Functional analyses of mammalian virus 5′UTR-derived, small RNAs that regulate virus translation

Abstract

Enterovirus A71 (EV-A71¹) RNA contains an internal ribosomal entry site (IRES) to direct cap-independent translation. IRES-dependent translation requires the host’s translation initiation factors and IRES-associated trans-acting factors (ITAFs). We previously showed that hnRNP A1, the mRNA stability factor HuR, and the RISC subunit Argonaute 2 (Ago2) are ITAFs that associate with stem loop II (SL-II) of the IRES and promote IRES-dependent translation. By contrast, the mRNA decay factor AUF1 is a negative-acting ITAF that also binds SL-II. Moreover, the small RNA-processing enzyme Dicer produces at least four virus-derived, small RNAs (vsRNAs 1–4) from the EV-A71 5′UTR in infected cells. One of these, vsRNA1, derived from SL-II, inhibits IRES activity via an unknown mechanism. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with SL-II. This presents a possible mechanism by which vsRNA1 could control association of ITAFs with the IRES and modulate viral translation. Here, we describe methods for functional analyses of vsRNA1-mediated regulation of IRES activity. These methods should be applicable to other virus-derived, small RNAs as well.

1. Introduction

Enterovirus A71 (EV-A71) poses a persistent global public health threat. Typical EV-A71 infections cause hand-foot-and-mouth disease (HFMD), herpangina, or flu-like symptom. EV-A71 infection can also lead to severe neurological complication and death in children worldwide [1–3]. In 1998, an EV-A71 outbreak occurred in Taiwan. Over 120,000 people were infected and 78 children died [4]. A recent EV-A71 epidemic in Vietnam caused 53,000 children to be hospitalized and six died [5]. The World Health Organization has included EV-A71, and the related EV-D68, as emerging, non-polio enteroviruses in its Blueprint List of Priority Diseases (https://www.who.int/blueprint/priority-diseases/en/). However, there is no FDA-approved antiviral agent or vaccine against EV-A71. The China FDA approved the first inactivated EV-A71 vaccine in 2015. However, there are challenges for worldwide use of this vaccine, such as vaccine effectiveness against different pandemic strains worldwide and quality control of vaccine production [6].

EV-A71, a positive-stranded RNA virus, belongs to the genus Enterovirus in the Picornaviridae family. The genome is approximately 7500 nucleotides (nts). The 5′-untranslated region (5′ UTR) is 745 nt and is predicted to fold into six stem-loops (SL). Stem loop I is a cloverleaf-like structure for viral RNA replication. Stem loops II–VI comprise an internal ribosomal entry site (IRES) for cap-independent translation [7]. Many cellular factors associate with the EV-A71 5′UTR and facilitate viral RNA synthesis and/or virus replication [8,9]. Our previous studies identified four cellular proteins – hnRNP A1, mRNA decay factor AUF1, mRNA stability factor HuR, and the RISC subunit Argonaute 2 (Ago2) – that associate specifically with stem loop II (SL-II) to regulate viral translation [10–15]. While hnRNP A1, HuR, and Ago2 promote IRES-dependent translation, AUF1 negatively regulates viral translation. Additionally, EV-A71-infected cells utilize Dicer to produce several virus-derived, small RNAs (vsRNAs) from the 5′UTR. One of these, SL-II–derived vsRNA1 (nt 105 to nt 133; 5′-GUAACUUAGAAGCUGUAAAUCAACGAUCA-3′) inhibits IRES-dependent translation and virus replication by unknown mechanisms [16,17]. In vitro RNA-binding assays revealed that vsRNA1 enhances association of Ago2, HuR, and AUF1 with SL-II. Here, we describe methods for analyses of interactions between SL-II, ITAFs, and vsRNA1 and a mechanism by which vsRNA1 controls association of ITAFs with the IRES to modulate viral translation and virus replication. It is important to note that many of the methods we present here can be applied to analyses of small virus RNAs derived from other RNA viruses, applicable RNA-binding proteins, and infected cell types.

2. Materials and methods

2.1. Cell culture, infection, and virus titers

2.1.1. Cell culture and infection

Appendix A contains a reagent and equipment list. SF268 (human glioblastoma) cells [18] are cultured at 37 °C in RPMI medium supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific). RD (human embryonal rhabdomyosarcoma) [19] and Vero (African green monkey kidney) cells [20] are cultured at 37 °C in Eagle’s minimum essential medium (MEM) supplemented with 10% FBS. EV-A71 (TW/2231/98) is propagated in RD cells. Cells are infected with EV-A71 at the specified multiplicity of infection (MOI) and then incubated at 37 °C for 2 h for adsorption. Unbound virus is removed by washing cells three times with ice-cold PBS (Sigma-Aldrich), and cells are fed fresh medium. Media from infected cultures are harvested at various times, and EV-A71 titers are determined by plaque assay using Vero cells.

2.1.2. Virus plaque assay

Vero cells are grown in 6-well plates (2 × 10⁶ cells per well) in a 37 °C incubator supplied with 5% CO₂ for ~16 h. Cells should be ~90–100% confluent. A serial 10-fold dilution of virus is made by mixing 200 μl of virus with 1.8 ml PBS, 0.1% BSA. Medium is aspirated from plates. Each well is inoculated with 0.5 ml diluted virus. Plates are incubated at 37 °C for 2 h for adsorption. Plates are tilted every 20 min to assure even infection. While the plates are incubating, prepare agarose-overlay medium by adding 1/10 vol of 3% agarose to MEM supplemented with 2.5% FBS, and maintain the overlay at 45 °C. At the end of adsorption, inoculum is removed from plates and 2 ml agarose-overlay medium is added to each well. Plates are incubated at 37 °C for 3 days. To stain cells, 2 ml 0.01% neutral red/PBS is added to each well and incubated at 37 °C for 4 h. Plaques are counted to determine virus titer.

2.2. Plasmid construction and in vitro transcription

2.2.1. Construction of plasmid containing 5′UTR/IRES for RNA synthesis

A plasmid containing a 5′UTR/IRES of interest must be constructed for synthesis of RNA to be used in protein-RNA binding assays. The method described here can be applied to any 5′UTR/IRES using relevant primers.

Plasmid pT7-EV-A71 5′UTR is constructed as follows: the 5′UTR of EV-A71 is amplified by PCR from a full-length, infectious cDNA [21] using forward 5′-GCCGGTAATACGACTCACTATAGGGAGATTAAAACA GCCTGTGGGT and reverse 5′-CATGTTTGATTGTGTTGAGGGTCAAAAT primers which contain the T7 promoter. PCR is performed in a final volume of 25 μl, containing 1 μl of template cDNA, 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse primer, and 12.5 μl of OneTaq Hot Start Quick-Load 2X Master Mix with Standard Buffer (NEB) and run in Multigene Mini Personal Thermal Cycler (Labnet). The thermal cycling conditions are as follows: initial denaturation: 94 °C 10 min; extension for 30 cycles: 94 °C 30 s; 55 °C 30 s; 68 °C 1 min. Final extension: 68 °C 5 min. Hold: 4 °C. The PCR product is purified using the QIAquick PCR Purification Kit (Qiagen). Five volumes of buffer PB is added to one volume of PCR reaction, mixed, loaded on a QIAquick column and centrifuged at 12,000 rpm for 60 sec. To wash, 750 μl buffer PE is added to the QIAquick column and centrifuged at 12,000 rpm for 60 sec. To elute DNA, 50 μl buffer EB (10 mM Tris·Cl, pH 8.5) is added to the center of the QIAquick membrane and centrifuged at 12,000 rpm for 1 min. Purified DNA is analyzed on a 1% agarose gel.

The purified DNA is inserted into plasmid pCRII-TOPO by TA cloning (Life Technologies). The ligation reaction is set up as follows: 2 μl of purified PCR product; 2 μl of 5× Express Link T4 DNA Ligase Buffer [50 mM Tris-HCl, pH 7.6, 50 mM MgCl₂, 5 mM ATP, 5 mM DTT, 25% (w/v) polyethylene glycol-8000]; 2 μl of pCRII vector (25 ng/μl); 3 μl of water; 1 μl of Express Link T4 DNA Ligase (5 units). The ligation reaction is incubated at room temperature for 30 min. For transformation, centrifuge the ligation reaction briefly and place it on ice; thaw one 50 μl vial of frozen One Shot competent cells on ice; pipet 2 μl of ligation reaction directly into the vial of competent cells and mix by finger-tapping the tube wall; incubate the vials on ice for 30 min; heat shock the cells for 30 sec at 42 °C; add 250 μl of room temperature S.O.C. medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl₂, 5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM dextrose) to the vial; shake the vial at 37 °C for 1 h at 225 rpm in a shaking incubator; spread 150 μl from the transformation vial onto LB agar plates containing X-Gal and 100 μg/ml ampicillin; incubate plates overnight at 37 °C. For identification of positive clones, pick 10 white colonies for plasmid isolation; grow colonies overnight in 3 ml LB broth (10% peptone, 10% yeast extract, 5% NaCl) containing 100 μg/ml ampicillin; isolate and analyze plasmids by restriction enzyme mapping.

2.2.2. Synthesis of 5′UTR/IRES RNA for protein-RNA interaction analyses

The T7 promoter–EV-A71–5′UTR fragment (Section 2.2.1), flanked by EcoRI sites, is excised from plasmid pCRII-TOPO by EcoRI digestion. There is no need to gel-purify the promoter-containing fragment. RNAs are synthesized using the MEGAscript T7 kit (Life Technologies). The reaction is assembled as follows: 2 μl of DNA template (0.5 μg/μl), 2 μl of 50 mM ATP solution, 2 μl of 50 mM CTP solution, 2 μl of 50 mM GTP solution, 2 μl of 50 mM UTP solution, 2 μl of 10X Reaction Buffer [400 mM Tris-HCl (pH 7.9 at 25 °C), 100 mM NaCl, 60 mM MgCl₂, 20 mM spermidine], 6 μl of nuclease-free water, 2 μl of T7 polymerase.

RNA can also be biotinylated for pull-down assays of protein-RNA complexes using streptavidin-sepharose (see Section 2.3.1.1). In this case, biotinylated RNA is synthesized in a 20-μl MEGAscript transcription reaction mixture, as above, by replacing nuclease-free water with 5 μl of 20 mM biotinylated UTP, Bio-16-UTP (Roche). The reaction mixture is incubated at 37 °C for 2 h. Template DNA is removed by addition of 1 μl TURBO DNase and incubated 15 min at 37 °C. Synthesized RNAs are purified using the RNeasy Mini Kit (Qiagen) and analyzed on 1% agarose gels. RNA quantitation is performed by UV light absorbance at 260 nm.

pT7-EV-A71 5′UTR can also be used as template in PCR reactions with appropriate primers to generate IRES subfragments containing various stem-loop regions. The primers should also contain the T7 promoter to permit RNA synthesis for protein-RNA interaction assays.

2.2.3. Construction of a plasmid for synthesis of bicistronic luciferase reporter RNA

The bicistronic reporter plasmid pRHF (which lacks an IRES) is constructed as follows: The Renilla luciferase gene (R) is inserted into EcoRI site of pTriEx-4 (Novagen); the firefly luciferase gene (F) is inserted downstream into the XhoI site of pTriEx-4; a hairpin (H) is inserted downstream of the Renilla luciferase gene to minimize ribosome read-through. This plasmid can serve as a template for synthesis of the negative control, reporter RNA as there is no IRES between the two luciferase open reading frames. The bicistronic reporter plasmid pRHFEV-A71–5′UTR (Fig. 1, upper panel), which contains the EV-A71 IRES inserted between the Renilla and firefly luciferase open reading frames, is constructed by ligating a NotI–EV-A71–5′UTR–NotI fragment into pRHF. The plasmid also contains a T7 promoter (downstream of the CMV promoter) to permit RNA synthesis for transfection. Note: insertion of the IRES in the antisense orientation (EV-A71 5′UTR-AS; Fig. 1, lower panel) permits synthesis of another negative control RNA. Plasmids pRHF-EV-A71–5′UTR and pRHF are linearized by DrdI digestion and used as templates for synthesis of RLuc-EV-A71–5′UTR-FLuc RNA and control RNA lacking an IRES, respectively, using the MEGAscript kit (Life Technologies) with inclusion of m⁷G(5′)ppp(5′)G (Ambion); this produces 5′-capped reporter RNAs [22].

Fig. 1.

The diagram depicts the bicistronic reporter plasmids used to synthesize RNA for transfections and dual-luciferase reporter assays. The expression of firefly luciferase is under the control of the EV-A71 5′UTR in the sense orientation (upper diagram). The EV-A71 5′UTR inserted in the antisense orientation (AS; lower diagram) serves as a negative control, bicistronic RNA.

2.3. Functional assays

The prior sections address building of tools needed for functional analyses of: (i) host protein-IRES RNA interactions, and (ii) structure–function analyses of virus IRES activity in cells. The methods in Section 2.3 utilize those tools, and others, for the aforementioned functional analyses and the effects vsRNAs on them.

2.3.1. Protein-IRES RNA interaction assays

We describe two assays for analysis of interactions between host RNA-binding proteins and viral IRES RNA: (i) streptavidin pull-down of protein-biotinylated RNA coupled with protein identification by Western blot analysis; and (ii) native ribonucleoprotein (RNP)-immunoprecipitation using antibodies to host RNA-binding proteins and detection/quantification of RNA by Northern blot or qRT-PCR. Schematic diagrams/workflows for these two assays are shown in Fig. 2.

Fig. 2.

Schematic diagrams/workflows of protein-IRES RNA interaction assays. (A) Streptavidin pull-down of protein-biotinylated RNA complexes. The biotinylated RNA in this example is the EV-A71 5′UTR. RNA not labeled with biotin serves as a negative control for the assay. The red box highlights SL-II from the IRES. For simplicity, proteins are shown only bound to SL-II. For the Western blots, the presence or absence of biotin in the RNAs are indicated by the plus and minus signs, respectively. The absence of a protein signal in the ‘minus’ lanes indicates that cellular proteins did not bind non-specifically to the paramagnetic particles, i.e., the detected proteins were purified via their association with RNA. (B) Immunoprecipitation of native RNP complexes from cell lysates. Native RNP complexes are incubated with an antibody directed against an RNA-binding protein of interest; non-immune antibody serves as a negative control. Dynabeads coupled to protein A permit magnetic purification of RNP–antibody–protein A-Dynabead complexes. Beads are washed and RNA is eluted and purified. Specific target RNAs associated with the protein of interest are detected by Northern blot or qRT-PCR.

2.3.1.1. Streptavidin pull-down of protein-biotinylated RNA complexes.

For preparation of cell extracts, SF268 cells are grown in RPMI medium supplemented with 10% FBS. Upon confluence, medium is removed and cells are washed with ice-cold PBS, removed from plates by scraping, pelleted by centrifugation at 2000 rpm for 10 min, washed three times with cold PBS, resuspended in CHAPS buffer (10 mM Tris-HCl, pH 7.4, 1 mM MgCl₂, 1 mM EGTA, 0.5% CHAPS, 10% glycerol, 0.1 mM PMSF, 5 mM β-mercaptoethanol), and incubated on ice for 30 min for swelling. Cells are lysed by centrifugation at 10,000g for 10 min at 4 °C, and the supernatants are collected, aliquoted, and stored at −70 °C. Protein concentrations of cell lysates are determined using the Bio-Rad protein assay (Bio-Rad) according to the manufacturer’s instructions.

The next step is to set up binding reactions using these cell extracts and biotinylated RNA, followed by streptavidin purification of protein-RNA complexes. Reaction mixtures contain 200 μg of cell lysate and 3 μg of biotinylated EV-A71 5′-UTR RNA or subfragments thereof, e.g., stem-loop 2 (SL-II), and the final volume is adjusted to 100 μl with RNA mobility shift buffer [5 mM HEPES (pH 7.1), 40 mM KCl, 0.1 mM EDTA, 2 mM MgCl₂, 2 mM dithiothreitol, 1 U RNasin and 0.25 mg/ml heparin]. Note: Preparation of biotinylated RNA is described in Section 2.2.2. The mixtures are incubated at 30 °C for 15 min and then added to 400 μl of Streptavidin MagneSphere Paramagnetic Particles (Promega). Binding is carried out at room temperature for 10 min. Beads, containing RNA-protein complexes, are washed five times with RNA mobility shift buffer lacking heparin. After the last wash, 15 μl of 6× SDS-PAGE sample buffer is added to the beads, incubated at room temperature for 10 min and boiled. Eluted proteins are fractionated by 10% SDS-PAGE. Specific proteins associated with the biotinylated RNA are detected by Western blot analyses.

The fractionated proteins are transferred to nitrocellulose membranes by Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio Rad). To assemble the transfer sandwich, filter paper, nitrocellulose membrane, and gel are pre-soaked in transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol) and assembled from bottom to top: filter paper, nitrocellulose membrane, gel, filter paper. The assembled sandwich is placed in the transfer cell and transferred at 15 V for 30 min. Membranes are blocked with phosphate-buffered saline (PBS) containing 5% fat-free dry milk (i.e., Blotto). Membranes are incubated with primary antibody and then washed with 0.2% Tween 20 in PBS. Membranes are incubated with goat anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (Promega). Reactions are developed using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific) and detected with Amersham Imager 680 blot and gel imager (GE Healthcare) or an equivalent system. For several primary antibodies we have used, the following dilutions or concentrations are: anti-AUF1 rabbit polyclonal, 1:15,000; anti-EV-A71 3C mouse polyclonal [23], 1:750; anti-Ago2 rabbit polyclonal (Abcam), 1:200; anti-HuR mouse monoclonal (Santa Cruz), 1:200; anti-hnRNP A1 mouse monoclonal (Abcam), 1:200; anti-hnRNP A2 mouse monoclonal (Abcam), 1:200; anti-β actin rabbit polyclonal (Abcam), 1:5,000. To permit sequential detection of different proteins, antibodies are stripped from blots by washing them with OneMinute stripping buffer (GM Biosciences).

2.3.1.2. RNP-immunoprecipitation.

Immunoprecipitation of endogenous, native protein-RNA complexes is used to assess association of ITAFs with two types of RNAs: (i) EV-A71 RNA in infected cells; or (ii) the EV-A71 IRES in cells transfected with bicistronic luciferase reporter mRNAs. This generally requires a highly specific antibody against a host RNA-binding protein of interest. However, co-transfection of a plasmid expressing the epitope-tagged RNA-binding protein of interest, for which epitope antibodies are commercially available, has also been utilized.

Cells are lysed with 1 ml PEB per T-75 flask. PEB buffer: 20 mM Tris-HCl, pH 7.4, 100 mM KCl, 5 mM MgCl₂, 0.5% NP-40. One ml of PEB buffer contains 40 μl protease inhibitor cocktail (Sigma-Aldrich), 1 mM PMSF (Sigma-Aldrich), 2.5 μl RNAse-OUT (Invitrogen), and 1 mM DTT. To pre-clear lysates for immunoprecipitation, lysates of 6 × 10⁷ mock-or EV-A71-infected SF268 cells (or equivalent numbers of reporter RNA-transfected cells) are incubated with rabbit non-immune serum (i.e., normal rabbit serum) (Sigma-Aldrich) for 45 min at 4 °C. Magnetic Dynabeads coupled to protein A (Invitrogen) are added for 30 min at 4 °C; beads are removed with a magnet. For immunoprecipitations, fresh beads are coated with antibody targeting the protein of interest or control, rabbit non-immune serum in NT-2 buffer [50 mM Tris-HCl (pH 7.4), 1 mM MgCl₂, 150 mM NaCl, and 0.05% Nonidet P-40] and washed. Pre-cleared cell lysates (1 mg protein) are incubated with 50 μl of coated beads in 200 μl of NT-2 buffer supplemented with 2.5 μl of RNase Out (Invitrogen) and 2 μl of 100 mM DTT for 3.5 h at 4 °C with constant rocking. Beads are washed eight times with ice-cold NT-2 buffer and two times with NT-2 buffer supplemented with 0.5 M urea. Proteins are digested with proteinase K (Promega), and RNAs are purified by phenol–chloroform extraction and ethanol precipitation.

RNAs are then analyzed by Northern blotting. RNA from each sample is denatured by the addition of formaldehyde and formamide (final concentrations of 2.2 M and 50%, respectively) and resolved on 1% agarose gel. The RNAs are then blotted onto a nylon membrane (GeneScreen Plus; Perkin Elmer). After UV crosslinking of RNA to the membrane, the blots are prehybridized for 2 h at 42 °C in Ultrahyb hybridization buffer (Ambion). A minus-strand oligodeoxyribonucleo-tide probe with a sequence corresponding to the RNA of interest is end-labeled with [γ−³²P]ATP and used to probe the RNAs on the nylon membrane by overnight hybridization. The blots are washed twice, each time for 15 min at 25 °C with 2xSSC (1xSSC is 0.15 M NaCl, 0.015 M sodium citrate, and 0.1% SDS), and then exposed for 24 h on a storage phosphor screen. The signals are detected using a Typhoon FLA 7000 PhosphorImager scanner (GE Healthcare) and analyzed by ImageQuant software.

Alternatively, levels of RNAs in precipitates can be quantified by qRT-PCR. Purified RNAs are reverse transcribed into cDNAs using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems), followed by SYBR green quantitative PCRs using specific forward and reverse primer sequences.

2.3.2. Determination of EV-A71 IRES activity

The day before transfection, SF268 cells are seeded in 12-well plates at a density of 2 × 10⁵ cells per well in 1 ml of RPMI with 10% FCS and cultured in an incubator at 37 °C, 5% CO₂. The cells should be about 80% confluence before transfection. On the day of transfection, 0.2 μg of RLuc-EV-A71–5′UTR-FLuc RNA is diluted with 75 μl of serum-free, antibiotic-free RMPI; 7.5 μl of SuperFect transfection reagent is added to the RNA solution, mixed by pipetting up and down five times, and incubated at room temperature for 15 min to allow formation of transfection-complex. While complex formation takes place, medium is removed from cell cultures and cells are washed once with 2 ml PBS; 400 μl of RPMI medium with serum and antibiotics is added to the transfection complexes, mixed by pipetting up and down three times, added to cells, and incubated at 37 °C for 4 h. Medium is removed, cells are washed once with 2 ml PBS, fresh RPMI medium (containing serum and antibiotics) is added, and incubated for 48 h.

Cells are lysed in 250 μl of 1× Passive Lysis Buffer (PLB; Promega) and homogenized by manually scraping the cells from the well and subjecting them to two freeze-and-thaw cycles. To prepare Luciferase Assay Reagent II (LAR II), the lyophilized Luciferase Assay Substrate is resuspended in 10 ml of Luciferase Assay Buffer II. To prepare Stop & Glo reagent, 1 vol of 50× Stop & Glo Substrate is added to 50 volumes of Stop & Glo Buffer in a glass tube. The measurements of firefly luciferase activity and Renilla luciferase activity are performed sequentially using one reaction tube. The 20/20 Luminometer (Turner Biosystems) is programmed to perform a 2-sec premeasurement delay, followed by a 10-sec measurement period for each reporter assay (DLR-O-INJ). 50 μl of LARII is dispensed into 1.5-ml microcentrifuge tubes. 10 μl of cell lysate is added to the tube and mixed by pipetting up and down twice. The tube is placed in the luminometer and the reading is initiated. After recording the firefly luciferase activity (FLuc), 50 μl of Stop & Glo reagent is added to the tube. Reading is initiated and the Renillia luciferase activity (RLuc) is recorded.

2.3.3. Knockdown of host RNA-binding proteins using RNAi

Knockdown of specific host RNA-binding proteins or Dicer using RNAi provides a genetic analysis of roles of these proteins, or vsRNAs, respectively, in IRES-dependent translation and virus replication. We have performed knockdown experiments for AUF1, Ago2, HuR, hnRNP A1/A2, and Dicer for functional analysis. Two micrograms of plasmid expressing control short hairpin (sh)RNA or shRNA against an RNA-binding protein/s are mixed in 100 μl serum-free MEM and combined with 10 μl SuperFect reagent (Qiagen). This mixture is incubated at room temperature for 15 min before addition to cell cultures. Knockdown efficiency is assessed 24 h later by Western blotting. Short hairpin plasmids: Control, pSilencer-U6-hygro/shCTRL; shAUF1, pSilencer-U6-hygro/shAUF1 [24]; shAgo2–1 and shAgo2–2 (kindly provided by Dr Shobha Vasudevan) [25]; shHuR, pcU6K/shHuR [26].

To knockdown expression of hnRNP A1 and hnRNP A2, SF268 cells are seeded in 12-well plates in antibiotic-free media. One hundred nmol of each siRNA targeting hnRNP A1 and hnRNP A2 (ON-TARGETplus SMARTpool L-008221-00-0005 or L-011690-01-0005, Dharmacon) is transfected with 3 μl Lipofectamine 2000 transfection reagent (Life Technologies) in 0.4 ml RPMI supplemented with 10% FCS following the manufacturer’s directions. Lipofectamine 2000 can be used to cotransfect shRNA plasmids targeting RNA-binding protein/s and siRNAs targeting hnRNPs A1 and A2, if desired. Knockdown efficiency is monitored by Western blotting.

2.3.4. Effects of vsRNA1 on viral protein synthesis and replication

SF268 cells are seeded into 12-well plates (2.5 × 10⁵/well) and incubated for 24 h. Cells are then infected with EV-A71 at an MOI of 10 or mock-infected. The virus is adsorbed for 2 h at 37 °C. After virus adsorption, the infected cells in each well are transfected with 160 pmol of synthetic vsRNA1 or scrambled RNA using 4 μl of Lipofectamine 2000 (Invitrogen). Medium is then replaced with methionine-free RPMI and incubated at 37 °C for 4 h. Medium is replaced with fresh medium containing ³⁵S-Met for labelling (50 mCi/ml; PerkinElmer). After 1 h of labelling, medium is removed and cell monolayers are washed with PBS and lysed with RIPA buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin]. Cell lysates are prepared by centrifugation at 12,000 × g for 10 min at 4 °C, and the supernatants are collected. Radiolabelled proteins are resolved by 10% SDS–PAGE, dried, and detected by autoradiography. The labelled viral proteins are identified according to their sizes. Viral 3C protease in the cell lysates is detected by Western blotting.

To examine the effects of vsRNA1 on EV-A71 replication, SF268 cells are infected with EV-A71 at an MOI of 10 or mock-infected. The virus is adsorbed for 2 h at 37 °C. After viral adsorption, the infected cells in each well are transfected with 160 pmol of synthetic vsRNA1 or scrambled RNA with 4 μl of Lipofectamine 2000 (Invitrogen). Media are collected at various time points post-infection. Viral titers are determined by plaque assays on Vero cells as described in Section 2.1.2.

For statistical analyses, data are compared using the unpaired two-tailed t test. P < 0.05 is considered significant. For data shown in Section 4, *P < 0.05; **P < 0.01; ***P < 0.001; N.S., not significant.

3. Results and discussion

3.1. Effect of EV-A71-derived vsRNA1 on association of ITAFs with IRES SL-II

SL-II within the EV-A71 IRES does not, by itself, function as an IRES. However, it is nonetheless important to examine the effects of virus-derived, small RNAs, e.g., vsRNA1, on the association of host ITAFs with SL-II. As noted already, the ITAFs hnRNP A1, AUF1, HuR, and Ago2 are key ITAFs that associate with SL-II to control virus translation. The protein-biotinylated RNA pull-down assay is key to determining the impact of vsRNA1 on association of the ITAFs with SL-II. The assay (Section 2.3.1.1) can be performed in two ways: (i) in vitro with cell extracts, biotinylated SL-II RNA, and synthetic vsRNA1, or (ii) by transfection of biotinylated SL-II and vsRNA1 into cells; pull-down assays are then performed using extracts of transfected cells.

For in vitro experiments, reactions are assembled as described in Section 2.3.1.1. In addition, increasing amounts of synthetic vsRNA1 mimic (5′-GUAACUUAGAAGCUGUAAAUCAACGAUCA-3′) or synthetic scrambled RNA (5′-AAUGCUAUGAGACUAAUGAUACCAAGACU-3′) (synthesized by Dharmacon) are added (0–10 pmol) to reactions. The amounts of vsRNA1 to be added are estimated based on the finding in our previous work that 2.5 × 10⁶ infected cells produced 1.5 pmol (6 h p.i.), 3 pmol (9 h p.i.), and 5 pmol (12 h p.i.) of vsRNA1 [16]. The amount of ITAF in the pull-down reaction is determined by Western blotting. Comparison of protein band densities revealed that vsRNA1 enhances AUF1, Ago2, and HuR association with SL-II by ~2- to 2.5-fold; by contrast, vsRNA1 has no effect on association of hnRNP A1/A2 with SL-II (Fig. 3). These results indicate that vsRNA1 specifically affects association of some proteins, but not all, to SL-II.

Fig. 3.

Effects of vsRNA1 on the association of AUF1, Ago2, HuR, and hnRNP A1/A2 with SL-II. The SF268 lysate and biotin-labeled SL-II were prepared as described in Sections 2.3.1.1 and 2.2.2, respectively. The pull-down reactions were set up with 200 μg of cell lysate and 12.5 pmol of biotin-SL-II RNA as described in Section 2.3.1.1. To examine the effects of vsRNA1 on binding of AUF1, Ago2, HuR, and hnRNP A1/A2 to biotin-labeled SL-II RNA, the indicated pmoles of synthetic mimic vsRNA1 or synthetic scrambled RNA were added to reaction mixtures. Proteins eluted from the streptavidin pull-down were analyzed by Western blot using antibodies to the indicated proteins. This figure is reprinted from Ref. [12].

To complement the in vitro binding assays, transfection-based assays can be performed. Biotin-labeled SL-II and various amounts of vsRNA1or scrambled RNA are co-transfected into SF268 cells for 24 h and extracts are prepared. Protein-biotinylated RNA pull-down assays are then performed as described in Section 2.3.1.1 to evaluate the effect of vsRNA1 on the association of ITAFs with SL-II. The transfection-based assay should be performed to lend validity to the in vitro binding assays.

3.2. Effect of vsRNA1 on ITAFs-EV-A71 5′UTR association and IRES activity in cells

Though informative, the experiments described in Section 3.1 utilize an RNA fragment that alone is not functional for translation. The experiment described here utilizes bicistronic reporter RNAs in which the EV-A71 5′UTR, i.e., IRES, is placed between two luciferase open reading frames; the control RNA lacks the IRES. The downstream open reading frame encodes firefly luciferase and requires an IRES for synthesis; the upstream open reading frame encodes Renilla luciferase for cap-dependent translation. The dual luciferase reporter RNAs (with and without an IRES) are described in Section 2.2.3. These translation-competent RNAs are co-transfected with various amounts of vsRNA1 or scrambled RNA into cells, where the reporter RNAs form native mRNPs associated with ITAFs. Extracts are prepared from these cells and association of specific ITAFs with the IRES can be assessed by mRNP-immunoprecipitation and comparing recovery of RNAs containing the IRES versus lacking the IRES (Section 2.3.1.2).

To assess the impact of vsRNA1 on ITAF-EV-A71 5′UTR interactions, SF268 cells are co-transfected with CMV-RLuc-EV-A71 5′UTR-FLuc RNA (or control RNA lacking the IRES) and various amounts of vsRNA1 (0–10 pmol). Cell lysates are prepared 24 h post-transfection and RNA-protein complexes are immunoprecipitated using control, non-immune antibody or an ITAF-specific antibody, for example AUF1. RNA isolated from the immunoprecipitates is quantified by qRT-PCR using luciferase-specific primers. For AUF1, an anticipated result is that, with increasing amounts of co-transfected vsRNA1, there would be increasing amounts of IRES-containing reporter RNA precipitated (i.e., increased binding by AUF1 with increasing concentrations of vsRNA1). This is anticipated since both vsRNA1 and AUF1 act to suppress IRES-dependent translation.

An important control is to analyze aliquots of immunoprecipitated samples by Western blot for the precipitated ITAF of interest to verify antibody-dependent recovery of the ITAF. Without this information, failure to detect a reporter RNA could be misinterpreted as failure of an ITAF to associate with the mRNP; however, if the antibody failed to immunoprecipitate the ITAF, there would of course be no RNA to detect.

A straightforward corollary experiment is to examine the effect of vsRNA1 on EV-A71 IRES activity (Section 2.3.2). The same dual luciferase reporter RNAs and vsRNA1 mimic/scrambled RNA are co-transfected into SF268 cells. After 24–48 h, cells are lysed and firefly and Renilla luciferase activities are measured. Based on the AUF1 example, increasing amounts of transfected vsRNA1 mimic should reduce IRES-dependent (firefly) luciferase translation/activity without affecting cap-dependent (Renilla) luciferse. Combining the ITAF-RNA binding and IRES activity assays offers a powerful combination to dissect the relationships between them. Furthermore, it is important to note that methods examining the effects of vsRNA1 on virus translation and replication, described in Section 2.3.4, further complement those in this section that examine specific contributions of SL-II and host ITAFs to IRES activity.

4. Notes and troubleshooting

4.1. To avoid carry-over of virus, a fresh pipette tip must be used for each serial dilution of virus for plaque assays.

4.2. It is critical to maintain the temperature of the overlay agarose medium at 45 °C when adding it to the cells. Cells will be killed if the temperature is higher than 45 °C. However, agarose will solidify if the temperature drops too much. We recommend keeping the overlay medium in a Styrofoam box filled with 45 °C water, and add the overlay medium to cells slowly.

4.3. The advantage of staining the cells with neural red for plaque assays is that, if the plaques are not yet formed, cells can be incubated for another 1–2 days. Cells can be fixed with 10% formamide and stained with 0.1% crystal violet for long-term storage.

4.4. Our experience with TA cloning is that, often after transformation, there are no positive (white) colonies on LB agar plates containing X-Gal and ampicillin. We still choose some colonies to grow and analyze by restriction enzyme digestion; we always obtain some positive clones.

4.5. To prevent degradation of RNAs synthesized in vitro, and for long term storage, we recommend adding RNAse inhibitor to the RNA and store at −80 °C.

4.6. According to the manufacturer (Thermo Fisher Scientific), it is not necessary to pre-clear lysates if magnetic Dynabeads are used for RNP-immunoprecipitation. However, if the background is high, we recommend pre-clearing cell lysates with non-immune serum. We have found pre-clearing lysates reduces the background markedly.

4.7. When using the bicistronic reporter system to study the effect of host RNA-binding proteins on EV-A71 IRES activity, it is important to transfect reporter RNA transcribed from this plasmid, rather than transfecting the plasmid. The reason is host proteins, such as hnRNP A1, may be involved in nuclear export of mRNA. In this event, knockdown of hnRNP A1 could inhibit translation indirectly by blocking nuclear export of reporter (and other) mRNAs. This of course would lead to misinterpretation of results.

4.8. To conserve reagents, we reduce the reaction volume of dual luciferase reporter assays by one-half. We find no effects on the assays.

4.9. We use Lipofectamine 2000 (Invitrogen) for transfection of siRNAs and vsRNA into cells. The manufacturer now has new reagent designed specifically for the delivery of siRNA and miRNA into cells, Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, catalog number: 13778075). We recommend at least testing it for siRNA and vsRNA transfection.

5. Conclusions

In an effort to identify essential factors that control EV-A71 replication, we found that the virus uses its 5′UTR to recruit host RNA-binding proteins [27]. We revealed that hnRNP A1, AUF1, HuR, and Ago2 proteins bind to SL-II of the viral 5′UTR to activate or repress IRES-dependent translation [10–12]. Disruption of these interactions genetically or through siRNA delivery significantly impairs EV-A71 replication by reducing translation. Moreover, we determined that a viral small RNA (vsRNA1) derived from Dicer cleavage of the EV-A71 5′UTR suppresses virus translation by interacting with SL-II of the 5′UTR [16]. The classic virology and biochemistry methods we describe here can be used to elucidate the mechanisms by which ITAFs and vsRNAs form complexes with the viral IRES to control translation. This will provide insight into protein-RNA and RNA-RNA interactions that contribute to EV-A71 pathogenesis. This will in turn lay a foundation for identification of novel antivirals that target the IRES of enteroviruses.

Abbreviations:

EV-A71

enterovirus A71

HFMD

hand-foot-and-mouth disease

IRES

internal ribosome entry site

ITAF

IRES trans-acting factor

MOI

multiplicity of infection

SL-II

stem-loop II

5′UTR

5′-untranslated region

vsRNA

virus-derived small RNA

siRNA

short interfering RNA

Appendix A. Reagent and equipment list.

Reagent/equipment	Supplier	Product number
30% (29:1) Acrylamide/Bis Solution	Bio-Rad	1610156
Agarose	Invitrogen	15510–02
Amersham Imager 680	GE Healthcare	29270769
BenchMark protein standard	Invitrogen	10747–012
Biotin-16-UTP (10 mM)	Roche	50-100-3411
Cap Analog [m7G(5′)ppp(5′)G]	Thermo Fisher Scientific	AM8050
Dual-Luciferase Reporter Assay System	Promega	E1910
Dynabeads Protein A	Invitrogen	10001D
Fetal bovine serum (FBS)	Thermo Fisher Scientific	26140079
GeneScreen Plus Hybridization	Perkin Elmer	NEF987001PK
High-Capacity cDNA Reverse	Applied Biosystems	4368813
Transcription Kit
Lipofectamine 2000	Invitrogen	11668019
20/20n Luminometer	Turner Biosystem	2030–002
MEGAscript T7 transcription Kit	Life Technologies	AM1334
Modified Eagle’s medium (MEM)	Thermo Fisher Scientific	11095080
MicroSpin G-25 Columns	GE Healthcare	27532501
Mini Trans-Blot Transfer Cell	Bio-Rad	1703930
Multigene Mini Personal Thermal Cycler	Labnet	TC-020–24
Normal rabbit serum	Sigma-Aldrich	NS01L
OneMinute stripping buffer	GM Biosciences	GM6001
OneTaq Hot Start Quick-Load	NEB	M0488S
Phosphate-buffered saline	Sigma-Aldrich	806552
Pierce™ ECL Western Blotting Substrate	Thermo Fisher Scientific	32106
PMSF	Sigma-Aldrich	P7626
Protease inhibitor cocktail	Sigma-Aldrich	P8340
Protein assay	Bio-Rad	5000006
QIAquick PCR Purification Kit	QIAGEN	28104
RMPI 1640 medium	Thermo Fisher Scientific	11875093
RNAse-OUT	Invitrogen	10777019
RNeasy Mini Kit	QIAGEN	74104
Streptavidin MagneSphere	Promega	Z5481
Paramagnetic Particles
SuperFect Transfection Reagent	QIAGEN	301305
TA Cloning Kit	Life Technologies	K203001
Typhoon FLA 7000 Phosphorlmager	GE Healthcare	n/a
Ultrahyb hybridization buffer	Themo Fisher Scientific	AM8670

Appendix B. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymeth.2020.02.008.