[PMC free article] [PubMed] [Google Scholar] 24. viral RNA-dependent RNA polymerase (RdRp) 3D, but also induced sumoylation and ubiquitination of the polymerase, which have been reported to facilitate its stability and boost viral replication (43). Taken together, our findings implied that m6A modification of EV71 RNA alpha-hederin constitutes an important process in the regulation of viral replication. MATERIALS AND METHODS Cell culture Vero (American Type Culture Collection (ATCC), Manassas, VA, USA; CCL-81), HEK293T (ATCC, CRL-11268)?and RD (ATCC, CCL-136) cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gibco) with 5% CO2 at 37C. Viruses EV71 (strain XF; Microorganisms & Viruses Culture Collection Center (MVCCC)) was obtained from the MVCCC, Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS). Viruses were amplified and titrated by 50% tissue culture infectious dose (TCID50) in Vero cells using the ReedCMuench formula (44). m6A-Methylated RNA immunoprecipitation (MeRIP) and Northern blotting Total RNA was extracted from Vero cells infected with strain EV71-XF at a multiplicity of infection (MOI) of 0.1 using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). EV71 RNA was transcribed from a cDNA plasmid (45) linearized by HindIII using the MEGAscript? T7 Kit (Ambion, Austin, TX, USA) according to the manufacturer’s protocols. SLC7A7 For MeRIP, 300 g of total RNA or 10 g transcribed EV71 RNA were incubated with an anti-m6A antibody (Synaptic Systems, Goettingen, Germany) or a IgG antibody in 300 l of immunoprecipitation (IP) Buffer (150 mM NaCl, 0.1% NP-40, 10 mM TrisCHCl, pH 7.4) for 2 h at 4C. The mixture was then incubated with 20 l of anti-rabbit antibody conjugated magnetic beads (NEB, Ipswich, MA, USA; S1432S), that have been cleaned 3 x with 500 l of IP buffer after that, followed by spinning for 2 h at 4C. Beads had been washed six situations with 500 l of IP buffer and incubated with 300 l of elution buffer (5 mM TrisCHCl, pH 7.5, 1 mM EDTA, pH 8.0, 0.05% sodium dodecyl sulfate (SDS), and 4.2 l of 20 mg/ml proteinase K) for 1.5 h at 50C. The eluted RNA was extracted with phenol/chloroform and precipitated with ethanol. All of the RNAs gathered from MeRIP had been separated on 1% agarose/2.2 M formaldehyde gels in working buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0) for 13 h in 28 V. The RNAs had been used in Hybond-N+ membranes in 20 SSC buffer (3.0 M NaCl, 0.3 M sodium citrate) overnight. UV-crosslinked to a membrane, and hybridized using a DIG-labelled EV71 probe (nt 1C7405). Probe recognition was performed using the Drill down Luminescent Detection Package II (Roche, Madison, WI, USA) based on the manufacturer’s guidelines. Signals had been developed on the ChemiDoc??MP imaging program (Bio-Rad Laboratories, Berkeley, CA, USA). MeRIP-Seq MeRIP-Seq from the EV71 methylome was completed regarding to a previously released process (46). In short, total mobile RNA extracted from EV71-contaminated Vero cells was fragmented by ZnCl2 accompanied by ethanol precipitation. Fragmented RNA was incubated with an anti-m6A antibody (Synaptic Systems, 1:300). MeRIP was executed as previously defined (46). The eluted RNA and insight had been put through high-throughput sequencing using regular protocols (Illumina, NORTH PARK, CA, USA). The MeRIP-Seq data had been analyzed as defined previously (32). Ultra-high functionality liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) EV71 share (1 L at 2 108 TCID50/ml) was focused by ultracentrifugation at 26 000 rpm within a SW28Ti rotor (Beckman Coulter, Brea, CA, USA) for 2 h at 4C. Viral RNA was extracted using an RNeasy mini package (QIAGEN, Venlo, HOLLAND). UHPLC-MS/MS evaluation was performed with an Agilent 1290 UHPLC program in conjunction with an alpha-hederin ESI-triple quadrupole mass spectrometer (G6410B or G6495, Agilent Technology, Santa Clara, CA, USA) regarding to previously released guidelines (47). Formaldehyde-crosslinked RNA-immunoprecipitation (RIP) Two 10-cm plates of 95% confluent RD cells had been used for alpha-hederin every sample. Cells had been crosslinked with the addition of phosphate buffered saline (PBS) filled with 1% methanol-free formaldehyde and incubated for 10 alpha-hederin min at 37C. Cross-linking was terminated with the addition of 2.5 M glycine to your final concentration of 0.125 M. Cells had been washed 3 x with ice-cold PBS and scraped from the.