First principle calculations verifies that such unique microstructure characteristics and high-level heteroatom doping nature can enhance Li adsorption stability, electronic conductivity and Li diffusion mobility of carbon nanomaterials. Therefore, the H-2D-HCA could be promising candidates for next-generation LIB anodes.In this work, high-level heteroatom doped two-dimensional hierarchical carbon architectures (H-2D-HCA) are developed for highly efficient Li-ion storage applications. The achieved H-2D-HCA possesses a hierarchical 2D morphology consisting of tiny carbon nanosheets vertically grown on carbon nanoplates and containing a hierarchical porosity with multiscale pore size. More importantly, the H-2D-HCA shows abundant heteroatom functionality, with sulfur (S) doping of 0.9% and nitrogen (N) doping of as high as 15.5%, in which the electrochemically active N accounts for 84% of total N heteroatoms. In addition, the H-2D-HCA also has an expanded interlayer distance of 0.368 nm. When used as lithium-ion battery anodes, it shows excellent Li-ion storage performance. Even at a high current density of 5 A g Then the solution was heated 100°C and kept for 12 h under magnetic stirring. After that, the temperature was decreased to 94°C and kept for another 12 h without stirring. The produced white powder was collected with vacuum filtration and freeze dried, then Mg-Al LDH was obtained. As-prepared Mg-Al LDH was calcined at 500°C for 3 h in a muffle furnace and ground to get Mg-Al LDO. To adsorption OII, 150 ml water was filled into a three-mouth-flask. After driving the dissolved air with Ar gas flow, 0.6 g Orange II  was dispersed into the DI water. Then, 1 g Mg-Al LDO was added to the solution to adsorb the dye for 48 h. Magnetic stirring and Ar gas flow were maintained during the overall adsorption process. Afterwards, the solution was vacuum filtrated and washed with water for several times, then the obtained powder (RLDH/OII) was freeze dried and ground. The obtained RLDH/OII was mixed with 0.6 g melamine , and the mixture was dispersed into 50 mL methanol in a beaker. After that, the solution was heated to 50°C under magnetic stirring. The temperature was kept until the methanol was totally evaporated, and then the RLDH-OII-melamine mixture (RLDH-OII-M) was collected. The carbonization of RLDH-OII-M was performed at 800°C for 2 h under Ar atmosphere with a heating rate of 2°C/min. Then the obtained black powder was washed with HCl (6M) and NaOH (2M) respectively at 70°C for several hours. After freeze drying process, the target product H-2D-HCA was obtained.Mg-Al layered double hydroxides (Mg-Al LDH) and Mg-Al layered double oxides (Mg-Al LDO) were prepared following our previous work  were carbonized and washed with the above-mentioned conditions to get the reference sample C-OII and high-level doped C-OII (H-C-OII), respectively.2 F30). A Rigaku D/max 2500/PC diffractometer (Cu-Kα radiation with λ = 1.5418 Å) was used to measure the powder X-ray diffraction (XRD) patterns of samples. A HORIBA Labram HR Evolution Raman spectrometer was used to measure the Raman spectra of the samples (the excitation wavelength of the leaser is 532 nm). The XPS analyses were carried out on a PHI 5000 VersaProbe II spectrometer using monochromatic Al K X-ray source. The N2 adsorption/desorption isotherms were measured by using a Micromeritics ASAP 2020 automated adsorption apparatus at 77 K. The specific surface areas was determined based on Brunauer-Emmett-Teller (BET) equation, and the pore-size distribution was calculated by utilizing density functional theory (DFT).The morphology observation and elemental mapping analyzing were conducted by using a field electron microscopy . The microstructures of the four samples were examined by a field high resolution transmission electron microscopy  was used as the electrolyte. Lithium foil was used as both the counter electrode and reference electrodes. Battery assembling was carried out in an Ar-filled glove box with both moisture and oxygen concentrations below 1 ppm. Cyclic voltammetry was measured using an electrochemical workstation (CHI 660E) in the potential window of 0.005–3 V vs. Li/Li+ with a scanning rate is 0.2 mV s−1. The cycling performance and rate capabilities were tested at in the potential window of 0.005–3 V vs. Li/Li+ using a LAND battery tester (CT2001A).Li-ion storage performance of these samples was tested with half-battery methods by using coin-type cells (CR2032). To prepare the working electrodes, a slurry consisted a mixture of 70 wt % of H-2D-HCA (or other three carbon samples), 20 wt % of poly (vinylidene fluoride) (PVDF) and 10 wt % of acetylene black in N-methyl pyrrolidone was coated on a copper foil. After being dried at 80°C for 12 h, the electrode was punched into disks with a diameter of 12 mm. The mass loading of active materials was ~ 1.2 mg cmH-2D-HCA was synthesized based on a template-assistant method that we reported before . Interestingly, H-2D-HCA has an amorphous nature and graphitic micro-crystallites with an average interlayer distance of 0.368 nm Figure , which iFigure X group (167.7 eV)  was utilized to evaluate the surface chemistry in H-2D-HCA. N and S heteroatom concentrations can be determined to be 15.5 and 0.9%, respectively  Figure . As demo) Figure . The ext) Figure . Obvious) Figure .2 adsorption/desorption isotherms of H-2D-HCA exhibit a combination of type I and type II characteristics, with a distinct hysteresis lop at relative pressure P/P0 ranging from 0.42 to 1  and partially disordered structures of the sp2 domains framework . After that, with the current density returns to 2,000, 1,000, 500, and 200 mA g−1, the discharge capacity recovers to the initial capacity values with only little fade.Li-ion storage properties of H-2D-HCA were evaluated using half-battery test methods. Cyclic voltammetry (CV) curves suggest that H-2D-HCA has similar Li-ion storage behaviors with other carbon nanomaterials. As shown in Figure −1 for two cycles, and then the current density was directly increased to 5 A g−1. The voltage profiles of these samples in the first two cycles are shown in Supplementary Figure −1, together with an initial columbic efficiency (ICE) of 56.4% , N-6 doped carbon (N-6/C), N-5 doped carbon (N-5/C), N-Q doped carbon (N-Q/C), and N, S co-doped carbon (N/S/C). For comparison, Eabs of above-mentioned carbon systems with an interlayer distance of 0.34 nm were also calculated. In the 0.368 nm spacing case, Eabs for the P/C, N-5/C, N-6/C, N-Q/C and N/S/C are −0.08, −2.74, −3.28, 0.58, and −1.68 eV, respectively  at the Fermi level. We calculated the DOS of carbon systems of pure one and N, S co-doped one  for highly efficient Li-ion storage. The hierarchical structure can alleviate the electrochemical active surface loss, and the porous structure provided rapid diffusion channels for Li ions. More importantly, the highly concentrated heteroatoms of 0.9% sulfur and 15.5% nitrogen are able to created abundant electrochemical active sties to storage Li ions. Also the increased N doping concentration improved the ICE of carbon nanomaterials. Furthermore, the expanded interlayer distance can promote insertion-extraction speed of Li ions. First principle calculations confirmed the enhanced Li absorption stability as well as the electronic conductivity by co-doping of N and S heteroatoms and the accumulated diffusion mobility of Li ions owing to expanded interlayer space. Benefiting from these unique microstructure characteristics and high-level heteroatom doping nature, H-2D-HCA exhibited enhanced Li-ion storage performance. Even at a high current density of 5 A gZW and YW did the materials preparation and characterization, as well as electrochemical tests. WW did the first principle calculations. BX and Y-BH supervised this work. All the authors discussed the results and wrote the manuscript.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.


CAPS binds phosphatidylinositol 4,5-bisphosphate P2) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming. We characterized purified soluble CAPS as mainly monomer in equilibrium with small amounts of dimer. However, the active form of CAPS bound to PC12 cell membranes or to liposomes containing PIP2 and Q-SNARE proteins was mainly dimer. CAPS dimer formation required its C2 domain based on mutation or deletion studies. Moreover, C2 domain mutations or deletions resulted in a loss of CAPS function in regulated vesicle exocytosis, indicating that dimerization is essential for CAPS function. Comparison of the CAPS C2 domain to a structurally defined Munc13-1 C2A domain dimer revealed conserved residues involved in CAPS dimerization. We conclude that CAPS functions as a C2 domain-mediated dimer in regulated vesicle exocytosis. The unique tandem C2-PH domain of CAPS may serve as a PIP2-triggered switch for dimerization. CAPS dimerization may be coupled to oligomeric SNARE complex assembly for vesicle docking and priming.Neurotransmitters and peptide hormones are secreted by regulated vesicle exocytosis. CAPS  is a 145-kDa cytosolic and peripheral membrane protein required for vesicle docking and priming steps that precede Ca CAPS2+-triggered dense core vesicle exocytosis in permeable neuroendocrine cells (–A) . B. B2+ elells (–A) –18. Muncls (–A) –, 19, 21.Caenorhabditis elegans indicated by the uncoordinated phenotype of unc-31(e714) and unc-31(ox299) strains that harbor point mutations in the C2 domain of the conserved worm CAPS ortholog UNC-31p P2- and SNARE-dependent liposome fusion (23–A) and could exhibit mutual regulation as in other tandem domain proteins . Treatment of the protein with SDS, which was effective at disassembling oligomeric molecular mass standards, collapsed the 700-kDa but not the 350-kDa form of the protein . This suggested that the 350 kDa band corresponds to a monomer, whereas the 700-kDa band is an oligomer. Consistent with this, zero length cross-linking partially converted the CAPS-Myc-His monomer to an oligomer that resisted SDS-induced disassembly . The minor amounts (∼6%) of oligomer in CAPS-Myc-His (n = 27) did not differ significantly from that detected in native cytosolic CAPS (n = 11), indicating that epitope tags were not responsible for oligomer formation (e.g. see C). The molecular mass of CAPS-Myc-His on SDS-PAGE is ∼145 kDa, which indicates that the 350-kDa CAPS-Myc-His form in blue native gels migrates as a non-globular protein with an oblate shape. To assess its native molecular mass independent of shape, purified CAPS-Myc-His was analyzed by analytical equilibrium ultracentrifugation, which showed that the major species of soluble CAPS-Myc-His is a ∼150-kDa monomer . Minor amounts of a higher molecular weight oligomer(s) were not well resolved by this method.We reported that native CAPS is a dimer, but this analysis was conducted with partially purified protein using indirect hydrodynamic criteria . HoweverFig. 1e.g. see C. The moSR) of ∼59 Å similar to a 350-kDa globular protein (A). This is consistent with a moderately elongated 145-kDa monomer with an SR/Rmin value of ∼1.7 . Long exposure of the Western blot revealed oligomer in the 0.06–0.12 Kav fractions (B), but analysis of these fractions 1 day later showed substantial conversion to monomer (C). We conclude that soluble CAPS-Myc-His is in dynamic monomer-oligomer equilibrium favoring monomer.Gel filtration studies indicated that the dominant form of CAPS-Myc-His migrates with a Stokes radius  or on the SLB lacking PIP2  was monomer. However, the inclusion of PIP2 in SLBs strikingly enhanced the amount of CAPS-Myc-His present as a dimer . The stimulatory effect of PIP2 on CAPS-Myc-His oligomerization was confirmed in native gel electrophoresis studies showing that CAPS-Myc-His oligomerized upon incubation with PIP2 micelles (C). PIP2 was the most effective phosphoinositide for inducing oligomerization (D). The results indicate that CAPS-Myc-His exists in two states and that PIP2 binding promotes the formation of oligomers that correspond to dimers.To further assess the oligomerization state of CAPS, we employed atomic force microscopy (AFM) and single-molecule microscopy. AFM measures protein volume and can be used to determine oligomer stoichiometry . The vol on mica A or on tIP2 B, top wa a dimer B, bottommicelles C. PI, and photobleaching steps were monitored by total internal reflectance fluorescence (TIRF) microscopy (F and G). As controls, monomeric mKate2 was shown to exhibit one-step bleaching, whereas dimeric mKate2 exhibited an increased number of two-step bleaches . CAPS-mKate2-His mainly exhibited 1-step bleaching, but ∼5% of the molecules exhibited two-step bleaching, indicating that a minor population of dimers is present . The inclusion of PIP2 markedly enhanced dimerization of the CAPS-mKate2-His protein . To exclude the role of the His tag, we prepared a CAPS-mKate2 protein, which exhibited similar PIP2-enhanced dimerization . Collectively, the AFM and single molecule studies indicate that CAPS oligomers correspond to dimers and that PIP2 enhances CAPS dimerization.Single molecule microscopy can be used to determine oligomeric state by monitoring the number of photobleaching steps for fluorescently tagged proteins . CAPS-mKFig. 3A (i)). Extensively washed permeable cells retain small amounts of bound native CAPS  or by direct electrophoresis into native gels (not shown). In contrast to native cytosolic CAPS, which was ∼5% dimer, the extracted membrane-bound CAPS was entirely dimer as analyzed on native gels . Similar studies were conducted by adding purified CAPS-Myc-His to the permeable cells ). The addition of purified CAPS to permeable PC12 cells stimulates Ca2+-dependent dense core vesicle exocytosis ). CAPS-Myc-His bound to the permeable cells was exclusively dimer  and exhibited ∼50% of the activity  of added CAPS-Myc-His  in promoting Ca2+-dependent secretion. Control studies showed that the activity of bound CAPS-Myc-His was inhibited by neutralizing CAPS antibodies but not by control IgGs . Strikingly, the bound CAPS-Myc-His dimer represented only ∼1% of the input CAPS-Myc-His  but accounted for 40–60% of the detected activity . The results indicate that dimer is the active membrane-bound form of CAPS associated with Ca2+-triggered exocytosis.To determine the functional significance of the oligomeric states of CAPS, we assessed whether there was a preferential association of CAPS monomer or dimer with PC12 cell membranes. CAPS is a soluble cytosolic protein that partitions onto membranes , 25, 36.Fig. 4ocytosis . We test2 is required for CAPS activity in exocytosis P2-containing liposomes purified by flotation (A) exhibited increased dimer compared with input (B and C). CAPS-Myc-His dimers were not evident in flotation studies with PC/PS-containing liposomes (B and C) or with liposomes containing only PIP2 (C). Although these results appeared to differ from the AFM studies with PIP2-containing SLBs (B), we attribute this to the dissociation of CAPS-Myc-His during flotation when bound at low affinity to PIP2-containing liposomes. The recovery of CAPS-Myc-His from liposomes containing only Q-SNAREs was also low (C) but corresponds to the synergy between PIP2 and Q-SNAREs for CAPS activity on liposomes Pocytosis , 19, so lotation A exhibitth input , B and Ciposomes , B and CIP2 C. Althouing SLBs B, we attalso low C but coriposomes . CollectA) corresponding to the unc-31(e714) and unc-31(ox299) alleles in C. elegans, respectively (B and C), and in the permeable PC12 cell assay to determine activity (E). The alleles at Leu-468 (L468E and L468K) had significantly reduced dimer and exhibited loss of function in the permeable cell assay . The deletion construct CAPS(ΔC2) also exhibited reduced dimerization as well as reduced activity . We also tested the CAPS(ΔC2) protein with PIP2 and found that PIP2-induced dimerization was similarly attenuated (D).Additional evidence for the importance of dimerization in CAPS activity and for the role of the C2 domain in dimer formation was provided by studies of CAPS C2 domain mutants. Mutant proteins assessed were rat CAPS(G476E) and CAPS(L468K) plus variants A correspectively . We alsoectively , B and Cactivity E. The altenuated D.B and C) but was strongly loss of function in the permeable cell assay (E). By contrast, a CAPS(G476A) variant was similar to wild-type protein (data not shown). Collectively, the data indicate that the C2 domain of CAPS, previously of unknown function, is involved in CAPS dimer formation and that altered dimerization inhibits CAPS function.Surprisingly, the CAPS(G476E) mutant exhibited strongly increased oligomerization , B and Cll assay E. By conA) were depolarized to promote Ca2+ influx. Vesicle fusion events were detected by the brightening of BDNF-EGFP in TIRF microscopy . The knockdown of CAPS in PC12 cells with shRNA . Full rescue was achieved by expressing a CAPS-TagRFP construct harboring silent mutations that bypass the shRNA . By contrast, CAPS(G476E), CAPS(ΔC2), or CAPS(L468K) proteins all failed to rescue Ca2+-stimulated exocytosis (D–F) despite normal expression  was solely attributable to the reduced number of vesicles in the TIRF field, the data were replotted as the number of fusion events normalized to the number of vesicles in the TIRF field. CAPS(G476E) was confirmed as strong loss of function for exocytosis as were the CAPS(L468K) and ΔC2 CAPS mutants . The live cell studies agree with the permeable cell activity assay results (E) and further indicate the essential nature of the C2 domain for CAPS function in vesicle exocytosis.We assessed the activity of the C2 domain mutants in regulated dense core vesicle exocytosis in live PC12 cells . Cells eFig. 7 in CAPS ) stronglpression . Intrigu results E and furD and G) but also interfered with the transit of vesicles to the plasma membrane. To further assess this, we determined the localization of CAPS(G476E) in cells by confocal microscopy. Expression of wild-type CAPS-EGFP or CAPS-TagRFP resulted in a global cytoplasmic distribution that largely obscured the dense core vesicle localization previously shown by TIRF microscopy (A and B). CAPS(L468K) and CAPS(ΔC2) proteins were expressed well and localized similarly to the wild-type protein as cytoplasmic. In striking contrast, CAPS(G476E) localized to large structures within the cells (A and C) that also contained dense core vesicle proteins synaptotagmin-1 and chromogranin B but not Golgi proteins TGN-38 or syntaxin-6 . This suggested that CAPS(G476E) expression induced the cytoplasmic clustering of mature dense core vesicles. We confirmed this by electron microscopy, which revealed cytoplasmic clusters of intact dense core vesicles in cells expressing CAPS(G476E) .CAPS in PC12 cells is cytoplasmic as well as bound to dense core vesicles . CAPSG4, D and Gcroscopy  Fig. 8,,D and G B) coupled with its binding to dense core vesicles (F). CAPS binding to dense core vesicles requires a C-terminal domain (A) (E). The results indicate that the G476E mutation enhances CAPS self-interactions even in trans across vesicle membranes. Such results are consistent with a role for the C2 domain in CAPS dimerization (F).Vesicle clustering by CAPS(G476E) may be due to its increased oligomerization B coupledvesicles F. CAPS bl domain A 3, 25), 25B) comain (A) E. The rerization F.Dimerization may be mediated by homotypic interactions between C2 domains. Structures of C2 domain homodimers have been reported for C2B of RIM1α, C2B of Rgp3, and C2A of Munc13-1 , 38. AnaA). Specifically, two types of interactions are conserved. Both C2 domains exhibit a characteristic partial chelation of a charged residue by three residues on the opposing domain and exhibit a lysine-glutamate electrostatic interaction (A). The positions of these latter residues are flipped between CAPS C2 and Munc13-1 C2A, which could function to prevent CAPS-Munc13-1 heterodimer formation. To test the sequence alignment predictions, we generated two charge reversal mutants in the CAPS C2 domain that are predicted to reduce dimerization. In accord with prediction, CAPS(K428D) and CAPS(D472K) exhibited impaired dimerization (B and C). Importantly, both CAPS C2 domain mutants showed reduced activity in the permeable PC12 cell assay (D). We suggest that the mechanism of C2 domain homodimerization is conserved between the CAPS C2 domain and the Munc13-1 C2A domain.Alignments revealed that residues involved in Munc13-1 C2A homodimerization are conserved in the CAPS C2 domain A. Speciferaction A. The porization , B and Cll assay D. We sugunc-31(e714) and unc-31(ox299) loss of function alleles in C. elegans that correspond to rat CAPS(G476E) and CAPS(L468K), respectively  allele has unusual properties that nonetheless support the idea of C2 domain-mediated oligomerization. CAPS(G476E) is strongly loss of function at the level of vesicle exocytosis (trans across membranes (F). Indeed, CAPS(G476E) exhibits a very strong tendency to oligomerize as dimers as well as possible tetramers (B). We cannot eliminate the possibility that the C2 domain in this mutant is locally misfolded by substitution of a small uncharged side chain by a charged side chain. The G476A allele, by contrast, exhibited wild-type properties. Gly-476 is highly conserved in CAPS and in other C2 domains (A), residing near the beginning of a flexible linker that connects two β strands. Whether the Gly to Glu substitution promotes local unfolding of the C2 domain or introduces additional electrostatic interactions is unclear, but the G476E substitution appears to strongly enhance C2-C2 domain interactions.The CAPSG476E) mutant corresponding to the ocytosis  and 7, a6E mutantocytosis . We inteembranes F. Indeedetramers B. We can domains A, residie.g. CAPS(ΔC2) and CAPS(L468K)) as well as increased (e.g. CAPS(G476E)) dimerization result in loss of CAPS function suggests that a cycle of dimerization and its reversal plays a role in CAPS function. CAPS dimerization may need to be locally activated with correct timing such that premature dimerization inhibits activity. Consistent with this, complete dephosphorylation inactivates CAPS and shifts it toward an oligomeric state . The PH domain is immediately adjacent to the C2 domain so that binding of PIP2 in the membrane could drive C2 domain-mediated dimerization. PH domains can also dimerize P2 binding and dimerization in CAPS function.The binding of PIPormation . Binding of CAPS  may indu of CAPS A. The PHdimerize , 55, whiiposomes . Thus, tRattus norvegicus) as the template to introduce L468A, L468E, L468K, G476A, G476E, K428D, or D472K single point mutations. For the ΔC2-CAPS, overlap extension PCR was used to delete amino acids 400–516 from the CAPS ORF. This allowed residue 399 to be directly fused to residue 517 without introduction of a restriction site. Primers used were f1 , r1 (AGAGTGCTTCATGTTTTGCTCCAAGGAGAAGGACAG), and r2 (CCTGAAATGTGTAATTTGATTTTCCAGCAGAACTCGG). CAPS-mKATE2-His constructs were created by cutting CAPS-Myc-His from pcDNA3.1 with XhoI and AfeI and ligating into a similarly cut mKate2-C  appended with a C-terminal His purification tag. All constructs were confirmed by Sanger sequencing.A site-directed mutagenesis kit  was used with pcDNA3.1 CAPS-Myc-His  with protease inhibitors and pelleted to remove cell debris. Supernatants were loaded onto a nickel-nitrilotriacetic acid column (Qiagen), washed, and eluted with 20 mm HEPES, 300 mm NaCl, 200 mm IDA. Eluates were diluted with 20 mm HEPES to 20 mm NaCl, 100 mm NaCl and concentrated to ∼10 μm CAPS.For purification of CAPS-Myc-His and CAPS-mKate2-His, CAPS was transfected into HEK293-FT (Invitrogen) cells using a standard calcium phosphate protocol. Two days after transfection, cells were washed and removed with PBS. Cells were resuspended in 0.5% Triton X-100, 20 mm HEPES, 10 mm NaCl, 10 mm imidazole, 5% glycerol, pH 7.4, were loaded onto a 6% polyacrylamide gel without SDS or DTT. Gels were run at 150 V for 2 h at 4 ºC. For immunodetection, native gels were subjected to a standard blotting protocol. Blue native PAGE was performed as described previously  and directly loaded into the gel. Gels were run at 150 V through the stack and then turned up to 250 V for 2 h. Gels were fixed in 40% methanol, 30% acetic acid, 2.5% glycerol followed by Coomassie G250 staining or subjected to immunoblotting. Western blotting was conducted with polyclonal antibodies generated against purified full-length rat CAPS or with Myc monoclonal 9E11 .For clear native PAGE, purified CAPS-Myc-His and mutants in 20 meviously . Brieflym) were analyzed. ∼100 μl of each sample was loaded into sectors of 1.2-cm charcoal-filled epon centerpieces with ∼110 μl of the final dialysate buffer in the reference sector. Data were collected at 4 °C with the gradients monitored at 279 nm. From the CAPS sequence, the partial specific volume was 0.733 ml/g and is uncorrected for temperature. The buffer composition was 100 mm NaCl, 1 mm EDTA in HEPES buffer at pH 7.4. The density at 4 °C was 1.0044 g/ml, as calculated from density increments (Laue diffraction) and corrected to 4 . CAPS molecular mass was computed as 150,613. An extinction coefficient of 138,660/m/cm was used based on Pace's average extinction (m. For data shown (D), the three lines for each color represent different rotor speeds for the high (green), medium (blue), and low (red) loading concentration.Three different CAPS concentrations , 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), and 1,2-dioleoyl-sn-glycero-3-phospho- P2), obtained from Avanti Polar Lipids  as chloroform stocks, were mixed at molar ratios of 75% phosphatidylcholine, 25% phosphatidylserine or 75% phosphatidylcholine, 22% phosphatidylserine, 3% PIP2. Chloroform was evaporated under a stream of nitrogen, and the lipids were rehydrated overnight in water (MilliQ) at a lipid concentration of 2 mg/ml. The lipid suspension was incubated at 65 °C for 30 min and then cooled to room temperature. CAPS, NaCl, HEPES-NaOH, and CaCl2 were added to produce a liposome suspension containing 10 nm CAPS in HEPES-buffered saline  and 1 mm CaCl2. The suspension was incubated at 37 °C for 30 min and then deposited onto freshly cleaved mica disks . After a 3-min adsorption, the sample was rinsed with HBS containing CaCl2 to remove unadsorbed liposomes and transferred to the atomic force microscope. AFM imaging was carried out at room temperature using a Bruker Multimode atomic force microscope equipped with an E-scanner and a Nanoscope IIIa controller, with an in-line electronics extender module . All images were collected using tapping mode in fluid with Micromasch AFM cantilevers NSC18/AL BS MikroMasch® . The cantilevers  were tuned to 10–20% below the peak of the resonance frequency, generally between 25 and 35 kHz. The drive amplitude was set to generate a root mean square amplitude of 2 V. The microscope was engaged with a 0-nm scan area to allow for tuning. The set point was applied to the sample. Images (2 × 2 μm) were captured at a scan rate of 2 Hz and with 512 scan lines/area. Data analysis was performed using commercially available software  or with ImageJ. Multiple images were taken from one supported lipid bilayer for each condition.CAPS was imaged by adding 100 p2-His were immobilized on glass by SiMPull  to remove dust and a BSA/antibody solution (FC protein (10 mg/ml), monoclonal His tag antibody (Penta-His (Qiagen)) concentration of 0.1 mg/ml), incubated for 10 min, and flushed with 5 chamber volumes of buffer, and 50 pm CAPS-mKate2-His was added to the chamber; protein was diluted in prelubricated 1.7-ml Eppendorf tubes to minimize nonspecific absorption to the plastic surface . Alternatively, CAPS-mKate2-His was immobilized on 5% PIP2-containing SLBs. CAPS-mKate2 lacking the His tag was prepared by cleavage with tobacco etch virus protease  and similarly bound to SLBs. Unbound protein was removed with 5 chamber volumes of buffer, and the bound protein was imaged by TIRF microscopy on a TE2000 inverted scope (Nikon) stationed on a floating table. A 514-nm laser line excited the fluorophore and was imaged with an Evolve digital monochrome EMCCD camera . 500-frame movies with a capture rate of 2 Hz were recorded. Movies were drift-corrected using the ImageJ plugin Descriptor-based registration (2d/3d +t); movies with significant drift were discarded. Movies were processed via a Matlab script generously provided by Dr. Aaron Hoskins . Photobleaching steps were manually recorded if a spot maintained uniform fluorescence before photobleaching, did not display x,y-drift, and underwent photobleaching in one frame.Single molecules of CAPS-mKate2-His, mKate2-His, or the dimeric (mKate2) SiMPull . BrieflyGel filtration of purified CAPS-Myc-His was conducted on a Sephacryl-300  prepacked column via FPLC. The column was calibrated by monitoring the elutions of thyroglobulin (670 kDa), ferritin (440 kDa), and aldolase (158 kDa). Purified CAPS-Myc-His was loaded onto the column, and 72 1.2-ml fractions were collected and analyzed by clear native gels and immunoblotting.m potassium glutamate, 20 mm potassium acetate, 20 mm HEPES, 2 mm EGTA, 1 mg/ml BSA, pH 7.2), and passed once through a ball homogenizer (10-μm clearance). 100 mm EGTA was added to a final concentration of 10 mm, and permeable cells were incubated on ice for 30–60 min and washed with KGlu/BSA to remove cytosolic proteins. Cell ghosts were divided into Eppendorf tubes, resuspended in 20 nm CAPS-Myc-His in priming solution (Mg-ATP/rat brain cytosol) for 12 min at 30 °C, and washed two times in 1 ml of KGlu/BSA. Bound CAPS was either extracted with 300 mm NaCl  or loaded directly onto native gels. 50% glycerol was added to a final concentration of 5% before loading onto native gels.PC12 cells were washed twice with PBS, removed with KGlu/BSA buffer P2. After resuspension, mixtures were rapidly diluted into 25 mm HEPES, 100 mm KCl, pH 7.4; dialyzed overnight into 25 mm HEPES, 100 mm KCl, 1 mm DTT, pH 7.4, with 1 g/liter Biobeads; and purified on an Accudenz medium  gradient. Liposomes were made identically with the omission of the SNARE proteins. The addition of 3H-labeled 1,2-dipalmitoyl phosphatidylcholine to liposomes was used to monitor lipid recovery, and SDS-PAGE densitometry of the Q-SNAREs was used to assess protein content. 1 μm CAPS-Myc-His was incubated with Q-SNARE proteoliposomes for 30 min at room temperature. Reactions were mixed 1:1 with 80% Accudenz and pipetted into a thin walled polycarbonate tube, overlaid with 30% Accudenz and a 20 mm HEPES, 100 mm KCl, pH 7.4, layer for centrifugation . Floated fractions were withdrawn and analyzed by either blue native or SDS-PAGE. For blue native analysis where indicated, Triton X-100 was added to a final concentration of 0.5% for several minutes followed by 2× blue native PAGE buffer  for 5 min. When quantification was required, floated fractions were monitored by the total amount of 3H-labeled 1,2-dipalmitoyl phosphatidylcholine.Proteoliposomes were prepared by co-micellization as described previously . Brieflym norepinephrine, 0.5 mm ascorbate. Cell ghosts were prepared as described above, washed in KGlu/BSA, and incubated in priming solution (rat brain cytosol at 1:10 dilution and 2 mm Mg-ATP) for 12 min at 30 °C, which generates PIP2 on the plasma membrane, washed, and resuspended in KGlu/BSA. Cell ghosts were added directly to the rotating disc electrode chamber with 10 nm CAPS, and Ca2+ was injected. A rotating carbon disc electrode with an applied potential of +500 MV detected norepinephrine release. The output was processed by a Dataq analog to digital data acquisition system and analyzed by WinDaq software.Rotating disc electrode voltammetry was used to detect norepinephrine secretion from PC12 cell ghosts as described previously  using PC2+-dependent exocytosis of BDNF-GFP-containing vesicles from live PC12 cells was monitored by TIRF microscopy as described previously  or an Evolve digital monochrome EMCCD camera (Photometrics) and analyzed by ImageJ software. Exocytic events were scored manually.The Caeviously . Brieflym sodium phosphate buffer (PB), pH 7.4, for ∼1 h at room temperature. Samples were rinsed 5 times for 5 min in 0.1 m PB. The rinsed cultures were post-fixed in 1% osmium tetroxide, 1% potassium ferrocyanide in PB for 1 h at room temperature. The samples were rinsed in PB followed by distilled water rinses three times for 5 min each to clear phosphates. Samples were en bloc stained in saturated aqueous uranyl acetate for 2 h at room temperature and rinsed in distilled water three times for 5 min each. Dehydration was performed at room temperature in a graded ethanol series  and transitioned in propylene oxide two times for 7 min each. Durcupan ACM  resin was used during infiltration and embedding. Increasing concentrations of accelerated  Durcupan were used for infiltration. All infiltration steps were done with the coverslips, cell side up, in aluminum weighing dishes in covered glass Petri dishes to minimize evaporation. The cultures were embedded in open aluminum weighing dishes at 60 °C in a drying oven overnight until polymerized. Samples were floated in concentrated hydrofluoric acid, glass side down, for 15 min to etch off the glass, revealing the embedded cells. Sections were viewed with a Philips CM120 transmission electron microscope, and images were documented with a MegaView III  side-mounted digital camera.PC12 cells were transfected with an Electroporator II instrument (Invitrogen) and plated on glass coverslips, and 48 h later, cells were fixed with 4% formaldehyde plus PBS for 20 min at room temperature. For immunostaining, fixed cells were extracted in 0.1% Triton X-100. Cells were washed with PBS and blocked for 30 min in 10% BSA plus PBS. Fixed cells were incubated with primary antibody for 1 h, washed, and incubated with secondary antibody for 1 h. After washes in PBS, cells were mounted and imaged on a Nikon C1 confocal microscope (TE2000-U). Primary antibodies used were chromogranin B monoclonal generously provided by W. B. Huttner , TGN38 monoclonal generously provided by K. E. Howell , syntaxin 6 monoclonal antibody , and synaptotagmin-1 N-terminal polyclonal antibody (Sigma-Aldrich). For electron microscopy, PC12 cells were transfected with pCMV CAPS-GFP or CAPS(G476E)-GFP for 48 h, sorted by FACS on a SORP BD FACSAria (University of Wisconsin Carbone Cancer Center Flow Cytometry Laboratory) to remove untransfected cells, and fixed. Cells on glass coverslips were fixed in a solution of 2.5% glutaraldehyde, 2.0% paraformaldehyde in 0.1 We used the C2 domain conserved protein domain family (CD00030) as a seed for C2 domain alignments. It is produced by an automated algorithm, which employs generic search parameters that invariably contain omissions and misalignments that require refinement. Only 32 of 97 C2 domain structures represented in the C2 domain PFAM (PF00168) were included . The missing structures were imported into Cn3D and aligned with the provided block alignment algorithm. The sequence alignment of every structure was manually refined by assessing the resulting spatial overlap between the C2 domain in question with all other C2 structures. After assessing every β strand for all structures, we were able to create a sequence alignment for all C2 domain structures that is based on a high degree of spatial overlap between all C2 domain residues. The aligned residues represent the core C2 domain residues that have an invariant spatial positioning between these 97 crystal structures. Using these core C2 residues as a search query, DALI identified all other C2 domain structures in the C2 PFAM, yielding an average root mean square deviation of 1 Å that accounts for 100 of the residues tested for spatial overlap. It is common to observe backbone atoms of two aligned C2 domains in these conserved regions with distances between 0.4 and 0.6 Å, less than that of an H–H bond.M. P., J. E., and T. F. J. M. designed the study and wrote the paper. M. P. conducted most of the experimental work; J. E. conducted single molecule, bioinformatic, and mutagenesis studies; G. K. conducted cellular studies on CAPS mutants; and S. M. conducted permeable cell secretion assays. H. T. and J. M. E. designed and conducted the AFM studies. All authors reviewed the results and approved the final version of the manuscript.


Human enterovirus B106 (EV-B106) is a new member of the enterovirus B species. To date, only three nucleotide sequences of EV-B106 have been published, and only one full-length genome sequence (the Yunnan strain 148/YN/CHN/12) is available in the GenBank database. In this study, we conducted phylogenetic characterisation of four EV-B106 strains isolated in Xinjiang, China. Pairwise comparisons of the nucleotide sequences and the deduced amino acid sequences revealed that the four Xinjiang EV-B106 strains had only 80.5–80.8% nucleotide identity and 95.4–97.3% amino acid identity with the Yunnan EV-B106 strain, indicating high mutagenicity. Similarity plots and bootscanning analyses revealed that frequent intertypic recombination occurred in all four Xinjiang EV-B106 strains in the non-structural region. These four strains may share a donor sequence with the EV-B85 strain, which circulated in Xinjiang in 2011, indicating extensive genetic exchanges between these strains. All Xinjiang EV-B106 strains were temperature-sensitive. An antibody seroprevalence study against EV-B106 in two Xinjiang prefectures also showed low titres of neutralizing antibodies, suggesting limited exposure and transmission in the population. This study contributes the whole genome sequences of EV-B106 to the GenBank database and provides valuable information regarding the molecular epidemiology of EV-B106 in China. Enterovirus, within family Picornaviridae, order Picornavirales, consists of 12 species: enterovirus A–H, J, and rhinovirus A–C (www.picornaviridae.com). Based on genomic characteristics, human enteroviruses (EV) can be divided into four species: EV-A, EV-B, EV-C, and EV-D, including more than 100 serotypes of polioviruses (PVs), Coxsackie A viruses (CVAs), Coxsackie B viruses (CVBs), echoviruses, and enteroviruses named with digital serial numbers since EV-D68 was designated1235The genus VP1 coding region containing a serotype-specific sequence and is known as the “molecular serotyping” method. In this method, strains possessing more than 75% VP1 nucleotide similarity (85% amino acids similarity) are considered to belong to the same serotype. In contrast, strains with less than 70% VP1 nucleotide similarity are considered to belong to different serotypes. VP1 nucleotide similarity between 70–75% is considered the “grey zone”, for which additional information such as neutralization testing with specific antiserum or sequences of other regions to assist with the identification is required2789EVs can cause a wide range of diseases such as acute flaccid paralysis (AFP), acute aseptic meningitis, encephalitis, myocarditis, hand-foot-and-mouth disease, and others. However, most EV infections are asymptomatic or lead to only mild symptoms resembling the common cold or slight febrile diseases. Classification of EVs is now based on variations in the P1 sequence of EV-B106  from Pakistan in 2010VP1 sequence of EV-B106  from Bolivia in 2003Enterovirus B106 (EV-B106) is a newly identified serotype within EV-B. The prototype strain (BAN2001-10634) was isolated in Bangladesh in 2001; however, sequences of the prototype strain were not released in the GenBank database. To date, there is only one EV-B106 full-length genome sequence in the GenBank database , isolated from an AFP patient in Yunnan Province, China in 2012VP4 sequences were amplified using the species-specific primer pairs EVP4 and OL68-1 and analysed using an online enterovirus genotyping tool14VP1 sequences of these four EV strains were analysed using the Basic Local Alignment Search Tool (BLAST) server by comparison with sequences available in the GenBank database; EV serotypes were determined using the online enterovirus genotyping toolVP1 sequences of the four Xinjiang strains showed the highest similarity with those of the EV-B106 strain in the GenBank database, which reached a percentage of as high as 92.8%; therefore, based on the EV molecular typing criteria, the four strains were identified as EV-B106.All four viral strains  were isolated from human rhabdomyosarcoma (RD) cells. After a complete EV-like cytopathic effect (CPE) was observed, the cell cultures were harvested. The four strains were confirmed to belong to EV-B when the The full-length genome sequences of the four Xinjiang EV-B106 strains were determined. The results showed that they were all 7421–7422 nucleotides in length with a single ORF of 6579 nucleotides encoding a single polypeptide of 2192 amino acids. The sequences were flanked by a non-coding 5′-UTR of 741–742 nucleotides and non-coding 3′-UTR of 101 nucleotides, with the latter preceding a long poly(A) tail. The overall compositions of the four stains  were 28.39–28.68% A, 23.73–24.01% C, 24.43–24.70% G, and 22.93–23.16% T, respectively. Because the full-length sequence of the prototype strain of EV-B106 was unavailable, we aligned the full-length genomes of the four Xinjiang strains with the Yunnan strain (12148/YN). The results showed that all four Xinjiang EV-B106 strains contained three nucleotide insertions at positions 7318, 7332, and 7425 and had one or two deletions compared to the Yunnan strain (12148/YN). Strain HTPS-QDH11F had two deletions at positions 95 and 7317; strain KS-KSH28F/XJ had one deletion at position 7317; strain KS-MGTH90F had two deletions at positions 95 and 7347; and strain AKS-AWT-AFP2F had one deletion at position 7347.P1 coding sequences of the four Xinjiang strains, 91.6–92.4% nucleotide identity and 96.9–97.5% amino acid identity were observed when compared with the Pakistan strain.Pairwise comparisons of the nucleotide sequences and the deduced amino acid sequences were conducted among the four Xinjiang EV-B106 strains with the Yunnan EV-B106 strain and Pakistan EV-B106 strain, as well as of other prototype strains with the EV-B species . The comVP1, P1, P2, and P3 coding region nucleotide sequences of the four Xinjiang EV-B106 strains, Yunnan EV-B106 strain, Pakistan EV-B106 strain (VP1 and P1 region sequences only), Bolivia EV-B106 strain , and other prototype strains of EV-B in the GenBank database  and Pakistan strain were 92.8%, 92.1%, 91.4%, and 92.2%, respectively. Based on the phylogenetic tree, the EV-B77 prototype strain (strain USA-TX97-10394) showed the highest homology with all EV-B106 strains with a bootstrap support value of 100%. The nucleotide identities of the VP1 region between the four Xinjiang strains and EV-B77 prototype strain reached percentages as high as 69.5%, 70.2%, 69.1%, and 69.9%, respectively.As shown in P2 and P3 coding regions, which suggesting that recombination between EV-B106 and other EV-B serotypes occurred. In the P2 coding region, the four Xinjiang EV-B106 strains clustered together and shared the highest similarity with the prototype strain EV-B87. In the P3 coding region, however, even the four Xinjiang strains did not cluster together, and the sequence similarities among their P3 regions varied widely from 82.9% (strain KS-KSH28F/XJ and KS-MGTH90F) to 97.8% (strain KS-KSH28F/XJ and HTPS-QDH11F). This unexpected finding suggested that the occurrence of recombination events among the four strains were different.Unexpectedly, the four Xinjiang EV-B106 strains did not form a lineage with the only Yunnan EV-B106 strain in the trees based on the To confirm the recombination events between the Xinjiang EV-B106 strains and other EV-B strains, similarity plots and bootscanning analyses were performed . The fouP1 coding region, the four Xinjiang strains showed the highest similarity with the Yunnan EV-B106 strain (12148/YN), as expected. However, in the other regions, the results revealed that the four Xinjiang strains clearly differed from the Yunnan EV-B106 strain. These results suggest that recombination events occurred in all four Xinjiang EV-B106 strains in the non-capsid regions. In the P2 region, no obvious donor sequence could be identified. In the P3 region, however, the four Xinjiang strains all recombined with other EV-B strains, but the donor sequences presented dissimilarities. One recombination event between three Xinjiang strains (excluding strain KS-MGTH90F) and EV-B100 prototype strain was identified from the 3′ end of the 3A region to the 5′ end of the 3C region. Bootscanning analysis also supported that strain KS-MGTH90F shared the highest similarity with EV-B86 followed by EV-B107 prototype strains in the 3D region, confirming the occurrence of recombination. Furthermore, recombination of a small fragment in the 5′-UTR region between strain AKS-AWT-AFP2F and Echovirus 9 prototype strain was also detected.In the P3 region sequences. We screened the P3 region sequences of the four Xinjiang strains by BLAST, and found that two strains, HTPS-QDH11F and KS-KSH28F, shared the highest nucleotides similarity in the P3 region with the EV-B85 strain, which was also isolated from Xinjiang, ChinaP3 region sequences and observed that strains HTPS-QDH11F and KS-KSH28F/XJ showed 89.9% similarity with the EV-B85 strain HYTY-ARL-AFP02F in the 3D region.We further evaluated the recombinant structure of the four Xinjiang EV-B106 strains because of their distinct 3D region were highly possible. The results also confirmed that the EV-B106 strains underwent extensive genetic exchanges with the other EV-B strains, including EV-B85 strains in Xinjiang, and that the unknown serotype EV-B donor sequence for the Xinjiang EV-B85 strain was also present in these Xinjiang EV-B106 strains.To confirm that recombination events between the Xinjiang EV-B106 strains and Xinjiang EV-B85 strain occurred, we conducted both similarity plots and bootscanning analyses on the four Xinjiang EV-B106 strains and Xinjiang EV-B85 strain HYTY-ARL-AFP02F along with other EV-B prototype strains  using thThe four Xinjiang EV-B106 strains were compared based on replication capacity at an elevated temperature (39.5 °C). A Xinjiang EV-B85 strain  was used for an experiment control. The results showed that all EV-B106 strains were temperature-sensitive with titre reductions of more than 2 logarithms at 36 °C/39.5 °C .A total of 50 serum samples were collected from newborns to 4-year-old children from Xinjiang, with 25 were collected from Kashgar prefecture and 25 from Hotan prefecture. From the samples surveyed, 21 were seropositive for EV-B106 (>1:8), with a total positive rate of 42%. The geometric mean titre (GMT) among the seropositive samples was 1:21.5. The composition ratios for the EV-B106 neutralization antibody titres of <1:8, 1:8–1:64, and >1:64 were 58%, 36%, and 6%, respectively. This suggests that compared with the seroepidemiology studies of other widely endemic EVs in China, such as EV-A71 and CV-A16, the positive rate and GMT of EV-B106 was lower42 = 0.082, p > 0.05, GMTs: p < 0.01).There were some differences between samples collected from the Kashar and Hotan prefectures in regards to the seroprevalence rates and GMTs. In the Kashar prefecture, the positive rate of EV-B106 neutralization antibody and GMTs were 44% and 1:12.4, respectively, while in the Hotan prefecture, they were 40% and 1:39.4 respectively. Although there was no significant difference between the positive seroprevalence rates of the two regions, the GMT in the Kashar prefecture was significantly higher than that in the Hotan prefecture  showing high nucleotide similarity with the Xinjiang EV-B106 strains in the The four Xinjiang EV-B106 strains were all temperature-sensitive, suggesting that their virulence is currently not high enough to have strong transmissibility16p < 0.05), suggesting that these EV-B106 strains widely circulated in the Kashar prefecture, particularly because two of the four EV-B106 strains were isolated in the Kashar prefecture. However, compared to the other prevalent EVs in China, such as EV-A71 and CV-A1617A seroepidemiological analysis of EV-B106 in the Xinjiang Autonomous Region of China was conducted. The results showed that GMTs were higher in the Kashar prefecture than in the Hotan prefecture  has been reported worldwide, indicating that this serotype is not widespread in China or in the world. Sequence analysis suggested that these four strains have high genetic diversity compared with the Yunnan EV-B106 strain, intertypic recombination in the non-structural protein region of all the four strains, and extensive genetic exchanges with the other EV-B circulating in Xinjiang. The high divergence among the EV-B106 strains reflects that this virus is not newly emergent, but has circulated in the environment for many years. In addition, the differences among the This study did not involve human participants or human experimentation; the only human materials used were stool samples collected from one AFP patient and three healthy children at the instigation of the Ministry of Health P. R. of China for public health purposes. Written informed consent for the use of their clinical samples was acquired from all individuals involved in this study. This study was approved by the Ethics Review Committee of the National Institute for Viral Disease Control and Prevention (NIVDC), Chinese Center for Disease Control and Prevention, all experimental protocols were approved by NIVDC, and the methods were carried out in accordance with the approved guidelines.The four EV-B106 strains  used in this study were isolated from stool specimens from one AFP patient and three healthy children in Hotan, Kashgar, and Aksu Prefectures of Xinjiang Autonomous Region, China. The samples were collected in 2011 during poliovirus surveillance activities.In the seroprevalence study of EV-B106 antibodies, 50 healthy children ≤5 years of age were surveyed. Fifty serum samples were collected in 2013 for seroepidemiological analysis of enteroviruses, with informed parental consents, by the Xinjiang Center for Disease Control and Prevention: 25 samples from Hotan and 25 samples from Kashgar, the two regions where the strains were isolated. The same serum samples were used previously for a EV-A89 seroepidemiology studyStool samples from the AFP patient and three healthy children were collected and processed according to standard procedures29VP1 coding region using the PrimeScript One Step RT-PCR Kit Ver.2  with primer pairs 490 and 492VP1 sequences were analysed with the BLAST server by comparing with the sequences available in the GenBank database and were determined using the EV Genotyping ToolViral RNA was extracted from the cell culture using a QIAamp Viral RNA Mini Kit . Reverse transcription polymerase chain reaction (RT-PCR) was conducted to amplify the partial The 5′ end of the genome sequence was amplified using the 5′-Full RACE Kit  according to the manufacturer’s instructions. The 3′ end sequence was obtained by using an oligo-dT primer (primer 7500A)The nucleotide and deduced amino acid sequences of the four Xinjiang EV-B106 strains were compared with those of the prototype EV-B strains by pairwise alignment using the MEGA program (version 5.03)2 incubators: 36 °C as the optimal temperature for virus propagation and 39.5 °C as the supraoptimal temperature. After absorption at 36 °C or 39.5 °C for 1 h, the unabsorbed virus inoculum was removed and 100 μL of maintenance medium was added to each well. The plates were incubated again at 36 °C and 39.5 °C and harvested at 8 time points post-infection  in succession. The 50% cell culture infectious dose (CCID50) was calculated by the end-point dilution method on monolayer RD cells in 96-well plates at 36 °C. Virus isolates showing more than a two-logarithm reduction in titre at different temperatures were considered to be temperature-sensitiveTemperature sensitivities of the four Xinjiang EV-B106 strains were assayed on monolayer RD cells in 24-well plates50 value among the four Xinjiang EV-B106 strains. Serum samples were inactivated at 56 °C for 30 min, and sample dilutions from 1:4 to 1:1024 were assayed. The mixture of virus samples (50 μL) with a CCID50 of 100 and appropriate serum dilution (50 μL) was then incubated at 36 °C in a CO2 incubator. After incubation for 7 days, the highest dilution of serum that protected 50% of the cultures was recorded according to the EV-like CPE. A serum sample was considered positive if the neutralization antibody titre was observed at a dilution of 1:8, and GMT was subsequently calculated.Neutralizing antibodies against EV-B106 were detected in a neutralization test using the human RD cell line as previously describedp < 0.05 were considered statistically significant.All statistical analyses were performed using SPSS Statistics software (version 19.0) . Chi-square test was used to compare the seroprevalence rates of the viral strains between Kashgar prefecture and Hotan prefecture. Mann-Whitney U test was used to compare the differences in GMTs. Titres below 1:8 were assumed 1:4 for calculation. Differences with an error probability of The full-length genomic sequences of the four EV-B106 strains  described in this study were deposited in the GenBank database under the accession numbers KX171334-KX171337, respectively.How to cite this article: Song, Y. et al. Phylogenetic Characterizations of Highly Mutated EV-B106 Recombinants Showing Extensive Genetic Exchanges with Other EV-B in Xinjiang, China. Sci. Rep.7, 43080; doi: 10.1038/srep43080 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Furthermore, the correlation between the SUVmax and PD-L1 expression has not been assessed. Thus, the purpose of this study was to investigate the correlation between PD-L1 expression and the SUVmax on 18FDG-PET and to examine the prognostic significance of PD-L1 expression and the SUVmax in surgical pulmonary SCC. Expression of PD-L1 was examined in 84 patients with resected SCC using immunohistochemistry. Positive PD-L1 expression in tumour cells was observed in 58.3% (49/84) of patients with SCC. High PD-L1 expression levels were significantly correlated with histological differentiation (P=0.006), and a high SUVmax was associated with histological differentiation (P=0.037), and lymph node metastasis (P=0.025). Spearman's test showed that there was a significant correlation between PD-L1 expression levels and the SUVmax. High PD-L1 expression levels and a high SUVmax were both independent risks factors for poor overall survival. Our results suggested that high PD-L1 expression levels and a high SUVmax was associated with poor prognosis in surgical pulmonary SCC. The existence of a statistically significant correlation between the SUVmax and PD-L1 expression levels justifies exploring the usefulness of the SUVmax as a predictor of PD-1/PD-L1 inhibitor activity.Programmed cell death-ligand 1 (PD-L1) expression is commonly observed in non-small cell lung cancer (NSCLC). The prognostic value of PD-L1 expression and the maximum standardized uptake value (SUVmax) on 18F-Fluorodeoxyglucose positron emission tomography ( Lung cancer, which can be broadly divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), is the most prevalent cancer worldwide . CertainThe programmed death 1 (PD-1) pathway is a major immune checkpoint. The binding of PD-1 ligand (PD-L1) to PD-1 induces activated T-cell apoptosis or exhaustion, and agents that block this inhibitory signal have been shown to enhance T-cells activity . Previou18FDG-PET) is an important noninvasive tool used for the diagnosing NSCLC and performing TNM staging in patients with the disease. The maximum standardized uptake value (SUVmax) on FDG-PET is widely used in clinical practice because of its simplicity [18F-Fluorodeoxyglucose positron emission tomography .The correlations between PD-L1 expression and the SUVmax and clinicopathological characteristics are presented in Table PD-L1-positive patients displayed significantly shorter DFS (P=0.007) and OS P=0.002) than PD-L1-negative patients. Furthermore, patients with a high SUVmax displayed shorter DFS (P=0.002) and OS P=0.014) than patients with a low SUVmax. for 60 minutes at room temperature. For antigen visualization, we immersed the sectionsin 3, 3-diaminobenzidine solution and counterstained them with hematoxylin.The PD-L1 immunostaining results were classified into two groups based on staining intensity and tumour cell positivity. Staining intensity was scored as follows: 0, negative staining; 1, weak staining; 2, moderate staining; and 3, strong staining. All cases in which more than 5% of tumour cells displayed a staining intensity ≥2 were considered positive. Cases with staining intensity <2 or less than 5% of tumour cells were considered negative. The 5% threshold used herein was chosen based on the results of a previous clinical trial –42.p values <0.05 were considered statistically significant. All statistical analyses were performed using SPSS software .The correlations between PD-L1 expression and clinicopathological characteristics were evaluated using chi-square tests or Fisher's exact test. Spearman’ s correlation coefficient (rho) was used for rank correlation. Disease-free survival and overall survival were calculated using by the Kaplan–Meier method, and comparisons were performed using the log-rank test. Univariate and multivariate regression was performed using the Cox proportional hazards model. Two-sided


PLOS Genetics' confidentiality standards. The error was corrected by removing the images in the HTML and PDF versions of this article on November 18th, 2016 and the corrected Fig. 1 is available in the supporting information of this article.The original Fig. 1 contained images incompatible with S1 FileRadiographic phenotypes of cases R14-123A and R08-269A & B. (A) A/P radiograph of the chest of R14-123ª showing small scapulae, 11 ribs, and abnormally formed hypoplastic humeri with radioulnar synostosis. (B) Hand radiograph showing oligodactyly, hypoplastic carpal bones and preaxial polydactyly. (C) Bilateral lower extremities showing hypoplastic or absent fibulae with small stippled calcanei. (D) A/P radiograph of R08-269A showing hypoplastic radii, oligodactyly, absent thumbs, thin fibulae, and club foot. (E) A/P radiograph of R08-269B showing 11 ribs, absent radii and ulnae.(TIF)Click here for additional data file.


CDKN2A and CDKN2B and the long noncoding RNA (lncRNA) known as ANRIL (or CDKN2B-AS), was associated with childhood acute lymphoblastic leukemia (ALL) susceptibility in several genome wide association studies (GWAS). However, the variants associated in the diverse studies were different. Recently, new and independent SNPs deregulating the locus function were also identified in association with ALL risk. This diversity in the results may be explained because different variants in each population could alter CDKN2A/B locus function through diverse mechanisms. Therefore, the aim of this study was to determine whether the annotated risk variants in the CDKN2A/B locus affect the susceptibility of B cell precursor ALL (B-ALL) in our Spanish population and explore if other SNPs altering additional regulatory mechanisms could be also involved. We analyzed the four SNPs proposed by GWAs and two additional SNPs in miRNA binding sites in 217 pediatric patients with B-ALL and 330 healthy controls. The SNPs rs2811712, rs3731249, rs3217992 and rs2811709 were associated with B-ALL susceptibility in our Spanish population. ALL subtypes analyses showed that rs2811712 was associated with B-hyperdiploid ALL. These results provide evidence for the influence of genetic variants at CDKN2A/B locus with the risk of developing B-ALL.The locus CDKN2A/B (9p21.3), which comprises the tumor suppressors genes  ARID5B), 7p12.2 (IKZF1) [CEBPE) [ARID5B) and B-hyperdiploid ALL [BMI1-PIP4K2A) [CDKN2A/B)[CDKN2A and CDKN2B and a long noncoding RNA (lncRNA) known as ANRIL.Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy , 2. The  (IKZF1) , 5 and 1 [CEBPE) , 6–8. Soloid ALL , 5. SubsPIP4K2A) , validatPIP4K2A) , but notPIP4K2A) , and 9p2CDKN2A/B), a regioCDKN2A. This association was replicated in several populations such as Germany, Canada [CDKN2A, rs2811709, with B-ALL in French children, a variant in low linkage disequilibrium (LD) with rs3731217 (r2<0.8). Then, in 2015, three independent studies using genotyping and imputation-based fine-mapping, pointed to rs3731249 in exon 2 of CDKN2A as the hit associated variant that conferred high risk for B-ALL in European and Hispanic children [ANRIL as an independent locus associated with B-ALL susceptibility in European and African-Americans [The region 9p21.3 is particularly noteworthy because independent association signals have been recently discovered at this locus in association with B cell precursor ALL (B-ALL) susceptibility. The first variant identified in children from the United Kingdom in 2010 was rs3731217 , which i, Canada  and Fran, Canada , Hispani, Canada  or Thai , Canada . In 2012mericans .G and CAGTAC) that may regulate alternative splicing of CDKN2A [CDKN2A which produces an alanine-to-threonine change in amino-acid-sequence, resulting in reduced tumor suppressor function of p16INK4A [ARF, where it creates a binding site for miR-132-5p and miR-4642 [CDKN2A and CDKN2B that disrupt or create microRNA (miRNA) binding sites have been described, but studies focused on SNPs in miRNA binding sites are almost absent. Finally, rs662463 in ANRIL regulates CDKN2B expression by disrupting a transcription factor binding site for CEBPB [These variants could alter the locus through diverse mechanisms. The alleles of rs3731217 create two overlapping cis-acting intronic splice enhancer motifs  and was carried out according to the Declaration of Helsinki. Written informed consent was obtained from all participants, or from their parents, prior to sample collection.A total of 231 European descent children diagnosed with B-ALL between 2000 and 2011 in the Pediatric Oncology Units of four Spanish hospitals  and 338 unrelated healthy controls were included in this study . This isETV6-RUNX1 gene fusion, were also analyzed. The other subtypes were not considered due to the low number of patients in our cohort. Sex and age data were systematically recorded  and B-lineage ALL bearing the t translocation leading to an recorded .http://hapmap.ncbi.nlm.nih.gov/) (The HapMap Data Coordination Center (DCC), Bethesda, MD) and Haploview software v.4.2 (http://www.broad.mit.edu/mpg/haploview/)  with an r2 threshold of 0.8. (ii) SNPs in miRNA binding sites of 3´UTR region of CDKN2A and CDKN2B with a MAF>10% identified using bioinformatics tools: Ensembl (http://www.ensembl.org/) , and miRNASNP (http://bioinfo.life.hust.edu.cn/miRNASNP2/index.php) . Of 47 SNPs identified in the 3´UTR region that disrupt or create miRNA binding sites  were genotyped across the plates. For rs3731249, the genotyping analyses were performed by using PCR followed by restriction analysis with To identify any deviation in Hardy-Weinberg equilibrium (HWE) for the healthy controls, a χ2 test was used. The association between genetic polymorphisms in cases and controls, as well as ALL subtypes and controls, was also evaluated using the χ2 or Fisher’s exact test. The effect sizes of the associations were estimated by the odds ratio from univariate logistic regression. The most significant test among codominant, dominant, recessive, and additive genetic models was selected. The results were adjusted for multiple comparisons using the false discovery rate (FDR). In all A total of 231 patients with B-ALL and 338 unrelated healthy controls were available for genotyping with GoldenGate Genotyping Assay. Successful genotyping was achieved for 217 patients with B-ALL and 330 controls (96.1%). Of the SNPs, 5/5  were genotyped satisfactorily . For rs3731249, 180 patients with B-ALL and 235 controls were available for genotyping with a genotyping rate of 97.6%. All of them were in HWE in the control cohort.CDKN2B displayed the most significant value under the log-additive genetic model (AA vs AG vs GG). The GG genotype showed a 1.98-fold increased risk of B-ALL . The second most significant association signal was found for rs3731249 at CDKN2A. In this case, the CT/TT genotypes produced a 2.61-fold increased risk of B-ALL . We also found AA genotype of rs3217992 associated with a decreased risk of B-ALL . Finally, rs2811709 AG/AA genotypes were associated with a 1.7-fold increased risk of B-ALL. All the SNPs remained statistically associated with B-ALL risk after FDR correction. The SNPs rs3731222 and rs1063192 were not associated with B-ALL susceptibility in our population.Of the 6 SNPs analyzed, we found 4 significantly associated with B-ALL risk . From thETV-RUNX1 ALL subtype, we found association between TT genotype of rs3731249 and B-hyperdiploid ALL , AA genotype of rs3217992 with ETV6-RUNX1 ALL  and GG genotype of rs2811712 with both B-hyperdiploid ALL  and ETV6-RUNX1 ALL   . After FIn the current study, we analyzed 6 SNPs at the CDKN2A/B locus in 217 children with B-ALL and 330 controls in a Spanish cohort. SNPs rs2811712, rs3731249, rs3217992 and rs2811709 were associated with B-ALL susceptibility. In the subtype analysis, rs2811712 was associated with the risk of developing B-hyperdiploid ALL.−4). This result is in line with most of the studies [2 = 0.83) and rs662463 (r2 = 1), both associated with B-ALL risk in European Americans [ANRIL. SNPs in lncRNAs may affect its expression or its structure by interfering with lncRNA folding or by modulating protein-lncRNA interactions [ANRIL has been shown to regulate CDKN2A and CDKN2B genes. Specifically, acting in cis, ANRIL binds various Polycomb proteins resulting in histone modification of the CDKN2A/CDKN2B locus, and in turn, silencing the cluster [ANRIL could be involved in the downregulation of the locus, contributing to increased susceptibility to B-ALL.The most significant finding was the association between GG genotype of rs2811712 and the increased risk of developing B-ALL . There is evidence that the variant p16INK4A (p.148T) is preferentially retained in the nucleus, compromising its ability to inhibit CDK4 and CDK6 in the cytoplasm [ARF, rs3731249 is in the 3´UTR region, where the risk allele creates a miRNA binding site for miR-132-5p and miR-4642 [ARF expression, and then, attenuate its function as cyclin inhibitor. Therefore, T allele of rs3731249 in CDKN2A could be involved in B-ALL through its effect on the function of both p16INK4A and p14ARF.The second most significant association was found for the CT/TT genotypes of rs3731249, which produced a 2.6-fold increased risk of B-ALL (P = 0.002). This association was also described recently by 3 independent studies, all of them pointing out the high impact of this variant, since it confers in all studies between two and three-fold increased risk of B-ALL susceptibility in children of European and Hispanic origin –17. Rs37ytoplasm  and favomiR-4642 . These mCDKN2B in which the A allele disrupts the binding for miR-138 and miR-205 [CDKN2B, explaining its protective role. As far as we know, this is the first time that this SNP is associated with B-ALL risk.The third finding was the association between the AA genotype of rs3217992 and a decreased B-ALL risk. This SNP is located in a miRNA binding site in  miR-205 . The losCDKN2B, with a decreased expression of CDKN2B mRNA for the risk allele [Regarding rs2811709, AG/AA genotypes were associated with an increased risk of B-ALL susceptibility in our population. This SNP was also associated with B-ALL risk in two previous studies of children of European origin . rs28117k allele , which c2 = 1), the SNP identified in the work performed by Sherborne et al. [On the other hand, we found no association between rs3731222 and rs1063192 and B-ALL susceptibility. One of them, rs3731222, is in high LD with rs3731217 . However, when we calculated the sample size needed to obtain an 80% of statistical power using the software OpenEpi  the estiIn conclusion, three of the variants previously proposed by the literature, rs2811712, rs3731249 and rs2811709, and a new variant, rs3217992, were associated with B-ALL susceptibility in our Spanish cohort. These results confirmed the implication of CDKN2A/B locus in the development of B-ALL since all these SNPs could act through different mechanisms that might alter the cluster. The identification of the specific causes that could lead to the development of B-ALL is clearly a worthwhile goal for prevention or early intervention of this disease.S1 FigIn bold, the SNPs significantly associated with B-ALL risk in our study.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableSNPs with a MAF>10% are in bold.(PDF)Click here for additional data file.S4 TableSNPs, single nucleotide polymorphisms; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; bp, base pairs.(PDF)Click here for additional data file.


The authors have stated that there was an error in the method used to subtract natural abundance of amino acid isotopologues. The authors have now re-calculated the results.There are errors in the “Calculations and statistical treatment of data” section in the Materials and methods. The authors have also added three new equations. Please see the corrected “Calculations and statistical treatment of data” section here:The amounts of N derived from the different N sources are reported in units of mg N in the tissue, calculated using the formula:The amount of N (μg) assimilated from the different N sources into AAs in the free AA pool was calculated in a stepwise manner.+1 and m+2):Initially, the natural abundance is subtracted from labelled samples. This is done by using correction factors calculated from non-labelled control samples (NLS) for each individual AA and isotopologue  of each isotopologue [AALS was calculated as:The concentration of N (μmol /g DW) derived from the labelled N source [N]TOT and MWN denotes the total DW of the biological replicate and the molecular weight of N, respectively.Lastly, the absolute amount of N (μg) derived from the labelled N source in each biological replicate was calculated as:Data from the individual isotopologues of L-His and L-Cys peaks were inconclusive and therefore data corresponding to these AAs were excluded.15N-amide-L-Gln and 15N-amine-L-Gln treatments were omitted from L-Gln calculations since L-Gln+1N synthesised de novo during the experiment cannot be discriminated from non-metabolised added tracer (15N-amide-L-Gln or 15N-amine-L-Gln).For the incorporation of N into the AA pool, the The results are presented as mean values ± the standard error of the mean (SE). Significant differences in the experiments were analysed by ANOVA with a Tukey HSD test using JMP Pro software . A P-value of <0.05 was defined as statistically significant.In Fig 5, the values and percentages of N originating from each N source are incorrect. Please see the corrected S2 Fig and S3 Fig have been updated after the re-calculation. Please see the corrected S2 Fig4+, NO3- and L-Gln .Fraction from each N source, NH(TIF)Click here for additional data file.S3 Fig4+, NO3-, amide-L-Gln and amine-L-Gln. (B) Fraction from the inorganic N sources. Each bar represents a mean ± SE; n = 9–10.(A) Fraction from each N source, NH(TIF)Click here for additional data file.


Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A.The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L  in vivo and that L* can substitute for 2-5A degrading enzymes produced by coronaviruses.Oligoadenylate synthetases (OAS) are a family of enzymes that produce 2’-5’ oligoadenylates (2-5A) from ATP. OAS accumulate as inactive enzymes in cells that have been exposed to interferon. When infection of these cells occurs, double-stranded RNA produced during virus replication activates OAS, which produce 2-5A. 2-5A act as second messengers. They bind a cellular endonuclease called RNase L, thereby triggering the dimerization and the activation of this RNase. Active RNase L degrades cellular and viral RNA molecules, ultimately killing the infected cell, thus preventing virus spread. Viruses have developed diverse mechanisms to escape RNase L activity. We report that Theiler’s murine encephalomyelitis virus, a virus that produces persistent infections of the central nervous system, evolved a novel mechanism to escape the OAS/RNase L pathway. The L* protein produced by this virus binds RNase L very close to the pocket known to sense 2-5A and thereby prevents 2-5A from activating RNase L. This mechanism contrasts with that used by Influenza (flu) virus, which sequesters double-stranded RNA and thereby also inhibits alternative antiviral pathways. We further showed that the RNase L antagonist activity of L* is effective  Cardiovirus in the family Picornaviridae. Persistent strains of TMEV, such as DA, BeAn or TO4 have a remarkable ability to establish persistent infections of the mouse central nervous system. In susceptible mouse strains, TMEV can persist lifelong, replicating mainly in oligodendrocytes, macrophages and microglial cells, in the white matter of the spinal cord [in vitro [in vivo [Theiler’s murine encephalomyelitis virus (TMEV or Theiler’s virus) belongs to the genus nal cord –5. Two anal cord , 5. The nal cord . It faciin vitro , 8 and t[in vivo , 10. L* [in vivo . We prev[in vivo . Interes[in vivo , whereas[in vivo .RNase L is the effector enzyme of the OAS/RNase L system, one of the best-characterized interferon-induced antiviral pathways . InSus scrofa RNase L [RNase L is composed of three domains: an N-terminal ankyrin domain formed of 9 ankyrin repeats (ANK R1-9) involved in 2-5A recognition, a central catalytically inactive pseudokinase domain contributing to RNase L dimerization, and a C-terminal ribonuclease domain responsible for target RNA cleavage . In the  RNase L .RNase L is active against many viruses such as West Nile virus  EncephalThis study aimed at unraveling the novel mechanism of RNase L inhibition involving TMEV L*. Our results show that L* binds RNase L ankyrin repeats very close to 2-5A binding pockets, thereby inhibiting 2-5A binding to RNase L. Using chimeric viruses, we further provide evidence that L* can substitute for another viral RNase L inhibitor acting upstream of L* in the OAS/RNase L cascade, namely the ns2 phosphodiesterase encoded by MHV.DA interacted with fusion proteins carrying mouse RNase L ANK R1-R2 but not with fusions carrying ANK R2 or ANK R1 alone  fusions were constructed that carry ANK repeats of mouse, rat and human RNase L upstream of the eGFP coding sequence . The ANKR1 alone . SpecifiNK R1-R2 . We concDA, we observed by fluorescent microscopy that the soluble pool of cytosolic L* was responsible for RNase L binding  and L* of the rat TMEV strain RTV-1 (L*RTV-1).The use of human/mouse RNase L chimeras does not allow identification of L* binding residues that are conserved between the RNase L proteins of the two species. Since we recently cloned rat RNase L and showed that L* from the rat TMEV strain RTV-1 inhibited rat but not mouse RNase L, we extended the analysis to rat/mouse RNase L chimeras . Rat andDA but not L*RTV-1, while rat RNase L was inhibited by and interacted with L*RTV-1 but not L*DA , these data show that residues 80 and 81 of ANK R2 contribute to L* binding to RNase L and L*-mediated RNase L inhibition.As expected, mouse RNase L interacted with and was inhibited by L*Structural and functional studies identified human RNase L residues that are involved in 2-5A binding , 29, 42.We next established an RNase L dimerization assay based on disuccinimidylsuberate (DSS)-mediated crosslinking. 293T cells overexpressing a C-terminally Flag-tagged RNase L (RNase L-Flag) and L* were gently lysed. Then, 2-5A molecules were added to the lysate to promote RNase L dimerization before addition of DSS. After incubation, a fraction of the sample was used for RNA extraction to monitor 2-5A-dependent (i.e. RNase L-mediated) RNA degradation. The remaining part of the lysate was used to monitor RNase L dimerization by immunoblotting (20).As can be seen in We next analyzed whether oligomers formed in the presence of 2-5A involved additional protein partners or whether they resulted from homooligomerization. Therefore, crosslinking was performed after 2-5A addition as above, using the reversible agent dithiobis(sulfosuccinimidyl propionate) (DTSSP) that is compatible with further mass spectrometry analysis. High molecular weight complexes were then separated by SDS-PAGE and analyzed by mass spectrometry . Except Collectively, these data show that 2-5A can trigger the dimerization and the homooligomerization of RNase L and that L* binding to RNase L inhibits these processes.ka = 2.1 x 105 M-1 x sec-1) with a relatively slow dissociation rate of 2.2 x10-3 sec-1 (6-L* immobilized on Ni-NTA sensor chip was then assessed. Consistent with our prior observations (12), we found that human RNase L did not bind to L* while mouse RNase L bound to L* with a KD of 1.3 x 10−6 M (50 = 6.0 x 10-7M), L* did not inhibit human RNase L from binding with 2-5A (50 = 1.32 x 10−8 M)  . Taken tK50) remained unchanged, the maximum velocity (Vmax) of the RNase L enzymatic activity was decreased by ~40% by L* (50: 9.99 μM vs 34.14 μM). Altogether, these results suggest that L* inhibits 2-5A binding to mouse RNase L, possibly because L* inhibits dimerization required for cooperative binding of 2-5A between the RNase L protomers [To further characterize the inhibition by L* of 2-5A-dependent RNase L activation, fluorescence energy transfer (FRET) assays based on the degradation of a labeled RNA probe  were per0% by L* . Next, m0% by L* , circlesrotomers , 45.in vivo, and in doing so, if L* could substitute for another antagonist of the OAS/RNase L pathway, acting upstream in the OAS/RNase L signaling pathway. L*-mediated inhibition of RNase L in vivo was not analyzed for TMEV, by comparing the infection of RNase L-deficient (RNase L-/-) and RNase L +/+ mice [b-restricted cytolytic T lymphocyte response [H126R-L*) or a truncated L*1–92 lacking anti-RNase L activity (ns2H126R-L*1–92) [We next asked whether L* displayed anti-RNase L activity +/+ mice  because response –48. We tThe replication ability of these viruses was first assessed in L2 fibroblasts in which IFN-β expression is not induced by MHV . WT, mutH126R-L* chimeric virus expressed L* in these cells , cells where MHV induces the expression of IFN, and in which ns2 PDE activity is required for an efficient replication , 49. We se cells , lane 4 se cells , lane 4.WT virus . Interesl titers , confirm-/- C57BL/6 mice were infected intrahepatically with 2000 PFU of WT A59, mutant and chimeric viruses and viral titers in the liver were measured by plaque assay on mouse L2 cells at 5 days post-infection [H126R) replication in the liver of WT mice was impaired as compared to that of A59 WT virus whereas ns2H126R-L* replicated to nearly WT levels. In contrast mutant L*1–92 was unable to confer robust replication when expressed from ns2H126R-L*1–92. All viruses showed the same robustness in the liver of RNase L-/- animals. These results show that the RNase L antagonistic activity of L* can replace the PDE function of ns2 in BMMs and in vivo, in the liver of WT C57BL/6 mice.Then, WT and RNase Lnfection Fig 8E)..-/- C57BThe OAS/RNase L pathway confers protection against many RNA viruses as well as some DNA viruses and, as a consequence, viruses developed numerous strategies to escape its antiviral effects . WeA fusion protein carrying ANK R1 and R2 but not either ANK repeat alone readily co-immunoprecipitated with L*, suggesting that these two repeats are both necessary and sufficient to mediate the interaction with L*. On the one hand, replacing two amino acids stretches (26–28 and 47–51) of human RNase L by the corresponding mouse RNase L residues was sufficient to trigger interaction with L* and efficient L*-mediated inhibition of human RNase L. On the other hand, rat/mouse RNase L chimeras showed that residues 80, 81 in ANK R2 were also critical for L* interaction with RNase L and, to a lesser extent, for RNase L inhibition. In general, results from interaction and functional studies were congruent. For some constructs, however, interaction was observed while inhibition was weak, suggesting that a strong affinity is required for RNase L inhibition. In summary, our data show that L* inhibits RNase L activation by associating with loops exposed at the surface of RNase L ANK R1 and R2 . The facUsing crosslinking experiments, we showed that L* prevented RNase L dimer and oligomer formation. Oligomerization of human RNase L was suggested by Han et al.  but was KD of 1.3 x 10−6 M. On one hand, increasing L* concentrations decreased mouse RNase L binding to immobilized 2-5A  was preserved  [It is intriguing to observe the variety of strategies developed by viruses to antagonize RNase L. The NS1 protein of influenza A virus sequesters dsRNA thus preventing sensing by OAS . DsRNA sse (PKR) , which lse (PKR) . Thus, ise (PKR) , 54.MHV, Rotaviruses and MERS-CoV also inhibit the OAS/RNase L pathway at the level of 2-5A by producing viral PDEs that degrade 2-5A , 37, 38.Using chimeric MHV expressing L*, we showed that L* could rescue the replication of MHV mutants lacking PDE activity, both in primary macrophages and in the liver of infected mice.in vivo, in infected mouse tissues. On the other hand, it suggests that proteins that act on distinct steps of the OAS/RNase L pathway can substitute for each other. ns2 triggers 2-5A degradation while L* prevents their association with RNase L. The fact that L* can substitute for ns2 therefore supports the idea that 2-5A does not play critical functions affecting viral replication other than activating RNase L.On the one hand, this result shows that L* readily antagonizes RNase L activity In conclusion, we identified a novel evasion mechanism of the antiviral OAS/RNase L pathway, which surprisingly involves interference of 2-5A binding to RNase L by a viral protein.This study was carried out in strict accordance as defined in the federal regulations set forth in the Animal Welfare Act (AWA), the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the guidelines of the University of Pennsylvania Institutional Animal Use and Care Committee. The protocols were approved (# 803711) by the Institutional Animal Care and Use Committee at the University of Pennsylvania.–/–mice as described previously [HeLa-M , and 293eviously  and cultExpression vectors used in this study are summarized in in vitro from plasmids carrying the corresponding full-length viral cDNA.TMEV derivatives were produced by electroporation of BHK-21 cells with the genomic RNA transcribed 1–92), named pSV31 was obtained by introduction of a stop codon mutation in pSV28. This mutation prevents the expression of full-length L* but does not affect the translation of the main viral open reading frame encoding the polyprotein. The corresponding viruses SV28 and SV31 were produced by reverse genetics and titrated by plaque assay. HeLa-M cells were typically infected at a MOI of 5 PFU per cell for 16 hours.Viruses were derived from the DA1 molecular clone (Genbank accession JX443418). A recombinant TMEV cDNA, named pSV28, was constructed, carrying the extra sequence coding for the HA epitope at the N-terminus of L* GGT ACC CGT ACG ACG TTC CGG ACT ACG CGC TGC TTG TAA GCA CGG between nucleotides 1081 and 1082 of pVV18 . DA1 mutant expressing a truncated L* . Additionally, two chimeric A59- ns2H126R -L* viruses were also assembled where L* is inserted in place of the nonessential NS4 protein. These viruses also expressed ns2H126R and either L*WT or another L* protein in which a stop codon (UAA) had been inserted at amino acid position 93 that renders it inactive (L*1–92) [.Chimeric MHV-L* viruses. A series of chimeric and recombinant viruses were constructed using the full-length infectious cDNA clone of MHV strain A59 as previously described , 58. The(L*1–92) . A sequePlasmid DNA were transfected using TransIT LT-1 transfection reagent according to the manufacturer’s protocol .Cells plated in 24 well-plates were transfected with 0.3 μg/mL polyI:C (Amersham-Pharmacia) with 2 μL Lipofectamine 2000 transfection reagent according to the manufacturer’s protocol .Flag-RNase L and N-terminally HA-tagged L* were coexpressed in 293T cells from transfected expression plasmids . CoimmunHeLa-M cells were grown on poly-L-lysine coated coverslips before transfection or infection. Twenty-four hours post-transfection or 16 hours post-infection, cells were fixed for 5 min with paraformaldehyde 4% in PBS then permeabilized for 5 min with Titron X-100 0.1% in PBS and unspecific antigens were blocked for 1h using 2% normal goat serum (Sigma-Aldrich S2007) in PBS. Cells were then incubated for 1 hour with primary antibodies at a dilution of 1:50 in the same buffer  or 1:25 . Cells were intensively washed with Tween 20 0.01% in PBS and incubated for 1 hour with species-matched AlexaFluor-conjugated secondary antibodies  at a dilution of 1:800 in the same buffer.For mitochondrial staining, cells were incubated for 45 min before fixation with 200 nM MitoTracker Red CMXRos . Coverslips were mounted on slides with Mowiol 4–88 medium  and analyzed by fluorescence microscopy using a spinning disk confocal microscope equipped with an Axiocam MRm camera (Zeiss). Intensity, contrast, and color balance of images were equilibrated using Zen (Zeiss) and Adobe Photoshop.Flag-RNase L and L* constructs  were coeth volume of the lysate as a control for RNase L activation and protein samples were prepared by addition of Laemmli buffer to the remaining lysate. RNase L activation was controlled by rRNA degradation analysis on RNA gel electrophoresis and RNase L dimerization was analyzed by immunoblotting using an anti-Flag antibody (Sigma-Aldrich M8823).To assess RNase L dimerization, RNase L-Flag expression vectors were transfected in 293T cells along with L* expression plasmids or equivalent empty vectors. C-terminally flagged RNase L were used because we noticed that detection of N-terminally flagged constructs by immunobloting was less consistent in this assay. Twenty four hours post-transfection, cells were washed with phosphate-buffered saline (PBS) and harvested in HEPES lysis buffer . Cells were disrupted by 10 passages through a 21G needle and lysates were centrifuged at 12,000x g for 5min at room temperature. 2-5A was then added in the supernatant at different final concentrations . After 10 min at 37°C, crosslinking of interacting proteins was performed by addition of 5mM disuccinimidyl suberate  for 30 min. Finally the crosslinking reaction was quenched with 50mM Tris-HCl pH7.5, RNA was extracted from 1/6For analysis of the oligomeric bands by mass spectrometry, the experiment was repeated using 2mM dithiobis(sulfosuccinimidyl propionate) , a cleavable protein crosslinking reagent. RNase L-Flag oligomers were then immunoprecipitated using an anti-Flag antibody (Sigma-Aldrich M8823) and protein samples were prepared by addition of a non-reducing protein buffer (Laemmli buffer without 2-mercaptoethanol).RNase L activity was determined using fluorescence resonance energy transfer (FRET) assays as described previously , 44. In After co-immunoprecipitation, samples were resolved using an 8% Tris-Glycine SDS-PAGE and proteins were visualized using Coomassie Blue . Bands of interest were cut out from the gel, reduced with DTT, alkylated with chloroacetamide and digested with chymotrypsin. The peptides were analyzed, as previously described , by capiE. coli TB1 . Bacteria were grown in LB containing 100 μg/ml ampicillin at 37°C, and shaked at 250 rpm until the OD600nm reached 0.7. The cultures were cooled on ice, induced using 0.5 mM IPTG and further grown for 6 h at 20°C. Cells were harvested and washed with ice cold 20 mM HEPES pH 8.2 containing 150 mM NaCl and lysed by sonication (20 x 10s) in buffer A  containing 10 units/ml benzonase, protease inhibitor cocktail (Roche) and 100μM PMSF. Lysates were clarified by centrifugation at 10,000 x g for 30 min at 4°C, diluted 5-fold and bound to SP Sepharose FF cation exchange resin (GE Lifesciences) packed in XK 15mm x 10mm column. The column was washed with binding buffer A until the basal OD280nm was constant. The bound proteins were eluted with a 0.2–2 M gradient of NaCl in buffer A. The fractions were analyzed by SDS page (L* eluted at >500 mM NaCl). The peak fractions containing L* were pooled and diluted 1:3 in buffer A and bound with Ni-NTA resin, washed with buffer A containing 10 mM imidazole and eluted with 150 mM imidazole in buffer A. Fractions were loaded on 12% SDS-PAGE and stained with Gel-Code Blue (Promega). The yield was ~0.25mg/L.The plasmid L*-pET15b was expressed in E. coli, as described [Human RNase L was produced as described . Murine escribed  and the 3(A2′p)nA, where n = 1 to ≥3) was enzymatically synthesized from ATP using hexahistidine-tagged and purified recombinant porcine 42-kDa 2-5A synthetase (OAS1). Individual 2-5A oligomers were purified (>95% purity) using a Dionex PA100 (22 mm × 250 mm) semi-preparative column interfaced with a Beckman system gold HPLC system under the control of 32-Karat workstation [3A2’p5’A2’p5’A, was used for RNase L activation. Biotinlyation of 2-5A was performed using periodate chemistry as described earlier [2–5A . Response units (RU), a measure of binding, were monitored as a function of time.6-L* protein was immobilized on Ni-NTA chips, according to the manufacturer’s protocol  and used for direct binding with human RNase L, mouse RNase L, or with mouse RNase L (2.5 μM) in the presence of different concentrations of 2-5A.In some experiments, biotin labeled 2-5A immobilized on streptavidin (SA)-chips was used with mouse RNase L or human RNase L in the absence or presence of different concentrations of L*. L* and 2-5A did not interact with each other as the signal was not influenced by L* in Alternatively, purified anti-GST monoclonal antibody clone P1A12  was immobilized on sensor CM5 chips  to achieve a surface equivalent to ~1000 RU. The purified recombinant GST-tagged murine RNase L (2.5 μM) supplemented with 0.005% surfactant P20 (HBS-P SPR buffer) was immobilized on anti-GST antibody coated sensor chips at a flow rate of 10 μl/min for 3 min at 25°C to achieve a surface equivalent to 1000 RU. An additional wash for 5 min at a flow rate of 20 μl/min was performed with buffer alone. L* protein  was used at 0.1 to 10 μM in SPR buffer HBS-P .ka/kd calculations. Fitting of sensorgram data was carried out according to global fitting, and the ka and kd values were calculated with a 1:1 Langmuir model.In all SPR experiments, analyte solutions of different concentrations were passed over the sensor chip with immobilized protein or 2-5A at a flow rate of 10 μl/min for 3 min, and association was monitored. Dissociation was monitored using SPR buffer for an additional 5 min. Data was normalized against a reference channel immobilized with GST alone. Analysis and fitting was performed with BIAEvaluation software, version 3.2 (Biacore Inc.), with the option for simultaneous Infection of BMM. Virus was added to cells at an MOI of 1 PFU/cell and allowed to adsorb for one hour at 37°C. Cultures were washed with PBS (3 times) and then fed with medium as described for each cell type. The culture supernatants were harvested at the times indicated for the specific experiments and titered by plaque assay on L2 cells .in vivo. Four- to five-week-old B6 mice or RNase L–/–mice were anesthetized with isoflurane  and inoculated intrahepatically with 2000 plaque forming units (PFU) of WT A59, ns2H126R or chimeric MHV-L* viruses in 20 μl of PBS containing 0.75% BSA. Organs were harvested at day 5 post infection and viral titers were determined by plaque assay on L2 cells [Infections of mice L2 cells .Statistics were done using the tests indicated in the figure legends using GraphPad Prism. Error bars represent standard error of the means. * indicates a statistically significant difference between indicated conditions (p< 0.05).S1 Fig1–92 was used as a negative control. A. Design of the experiment. B. Visualization of Flag-muANK R1-2-eGFP fluorescence (green). L*1–92  expression did not induce any relocation of the fusion protein. L* WT expression led to Flag-muANK R1-2-eGFP relocation to the cytoplasmic compartment. C. L* did not concentrate the fusion protein at the mitochondrial surface. Upper panels: visualization of Flag-muANK R1-2-eGFP fluorescence , HA-L*  and mitochondria . Lower panels: merge of Flag-muANK R1-2-eGFP (green) and mitochondria  (left), merge of HA-L* (blue) and mitochondria  (middle), and merge of Flag-muANK R1-2-eGFP (green), L* (blue) and mitochondria  (right).HeLa-M cells were transduced to stably express Flag-muANK R1-2-eGFP. Transduced cells exhibited a diffuse cytoplasmic and nuclear green fluorescence. L* was then introduced in the cells either by transfection or by infection to assess whether L* expression would trigger the relocation of Flag-muANK R1-2-eGFP to the cytosol and/or the mitochondrial surface. The truncated inactive L*(PDF)Click here for additional data file.S2 FigDA. Values under the lanes indicate the extent of rRNA degradation inhibition compared to that of WT mouse RNase L. B. Inhibition of mouse RNase L carrying indicated rat RNAse L residues (left) and of rat RNase L carrying indicated mouse RNAse L residues (right) by L*DA and L*RTV-1. Values under the lanes indicate the extent of rRNA degradation inhibition by L* compared to that mediated by L*DA on mouse RNase L (left panel) or compared to that mediated by L*RTV-1 on WT rat RNase L (right panel).Analysis of RNase L-mediated RNA degradation in HeLa-M cells overexpressing indicated Flag-RNase L chimera and HA-L*. RNA samples extracted 7 hours after polyI:C transfection were analyzed by RNA chips and quantified. A. Inhibition of mouse RNase L carrying indicated human RNAse L residues (left) and of human RNase L carrying indicated mouse RNAse L residues (right) by L*(PDF)Click here for additional data file.S3 Fig6-L* was immobilized on Ni-NTA chips and human RNase L was injected at the indicated concentrations. B. His6-L* was immobilized on Ni-NTA chips and mouse RNase L was injected at the indicated concentrations. C. GST-mouse RNase L was immobilized on anti-GST CM5 sensor chip and L* was injected at the indicated concentrations. RU: response units.Surface plasmon resonance (SPR) experiments were performed using Biacore technology with purified recombinant L*, mouse RNase L and human RNase L. A. His(PDF)Click here for additional data file.S4 FigA. Flag-L* expression in infected BMM was detected by immunoblotting using an anti-Flag antibody. B. Analysis of RNase L-mediated RNA degradation was performed by RNA chip.(PDF)Click here for additional data file.