed with each other for binding to hEGFR, suggesting that they all bind to a common hot spot on hEGFR. Importantly, E11.8 and E18.6 did not compete with each other, indicating that they recognize two distinct epitopes on hEGFR (C). Together, these experiments demonstrate that rcSso7d can be engineered for binding to different epitopes on natively folded hEGFR on human cancer cells with high affinities. Moreover, the interaction with the cells is highly specific for EGFR because binding is efficiently blocked by preincubation with either non-tagged rcSso7d-based binders or with the clinically approved antibody cetuximab.To investigate whether the isolated hEGFR binders bind to overlapping epitopes, we performed competition experiments on A431 cells. Briefly, cells were preincubated with non-tagged rcSso7d mutants or cetuximab followed by His-tagged binders, which were detected with an anti-Hisantibody C. In agron hEGFR C. TogethA) as opposed to MSA libraries, which converged to one prominent clone family (A).To evaluate the distribution of amino acids in the rigid binding surfaces of rcSso7d-based binders, we performed deep sequencing analysis of EGFR-selected rcSso7d-11 and rcSso7d-18 libraries. We chose the EGFR libraries because they contained distinct sequence families A as oppoe family A.A, left). Surprisingly, there was little to no correlation between the amino acid frequencies in rcSso7d-based binding surfaces and in antibody CDR-H3 loops . In contrast, the amino acid distribution in rcSso7d-based binders shows similar trends compared with that at antibody hot spots . For example, in both antibody hot spots and binding surfaces from library rcSso7d-11, the two most frequent residues are W and Y. A similar trend was observed for rcSso7d-18-derived binders with the top six amino acids  closely resembling the top six in antibody hot spots . This agreement with data from a computational alanine scan was confirmed by comparing the frequencies of amino acids in rcSso7d-based binders with experimentally determined enrichments in PPI hot spots   compared with the R2 values obtained from correlations with overall distributions in antibody CDR-H3 loops .All reads from deep sequencing were first grouped into six and 13 sequence families for rcSso7d-11 and rcSso7d-18, respectively. Reads within a family contained similar sequence patterns with only few variations at the nine binding site positions. Subsequently, each family was treated as one clone for further analysis to avoid over-representing the most abundant clones. Finally, the frequency of amino acids in the binding surface of hEGFR binders was analyzed and plotted against that in antibody CDR-H3 loops   with R2, right) . Again, B). The MSA binders discussed above  as well as the two other aromatic amino acids, Tyr and Phe. Remarkably, analysis of the sequence families obtained by deep sequencing showed that the vast majority of hEGFR binders contain three to five of these aromatic amino acids in their nine-residue binding surfaces, and no binder contained less than two of these residues B. The MSed above  also conA), suggesting they are monomeric. Importantly, no aggregation was observed for any of these binders except for E18.7, which contains three Trp and two Phe residues in the binding surface. Moreover, the majority of binders are highly stable with mT values above 70 °C as shown by DSC analysis . Only a single sequence family , containing two aromatic amino acids, is less stable with mT values of around 60 °C. Together, these results demonstrate that the nine-residue binding surface tolerates up to four aromatic side chains, yielding highly stable binders with SEC elution profiles very similar to wild-type Sso7d.To investigate whether the high frequency of aromatic amino acids negatively impacts the biophysical properties of the resulting binders, we analyzed the diverse set of hEGFR binders shown in Fig. 6C). Moreover, comparing the net formal charges of affinity-matured binders with that of their parental clone demonstrated that the antigen-dependent charge difference was further enhanced during affinity maturation with MSA binders becoming more positive and hEGFR binders becoming more negative . Thus, there was a clearly different requirement for positively or negatively charged residues depending on the charge environment of the antigen that was used during selection as will be discussed below.Finally, to evaluate how the overall charge of the binders evolved during selection, we calculated the net formal charges for all sequences shown in Fig. 5mT of 95.5 °C), and its net formal charge was reduced from +7 to +1. Because one Lys and one Arg residue were part of the binding surface that was randomized in the libraries, the final net formal charge of the library scaffold was −1. In addition, our improvement of the Sso7d scaffold also involved flattening the binding surface. To remove the protuberance at position Lys-39 and thereby avoid clashes with interacting antigens, this residue was mutated to the small side chain of alanine.In this study, we engineered potential improvements to the hyperstable Sso7d protein in an attempt to make it a better scaffold for molecular recognition. First, we charge-neutralized this highly positively charged DNA-binding protein by deleting the two C-terminal lysines and mutating four lysines to neutral amino acids. The resulting mutant rcSso7d maintained high thermal stability , would have yielded high affinity binders against this positively charged epitope on hEGFR.Second, we hypothesized that the high concentration of positively charged residues around the binding surface would hinder selection of binders against positively charged epitopes, whereas a charge-neutralized scaffold would be able to easily adapt to different charge-environments of different epitopes. Indeed, we observed strong antigen-dependent charge differences in the selected binders. Selection for MSA binding resulted in positively charged binders, whereas hEGFR binders were negatively charged. This difference was further enhanced during affinity maturation. Thus, in the case of MSA selections, the binders adapted to the negatively charged antigen  of 5.5) by enriching positively charged amino acids. Interestingly, the extracellular domain of hEGFR is almost neutral in charge, not explaining the trend toward negatively charged hEGFR binders. However, the crystal structure of the antigen-binding fragment (Fab) of cetuximab bound to the extracellular domain of hEGFR shows that the cetuximab epitope on hEGFR involves three positively charged amino acids  but no negatively charged side chains . MoreoveFig. 1Because yeast display levels have been shown to correlate with protein fitness, in particular with thermal stability –27, we a–m were isolated from the naïve libraries without any affinity maturation. Although affinity maturation of these hEGFR binders resulted in increased binding signals when measured on EGFR-positive A431 cells, the improvements in dK were moderate. One possible explanation for the moderate improvement in affinity could be the enrichment of negative charges during affinity maturation, possibly leading to increased repulsion from the anionic mammalian cell surfaces.The rcSso7d-11 and rcSso7d-18 libraries yielded high affinity clones for both MSA and hEGFR. After two rounds of affinity maturation, MSA binders with single digit nanomolar affinities were obtained, which is striking given the small binding surface that only contains nine amino acids. Moreover, hEGFR binders against three different epitopes with affinities up to 20 nApart from charge-neutralizing and flattening the binding surface, we also mimicked the amino acid distribution found in natural PPIs. We restricted diversity to a set of 11 amino acids (library rcSso7d-11) that show high enrichment in PPI hot spots  and/or hTo investigate the amino acid preferences of the small and rigid binding surface of rcSso7d-based binders, the more diverse populations of hEGFR binders were analyzed by deep sequencing. Surprisingly, there was hardly any correlation between the frequency of amino acids in rcSso7d binding surfaces and the distribution in antibody CDR-H3 loops. However, the distribution of amino acids in the binding sites of rcSso7d-based binders closely matched the frequency of residues that strongly contributed to binding  in a computational alanine scan of 227 antibody-antigen complexes . OverallA, it is apparent that Tyr is highly frequent in all of the described data sets. In contrast, the other aromatic residues, Trp and Phe, are present only at low frequencies in antibody CDR-H3 loops but strongly enriched among the residues that contribute ≥0.8 kcal/mol to antibody binding in the computational alanine scan as well as in binding surfaces of rcSso7d mutants . Conversely, the small, flexible amino acids Gly and Ser, which are highly frequent in antibody CDR-H3s, are much less frequent in rcSso7d-based binders. Consistent with the low frequency in hEGFR binders, Ser and Gly are almost never found at antibody hot spots. Thus, it appears that Gly and Ser play an important role in loop-based binding surfaces where flexibility is important for adapting the right antigen binding conformation, but they do not directly contribute to the binding energy as was also suggested by Sidhu and co-workers  was Trp. This strong enrichment for Trp was also observed in computational  and expeIt should also be noted that Trp, Tyr, and Phe did not show any preference for particular positions within the binding surface. Deep sequencing data showed high frequencies of aromatic amino acids at all nine binding site positions (data not shown).Importantly, the binders isolated from the rcSso7d libraries were highly stable and monomeric despite some containing up to four aromatic amino acids of their nine-residue binding surface. We hypothesize that the high thermal stability of rcSso7d, paired with the rigidness of its binding surface, prevents the hydrophobic amino acids from interacting nonspecifically and aggregating. Consistent with this hypothesis, it has been reported that disulfide bonds and salt bridges can stabilize aggregation-prone regions in their native conformations, thereby avoiding the structural rearrangements necessary for aggregation and misfolding .in vivo half-life extension (MSA). Finally, sequence analysis of the isolated binders generated design principles for small and rigid binding surfaces as distinct from the thoroughly explored design principles for antibody CDR loops.Overall, we have demonstrated that libraries based on the charge-neutralized and flattened Sso7d variant rcSso7d yield binders against multiple antigens and epitopes with low nanomolar affinities and beneficial biophysical properties. Recently, rcSso7d-based binders generated in this study were used for the development of highly efficient reagentless biosensors , and a b1SSO)  . Terminain silico protein design, a previously described algorithm was used  + chloramphenicol (34 μg/ml) and incubated at 37 °C. When an A600 of 2 was reached, expression was induced by addition of 1 mm isopropyl β-d-1-thiogalactopyranoside. After overnight expression at 20 °C, the cells were centrifuged, resuspended in sonication buffer , sonicated, and centrifuged again. Subsequently, the His6-tagged SUMO fusion proteins were purified from the sonication supernatants using TALON metal affinity resin (Clontech). After addition of 10 mm imidazole, the sonicated supernatants were applied onto the resin twice followed by washing with equilibration buffer . For the last washing step, 15 mm imidazole was added to reduce nonspecific binding to the resin. Finally, proteins were eluted with elution buffer (equilibration buffer + 250 mm imidazole) and buffer-exchanged to phosphate-buffered saline, pH 7.4 (PBS).All Sso7d mutants were expressed as fusion proteins consisting of an N-terminal hexahistidine tag followed by small ubiquitin-like modifier (SUMO) and Sso7d using the pE-SUMO vector . This expression system offers the advantage of generating Sso7d mutants without any attached tags by subsequent digestion with SUMO protease 1 and purification of His-tagged SUMO and SUMO protease. Briefly, Rosetta 2 (DE3) m NaCl, 20 mm Tris-HCl, pH 8.8. Subsequently, they were loaded onto the resin and washed/eluted with increasing concentrations of NaCl (up to 900 mm). The resulting protein samples were buffer-exchanged in PBS again.In the case of Sso7d-WT and its point mutants, with which DNA was co-purified, the proteins were further purified by anion exchange chromatography using Q Sepharose Fast Flow resin . Samples were buffer-exchanged to 50 m6-SUMO tag right before the N terminus of Sso7d. After overnight digestion at 22 °C, the digestion product was purified using TALON metal affinity resin. Digested SUMO, non-digested SUMO fusion proteins, and SUMO protease 1  bound to the resin and the flow-through, only containing Sso7d, were collected.For biophysical analysis (DSC and SEC), the SUMO fusion proteins were digested with the protease Ulp1 (SUMO protease 1), resulting in cleavage of the N-terminal Hism glycine, pH 3.5. The proteins were directly eluted into 0.8 ml of 1 m Tris, pH 8.0, to minimize the incubation time at pH 3.5.Human IgG1-Fc and hEGFR-Fc were cloned into the gWIZ vector  with a hexahistidine tag at the C terminus. FreeStyle HEK293F cells (Life Technologies) were cultured in FreeStyle 293 expression medium (Life Technologies) and transiently transfected using polyethylenimine (PEI). After 1 week, the cultures were centrifuged and filtered. 10× PBS was added to the supernatants to achieve a 1× PBS concentration. The resulting solution was applied onto Protein A resin , washed four times with PBS, and finally eluted with 8 ml of 0.1 Fatty acid-free MSA was purchased from Alpha Diagnostic International . All antigens  were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Life Technologies), and subsequently monomeric protein was purified by size exclusion chromatography using a HiLoad 16/600 Superdex 200 column .XXXGTGXXXCGTXXXGGCCAGXXXATTXXXTTTXXXTATGATGAAGGTGGTGGTGCC-3′, and the sequence of ran_rev_11 and ran_rev_18 was 5′-GCAGTTCTTTCGGTGCATCTTTTTCGCTCACXXXACCXXXACCXXXGGCACCACCACCTTCATCATA-3′; “XXX” is a mixture of 11 different codons for library rcSso7d-11  and a mixture of 18 different codons for rcSso7d-18 . The ran_rev oligonucleotides are reverse primers, and therefore the XXX codons are antisense codons in those oligonucleotides. The resulting fragment was elongated by PCR twice using the following oligonucleotides: Sso_lib_nest_fwd (5′-GCAACCGTGAAATTCACATACCAAGGCGAAGAAAAACAGGTGGATATTAGCAAAATCAAG-3′) and Sso_lib_nest_rev (5′-TTGCTTTTCCAGCATCTGCAGCAGTTCTTTCGGTGCATCTTTTTCGCTCAC-3′) for the first elongation PCR; CON2_Sso6T8Q_fwd (5′-GGCTCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGGCTAGCGCAACCGTGAAATTCACATACCAAGGCG-3′) and CON2_Ssoshort_back (5′-CTATTACAAGTCCTCTTCAGAAATAAGCTTTTGTTCGGATCCTTGCTTTTCCAGCATCTGCAGCAGTTC-3′) for the second elongation PCR. Both sides of the final PCR product contained regions that were homologous to the NheI/BamHI-linearized pCTCON2 vector, facilitating homologous recombination in yeast. The final construct encoded for the fusion protein Aga2p-HA tag-(Gly4Ser)3 linker-rcSso7d-c-MYC tag. Saccharomyces cerevisiae strain EBY100 was transformed by electroporation with the linearized vector and PCR fragments as described previously . For each library, two self-annealing degenerated oligonucleotides were synthesized: ran_fwd_11 and ran_rev_11 for library rcSso7d-11 and ran_fwd_18 and ran_rev_18 for rcSso7d-18. The sequence of ran_fwd_11 and ran_fwd_18 was 5′-GGCGAAGAAAAACAGGTGGATATTAGCAAAATCAAGeviously . The twom concentration each of 8-oxo-dGTP and dPTP. 15 epPCR cycles were performed using the primers epSso_fwd (5′-GGCTCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGGCTAGC-3′) and epSso_rev (5′-CTATTACAAGTCCTCTTCAGAAATAAGCTTTTGTTCGGATCC-3′). A detailed protocol is provided elsewhere ). For all MSA selections and MSA titrations, PBSO  was used instead of PBSA to avoid blocking potential cross-reactive MSA binders with BSA.Yeast display experiments were performed as described previously –46. Brie–Bead selections were conducted using biotinylated antigen and streptavidin-coated Dynabeads (Life Technologies) as described previously , 43. For6 . In this case, the c-MYC tag was detected with chicken anti-c-MYC  followed by goat anti-chicken Alexa Fluor 488 (Life Technologies). Cells were either sorted on a FACSAria IIU or analyzed on an Accuri C6 flow cytometer (both from BD Biosciences).For FACS experiments, washed cells were incubated with biotinylated antigen and mouse anti-c-MYC (clone 9E10) followed by incubation with Streptavidin-Alexa Fluor 647 and goat anti-mouse Alexa Fluor 488 . For hEGFR selections, we alternated between use of biotinylated and non-biotinylated antigen. Non-biotinylated hEGFR-Fc was detected with allophycocyanin (APC)-labeled mouse anti-Hism His6-SUMO-Sso7d mutants in PBS followed by detection of surface-bound proteins with mouse anti-His6-APC . During the last washing step before FACS analysis, the cells were stained with propidium iodide (2 μg/ml), and only negative cells (i.e. non-necrotic cells) were included in the final analysis.HeLa cells were cultivated in DMEM  supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies). Cells were harvested by incubating them in trypsin/EDTA . After washing in PBS, cells were incubated with 1 μ6-SUMO-Sso7d mutants in PBSA followed by three washing steps with PBSA and detection with anti-penta-His-Alexa Fluor 647 . For the competition assay, cells were preincubated with blocking reagent (1.5 μm rcSso7d mutants without the His6-SUMO tag or 300 nm cetuximab). After 90 min, 20 nm His6-SUMO-rcSso7d mutants was added, resulting in a final concentration of 1.0 μm for the blocking rcSso7d mutants and 200 nm for cetuximab. Finally, the His6-SUMO-tagged proteins were detected with anti-penta-His-Alexa Fluor 647.A431 cells (ATCC) were cultivated in DMEM + 10% heat-inactivated fetal bovine serum and detached using trypsin/EDTA. To determine EGFR affinities, A431 cells were incubated with various concentrations of HisAll mammalian cell experiments were analyzed on an Accuri C6 flow cytometer. To avoid endocytosis, all incubation steps were done at 4 °C.m in PBS. Samples were heated from 20 to 120 °C with a heating rate of 1 °C/min. Buffer baselines were subtracted followed by normalization for protein concentration and fitting with a non-two-state thermal unfolding model. For analytical SEC analysis, 100 μl of a 35 μm solution were loaded onto a TSKgel G2000SWxl column and eluted at a flow rate of 0.7 ml/min using a buffer containing 100 mm phosphate and 150 mm NaCl, pH 6.8.All mutants were analyzed on a VP-DSC capillary cell microcalorimeter  at a concentration of 30 μm) followed by measuring dissociation in buffer. Buffer baselines (MSA-loaded tips without addition of binder) were subtracted from the data followed by global fitting to a 1:1 binding model. dK values were obtained from steady state binding analysis.Samples were analyzed on an Octet RED96 instrument  using PBS supplemented with 0.1% ovalbumin and 20 μl/liter Tween 20. Biotinylated MSA was immobilized onto streptavidin-coated BLI tips . Subsequently, association was analyzed at various concentrations of SUMO-rcSso7d fusion proteins (1:3 dilutions starting from 1000 to 1.37 nPlasmids were isolated from yeast libraries that had been selected for hEGFR binding (two rounds of bead selection and five rounds of FACS of which one FACS round was a negative selection against hIgG1-Fc). hEGFR-binding rcSso7d mutant genes were amplified by PCR with primers containing barcode sequences, facilitating pooling of multiple samples in one sequencing run. Subsequently, the pooled sample was analyzed on an SMRT cell using an RSII instrument .Analysis of deep sequencing data was performed in Matlab. For all members of the library, pairwise sequence distances were calculated based on the Gonnet substitution matrix. The results were then hierarchically clustered, and sequence families within the phylogenetic tree were defined as sets of sequences that were connected by a path length less than a specified cutoff. Only sequence families that were present at a frequency of at least 0.4% were considered for further analysis. Subsequently, the amino acid frequencies at the nine positions of the binding surface were calculated for each sequence family followed by normalization of the frequency of each sequence family to 1 to avoid over-representation of amino acids that were present in highly frequent sequence families. Finally, the average amino acid frequency in the binding sites among all sequence families was calculated.in silico experiments. A. W. T. processed deep sequencing data. M. W. T., R. R. S., N. J. Y., and K. D. W. wrote the paper.M. W. T. and K. D. W. designed the study. M. W. T., J. D. K., E. L., and N. K. M. performed experiments. R. R. S. performed and B. T. designed


In silico analysis predicted direct binding sites between PRINS and the mRNAs, which was confirmed by an in vitro binding assay and on cellular level. Our results indicated that PRINS binds directly to the mRNAs of IL-6 and CCL-5 at specific binding sites and eventually destabilizes these mRNAs, leading to a decrease in their expression and secretion of the corresponding proteins. These results may indicate a restrictive role for PRINS in inflammatory processes.Cytosolic DNA fragments are recognized as pathogen- and danger-associated molecular patterns that induce a cascade of innate immune responses. Moreover, excessive cytosolic DNA can enhance chronic inflammation, predominantly by activating inflammasomes, and thereby contributing to the pathogenesis of chronic diseases, such as psoriasis. Psoriasis associated non-protein coding RNA induced by stress (PRINS) is a long non-coding RNA, which has been shown to be associated with psoriasis susceptibility and cellular stress responses; however, the precise mechanism of its action has not been determined. Here, we provide evidence that, in addition to inflammasome activation, cytosolic DNA induces intracellular inflammatory reactions while decreasing PRINS gene expression. Furthermore, PRINS overexpression can ameliorate the inflammatory-mediator production of keratinocytes induced by cytosolic DNA. Overexpression of PRINS resulted in decreased interleukin-6 (IL-6) and chemokine (C–C motif) ligand 5 (CCL-5) expression and secretion.  The skin provides the first line of defense against a variety of environmental, chemical, and physical stimuli and acts as an active member of the innate immune system. Double-stranded DNA (dsDNA) fragments are known to induce antiviral responses in keratinocytes , 2 as weIn the last decade, genome-scale transcription studies have uncovered non-coding RNAs as previously unrecognized players in the dysregulation of inflammatory reaction in psoriatic skin , 10. TheAccording to our results, PRINS can ameliorate dsDNA-induced keratinocyte immune responses. Bioinformatic analysis of the PRINS lncRNA sequence revealed putative interaction sites for interleukin-6 (IL-6) and CCL-5 mRNAs. Destruction of the putative interacting region resulted in the loss of the ability of PRINS to bind to the IL-6 mRNA. In addition, our functional studies confirmed the regulatory role of the interaction between PRINS and IL-6 mRNA. Our results suggest a general anti-inflammatory function for PRINS and provide insight to the role of high PRINS expression in psoriatic non-lesional skin.Normal human epidermal keratinocytes used for the experiments were separated from skin specimens obtained from the Plastic Surgery Unit of our department. Written informed consent was obtained from all investigated individuals. The study was approved by the Human Investigation Review Board of the University of Szeged and complied with the ethical standards of research and in accordance with the Helsinki Declaration.2 cell-culture flasks and were maintained in keratinocyte serum-free medium, containing EGF and BPE  and supplemented with 1% antibiotic/antimycotic solution  and 1% l-glutamine (PAA Laboratories), at 37°C in a humidified atmosphere with 5% CO2. The medium was changed every 2 days.Epidermis was separated from the dermis by overnight incubation in Dispase , and keratinocytes were obtained after maceration in 0.25% trypsin. Cells were grown in 75 cmThird passage keratinocytes were seeded into 6-well plates. Cells were primed in supplement-free medium by addition of 5 ng/ml tumor necrosis factor-α (TNF-α) and 5 ng/ml interferon-γ (IFN-γ). After 24 h, cells were transfected with 0.666 µg/ml polydeoxyadenylic acid–polydeoxythymidylic acid double-stranded homopolymer  using the X-tremeGENE 9 DNA transfection reagent (Roche Diagnostics). Cells were harvested 12 h after poly(dA:dT) transfection.For overexpressing PRINS, the AK022045 cDNA sequence  was cloned into a pcDNA3.1(+) vector. The empty pcDNA3.1(+) vector served as a control.The ΔPRINS construct was created by replacing the AK022045 region (position 538–622) with the following scrambled sequence: GTGCGTGGCGGAGACGTGGTGGTAGACCGAATTGAGGAGGATCCGAAGGTTAGACGTAGGCGATCGCCGCTTCGGACGCGGTCGC. The ΔPRINS sequence was created by GeneArt gene synthesis (Thermo Scientific), and cloned into a pcDNA3.1(+) vector.Transient transfection of NHEK cells was carried out at approximately 70% confluency in parallel to cytokine priming, using the X-tremeGENE HP DNA transfection protocol, as described by the manufacturer (Roche Diagnostics). The transfection efficiency was 85% on average, as determined by the transfection of a GFP reporter construct  and analysis of GFP expression by flow cytometry. The effectiveness of overexpression was investigated with real-time RT-PCR .Sequence complementarity between PRINS AK022045) and the mRNA of IL-1α (M28983.1), IL-1β (NM_000576.2), IL-6 (NM_000600.4), IL-8 (NM_000584.3), TNF-α (NM_000594.3), and CCL-5 (NM_002985.2) was analyzed using two algorithms: RIsearch 45 and th–23, whic® Reagent , following the manufacturer’s instructions. Turbo DNA-free Kit  was used for the removal of contaminating DNA. cDNA was synthesized from 1 µg total RNA using the iScript cDNA Synthesis Kit . Real-time RT-PCR experiments were carried out with the Universal Probe Library system (Roche Diagnostics) using a C1000 Touch Thermal Cycler (Bio-Rad Laboratories) and the primers listed in Table S1 in Supplementary Material. The expression of each gene was normalized to the 18S rRNA gene. Relative mRNA levels were calculated by the ΔΔCt method.Total RNA was isolated from cells using TRIzolCell supernatants were centrifuged  to pellet cell debris, and the amount of cytokines IL-1α, IL-1β, IL-6, IL-8, CCL-5, and TNF-α was determined by ELISA , according to the manufacturer’s instructions.in vitro transcription from pcDNA3.1(+) containing the AK022045 or ΔPRINS cDNA sequence, using Transcript Aid T7 In Vitro Transcription Kit (Thermo Scientific). Products were purified by the GeneJET RNA Purification Kit (Thermo Scientific). Quality and sequence-length analyses were carried out on reducing agarose gel-electrophoresis. The single-stranded RNA products were used in a fluorescent binding assay. The fluorescently labeled RNA sequence 5′6-carboxyfluorescein(6-FAM)/GAAGCUCUAUCUCCCCUCCAGGAGCCCAGCUAUGAACUCCUUCUCCACAAGCGCCUUCGGUCCAGUUGCCUUCUCCCUGGGGCUGCUCCUGGUGUUGCCUGCUGCCUUCCCUGCC-3′, comprising positions 91–205 of the IL-6 (NM_000600.4) mRNA sequence, was produced by Integrated DNA Technologies.PRINS and ΔPRINS RNA sequences were produced by in vitro binding assay was carried out on a Monolith NT.115 Pico MicroScale Thermophoresis instrument , in nuclease-free water, at 25°C, with 80% Laser Power, 10% LED Power, by 2bind GmbH, Regensburg, Germany. Fluorescence enhancement of the 6-FAM labeled specific truncated IL-6 RNA sequence or a 6-FAM labeled unspecific DNA, as negative control, was measured after addition of PRINS or ΔPRINS. The concentration of fluorescently labeled molecules was 10 nM constantly, while unlabeled RNA concentration ranged from 126.75 nM to 61.9 pM. Initial fluorescence was analyzed for binding curves; the formulation used is displayed in Figure S2 in Supplementary Material.An t-test was used to compare two groups. Statistical analysis was carried out using R software, version 3.2.2., and the significance level was set at p ≤ 0.05.Experiments were carried out in triplicate with at least three biological repeats, as indicated in figure legends. For statistical analysis, one-way ANOVA was used to compare more than two groups, and one-tailed, paired To study how dsDNA influences NHEKs immune function, we analyzed the expression Figure  and secrWe confirmed that poly(dA:dT) significantly induced the expression and secretion of IL-1β in keratinocytes, as described previously . In addiTo gain pronounced inflammatory reaction in NHEKs, a priming step using TNF-α and IFN-γ is often applied before poly(dA:dT) treatment , 4; howeIn a manner similar to mRNA expression, poly(dA:dT) transfection induced the secretion of the investigated cytokines, and the priming step significantly enhanced the amount of secreted IL-1α, IL-1β, and IL-8 in the keratinocyte supernatant Figure .As we and others have demonstrated altered PRINS expression upon exposure to inflammatory molecules , 16, we The change in PRINS expression upon inflammatory stimuli suggests the possible contribution of PRINS to inflammatory responses of NHEK cells. To determine whether PRINS can regulate inflammatory cytokine expression, cells were transfected with a construct for overexpressing PRINS during the combined priming and poly(dA:dT) treatment. Expression and secretion of IL-6 and IL-8 was significantly decreased by PRINS overexpression Figure , whereasThe mRNA of the chemokine CCL-5 was previously predicted to interact with PRINS; however, it was not reported whether this interaction affects the stability of the CCL-5 mRNA . Therefoin silico analysis using INTARNA  were also included. The regions predicted by both algorithms were considered putative interacting sites. Putative interaction sites were not predicted for the cytokines not affected by PRINS overexpression  in this analysis. PRINS interaction regions were only predicted for the IL-6 and CCL-5 mRNAs  and the mRNAs of IL-6 (NM_00600.4) and IL-8 (NM_000584.3), we performed an 045 and t INTARNA  and RIse INTARNA , we alsoin vitro binding experiment was carried out using the PRINS lncRNA and the IL-6 mRNA. Binding affinity was determined using the single-stranded PRINS RNA and a fluorescently labeled, truncated IL-6 mRNA sequence containing the predicted interacting sequence. The ΔPRINS sequence, in which the predicted interaction site to IL-6 was replaced by scrambled sequence, was used as a control. As a negative control, a fluorescently labeled DNA sequence with no similarity to either PRINS or IL-6 RNAs was used. While PRINS exhibited a very high binding affinity to the labeled IL-6 mRNA .To validate the predicted interaction site, an A Figure B. This rin silico predicted and in vitro determined IL-6 mRNA interacting region in the PRINS sequence, we performed the overexpression experiments in NHEKs with vectors containing the wild-type PRINS or ΔPRINS (with scrambled IL-6 binding site) sequences, during the combined priming and poly(dA:dT) treatment. IL-6 expression was not affected by overexpression of ΔPRINS but was, in contrast, significantly decreased by PRINS overexpression , which was reported to induce inflammasome activation in these cells , 28 and in silico analysis, regions in the PRINS sequence were predicted to interact with CCL-5 and IL-6 mRNA. As inhibition of transcription by DNA:lncRNA triplex formation or posttranscriptional destabilization of the mRNA by mRNA–lncRNA duplex formation has been reported (in vitro: PRINS showed a very high binding affinity (Kd = 10.3436 nM) to IL-6 mRNA, and the destruction of the predicted binding site abolished the ability of PRINS to bind to the IL-6 mRNA. The binding site of PRINS lies within the 5′ UTR of the IL-6 mRNA, which is a rare phenomenon. The majority (~40%) of lncRNAs bind to the 3′ UTR of their target mRNAs, while only around 5% of lncRNAs is able to bind to the 5′ UTR of their target (PRINS was recently described to potentially interact with the CCL-5 mRNA , but itsreported , we analreported , making r target . The funSimilar to IL-6, PRINS overexpression decreased the expression of IL-8, but no interaction site could be predicted. We hypothesize that this is a secondary effect ; howeverMany lncRNAs which were reported to regulate mRNA expression were found to act as primary transcripts for miRNAs . We haveLike many other non-coding RNAs, PRINS seems to have multiple cellular functions . It bindOur studies were performed using a non-professional immune cell type, keratinocytes; however, the same mechanisms might be also relevant in professional immune cells. Further studies on the same PRINS-related cellular events upon additional stressors such as UVB or microbial components may widen our knowledge on the cellular functions of PRINS and, in general, about lncRNAs.JD and MS designed the study, JD and AG prepared laboratory work and analyzed the data. DJ, AG, ZB-C, LK, and MS interpreted the data and drafted the manuscript. All authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-glycosylation sites. Glycosylation of human CBGinfluences its steroid-binding activity, and there are N-glycosylationsites in the reactive center loops (RCLs) of human and rat CBGs. Proteolysis of the RCL ofhuman CBG causes a structural change that disrupts steroid binding. We now show thatmutations of conserved N-glycosylation sites at N238 in human CBG andN230 in rat CBG disrupt steroid binding. Inhibiting glycosylation by tunicamycin alsomarkedly reduced human and rat CBG steroid-binding activities. Deglycosylation of fullyglycosylated human CBG or human CBG with only one N-glycan at N238 withEndo H-reduced steroid-binding affinity, while PNGase F-mediated deglycosylation does not,indicating that steroid binding is preserved by deamidation of N238 when itsN-glycan is removed. When expressed inN-acetylglucosaminyltransferase-I-deficient Lec1 cells, human and ratCBGs, and a human CBG mutant with only one glycosylation site at N238, have higher(2–4 fold) steroid-binding affinities than when produced by sialylation-deficientLec2 cells or glycosylation-competent CHO-S cells. Thus, the presence and composition ofan N-glycan in this conserved position both appear to influence thesteroid binding of CBG. We also demonstrate that neutrophil elastase cleaves the RCL ofhuman CBG and reduces its steroid-binding capacity more efficiently than does chymotrypsinor the Pseudomonas aeruginosa protease LasB. Moreover, whileglycosylation of N347 in the RCL limits these activities, N-glycans atother sites also appear to protect CBG from neutrophil elastase or chymotrypsin.Corticosteroid-binding globulin (CBG) is a plasma carrier of glucocorticoids. Human andrat CBGs have six  N-glycansthat account for about 30% of its overall mass (N-glycosylation are in conserved positions.Corticosteroid-binding globulin (CBG) is a plasma glycoprotein produced by the liver. Itbinds glucocorticoids and progesterone preferentially and determines the amounts of thesesteroids that are non-protein bound and accessible to target cells  and proteases  clade A familymembers, human CBG (SERPINA6) does not inhibit proteases  and complementary pairs of mutagenicoligonucleotide primers (mutated nucleotide(s) in lower case), as indicated: human CBGN238D (5′-GATGAACTACGTGGGCgATGGGACTGTCTTCTTC); human CBG N238+N347 produced bymutating the human CBG N238 expression plasmid(5′-CTCCACTGGGGTCACCCTAaAcCTGACGTCCAAGCCTATCATC) and rat CBG N230D(5′-AGATGGACTATGTGGGAgATGGAACTGCCTTCTTCATTC). The mutated cDNAs were sequenced toensure only the targeted mutations had occurred and were expressed after stabletransfection of Chinese Hamster Ovary (CHO-S) cells (Gibco #11619-012) or CHO cell lines,Lec1 (ATCC CRL-1735) and Lec2 (ATCC CRL-1736), with defined defects  to generate unglycosylated CBG . To accocharides  to test 3H]cortisol or [3H]corticosterone as the labeled ligands for human and rat CBGs, respectively,and dextran-coated charcoal to separate bound from free [3H]-labeled steroids and transferred to polyvinylidene fluoride membranes. Westernblots were incubated with 1:5,000 dilutions of rabbit anti-human CBG antiserum  that specificallytarget the CBG RCL . The amoP value <0.05 wasconsidered significant.One-way ANOVA followed by Tukey’s or Dunnett’s multiple comparisons testsor two-way ANOVA followed by Bonferroni tests were performed as indicated using GraphPadPrism 5 software (GraphPad Software). A N-glycosylation sites in human and ratCBGs are compared, only one of the sites is not in a conserved position; N154 in human CBGand N194 in rat CBG  bound steroidappropriately in relation to its immunoreactivity on a Western blot , orlack the CMP-sialic acid Golgi transporter required for sialylation (Lec2 cells), weredetermined by Scatchard analysis after FPLC chromatographic purification (P < 0.05) and ~2-fold(P < 0.05) higher binding affinities for cortisol,respectively, when compared to their counterparts produced in CHO-S or Lec2 cells. Thesedata indicate that the composition of an N-glycan at N238 specificallyinfluences the steroid-binding activity of human CBG. Moreover, when rat CBG was examinedin this way, a similar ~2-fold (P < 0.05) increasein its affinity for corticosterone was observed when expressed in Lec1 cells vs CHO-S orLec2 cells  in steroid-binding capacity, its high steroid-binding affinity was retained  are positioned close to a conserved Arg  thatcontributes to the intra-molecular interactions required for the formation of a functionalsteroid-binding site  arerequired for high affinity steroid-binding activity  utilized  and rat(N230) CBGs are necessary for steroid binding, and this may explain why anN-glycosylation site is present in the same relative position of allmammalian CBG sequences. Importantly, our experiments provide insight into how quantitativeand qualitative differences in N-glycosylation influence CBGsteroid-binding activity and susceptibility to proteolysis. Our results also imply thatN-glycosylation acts globally to limit susceptibility to proteolysisand/or restrict cleavage to functionally relevant sites within the RCL. The possibility thatspecific N-glycans associated with CBG influence its function andrecognition by other proteins has implications in terms of the actions of CBG duringinfectious and inflammatory diseases or its detection by antibodies in immunoassays.In conclusion, The authors declare that there is no conflict of interest that could be perceived asprejudicing the impartiality of the research reported.This work was supported by an operating grant (MOP-111102) from the Canadian Institutesof Health Research (G L H), a Canada Research Chair in Reproductive Health (G L H), and apostdoctoral fellowship from the Fonds de Recherche du Québec en Santé andthe Michael Smith Foundation for Health Research (M S).Conceived experimental design and hypotheses: M S and G L H; performed experiments: M Sand C U; analyzed data: M S and C U; wrote the paper: M S and G L H.


Rhabdoviridae and the genus Ephemerovirus. BEFV causes an acute febrile infection in cattle and water buffalo. In this study, a recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of BEFV (rL-BEFV-G) was constructed, and its biological characteristics in vitro and in vivo, pathogenicity, and immune response in mice and cattle were evaluated. BEFV G enabled NDV to spread from cell to cell. rL-BEFV-G remained nonvirulent in poultry and mice compared with vector LaSota virus. rL-BEFV-G triggered a high titer of neutralizing antibodies against BEFV in mice and cattle. These results suggest that rL-BEFV-G might be a suitable candidate vaccine against BEF.Bovine ephemeral fever (BEF) is caused by the arthropod-borne bovine ephemeral fever virus (BEFV), which is a member of the family  Ephemerovirus of the family Rhabdoviridae [Rhabdoviridae includes members of the genera Lyssavirus , Vesiculovirus , and Ephemerovirus  and 10 other genera  [Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that belongs to the genus oviridae  and causoviridae . The famviruses) , 29. BEFviruses) . It is cviruses) . Althougviruses) .The BEFV G protein is the virion envelope glycoprotein, which serves as a protective antigen , 20, 44.Newcastle disease virus (NDV) has been used in vaccine vectors for research on the characteristics of oncolytic and foreign antigens , 42, 43.Baby hamster kidney BHK-21) and Madin–Darby bovine kidney (MDBK) cells were grown in Dulbecco’s modified Eagle’s medium containing 5 % fetal bovine serum. NDV LaSota as a vector virus was rescued from the genomic cDNA of the NDV LaSota vaccine strain (GenBank accession no. AY845400.2) with additional help from MVA-T7 as reported previously  and Madi. The rec50) per cell. The supernatant was harvested, and BEFV genomic RNA was extracted using a Total RNA Extraction Kit . The G gene was amplified by RT-PCR using the following primer pair: 5′-GACTGTTTAAACTTAAGAAAAAATACGGGTAGAAGTCTGGCCACCatgttcaaggtcctcataattacc-3′ and 5′-GACTGTTTAAACttaatgatcaaagaatctatc-3′, in which the gene end and gene start sequences of NDV (underlined), an optimal Kozak sequence , and PmeI restriction sites (bold) were introduced. The amplified BEFV G gene was sequenced and inserted into the LaSota genomic cDNA between the P and M genes. The resultant plasmid (designated as pLa-BEFV-G) was used for virus rescue as described previously [pBR322 containing NDV LaSota genomic cDNA has been described previously . The opeeviously . The resg. The supernatant was stored at −70 °C until used for western blotting. Western blotting was performed as described previously [2-peroxidase antibody . The primary NDV antibody was produced in a chicken.BHK-21 cells were infected with rLaSota or rL-BEFV-G at MOI 1. After 24 h, the total cellular proteins were extracted with lysis buffer  on ice for 5 min, and collected in 1.5-ml Eppendorf tubes, followed by centrifugation for 2 min at 15,000 × eviously , except For confocal assay, BHK-21 cells were plated on coverslips in 35-mm-diameter dishes and infected with rLaSota or rL-BEFV-G at an MOI of 0.01. The experimental procedure was performed as described previously , except 4 times the 50 % egg infective dose (EID50) in a volume of 100 μl. At 24, 48, 72 and 96 h, six chick embryos were randomly picked and allantoic fluid was used to measure the EID50. Monolayers of MDBK cells were infected with either rLaSota or rL-BEFV-G at an MOI of 0.01. After replacement of the medium with fresh medium, the infected cells were incubated at 37 °C in the absence or presence of TPCK trypsin (1 μg/ml). At 24, 48, 72, 96, and 120 h, the samples were collected. The virus was titrated on MDBK cells.To compare the growth kinetics in SPF chicken embryonated eggs, the rL-BEFV-G and parental strain rLaSota were inoculated into the allantoic cavity of 10-day-old embryonated chicken eggs at 107 TCID50 (30 or 100 μl). At 5 days after inoculation, tissues were collected and homogenized from five mice of each group. Viral titers in tissue were tested by indirect immunofluorescence assay (IFA) as described previously [The intracerebral pathogenicity index (ICPI), intravenous pathogenicity index (IVPI), and mean death time (MDT) in chicken embryos were determined using the method recommended by the Office International Des Epizooties (OIE). To assess the pathogenicity of recombinant viruses in mice, 4-week-old female mice (BALB/c)  were inoculated intramuscularly (n = 10) and intracerebrally (n = 10) with rL-BEFV-G at 10eviously  and RT-P7 TCID50). Ten mice in the rL-BEFV-G group were immunized with inactivated BEFV vaccine by intramuscular injection . An equal number of mice were inoculated intramuscularly with rLaSota . Ten mice were mock-infected with PBS (100 μl). Booster immunization was performed at 3 weeks after primary immunization. Blood samples were collected every week.Forty 4-week-old female BALB/c mice were divided randomly into four groups, and the groups were named rL-BEFV-G, rLaSota, inactivated BEFV vaccine , and phosphate-buffered saline (PBS). Ten mice in the rL-BEFV-G group were immunized with rL-BEFV-G by intramuscular injection  or 4 ml of inactivated BEFV vaccine. At 3 weeks after initial vaccination, the cattle received a second immunization at the same dose. Blood was collected 3 weeks after the first inoculation and 2 weeks after the second.Eight Holstein calves that were seronegative for BEFV were injected intramuscularly with 4 ml of allantoic fluid for rL-BEFV-G .Data on virus and antibody titers were analyzed by Student’s The BEFV G gene ORF was cloned between the P and M genes of the NDV genome at the PmeI site, using the NDV LaSota virus reverse genetic system established by Ge et al. –15 in whBHK-21 cells were infected with rLaSota or rL-BEFV-G at an MOI of 0.05. At different times post-inoculation, cells were fixed and stained with fluorescein. rLaSota was observed to infect individual cells, but the infection did not spread to adjacent cells. At 24 h, cell-to-cell spread was observed in cells infected with rL-BEFV-G  antibodies against BEFV was 1:6 in the rL-BEFV-G group and 1:16 in the inactivated BEFV group Fig. A. At 2 w7 TCID50 by intramuscular injection. The cattle in the inactivated BEFV vaccine group were immunized with commercial inactivated vaccine by the same route of administration. The SN antibodies in the rL-BEFV-G and inactivated BEFV groups were titrated after the first and second immmunizations. After the first dose, most cattle produced detectable SN antibody (Table Eight 1-year-old BEFV-seronegative Holstein calves were allotted randomly to the rL-BEFV-G and inactivated-BEFV-vaccine groups. The cattle in the rL-BEFV group were immunized with 4 ml of allantoic fluid with 2 × 10BEFV causes an acute febrile infection in cattle and water buffalo  and ofteViruses can spread by two fundamentally distinct modes, either by diffusion through the extracellular space or by direct cell–cell contact , 35, 45.rL-BEFV-G induced a good immune response in mice and cattle. The titers were 1:388 and 1:64–128, respectively. Other live-vector vaccines for BEFV have been reported. Vaccinia virus expressing BEFV G induced neutralizing antibody with a titer of ~1:100 after the second inoculation and provided protection against experimental BEFV infection in cattle . BEFV G As a result of the instability of BEFV, it was difficult to carefully regulate the challenge dose prior to the trial and successfully duplicate clinical symptoms. In this study, we did not perform a challenge test, but this needs to be done in the future.In conclusion, our results demonstrate that rL-BEFV-G is safe in mice and chickens. rL-BEFV-G induces high levels of neutralizing antibodies in mice and cattle, and thus probably confers good protection against BEFV challenge. rL-BEFV-G appears to be a promising candidate vaccine against BEFV.


Here, we show that another eIF4E family gene, nCBP, acts as a novel recessive resistance gene in Arabidopsis thaliana toward plant viruses in Alpha- and Betaflexiviridae. We found that infection by Plantago asiatica mosaic virus (PlAMV), a potexvirus, was delayed in ncbp mutants of A. thaliana. Virus replication efficiency did not differ between an ncbp mutant and a wild type plant in single cells, but viral cell-to-cell movement was significantly delayed in the ncbp mutant. Furthermore, the accumulation of triple-gene-block protein 2 (TGB2) and TGB3, the movement proteins of potexviruses, decreased in the ncbp mutant. Inoculation experiments with several viruses showed that the accumulation of viruses encoding TGBs in their genomes decreased in the ncbp mutant. These results indicate that nCBP is a novel member of the eIF4E family recessive resistance genes whose loss impairs viral cell-to-cell movement by inhibiting the efficient accumulation of TGB2 and TGB3.One of the important antiviral genetic strategies used in crop breeding is recessive resistance. Two  About half of the determinant loci from natural resistant crop cultivars are recessively inherited2Arabidopsis thaliana59in vitro and in vivo9The most common recessive resistance genes encode eukaryotic translation initiation factor 4E and eIF4G, components of eIF4FeIF4E or eIFiso4E genes in Capsicum spp., Solanum lycopersicum, and A. thaliana conferred full resistance against potyviruses4141617eIF4E in melon also resulted in an immunity type of resistance against a carmovirus, Melon necrotic spot virus (MNSV)19Barley yellow mosaic virus and Barley mild mosaic virusA. thaliana mutants of the eIF4E gene were also partially resistant to Cucumber mosaic virus 21eIF4E and eIFiso4E are known to be one of loss-of-susceptibility determinants to many plant viruses2121314Alpha- and Betaflexiviridae are groups of flexuous, filamentous viruses that predominantly infect plants, and encode an RNA-dependent RNA polymerase (RdRp), a 30K-type movement protein (MP) or triple-gene-block (TGB)-type MPs, and a coat protein (CP). Some viruses in Alpha- and Betaflexiviridae encode additional proteins in their genome. The most extensively studied of these plant viruses are members of the genus Potexvirus in the family Alphaflexiviridae, which have one genomic RNA with a cap and poly(A) tail23242629Potato virus X (PVX)26A. thaliana mutant of another member of the eIF4E family gene, nCBP, was resistant to a potexvirus, Plantago asiatica mosaic virus (PlAMV). Cell-to-cell movement of PlAMV was delayed in an ncbp mutant compared to the wild type, although viral accumulation in single cells of the mutant and the wild type did not differ. The accumulation of TGB2 and TGB3 was decreased in the ncbp mutant compared with the wild type.In this study, we found that an A. thaliana has three types of eIF4E isoforms, eIF4E, eIFiso4E, and nCBP. Two homologs of eIF4E, eIF4E1b and eIF4E1c, are also encoded by A. thalianaA. thaliana mutant lines bearing a T-DNA insertion or a point mutation in an eIF4E family gene were mechanically inoculated with PlAMV-GFP, a GFP-expressing variant of PlAMV. The inoculated leaves were examined for fluorescence from PlAMV-GFP at 4 days post inoculation (dpi). Infection foci on the inoculated leaves of eif4e, eif4e1b, eif4e1c, and eifiso4e mutants were nearly the same size as those on the A. thaliana ecotype Columbia-0 (Col-0) leaves , the GFP signal was observed in stems and upper leaves of Col-0, but no GFP signal was apparent in the upper leaves of the ncbp-1 mutant  were obtained and analyzed for their expression of nCBP. While we failed to detect the nCBP protein in the ncbp-1 mutant, three transgenic lines expressed it , and the amount of viral RNA was analyzed by RT-qPCR using the cotransfected GFP gene as an internal control. RT-qPCR analysis revealed that the accumulation level of viral RNA in the ncbp-1 mutant protoplasts was similar to that in Col-0 protoplasts  and a lolavirus (Lolium latent virus [LoLV]). Furthermore, a carlavirus (Potato virus M [PVM]) in Betaflexiviridae, a potyvirus (Turnip mosaic virus [TuMV]) in Potyviridae, and a tobamovirus (Youcai mosaic virus [YoMV]) in Virgaviridae were also investigated. The inoculated leaves were harvested at 4 dpi, and total RNA extracted from each leaf was analyzed using RT-qPCR with primers specific to each virus. Accumulation of AltMV, CymMV, LoLV, and PVM in the ncbp-1 mutant decreased drastically to 14%, 18%, 11%, and 42% of that in Col-0, respectively  can act as recessive resistance genes.We identified eIF4E family gene products, eIF4E or eIFiso4E, by plant viruses163134eIF4E family gene products during their infectioneIF4E family gene products may be correlated with the unique translation initiation strategies of plant viruses. In fact, a large number of plant viruses do not possess the cap and/or poly(A) structures in their genomic RNAcis-acting RNA elementsUmbravirus, and Luteovirus. Each virus recruits specific eIFs to the 3′ CITE to facilitate the translation of viral proteins419ncbp mutant plants. Our results showed that eIF4E family genes can serve as recessive resistance genes against viruses with cap and poly(A) structures.Recessive resistance exhibited by eIF4E deficiency is thought to be caused by the specific use of ncbp mutant , eif4e1c (SALK_053503C), ncbp-1 (SALK_131503C), and ncbp-2 (SALK_146604), were purchased from the Arabidopsis Biological Resource Center . The dSpm insertion line, eifiso4eA. thaliana was maintained in the growth chamber with 16-h-light/8-h-dark conditions at 22 °C throughout the assays.Seeds of 4e cum1-1, eif4e1bc SALK_0503C, ncbpEscherichia coli, using a pET30a vector, and the purified recombinant protein was used as an antigen. Anti-TGB1, CP and RdRp antibodies were prepared as described previously3243Anti-TGB2 and TGB3 antisera were raised in rabbits using purified peptides  as antigen . The nCBP protein was expressed in nCBP gene with putative promoter and terminator sequences was PCR-amplified with primers Sl-At5g18110-up1374F and Nt-At5g18110-down1011R using total DNA extracted from Col-0 as template  and Sol Genomics Network (http://solgenomics.net). Predicted cDNA sequences of AteIF4E1b and AteIF4E1c were obtained from The Arabidopsis Information Resource . Amino acid alignments of the core regionHomo sapiens eIF4E) were preformed using ClustalW software. Phylogenetic trees were constructed from nucleotide alignments of the core region using the neighbor-joining and boot-strapping algorithms within the Mega 6.0 software.Sequences of sGFP mRNA, and actin2F and actin2R for actin mRNA. For RT-PCR to detect PlAMV-GFP, we used Pr-det-F and Pr-det-R using cDNA described above as a template.Total RNA was extracted from inoculated leaves or transfected protoplasts using Sepasol-RNA I solution . Total RNA was subjected to DNase treatment  followed by reverse transcription using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). For RT-qPCR, viral RNA was amplified using the Thermal Cycler Dice Real Time System  with SYBR Premix Ex Taq II (TaKaRa). We used specific primers, PlRep-F3 and PlRep-R3 for PlAMV, AltMV_rt_2280F and AltMV_rt_2425R for AltMV, CymMV-realt6F and CymMV-realt6R for CymMV, LoLV_realt7F and LoLV_realt7R for LoLV, PVM-realt-9F and PVM-realt1R-9R for PVM, TuMV-rt1F and TuMV-rt1R for TuMV, YoMV_rt2F and YoMV_rt2R for YoMV, sGFP-379F and sGFP-486R for BamHI restriction enzyme and transcribed with T7 RNA polymerase. The intensities of RNA bands were quantitated using ImageJ software v1.40 .Total RNA (1 μg) was analyzed with the digoxigenin (DIG) system (Roche). To produce probes for plus- and minus-strand viral RNA detection, pCR-Pr-1 and pCR-Pr-2 were digested with Particle bombardment was performed using a Biolistic PDS 1000/He Particle Delivery System , as described previouslyArabidopsis protoplast preparation and transfection were performed as described previously6 protoplasts/mL).TM, EDTA-free Protease Inhibitor Cocktail (Roche) was denatured in gel sample buffer (50 mM Tris-HCl (pH 6.8), 2 w/v% SDS, 10% glycerol, 100 mM DTT). Preparation of S30 and P30 fractions was performed as described previouslyThe agroinfiltrated leaves and transfected protoplasts were harvested at 4 and 3 dpi, respectively. Total protein extracted using RIPA buffer (50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5 w/v% sodium deoxycholate, 0.1 w/v% SDS, 1.0 w/v% NP-40, 100 mM DTT) with cOmpleteHow to cite this article: Keima, T. et al. Deficiency of the eIF4E isoform nCBP limits the cell-to-cell movement of a plant virus encoding triple-gene-block proteins in Arabidopsis thaliana. Sci. Rep.7, 39678; doi: 10.1038/srep39678 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.