Materials and Reagents
Human embryonic kidney 293T (HEK293T) cells, human rhabdomyosarcoma (RD) cells, African green monkey kidney Vero cells, human neuroblastoma SK-N-SH cells, human cervical epithelial Hela cells, human lung fibroblast MRC-5 cells, and Human oral epidermoid carcinoma KB cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell lines were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). EVA71 (FJ08089 strain), CVA10 (SHAPHC798F/SH/CHN/2010) and the rescued CVA10 (AY421767.1) were propagated in RD cells. Viral titer was determined in RD cells by plaque assay.
Mouse monoclonal antibody against EV71 capsid protein VP1 was from Abcam (Cambridge, UK). The secondary antibody conjugated to Allophycocyanin (APC) was from BD Biosciences (Franklin Lakes, CA, USA). AlexaFluor594-Conjugated AffiniPure Goat Anti-Mouse IgG (H + L) was from ZSGB-BIO (Beijing, China). Fluoroshield Mounting Medium with DAPI was from Abcam. Mouse anti-Flag monoclonal antibody was from Sigma (M2 mAb, St Louis, MO, USA). Rabbit anti-GAPDH and mouse anti-3A monoclonal antibody were from Youke (Shanghai, China). Mouse anti-dsRNA antibody was from SCICONS (J2 mAb, Budapest, Hungary). Rabbit anti-Myc monoclonal antibody was from Cell Signaling Technologies (Danvers, MA, USA). Luciferase assay system was from Promega (E1500, Madison, WI, USA).
Three segments (1–2436, 2437–5064, 5065–7409) covering the CVA10 (AY421767.1) genome were synthesized and cloned in the pUC57 vector. The cloning backbone PL451 and the three segments of CVA10 carrying overlapping end (15–20 bp) were amplified and seamlessly linked together in the transformed E. coli by recombination according to HieffCloneTMMultiOneStep Cloning kit (YEASEN, Shanghai, China). The CVA10 capsid segments were seamlessly cloned into pcDNA6.0-EGFP to form the CVA10 capsid expresser. The construct of CVA10 replicon was derived from the plasmid of EVA71 replicon. Firstly, the original EVA71 5′-untranslated region (UTR) was replaced by CVA10 5′-UTR. Secondly, the non-structural gene segments of EVA71 (from 2A to 3′UTR) were replaced by that of CVA10. The cloning strategy and vector maps were illustrated in Supplementary Figure S1.
In Vitro Transcription
The plasmids of CVA10 infectious clone and replicon were linearized by SalI (NEB, Ipswich, MA, USA), and then purified by TIANquick Midi Purification Kit (TIANGEN, Beijing, China). The linearized plasmids were transcribed by T7 polymerase following the manual of MEGAscript T7 in vitro transcription kit (Cat. AM1334, Invitrogen, USA). The RNA quality was confirmed by agarose gel electrophoresis.
CVA10 mRNA was introduced into RD cells using Lipofectamine 3000 (Life Technologies, Carlsbad, USA) according to the manufacturer's instruction. RD cells were seeded at 3 × 105 cells/well in 12-wells plates. Next day, the mixture containing 0.5 μg of in vitro transcribed RNA and transfection reagents was added into RD monolayers (90% confluence) and incubated for 6 h at 37 ℃. The medium was then changed and cells were maintained in fresh culture medium.
Virus Next Generation Sequencing (NGS)
Virus NGS was conducted at Shanghai Tanpu Biotechnology Co., Ltd. Briefly, viral RNA was extracted from culture supernatants using the TIANamp RNA kit (TIANGEN, Beijing, China), and then the first strand cDNA synthesis was conducted using NEBNext Ultra RNA Prep Kit with random primers. Second Strand Master Mix was added into the original reaction mixture to synthesize the second strand. NGS libraries were prepared using the Nextera DNA sample preparation kit (Illumina, CA, USA) for Illumina sequencing according to the manufacture's instructions. After quality control, trimming and adapter clipping, data filtering and de-novo assembly, whole genome sequence was obtained and analyzed as described previously (Watson et al. 2013).
Viral Infection and Plaque Assay
The plaque assay was performed using 6 or 12-wells plates containing RD cell monolayers. Ten-fold series of viral dilutions were added and the plate was shaken every 15 min for 1 h. Then the inoculums were removed and 2 or 1 mL of DMEM containing 2% FBS and 1% low melting point agarose (Promega, Madison, USA) was added to each well, before incubation at 37 ℃. After cultured for 3–5 days, the plates were stained with 0.1% crystal violet (Sigma, St. Louis, USA) containing 10% formaldehyde and plaques were counted to measure the viral titer.
Transmission Electron Microscopy (TEM)
The CVA10 culture supernatant was harvested and the cell debris was removed by passage through a 0.45 μm filter (Pall, Puerto Rico). The virus sample was precipitated by incubation with 8% polyethylene glycol (PEG) 8, 000 in PBS (pH 7.4) at 4 ℃ for 12 h. After one more centrifugation, the virus resuspended in PBS and subsequently loaded onto a 10%–50% continuous sucrose gradient for ultracentrifugation in Beckman SW41 Ti rotors at 175, 000 g for 3 h. The fractions (60 mL per fraction) at 20% to 40% sucrose were collected and individually concentrated by diafiltration using an Amicon 100 K tube (Millipore, Belerica, MA USA) at 4000 g for 30 min. The concentrated virus was stored at 4 ℃. Characterization of virus particles was analyzed by negative staining electron microscopy. Briefly, virus was inactivated by 1/4000 (v/v) formalin at 37 ℃ for 3 days and then absorbed onto 200-mesh carbon-coated copper grid for 20 min at room temperature. The grids were washed twice with ddH2O and subsequently negatively stained with 2% phosphotungstic acid (pH 6.4) for 2 min. The stained grid was dried for 3 days and observed under a FEI Talos F200 transmission electron microscope (ThermoFisher, USA).
RD cells were seeded in 12-well plate (3 × 105 cells/well) with coverslips. The next day, RD cells were incubated with EV71, CVA10 or CVA10-Myc (MOI = 1) for 6 h. Then, RD cells were washed with cold PBS, fixed with 4% paraformaldehyde (Sigma, St Louis, USA) for 15 min, permeabilized with 0.05% Triton X-100 in 2% FBS/PBS, and then stained with mouse anti-dsRNA antibody (1:250 dilution, J2 clone) (SCICONS, Budapest, Hungary), mouse anti-3A antibody (1:1000 dilution, YOUKE) or rabbit antiMyc antibody (1:250 dilution, CST) for 1 h at room temperature. Three washes with PBS were followed by 30 min-incubation with the secondary antibodies. After washes with PBS, coverslips were stained by mounting medium with DAPI. Immunofluorescent imaging was taken on EVOS® FL Color Imaging Systems (Life technology, Grand Island, NY, USA).
Viral RNA Quantification
Viral RNA was extracted from infected cell lysates using the TIANamp RNA kit (Cat.SD101, TIANGEN, Beijing, China). Quantification of viral RNA was performed by quantitative real-time RT-PCR assay using specific primers for VP1. To make a standard curve, a fragment of CVA10 capsid from nt 2007 to 2145 was cloned into pcDNA6.0. The plasmid was linearized with SalI (NEB), in vitro transcribed using the MEGAscript T7 transcription kit (Invitrogen, USA), and DNase treated to generate the RNA standard.
Cells were harvested and lysed in Radioimmunoprecipitation assay buffer with a mixture of proteinase inhibitors (MedChem Express, Monmouth Junction, NJ, USA). The concentration of proteins was quantified by bicinchoninic acid protein assay (Biosharp, China) and equal amounts of proteins were loaded and separated by SDS-PAGE, then transferred to a nitrocellulose membrane. The membrane was blocked with blocking buffer (0.1% Tween-20 in PBS containing 5% BSA) and then was incubated overnight with primary antibody diluted in blocking buffer at 4 ℃. The membrane was then washed three times in 0.1% Tween-20/PBS and incubated with anti-mouse IgG conjugated to AlexaFluor790 (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. The immunoblots were visualized using an Odyssey Fc Imager (Lincoln, NE, USA).
SRIPs of CVA10, CVA10 HHRib and EVA71 were generated by sequential transfection of HEK293T cells with the capsid expresser and the CVA10 replicon mRNA. The capsid plasmid (1 lg) was firstly transfected into HEK293T cells at 60%–80% confluence. 24 h later, 1.5 lg of replicon RNA was then transfected using lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The pseudovirus was harvested 24 h post-RNA transfection with two rounds of freeze–thaw cycle. Then 200 μL of pseudovirus mixed with 800 lL of fresh DMEM was applied to RD cells in 12-well plates. The SRIPs were scored by assay of luciferase activity after 12 h infection at 37 ℃. Briefly, the inoculums were removed after 12 h incubation, and then the cells were washed twice by PBS and lysed directly on the plates by the addition of 150 lL of cell culture lysis reagent. Ten microliters of the cell lysate were then used to assay luciferase activity by using a luciferase assay kit (E1500, Promega, Madison, WI, USA). Luciferase activity was expressed as relative luciferase units (RLU).
Significance of differences was evaluated using Student's t test. Statistical analysis was performed with GraphPad Prism version 6.0 (La Jolla, CA, USA). The P values (P) less than 0.05 were considered statistically significant.