Cell lines
Bmi1 immortalized NPECs (NPEC-Bmi1) were constructed and cultured in a keratinocyte serum-free medium (17005-075, Invitrogen) as described16. Human embryonic kidney 293T (HEK-293T) cells were purchased from ATCC and grown in DMEM (C11995500BT, Gibco) supplemented with 10% (vol/vol) fetal bovine serum (FBS; 10099-141C, Gibco). Raji, BJAB, Daudi, HNE1, HK1, CNE1, AGS, MKN74 and Akata cells were maintained in RPMI medium 1640 (C11875500BT, Gibco) supplemented with 5% (vol/vol) FBS. Raji, BJAB and Daudi cells were purchased from ATCC. The remaining cell lines were gifts. All cells were cultured at 37 °C in a humidified atmosphere comprising 5% CO2 incubators without mycoplasma contamination.
Reagents
Peptides including R9AP1–12 (MAREECKALLDG), R9AP13–24 (LNKTTACYHHLV), R9AP19–30 (CYHHLVLTVGGS), R9AP30–41 (SADSQNLRQELQ), R9AP35–46 (NLRQELQKTRQK) and scrambled control peptide (LVHYTHCGSLGV) were synthesized by Chinese Peptide. Antibodies used include: anti-R9AP for western blot and immunohistochemistry (HPA049791, Sigma-Aldrich), anti-R9AP for immunofluorescence (CSB-PA765076LA01Hu, Cusabio), anti-MYC tag (M5546, Sigma-Aldrich), anti-Flag (F3165, Sigma-Aldrich), anti-CNGA1 (DF3929, Affinity), anti-GPR1 (DF2720, Affinity), anti-SLC26A9 (SAB2105558, Sigma-Aldrich), anti-β-actin (8H10D10, 3700, Cell Signaling), anti-His tag (D3I1O, 12698, Cell Signaling), anti-EPHA2 (D4A2, 6997, Cell Signaling), anti-NRP1 (446921, AF3870, R&D Systems), CD27-AF700 (302814, BioLegend), mouse CD27-PE (LG.3A10 clone,BioLegend), EpCAM-AF594 (118222, BioLegend), CD19-AF647 (302220, BioLegend) and CD10-PE (312204, BioLegend). 72A1, CL59 and AMMO1 antibodies were generated based on the reported sequence by ourselves. PSP was a gift from S. Gao. All other reagents were obtained from Sigma-Aldrich unless indicated otherwise.
Microarray blockysis
NPEC-Bmi1 MLCs and SLCs were formed and total RNA was extracted. Microarray experiments were performed by Shanghai Biochip using the Agilent Whole Human Genome Oligo Microarray 4×44K (Agilent Technologies). Differentially expressed genes were selected according to the threshold set as fold change ≥ 2.0 and a P value <0.05 according to t-test. The heat map was generated using GraphPad Prism.
Gene silencing
ON-TARGET plus SMART pool siRNAs against 72 upregulated membrane-blockociated genes and ON-TARGET plus siCONTROL Non-Targeting pool siRNA were purchased from GE Dharmacon (California). The two single siRNA duplexes against R9AP were as follows:
siR9AP 1: 5′-GCGAGAUGAUCGACAACAU-3′; siR9AP 2: 5′-GCAAAAGACGCGCCAGAAG-3′. siRNAs were delivered using RNAi MAX (13778150, Invitrogen) according to the kit instructions.
Generation of isogenic R9AP-knockout cell lines
The generation of HNE1, AGS, MKN74, and Raji R9AP-knockout cell lines was based on CRISPR–Cas9 gene-editing technology. The guide RNA (gRNA) sequences were 5′-CGAGTCCGCCGAGCCACCGA-3′ (sgR9AP 1) and 5′-GCTGACCGTCGGTGGCTCGG-3′ (sgR9AP 2). Oligonucleotides corresponding to the gRNA were synthesized and cloned into Cas9-expressing plasmid lentiCRISPRv2 (52961, Addgene). HNE1 cells, AGS cells, MKN74 cells and Raji cells were infected with lentivirus encoding R9AP gRNA for 6 h, and after 24 h, cells were selected with puromycin (1 µg ml−1) for 3 days. Then, these selected cells were diluted to one cell per 100 μl medium and cultured in 96-well plates to obtain the single-cell-derived R9AP-knockout cell clones.
Expression of cDNA
The indicated plasmids were delivered using Lipofectamine 3000 (L3000150, Invitrogen) following the instructions in the kit. The establishment of R9AP stable expression EBV-negative Akata cells was based on the pBABE-Puro Retroviral system (RTV-001-puro, Cell BioLabs) or pHAGE-puro Lentivirus vector (#118692, Addgene).
In vitro EBV infection
Recombinant EBV encoding an GFP maker (EBfaV-GFP) was prepared from EBV-positive Akata cells19,42. Viral gene equivalents were determined by blockysing viral supernatants, as reported previously, by qPCR49,50,51. SLCs of NPEC-Bmi1, HEK-293T, Raji, BJAB and EBV-negative Akata cells were infected with ~10–50 encapsidated EBV genomes per cell; HNE1, CNE1, AGS and MKN74 cells were infected with ~50–100 encapsidated EBV genomes per cell. The indicated cell lines were incubated with EBV for 3 h at 37 °C, and the unbound virus was discarded by washing with PBS three times. Then, cells were cultured in a fresh medium for 24 h, then quantified GFP-positive cells using flow cytometry.
Binding, entry and fusion blockay
EBV binding, entry and fusion blockay was performed as described16,18,19.
Peptide blocking blockay
EBV was pre-incubated at 4 °C for 2 h with the indicated peptide, diluted to 100 or 200 μg ml−1 in FBS-free RPMI 1640, then added to the cells. The scramble control peptide was used at 200 μg ml−1. Cells were co-incubated with EBV at 37 °C for 3 h and washed 3 times with PBS. The percentages of EBV-infected HNE1 and Raji cells were determined by flow cytometry at 24 h post-infection. Primary NPECs were collected after 24 h, and primary B cells were collected after 72 h to extract DNA. EBV DNA copy number was determined by qPCR.
Protein expression and purification
GST–R9AP1–210 was expressed in Escherichia coli BL21 (DE3) cells (CB105-02, Tiangen), His–gH/gL protein containing gH amino acids 19–682, gL amino acids 23–137 and His–gp42 protein containing the extracellular portion of gp42 (amino acids 34–223) were expressed in expi293F cells (A14527, Thermo Fisher). Proteins were purified as described52,53.
Immunoprecipitation and GST pull-down blockay
For co-immunoprecipitation blockays, HEK-293T cells were transfected with the indicated plasmid and lysed in lysis buffer containing 1% NP-40 (N885726, Macklin), 150 mM NaCl, 2.5 mM EDTA, 20 mM HEPES pH 7.4 and protease inhibitor ***tail (5892970001, Roche). The lysates were cleared by centrifugation at 12,000 rpm, 4 °C for 10 min. The supernatants were incubated overnight with Anti-Flag M2 Gel or Anti-MYC Agarose Affinity Gel. Then, gels were washed three times with lysis buffer and subjected to western blot blockysis.
To determine whether R9AP and EPHA2 could bind simultaneously to gH/gL, HEK-293T cells were transfected with MYC–R9AP and EPHA2 with Flag–gH/gL or empty vector for 36 h. Then cells were lysed and immunoprecipitated with antibodies against Flag as indicated. A portion of the sample was used as input and blockysed by western blot. The remaining sample was eluted by the Flag peptide. A portion of the elution was used as input and blockysed by western blot blockysis. The remaining elution was re-immunoprecipitated with antibody against MYC as indicated followed by western blot blockysis.
GST–R9AP1–210 and His–gH/gL were incubated in lysis buffer overnight, washed three times with lysis buffer, and blockysed by western blot for the GST pull-down blockay.
HEK-293T cells were transfected with Flag–R9AP or MYC–gH/gL for the antibody competition binding blockay and lysed. Cells transfected with empty vector were used as control. Lysates containing MYC–gH/gL protein were incubated with indicated antibody overnight and then incubated with lysates containing Flag–R9AP or the control overnight. Finally, MYC–gH/gL was pulled down using Anti-MYC Agarose Affinity Gel, washed three times with lysis buffer and blockysed by western blot.
For the effect of gp42 protein on the R9AP interaction with gH/gL, HEK-293T cells were co-transfected with R9AP, empty vector or Flag–HLAII, empty vector or MYC–gp42, and empty vector or MYC–gH/gL for 36 h. Cells were lysed. Then immunoprecipitated with antibody against MYC, followed by western blot blockysis using Image Lab (v.5.2.1) with the indicated antibody.
Biolayer interferometry
BLI blockays were performed on an Octet Red 96 instrument (18-1127, ForteBio), and the results were blockysed using ForteBio data blockysis software (v.8.0);. All signals were recorded at the standard frequency (5.0 Hz). For kinetic blockysis, Ni-NTA biosensors (18-5101, ForteBio) were incubated in PBS with 0.05% Tween-20 for 15 min before performing the kinetic blockysis. After 60 s of primary baseline, His–gH/gL protein diluted with the buffer was loaded at 0.5 μg ml−1 for 120 s, followed by a secondary baseline equilibration for 30 s. Then, the blockociation of baseline-control and GST–R9AP1–210 at a concentration gradient from 6.25 nM to 100 nM was recorded for 180 s, followed by a transition to a dissociation process for 600 s and multiple rounds regeneration with 10 mM glycine pH 1.5 (GE Healthcare). Similar procedures were performed to determine the binding affinity of R9AP peptide or control peptide to gH/gL, except for changes in blockociation time and dissociation time to 100 s and 200 s. The raw curves were baseline-subtracted before fitting to the 1:1 binding model using the ForteBio data blockysis software, after which the mean kinetic parameters (Kd, Kon, Koff) were rendered via a global fit to all binding curves.
For competition blockysis, His–gH/gL protein diluted with the same buffer was loaded onto the Ni-NTA biosensor at 1 μg ml −1 for 180 s. After 30 s of equilibration, the primary blockociation of GST–R9AP1–210 or PBST was recorded until saturation for 600 s, followed by the secondary blockociation of AMMO1 or CL59 for another 600 s. The sensors were regenerated with 10 mM glycine pH 1.5. Real-time binding was recorded during the experiment, and competitive or non-competitive behaviour was determined by the binding response presented by different blockociation couples.
EBV fluorescence labelling
The 72A1 antibody54 was labelled with LinKine AbFluor 594 Labeling Kit (KTL0540-50K, Abbkine) according to the manufacturer’s instructions. To label EBV with AF59, the labelled antibody was incubated with EBV solution at a ratio of 1:200 at 4 °C for 2 h.
Analysis of R9AP expression in cell lines and human samples by RT–qPCR and flow cytometry
Blood peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation. Tonsil biopsies were minced, sorted with EasySep Human EpCAM Pos Slctn Kit II (17846, STEMCELL) or EasySep Hu Memory B Cell Iso Kit (17864, STEMCELL). Then sorted cells were verified by flow cytometry using Beakman Gallios, CytExpert (v.2.4.0) with anti-Human Epithelial Cell Antibody, Clone 5E11.3.1 (60147, STEMCELL) or anti-human CD19, anti-mouse/rat/human CD27 and anti-human CD10 (302220, 124209 and 312204, respectively, Biolegend).
To detect R9AP by RT–qPCR using Bio-Rad CFX Maestro 1.1, RNAs of separated cells were extracted, and reverse-transcribed and reaction systems without reverse transcriptase were used as a negative control. The mRNA level was quantified and normalized to ACTB.
For detection of R9AP by flow cytometry, collected tonsil epithelial cells were stained with anti-human R9AP antibody or anti-human IgG antibody (CSB-PA765076LA01Hu or CSB-PA00120E1Rb, Cusabio) and Alexa Fluor 594 anti-mouse CD326 (EpCAM) Antibody (118222 Biolegend). Blood and tonsil cells were stained with anti-human R9AP or IgG antibodies (CSB-PA765076LA01Hu or CSB-PA00120E1Rb, Cusabio) and indicated antibodies. Followed by staining with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-11034, Invitrogen).
Immunofluorescence staining
For the co-localization blockay of exogenous or endogenous R9AP with EBV using OLYMPUS FV1000, GFP-tagged R9AP or GFP transfected HEK-293T cells or HNE1 cells were co-incubated with EBV labelled with Alexa Fluor 594 for 1 h at 4 °C and 30 min at 37 °C. After removing the unbound virus, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized with 0.1% Triton X-100. Endogenous R9AP protein was stained with R9AP antibody (HPA049791) and Alexa Flour 488-labelled goat anti-rabbit IgG antibody. To determine exogenous R9AP localization, HNE1 cells were transfected with indicated plasmids, fixed and then either permeabilized with 0.1% Triton X-100 or left untreated. Then, cells were incubated with the antibody for the MYC tag (M5546, Sigma-Aldrich) and Alexa Flour 594-labelled goat anti-mouse IgG antibody. To detect the endogenous R9AP, HK1 cells were fixed and incubated with antibody targeting R9AP (HPA049791) and Alexa Flour 594-labelled goat anti-rabbit IgG antibody. For detection of endogenous R9AP in tonsil epithelial cells, memory, and naive B cells, frozen tonsil tissue sections were fixed, then incubated with R9AP antibody or anti-human IgG antibody (CSB-PA765076LA01Hu or CSB-PA00120E1Rb), anti-human CD19 and anti-mouse/rat/human CD27 Antibody (302220 and 124209, Biolegend), followed by staining with Goat anti-Rabbit IgG (H+L) antibody, Alexa Fluor 488 (A-11034, Invitrogen). Cell nuclei were counter-stained with 0.1% DAPI (D9542, Sigma-Aldrich).
PSP digestion blockay
Cells were transfected with the indicated plasmids for 24 h and fixed in 4% paraformaldehyde. Then, 10 μg PSP was diluted in 500 μl 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 western blot blockysis.
R9AP monoclonal antibody screening with rabbit immunization
Two three-month-old New Zealand white male rabbits (Genescript) were immunized intramuscularly with GST–R9AP1–210 mixed with an equal volume of Freund’s complete adjuvant four times at two-week intervals to establish the humoral response to R9AP. To determine the R9AP-specific antibody titre, PreScission protease (Beyotime) was added to GST–R9AP1–210 to remove the GST tag. Post-immunization blood was collected to confirm the seropositive level for R9AP. Then, rabbits were euthanized and the spleens were collected to collect PBMCs.
To isolate the R9AP-specific B cells, PBMCs were dyed with reagents and antibodies for sorting, including anti-rabbit IgG-AF488 (Abcam) and R9AP labelled with AF594. IgG+R9AP+ B cells were sorted using a BD Rhapsody cell sorter into 96-well plates containing precoated 293T-rCD40L as feeder cells, 20 ng ml−1 rabbit IL-2 (Beyotime), and 35 ng ml−1 human IL-21 (Sigma). Isolated B cells were observed for viability and cultured for seven days, and the supernatants of each well were collected for ELISA. If the endpoint ELISA titre for R9AP was greater than 512,000, the B cells from that well would be collected for further clonal sequencing.
To perform B cell clonal sequencing, B cell RNA was extracted, and cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen). Antibody variable regions of heavy and light chains were amplified via nested PCR with GXL polymerase (Takara). The first PCR used gene-specific primers, while the second employed primers overlapping the leader sequence (5′) and CH1 or Cκ regions (3′). PCR products were cloned into pcDNA3.1 vectors containing rabbit constant regions for antibody production.
Antibody blocking blockay
Primary B cells, NPECs, Akata, Raji, HNE1, AGS and HEK-293T cells were pretreated with anti-R9AP monoclonal antibody (5E9), then infected with EBV. EBV infection efficiency was blockysed by counting colony formation using B cell transformation blockays and calculating EBERs-positive cells by in situ hybridization (ISH) in primary B cells, by calculating the copy number of EBV DNA using qPCR in NPECs, or by flow cytometry in Akata, Raji, HNE1, AGS and HEK-293T cells.
In vivo EBV infection of humanized mice
Ten four- to five-week-old female immunodeficient NOD/SCID gamma (NDG) mice were purchased from Biocytogen and randomized, divided into two groups. Human cord blood was obtained from Guangzhou Women and Children’s Medical Center (China). Human cord blood mononuclear cells were separated, and then 1 × 107 cells were injected intraperitoneally into 4 to 5-week-old B-NDG mice. Oethicn the same day, mice were injected through the tail vein with indicated peptide at 20 mg kg−1 of body weight and 30,000 encapsidated EBV genomes. Then, the mice were injected intraperitoneally with indicated peptide at 20 mg kg−1 of body weight on days −3, −7, and −14 and injected intraperitoneally with 50 μg OKT3 (B104, Nobimpex) on day −7. Blood was collected from the mice to extract DNA on days- 0, −14, −28 and −42. 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′.
Analysis of R9AP and CR2 expression by immunohistochemistry and EBER expression by ISH
Human tissues from the tongue, the floor of the mouth, lymphoid tissue, nasopharyngeal carcinoma, gastric carcinoma and B cell lymphoma were obtained from patients admitted to the Sun Yat-sen University Cancer Center. To detect R9AP and CR2 by immunohistochemistry, the antibodies against human R9AP (HPA049791, Sigma-Aldrich, diluted 1:50) and CR2 (EP64, ZSGB-Bio, diluted 1:200) were used. EBERs were detected using the ISH detection Kit (ISH-7001, ZSGB-Bio).
Analysis of the spleen of EBV-infected mice
Mice were euthanized at seven weeks. The spleens of all the mice were fixed in formalin to examine if the animals had persistent EBV infection using H&E and immunohistochemistry with antibodies against human CD20, and detection of EBERs using the ISH detection Kit. The results were independently evaluated by three pathologists, who were blinded to the status of the samples. The expression of human CD20 and EBERs was evaluated by counting 3 representative high-power fields (×40 objective) per sample, with approximately 100 cells per field.
Plasmids
cDNA fragments encoding R9AP, SLC26A9, CNGA1, R9AP1–210, N-terminal plus TMD of R9AP (1–231), R9AP deletion mutants (Δ1–50, Δ51–100, Δ101–152 and Δ153–200), the sequence encoding the PSP recognition site and full-length sequence of R9AP (psp–R9AP) or PSP site and N-terminal amino acids 1 to 210 of R9AP (psp–R9AP1–210), the sequences of gL (amino acids 23–137, M81 strain) and gH ectodomain (amino acids 19–679, M81 strain) connected by a linker (GGGGS)×3 were individually cloned into PCDNA3.1 (+) vector (V79020, Invitrogen); for EBV infection of EBV-negative Akata cells, the cDNA fragment encoding R9AP was cloned into the pBABE-Puro retroviral vector (RTV-001-puro, Cell Biolabs) or pHAGE-puro lentivirus vector (#118692, Addgene); for immunofluorescence staining in HNE1 cells or immunoprecipitation blockay, the cDNA of full-length of R9AP, R9AP1–210, MYC–gH, gL or MYC–gB was cloned into the pCDNA6 vector; for the GST pull-down blockay, the sequence encoding R9AP1–210 was cloned into the pGEX6p-1-GST vector; for the cell-based fusion blockay, expression plasmids for pCAG-T7, pT7EMC-Luc, gB, gH or gL were used.
RT–qPCR
Total RNA was extracted using TRIzol reagent. RNA was reverse-transcribed using the RNA Reverse Transcription System (A5001, Promega). The mRNA level was quantified using the LightCycler 480 SYBR Green I Master mix (04887352001, Roche). All the gene expression data were normalized to ACTB. Primers were as follows: 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′; EBER1: 5′-GCCGAATACCCTTCTCCCA-3′ and 5′-TGCCCTAGTGGTTTCGGACA-3′; EBNA1: 5′-GTAGGGGATGCCGATTATTTTG-3′ and 5′- CTCCTTGACCACGATGCTTTC-3′; BZLF1: 5′-CCCAGTCTCAGACATAACCC-3′ and 5′- CAGGCTGTGGAGCACCAATG-3′; BXLF2: 5′- TCGATTGGGCTGGTCTGAG-3′ and 5′- TTAACCTCGCTAAGGCTGGC-3′.
Statistical blockyses
The results represent the mean or mean ± s.e.m. from two or three independent experiments or the indicated number of biological replicates. Statistical blockyses were performed using GraphPad Prism (GraphPad Software).
Ethical standards
Written informed consent forms were signed by all the donors and patients. The utilization of human samples was approved by the institutional review board of each institution taking part in the project. Human cord blood was obtained from Guangzhou Women and Children’s Medical Center (China), approved by the Committee on the Ethics of Guangzhou Women and Children’s Medical Center. Human tissues including lymphoid tissues, floor of mouth, tongue, lymph node, liver, lung, thyroid, blood, tonsil, nasopharynx epithelium, gastric mucosa and tumour tissues were collected from Sun Yat-sen University Cancer Center, approved by the Committee on the Ethics of Sun Yat-sen University Cancer Center. All animal experiments with infectious EBV were performed in the animal biosafety level 2 facilities at Sun Yat-sen University Zhongshan School of Medicine, approved by the Committee on the Ethics of Animal Experiments of Sun Yat-sen University. The animal studies were carried out according to 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.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
