Cell lines. Bmi1 immortalized nasopharyngeal epithelial cells (NPECs-Bmi1) were cultured in keratinocyte serum-free medium (17005-075; Invitrogen, California). The NPECs-Bmi1 monolayer cells (MLCs) and sphere-like cells (SLCs) were formed according to a published protocol27. Briefly, 5 × 104 or 5 × 105 NPECs-Bmi1 cells were seeded into 48-well plates and cultured for 36 h. Cells seeded at 5 × 104 per well turned to grow as MLCs, while cells seeded at 5 × 105 per well grew as SLCs. Human embryonic kidney 293T (HEK-293T) cells were grown in DMEM (C11995500BT; GIBCO, California) supplemented with 10% (vol/vol) FBS (10099-141C; GIBCO, Australia). HNE1, HK1, CNE1, AGS, Raji, MKN74 and Akata cells were maintained in RPMI medium 1640 (C11875500BT; GIBCO, California) supplemented with 5% (vol/vol) FBS. All cells were cultured at 37 °C in a humidified atmosphere comprising 5% CO2 incubators. HEK-293T cells were purchased from ATCC; NPECs-Bmi1 were established by our laboratory as described before (26); CNE1 cells were a kind gift from Professor Quen-Tin Liu (Sun Yat-sen University, Guangzhou, China); HNE1 and HK1 cells were a kind gift from Professor Sai-Wah Tsao (University of Hong Kong, Hong Kong SAR); AGS cells were a kind gift from Professor Qian Tao (Chinese university of Hong Kong, Hong Kong SAR); MKN74 cells were a kind gift from Professor Xu Rui-Hua (Sun Yat-sen University, Guangzhou, China); EBV-positive Akata cells were a kind gift from Professor Maria G. Masucci (Karolinska Institute, Sweden).
Reagents: Peptides based on amino acids 1 to 50 of R9AP, including R9AP1-12 (MAREECKALLDG), R9AP13-24 (LNKTTACYHHLV), R9AP19-30 (CYHHLVLTVGGS), R9AP30-41 (SADSQNLRQELQ), and R9AP35-46 (NLRQELQKTRQK), as well as a scrambled control peptide (LVHYTHCGSLGV) were synthesized by CHINESE PEPTIDE (China). The rabbit anti-R9AP antibody used for Western blotting (WB), immunofluorescence staining and immunohistochemistry staining was purchased from Sigma-Aldrich (HPA049791, Germany). AMMO1 and CL59 antibodies were a kind gift from Professor Andrew T. McGuire (University of Washington) and Professor Richard Longnecker (Northwestern University), respectively. PreScission protease (PSP) was a kind gift from Professor Song Gao (Sun Yat-sen University Cancer Center). All other reagents were obtained from Sigma-Aldrich, unless indicated otherwise.
Microarray analysis. NPECs-Bmi1 MLCs and SLCs were formed, then, total RNA was extracted using TRIzol reagent (T9424; Sigma-Aldrich, Germany), according to the manufacturer’s instructions. The integrity of the RNA was checked on a Bioanalyzer 2100 (Agilent Technologies, USA). The microarray experiments were performed by Shanghai Biochip Corporation (Shanghai; China) using the Agilent Whole Human Genome Oligo Microarray 4×44K (Agilent Technologies, California). The cRNA synthesis, cRNA labelling, sample fragmentation, and microarray hybridization were performed based on the manufacturer’s standard protocols. The arrays were scanned using the Agilent Scanner G2505B (Agilent Technologies, California). Feature Extraction software (version 126.96.36.199, Agilent Technologies) was used to obtain raw data, which were normalized using the quantile algorithm in Genespring (version 9, Agilent Technologies). Differentially expressed genes were then selected according to the threshold set as fold change > = 2.0 and a P value < 0.05 according to the t-test. The identified differentially expressed genes were then subjected to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis. The microarray data were deposited with the NCBI GEO depository under the accession number of GSE159958. The zipped data includes raw data from the microarray for each sample, the normalized expression matrix of genes from all samples, and per-sample metadata. Differentially expressed genes were selected according to the threshold set as fold change >2.0 and a P value <0.05 by t-test. The bioinformatics analysis of differentially expression genes were conducted by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. The heatmap was generated using GraphPad Prism software.
Gene silencing. ON-TARGET plus SMART pool siRNAs against 72 upregulated membrane-associated genes and ON-TARGET plus siCONTROL Non-Targeting pool siRNA which was used as control were all purchased from GE Dharmacon (California). The two single siRNA duplexes against R9AP were as follows:
R9APsi1#:5’-GCGAGAUGAUCGACAACAU-3’; R9APsi2#:5’-GCAAAAGACGCGCCAGAAG-3’; All the siRNAs were delivered using RNAi MAX (13778150; Invitrogen, California) according to the kit instructions.
Generation of isogenic R9AP knockout cell lines. The generation of R9AP HNE1 and Raji knockout cell lines was based on CRISPR-Cas9 gene editing technology. The guide RNA (gRNA) sequences were 5’- CGAGTCCGCCGAGCCACCGA-3’ (R9APsg1#) and 5’- GCTGACCGTCGGTGGCTCGG-3’ (R9APsg2#). Oligonucleotides corresponding to the gRNA were synthesized and cloned into Cas9-expressing plasmid PX459 (48139, Addgene, Massachusetts) or lentiCRISPRv2 (52961, Addgene, Massachusetts). HNE1 cells were transiently transfected with R9AP gRNA cloned into pX459 using Lipofectamine 3000 transfection reagent (L3000015; Invitrogen, California). After 24h, transfected cells were selected using puromycin (60210ES80; Yeasen, China) at 1µg ml-1 for 3 days. Raji cells were infected with lentivirus encoding R9AP gRNA, and after 24h, the infected cells were selected using puromycin (1 µg ml-1) for 3 days.
Expression of cDNA. The indicated plasmids were delivered using Lipofectamine3000 following the instructions. The establishment of R9AP stable expression EBV-negative Akata cells was based on the pBABE-Puro Retroviral system (RTV-001-puro, Cell BioLabs, California).
In vitro EBV infection. The utilized EBV containing eGFP (EBfaV-GFP), was prepared in EBV-positive Akata cells36. EBV production, labeling, and determination of MOI and infectious particles were performed as reported previously27,28. SLCs of NPECs-Bmi1 and HEK-293T were infected with EBV at an MOI of ～300; HNE1, HK1, CNE1, AGS, MKN74, Raji and EBV-negative Akata cells cells were infected with EBV at an MOI of ～1000. The indicated cell lines were incubated with EBV for 3h at 37°C, and unbound virus was discarded by washing with PBS three times. Then, cells were cultured in fresh medium for 24h, followed by quantification of eGFP positive cells using flow cytometry (cytoFLEX; Beckman).
Fusion assay. The virus-free cell fusion assay was performed according to previous published protocol (30). Effector HEK-293T cells were transfected with T7 luciferase reporter plasmids with a T7 promoter, gB, gH and gL. Renilla luciferase was used as internal control. To examine the effects on fusion after expressing R9AP, target HEK-293T cells were transfected with the T7 DNA polymerase expression plasmid together with the R9AP expression plasmid or empty vector. To test the effects on fusion after R9AP knockdown, target HEK-293T cells were transfected with R9AP siRNA or the control siRNA, followed by transfected with the T7 DNA polymerase expression plasmid 36h later. After 24h, 2.5× 105 effector HEK-293T cells were mixed with 2.5× 105 target HEK-293T cells in 24-well plates and cultured for another 24h. The luciferase activity was measured via a dual-luciferase reporter assay system (E2920, Promega, Wisconsin) using the GloMax-96 Microplate Luminometer. The ratio of firefly luciferase activity to Renilla luciferase activity was used as the relative fusion activity.
Peptide blocking assay. EBV was pre-incubated at 4°C for 2h with either R9AP1-12, R9AP13-24, R9AP19-30, R9AP30-41 or R9AP35-46 peptide which was diluted to the concentration of 100 and 200μg mL-1 in FBS-free RPMI 1640, and then added to the cells. The scramble control peptide at 200μg mL-1 was used as control. Cells were treated with EBV at 37°C for 3h and subsequently washed three times by PBS. The percentage of EBV-infected cells was determined by flow cytometry at 24h post infection.
Protein expression and purification. GST-R9AP1-210 was expressed in E.coli BL21 (AI) cells (CB105-02; Tiangen, China). The cells were cultured on TB plates containing 0.1mg/L of ampicillin (A010-10g; MDbio, China) at 37°C overnight. A single colony was picked and transfected into liquid TB medium supplemented with ampicillin for expansion until an appropriate OD value (0.6-0.8) was reached. The culture was then cooled down to 18 °C and isopropyl-thio-β-D-galactopyranoside (IPTG; 1122GR025; BioFroxx, China) was added to a final concentration of 0.2mM to induce protein expression, which was continued for 8 hours at 18°C. The cells were then collected by centrifugation and resuspended in PBS, pH 7.4 containing Roche’s complete Protease Inhibitor (EDTA-free) (11697498001; Roche, Switzerland). Then, the cells were lysed by pressured crushing at 4°C, after which the supernatant was collected by centrifugation and filtered through a 0.22μm pore-size syringe filter. The recombinant protein was purified using a gravity column with Glutathione Sepharose 4B resin (17075601; GE healthcare, Massachusetts) which was washed with the re-suspension buffer 3 times and eluted with PBS, pH 7.5 containing 10mM GSH (G4251-25G, Sigma-Aldrich, Germany) at 4°C. Then, the samples from cell re-suspension, lysate, supernatant, column flow-through, resin wash buffer and resin elution buffer were analyzed by SDS-PAGE to identify the collected protein. The elution fractions were combined and concentrated to 1mL using a 10kD molecular weight cutoff ultracentrifuge tube (UFC901096; Millipore, Germany) and loaded onto a size exclusion chromatography Superdex200 10/300GL column (28-9909-44; GE Healthcare, Massachusetts), which was eluted with PBS, pH 7.4 at room temperature. Fraction size was set to 0.5mL and each fraction was analyzed by SDS-PAGE. The SEC-purified protein was shock-frozen in liquid nitrogen and stored in -80 °C.
His-gH/gL was expressed in HEK-293T cells, which were transfected with the expression plasmid using polyethylenimine (PEI; 23966-1, Polysciences, Pennsylvania ) to HEK-293T cells at a ratio of 1mg plasmid/ 5mg PEI / 1×106 cells / 1L. The plasmid and PEI were diluted with HEK-293T medium (UP1000, Union, China) to 0.05mg mL-1 and 0.25mg mL-1 individually and mixed together for 15 minutes at room temperature before adding to adequate amount of cells. The transfected cells were cultured for 7 days under shaking at 120rpm. The supernatant was collected through centrifugation and filtered through a 0.22μm pore-size membrane. The following purification procedures were similar to those for protein expression in E.coli. His-resin (88229; Thermo, California) was used for affinity chromatography, washed by PBS pH7.4, 30mM imidazole (1460GR500; Biofroxx, Germany) and eluted by PBS pH7.4 (ZLI-9062; OriGene, China), 300mM imidazole. During SEC, PBS pH7.4 was used as running buffer for SEC.
Immunoprecipitation and GST pull-down assay. For the co-immunoprecipitation assay, HEK-293T cells were transfected with the indicated plasmid and lysed in lysis buffer containing 1%NP-40 (N885726; Macklin, China), 150mM NaCl (13423-6X1KG-R; Invitrogen, California), 2.5mM EDTA (E6758; Sigma-Aldrich, Germany), 20mM HEPES (H7523; Sigma-Aldrich, Germany) pH7.4 and protease inhibitor cocktail at 36h post transfection. The lysates were cleared by centrifugation at 12000 rpm and 4°C 10min . The supernatants were incubated with ANTI-FLAG M2 Gel (A2220, Sigma-Aldrich, Germany) or Anti-c-Myc Agarose Affinity Gel (A7470, Sigma-Aldrich, Germany) overnight. Then, the gels with bound protein were washed three times with lysis buffer and subjected to WB analysis. For the GST pull-down assay, GST-R9AP1-210 and His-gH/gL were incubated in lysis buffer overnight, then washed three times with lysis buffer and analyzed by WB. For the antibody competition binding assay, HEK-293T cells were transfected with plasmids encoding FLAG-R9AP or Myc-gH/gL for 36h, and lysed in lysis buffer. HEK-293T cells transfected with empty vector were used as control. Lysates containing Myc-gH/gL protein were incubated with 5mg IgG control, 5mg Ammo1, 10mg Ammo1, 5mg CL59 or 10mg CL59 overnight, and then incubated with lysates containing FLAG-R9AP or the control overnight. Finally, Myc-gH/gL was pulled down using Anti-c-Myc Agarose Affinity Gel which was then washed three times with lysis buffer and subjected to WB analysis.
Biolayer Interferometry (BLI). BLI assays were performed on an Octet Red 96 instrument (18-1127; ForteBio, California) at 30°C with shaking at 1000rpm. All signals were recorded at standard frequency (5.0Hz). For kinetic analysis, Ni-NTA biosensors (18-5101; ForteBio, California) were incubated in PBS with 0.05% Tween20 (P7949; Sigma-Aldrich, Germany), the dilution buffer used throughout the whole assay, for 15min before performing the kinetic analysis. After 60s of primary baseline, His-gH/gL protein diluted with the buffer was loaded at 0.5mg mL-1 for 120s, followed by a secondary baseline equilibration for 30s. Then, the association of baseline-control and GST-R9AP1-210 at a gradient of concentration from 6.25nM to 100nM was recorded for 180s, followed by a transition to a dissociation process for 600s and multiple rounds of regeneration with 10mM Glycine pH1.5 (GE healthcare). Similar procedures were performed for determination of binding affinity of R9AP peptide/control peptide to gH/gL, except changes of association time and dissociation time to 100s and 200s.The raw curves were baseline-subtracted before fitting to the 1:1 binding model using the ForteBio data analysis software, after which the mean kinetic parameters (kD, kon, koff etc.) were rendered via a global fit to all binding curves. For competition analysis, His-gH/gL protein diluted with the same buffer was loaded onto the Ni-NTA biosensor at 1μg mL -1 for 180s. After 30s of equilibration, primary association of R9AP or PBST was recorded until saturation for 600s, followed by the secondary association of AMMO1 or CL59 for another 600s. The concentrations of GST-R9AP1-210, AMMO1 antibody and CL59 antibody used in the assay were 200nM, 100nM, and 100nM, respectively. The sensors were regenerated with 10mM Glycine pH1.5. Real-time binding was recorded during the experiment and competitive/non-competitive behavior was determined by the binding response presented by different association couples.
Immunofluorescence staining. For the co-localization assay of R9AP with Alexa Flour 594-labelled EBV, HNE1 cells were transfected with a plasmid expressing eGFP-tagged R9AP for 24h, then co-incubated with EBV labeled with Alexa Fluor 594 (R37117, Molecular Probes, California) for 1h at 37°C. After removing the unbound virus, cells were fixed with 4% paraformaldehyde (N1012, NCM biotech, China) in PBS for 20min at room temperature, and permeabilized with 0.1% Triton X-100 (0219485480; MPbio, California). The cell nuclei were counter-stained with 0.1% DAPI (D9542; Sigma-Aldrich, Germany). For the determination of exogenous R9AP localization, HNE1 cells were transfected with the indicated plasmids for 36h, then fixed with 4% paraformaldehyde in PBS, and either permeabilized with 0.1% Triton X-100 or left untreated. Then, the cells were incubated with an antibody specific for the Myc tag (M5546; Sigma-Aldrich, Germany) and an Alexa Flour 594-labelled goat anti-mouse IgG antibody. The cell nuclei were counter-stained with 0.1% DAPI. To detect the endogenous R9AP localization, HNE1 cells were fixed with 4% paraformaldehyde in PBS and incubated with an antibody targeting R9AP (HPA049791, Sigma-Aldrich, diluted 1:100) and Alexa Flour 594-labelled goat anti-mouse IgG antibody. The cell nuclei were counter-stained with 0.1% DAPI.
PSP digestion assay. Cells in 12-well plates were transfected with the indicated plasmids for 24h, and then fixed in 4% paraformaldehyde. Then, 10μg PSP was diluted in 500μl PBS and incubated with the fixed cells at 4°C for 8 h. The treated cells were washed three times with PBS and collected for WB analysis.
In vivo EBV infection of humanized mice. Ten immunodeficient B-NDG mice were purchased from BIOCYTOGEN (China), and divided into two groups. Human cord blood was obtained from Guangzhou Women and Children’s Medical Center (China) after obtaining informed consent. Human cord blood mononuclear cells were separated using Ficoll-Hypaque density gradient, and then 1×107 cells were injected i.p. into 4 to 5-week-old B-NDG mice. At the same day, mice were injected through the tail vein with R9AP19-30 peptide or control peptide at 20mg/kg of body weight, together with 30,000 infectious EBV particles. Then, the mice were injected intraperitoneally with R9AP19-30 peptide or control peptide at 20mg/kg of body weight on days -3, -7 and -14, and injected intraperitoneally with 50 mg OKT3 (B104; Nobimpex, Germany) on day -7. Blood was collected from the mice to extract DNA from mice on days- 0, -14, -28 and -42.
Measurement of EBV titers in mouse blood. To quantify the EBV DNA copy number in mouse blood, qPCR was used to detect the BamHI-W fragment of the EBV genome using the primers 5’-CCCAACACTCCACCACACC-3’ and 5’-TCTTAGGAGCTGTCCGAGGG-3’. A calibration curve was made based on the EBV Nucleic Acid standards (BDS).
Analysis of R9AP and EBERs expression in human tissues. Samples of human tissue from the tongue, floor of the mouth, lymphoid tissuse, nasopharyngeal carcinoma, gastric carcinoma and B cell lymphoma were all obtained from patients who were admitted to the Sun Yat-sen University Cancer Center, and signed written informed consent forms. Samples of tissue from the tongue, floor of the mouth, and lymphoid tissues were analyzed by hematoxylin-eosin staining (H＆E) and R9AP antibody staining. To detect R9AP, the rabbit polyclonal antibody against human R9AP (HPA049791, Sigma-Aldrich, diluted 1:50) was used as primary antibodies, which was incubated overnight at 4°C. After washing three times in PBST, the tissue sections were incubated with anti-rabbit secondary antibody (1:1000, Zymed, California), and then treated with 3-diaminobenzidine tetrahydrochloride for 10 seconds, followed by staining with 10% Mayer’s hematoxylin (ZSGB-Bio). Staining of paraffin-embedded tissue sections for EBV EBERs was performed using the ISH detection Kit (ISH-7001, ZSGB-Bio, China).
Analysis of the spleen of EBV-infected mice. After the death of all the ctrl-treated mice, the R9AP19-30-treated mice were euthanized. The spleens of all the mice were fixed in formalin to examine if the animals had persistent EBV infection using H＆E staining, IHC staining with antibodies against human CD20 (B cell marker), and detection of EBV EBERs using the ISH detection Kit. The results were independently evaluated by two pathologists, who were blinded to the status of the samples. The expression of human CD20 and EBV EBERs was evaluated by counting 3 representative high-power fields (×40 objective) per sample, with approximately 100 to 200 cells/field. The proportion of EBER-positive cells compared to human CD20-positive cells in the spleens of EBV infected humanized B-NDG mice was then calculated.
Plasmids. For EBV infection of HEK-293T or epithelial cells, cDNA fragments encoding R9AP, SLC26A9, CNGA1, the N-terminal amino acids 1 to 210 of R9AP (1-210), N-terminal plus transmembrane domain of R9AP (1-231), deletion of amino acids 1 to 50 of R9AP (△1-50), deletion of amino acids 51 to 100 of R9AP (△51-100), deletion of amino acids 101 to 152 of R9AP (△101-152) and deletion of amino acids 153 to 200 of R9AP (△153-200) were individually cloned into PCDNA3.1 (+) vector (V79020, Invitrogen, California); for EBV infection of EBV-negative Akata cells, the cDNA fragment encoding R9AP was cloned into the pBABE-Puro retroviral vector (Cell Biolabs, RTV-001-puro, California); for immunofluorescence staining in HNE1 cells, the cDNA of full-length of R9AP (R9APFL) or N-terminal amino acids 1 to 210 of R9AP (R9AP1-210) was cloned into the pCDNA6-Myc vector (V221-20, Invitrogen, California); for the PSP digestion assay, the sequence encoding the PSP recognition site and full-length sequence of R9AP (psp-R9APFL) or PSP site and N-terminal amino acids 1 to 210 of R9AP (psp-R9AP1-210) were cloned into the pCDNA3.1 (+) vector; for the immunoprecipitation assay, cDNA of R9AP was cloned into pCDNA3.1 (+) vector, myc-gH, gL or myc-gB were cloned into the pCDNA6-Myc vector; for the GST pull-down assay and protein expression, the sequence encoding N-terminal amino acids 1 to 210 R9AP was cloned into the pGEX6p-1-GST vector, which was a kind gift from Professor Song Gao (Sun Yat-sen University Cancer Center), and the sequences of gL (23-137AA, M81 strain) and gH ectodomain (19-679AA, M81 strain) connected by a linker (GGGGS)x3 were cloned into the pCDNA3.1(+) vector; for the cell-based fusion assay, expression plasmids for pCAG-T7, pT7EMC-Luc, gB, gH or gL, which were a kind gift from Wolfgang Hammerschmidt (Helmholtz Zentrum Munich) and Professor R. Longnecker (Northwestern University), were used.
Quantitative real-time PCR. Total RNA was extracted using TRIzol reagent (T9424; Sigma-Aldrich, Germany). To analyze gene expression, 1µg of RNA was reverse-transcribed using the RNA Reverse Transcription System (A5001; Promega, Wisconsin). The mRNA level was quantified using the LightCycler 480 SYBR Green I Master mix (04887352001; Roche, Switzerland) on a Roche LightCycler 480 instrument. All the gene expression data were normalized to the housekeeping gene beta actin (ACTB). Primers are listed below:
CNGA1: 5’-AAGGGAGGACCATCACAGA-3’ and 5’-TTCTGGTTCCTGGTCCTTA-3’
GPR1: 5’-TCTTATCTCATCGGCATCG-3’ and 5’-GCTTCGCTTCTTCACCTT -3’
SLC26A9:5’-CAACAAGCACGGCTACGAC-3’ and 5’-TTGAGGGAGTTCTTGAGATTGA-3’
R9AP: 5’-ATGAAGAGCGTTCGTGCCG-3’ and 5’-GCACGCAGTCGTCTTGTTGAG-3’
Statistical analyses. The results represent the means ± SEM from three independent experiments. The two-tailed unpaired Student’s t-test was used for statistical analysis involving two group comparisons for all in vitro experiments and detection of EBV DNA copy numbers in mice at the 6th week (ns, not significant; *P< 0.05, **P< 0.01, ***P< 0.001). A Log-rank test was used for statistical analysis involving two group comparisons for survival in the in vivo experiments. Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA).
Ethical standards. Written informed consents forms were signed by all the patients. All the utilization of human samples was approved by the institutional review board of each institution taking part in the project. All animal experiments with infectious EBV were performed in the animal biosafety level 2 facilities at Sun Yat-sen University Zhongshan School of Medicine, which was approved by the Committee on the Ethics of Animal Experiments of Sun Yat-sen University. The animal studies were carried out in strict accordance with the recommendations promulgated in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China.