Cells, viruses and reagents
RD cells, 293 T cells, HeLa cells and ADAR1 stable knockdown cell line (ADAR1 KD) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, USA) with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin and streptomycin (HyClone, USA) at 37 °C with 5% CO2. EV-D68 (Fermon strain) was kept in our lab and propagated in RD cells. HPIV3 (NIH47885) was kindly granted by Professor Mingzhou Chen of Wuhan University and propagated in HeLa cells by inoculation at a multiplicity of infection (MOI) of 0.1.
Plasmids mediated gene expression and siRNA mediated gene targeting
Plasmids pCAGGS, pCAGGS-DDK-ADAR1p150 (termed as ADAR1p150 or DDK-ADAR1p150), plasmids M07, M07-HA-ADAR1p110 (termed as ADAR1p110 or HA-ADA1p110), M07-HA-ADAR1p110-H910A, M07-HA-ADAR1p110-C996A and M07-HA-ADAR1p110-C1036A (termed as p110-H910A, p110-C996A and p110-C1036A) were described previously . And pCAGGS plasmids were used as control. The plasmids used for expressing dsRBDs deleted ADAR1p110 (termed as p110-△RBD) were generated by amplifying plasmid M07-HA-ADAR1p110 with the primers ADAR1-Drbd-F: ATTGGGGAGAACGAGAAGGCA and ADAR1-Drbd-R: CTTCTCGTTCTCCCCAATGTTCTTCAGCTGGCACTCTG, then ligated the PCR product by using seamless ligase kit (Biorun, China) and transformed to the DH5α competent cells. To generate the dual-luciferase reporter plasmids (termed as pSiCheck2-5′UTR), the 5′-UTR, including the first 19 amino acid coding regions in the ORF of EV-D68 (Fermon strain), was amplified using a primers pair, 5UTR-F: AAAGCTCTTCATAGTTAAAACAGCTCTGGGGTTG and FermVP4-R: AAAGCTCTTCAGGCGGTGGCTAGCGCAATGTTAGCATTCTCA. pSiCheck2 plasmids were amplified using the primers, Fluc-F: AAAGCTCTTCAGCCGATGCTAAGAACATTAAG and Rluc-R: AAAGCTCTTCACTAGAATTACTGCTCGTTCTTCAGCA. The two fragments were ligated based on the protocols of Golden Gate clone shuffling method [21, 22]. Small interfering RNA targeting ADAR1 (si-ADAR1) was purchased from Guangzhou RiboBio (RiboBio, China). All the plasmids and siRNAs were transfected into cells using Lipofectamine 3000 reagent (Invitrogen, USA) following the manufacturer’s instructions.
Viral infection and viral titers measurement
RD cells were seeded in 6-well plates. When the cell density reached 40–50%, EV-D68 inoculum was added and incubated at 37 °C with 5% CO2. After 1.5 h, the supernatant was replaced by DMEM containing 4% FBS. According to different experimental requirements, cell samples were collected after indicated hours. For HPIV3 infection, the same operations were performed on HeLa cells. The titer of virus was determined by TCID50 method. Specifically, RD cells were cultured in 96-well plates. When cell density reached 30–40%, the original medium was discarded and the cells were washed by PBS for two times. Meanwhile, the virus stock for testing was diluted in a tenfold gradient in DMEM medium, from 10–3 to 10–8. The PBS medium in the wells of 96 plates was discarded and then the diluted viral inoculums were added into the wells in order. Each dilution was replicated for 3 wells. Two hours later, the supernatant was replaced with DMEM medium contains 4% FBS. The cells were observed for 3–5 consecutive days to record the cytopathic effect (CPE). TCID50 was calculated by Spearman-Karber method. The formula is lgTCID50 = L + D (S-0.5). L is the logarithm of the lowest dilution, D is the dilution factor and S is the ratio of positive wells combined.
The infected or transfected cells were washed and harvested with pre-chilled phosphate buffered saline (PBS), then centrifuged at 13,000 rpm for 1 min, thereafter whirled with TNE buffer (50 mM Tris–Cl [pH 7.4], 150 mM NaCl, 2 mM EDTA [pH 8.0], 0.1% 2-mercaptoethanol and protease inhibitor cocktail) to lyse cell debris for 30 min. Cell lysates were centrifuged at 13,000 rpm for 30 min at 4 °C. The protein-containing supernatant was mixed with 5 × SDS-PAGE loading buffer, boiled at 100 °C for 10 min, then subjected to a 10% sodium dodecyl sulfate–polyacrylamide gel and electro-blotted the samples onto a PVDF membrane. Skim milk was dissolved with PBS with Tween 20 (1/1000 Tween 20) and the membrane was blocked for 30 min at room temperature. After that, the PVDF membrane was incubated with primary antibody for 1.5 h and secondary antibody for 45 min. The primary antibodies used were donkey anti-HPIV3 (1:2500, Abcam, United Kingdom), rabbit anti-ADAR1 (1:500, Santa Cruz, USA), rabbit anti-HA(1:4000, Proteintech, China), rabbit anti-DDK(1:4000, Proteintech, China), rabbit anti-beta-actin (1:1000, Proteintech, China), mouse anti-PKR (1:4000,huabio, China), mouse anti-P-PKR (1:2000,huabio,China), mouse anti-eIF2α(1:2000, huabio, China) and mouse anti-P-eIF2α(1:2000,huabio, China). HRP-conjugated goat anti-mouse IgG (1: 5000, Sigma, USA), HRP-conjugated goat anti-rabbit IgG (1:5000, Sigma, USA) and HRP-conjugated goat anti-donkey IgG (1:5000, Sangon, China) were used as secondary antibodies.
RD cells were digested with TRIzol reagent (Beyotime, China) and total cellular RNA was extracted. Total RNA was reverse transcribed into cDNA using PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan). The amount of ADAR1 and beta-actin were quantified by using BioRun ChemoHS qPCR Mix (SYBR) (Biorun, China) and Light Cycler (Roche, Switzerland). Data shown are the relative abundance of ADAR1 RNA with normalization to the housekeeping gene beta-actin. The primers used for RT-qPCR were as follows: Q-actin-F: TGAAGTGTGACGTGGACATCCG, Q-actin-R: GCTGTCACCTTCACCGTTCCAG and primers set for ADAR1 were described before .
HeLa cells were grown to 30–40% confluency in 12-well plates plated with cell slides and transfected of ADAR1 expression plasmids or infected with EV-D68 as described above. 24 h later, slides were taken out and washed three times with 4 °C PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 25 min, and blocked with 3% bovine serum albumin (BSA) for 30 min. Primary antibody was incubated for 1.5 h at room temperature. The primary antibody were rabbit anti-HA tag (1:100, Proteintech, China), rabbit anti-DDK tag (1:50, Proteintech, China) and mouse J2 anti-dsRNA (1:200, Scicons, Netherlands). Cells were subsequently washed 3 times with 1% BSA and incubated with secondary antibody for 45 min at room temperature. The secondary antibody used were Alexa Fluor 488 conjugated goat anti-rabbit IgG and Alexa Fluor 568 conjugated goat anti-mouse IgG (1:1000, Thermo, USA). Nuclei were stained with DAPI (SolarBio, China). Cells were observed by immunofluorescence microscopy (Nikon Ts2-FL, Japan).
The luciferase activity assay was performed according to the kit instructions (Promega, USA). Briefly, 24 h after transfection of pSiCheck2-5’UTR with firefly and renilla reporter gene luciferase expression plasmid, medium was removed and 400 μL of 1 × Lysis Reagent was dispensed into each cell. Pellet the debris by centrifugation at 12,000 rpm for 1 min and transfer the supernatant to a new tube. Mix 20 μL of cell lysates with 100μL of luciferase detection reagent and measure the resulting light intensity using the SpectraMax-iD5 (Molecular Devices, USA).
Sequencing of EV-D68 5’-UTR region
After infection with EV-D68, cellular total RNA was extracted with Trizol reagent (Beyotime, China) and reverse transcribed was performed by using reverse transcription kit (Takara, Japan). Thereafter, the EV-D68 5’-UTR region was amplified by using the 2 × PFU MasterMix (TIANGEN, China). A primer set complementary to the corresponding region was as follows: 5UTR-SF: TAAAACAGCTCTGGGGTTG, 5UTR-SR: GCATTCTCATGAGTTCCAG. At last, PCR products were analyzed by Sanger sequencing.
The amounts of protein in cell lysates were estimated based on the density of protein bands using Bio-Rad software Quantity One-4.6.2. The amount of ADAR1/beta-actin, VP1/beta-actin, HN/beta-actin in the corresponding lysates are normalized to the value obtained in the control group which is set to 1.0. The phosphorylation level of either PKR or eIF2a were quantified as the amount of p-PKR/PKR or p-eIF2a/eIF2a. The results are shown as average with standard error of mean (SEM), and GraphPad Prism 9.0 was used to analyze the statistics. Statistical analysis was performed using Student’s t-test, and p values less than 0.05 were considered significantly different and indicated by asterisks in the figures.