Plant RNA viruses exploit nonorthodox strategies, such as the use of internal ribosomal entry sites (IRES), to express multiple genes from a single RNA species. IRES elements have been reported in tobacco etch virus (TEV), crucifer infecting tobamovirus (crTMV), hibiscus chlorotic ringspot virus (HCRSV), and many other animal and plant RNA viruses. In this chapter, the methodology used to identify and characterize a plant virus IRES element, including construction of a translation reporter vector for testing the IRES activity, testing the IRES activity in coupled in vitro transcription and translation systems and mammalian cells analysis of RNA stability, and sucrose gradient analysis and polysome profiling, is presented.
In eukaryotes, translation initiation involves recruitment of 40S ribosomal sub units at either the 5′ m7G cap structure or internal ribosome entry sites (IRES). The ribosomal subunit together with other factors locates a start codon and protein synthesis begins following binding of the 60S ribosomal subunit, terminating at a stop codon (
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IRES elements, most well studied in picornaviruses, share common features that are responsible for activity. Most of the known viral IRESs are located in 5′ UTRs, highly structured and contain multiple conserved AUGs. Parts of the secondary structure of IRES elements associated with activity include sequences that form part of double-stranded regions (
Acetic acid. pBluescript®. Coommasie blue R-250 (Sigma). T7 RNA polymerase. A bicistronic construct containing GFP gene as the 5′ cistron and the envelope (E) protein gene of coronavirus infectious bronchitis virus (IBV) as the 3′ cistron. [α-{su32}P]UTP. [{su35}S]methionine. Phenol (pH 8). 0.5M NH{sb4}OAc. tRNA(10mgmL{su-1}). Chloroform. 10–30% (w/v) linear sucrose gradient. 25 mM Tris-HCl (pH 7.6). l00mM KC1. 5mMMgCl{sb2}. Ultrahigh speed centrifuge and rotors. Absolute ethanol. 1% formaldehyde agarose gel. Cos-7 cells. β-tubulin antibody. Coupled in vitro transcription translation(TnT) kit (Promega). 30 ²C heat block. Complete Dulbecco's modified Eagles medium (Gibco Life Technologies). 10% bovine calf serum (Hyclone). 1%penicillin/streptomycin (Gibco Life Technologies). X-ray film and cassette. X-ray film developer. 0.1% SDS (v/v). Recombinant vaccinia virus (vTF7-3). DOTAP transfection reagent (Roche). Humidified 5% CO{sb2} incubator. GS-710 calibrated imaging densitometer (Bio-Rad). Molecular Analyst computer software (Bio-Rad). 1X Laemmli's sample buffer; 24mM Tris-HCl pH 6.8, 100mM DTT, 2% SDS, 0.1% bromophenol blue, 20% glycerol. Protein gel running buffer: 2.88% glycine (w/v), 0.6% Tris-HCl, and 0.1% SDS (v/v). Amplify™ (Amersham Pharmacia Biotech). Nitrocellulose membrane (Stratagene). Semidry transfer cell (Bio-Rad Trans-Blot SD). Western blot blocking buffer: 5% skim milk powder in TBST including 20mM Tris-HCl (pH 7.4), 150mM NaCl, 0.1% Tween 20. IgG conjugated with horseradish peroxidase (Dako). Chemiluminiscence detection kit (ECL, Amersham Pharmacia Biotech).
Plasmid DNAs (5μg) are linearized and extracted by phenol/chloroform and ethanol precipitated before adding to the 50μL reaction mix containing the following components:
Linearized DNA 2 μg 5X transcription buffer (Promega) 10 μL 100mMDTT5μL NTPs (2.5 mM each of ATP, GTP, CTP, TTP) 10 μL RNase inhibitor (40U μL−1) 0.5μL T7 RNA polymerase 1 μL Sterile water to 50μL Incubate the reaction mix at 37 °C for 1 h. Check the integrity of the transcripts by agarose gel electrophoresis. In vitro translation reaction includes the following components:
1 μg RNA template (after in vitro transcription) 2μL Wheat germ extracts 35 μL Minus methionine amino acid mixture 1 μL 10 mM methionine 1 μL RNase inhibitor (40 U μL-1) 1 μL 50μCi of [35S] methionine 1 μL Add sterile water to 50 μL Incubate the reaction at 30 °C for 1 h. Reaction products are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by autoradiography.
Culture Cos-7 cells (ATCC-CRL-1651) in complete Dulbecco's modified Eagles medium (Gibco Life Technologies) supplemented with 10% bovine calf serum (Hyclone) and 1% penicillin/streptomycin (Gibco Life Technologies) at 37 °C in humidified 5% CO2 incubator. Infect Cos-7 cells with 10 PFU/cell of recombinant vaccinia virus (vTF7-3) for 2 h at 37 °C prior to transfection. Transfect the vTF7-3-infected cells with the plasmids (see After 20–24h post-infection, harvest the cells. Lyse transfected Cos-7 cells Mix total proteins with 2X SDS loading buffer and subjected to SDS-PAGE.
Discontinuous polyacrylamide gel electrophoresis system ( Typically, an aliquot of the translation reaction is added to the IX Laemmli's sample buffer and boiled at 100°C for 2min and cooled on ice before loading on a SDS-polyacrylamide gel. In a reservoir of protein gel running buffer, gels are run vertically at 20mA until the bromophenol blue dye reached the bottom of the gel. After electrophoresis, gels containing [35S]-methionine are fixed in 50% methanol and 10% acetic acid for 30min. The signal is enhanced with the use of Amplify™ (Amersham Pharmacia Biotech) for 15min. Gels are dried under vacuum at 80 °C for 1 h and later exposed to X-ray film (Biomax, Kodak) for autoradiography at −80 °C overnight. Gels containing unlabeled polypeptides are stained in 50% methanol, 10% acetic acid and 0.05% coommasie blue R-250 (Sigma) for 30min at RT°C and destained in 50% methanol, 10% acetic acid before drying.
After SDS-PAGE, proteins are transferred to nitrocellulose membrane (Stratagene) by a semidry transfer cell (Bio-Rad Trans-Blot SD) and blocked overnight at 4 °C in blocking buffer. The membrane is incubated for 2h at room temperature in a dilution (1:1000) of a specific antiserum against the reporter gene in blocking buffer. After three washes for 15min each with TBST, the membrane was blocked for 20min before it is incubated with either antirabbit or antimouse IgG conjugated with horseradish peroxidase (Dako) diluted 1:2500 in blocking buffer for 1 h at room temperature. After three washes with TBST, the membrane was treated using a chemiluminis-cence detection kit (ECL, Amersham Pharmacia Biotech) accordingly.
The intensities of the protein bands are measured by a GS-710 calibrated imaging densitometer (Bio-Rad) and analyzed using Molecular Analyst computer software (Bio-Rad). The protein band of interest is normalized accordingly to the intensities of the corresponding p28 band or a consistent background band.
Equal amounts of templates are linearized for in vitro transcription in the presence of [α−32P]UTP. Equal amounts of the labeled RNA (2μL of the 25 μL reaction) are added directly into the in vitro translation reaction mixture. At specific time intervals, a portion (5 μL) is withdrawn from each translation reaction mixture. Prior to adding 100μL of phenol (pH 8), 94 μL 0.5 M NH4OAc and 1 μL tRNA (l0 mg mL−1) are added to the reaction mixture. The sample is vortexed and centrifuged at 20,000 g for 5 min. The supernatant is added to 100μL chloroform, vortexed and centrifuged at 5,000 g for 5min. The RNA is precipitated by transferring the supernatant into 300 μL absolute ethanol and placed in −80 °C for 30 min before obtaining the pellet. The extracted RNAs are resolved in a 1% agarose gel containing 0.1% SDS before autoradiography.
The integrity of mRNA derived from a test construct is analyzed by sucrose gradient analysis as described by Pelletier and Sonenberg ( The DNA template is linearized for in vitro transcription in the presence of [α−32P]UTP. The in vitro synthesized RNA is extracted with phenol/chloroform, precipitated with ethanol, and incubated in a 50 μL translation reaction containing 35 μL of wheat germ extract at 30 °C for 10 min. The translation mixture is cooled on ice and layered onto a 10–30% (w/v) linear sucrose gradient (2mL) in buffer containing 25mM Tris-HCl (pH 7.6), lOOmM KC1, and 5mM MgCl2. The gradient was then subjected to centrifugation at 259,000 g in an appropriate rotor at 4°C for 60 min. Fractions of 200 μL are collected from the top of the gradient and measured at absorbance 260 nm to obtain the polysome profile. Based on the polysome profile, fractions are pooled and RNA extracted by phenol/chloroform and ethanol precipitation. The transcripts are resolved on a 1% formaldehyde agarose gel and analyzed by autoradiography.
To test if the plant virus IRES element is functional in mammalian cells, the GFP gene is cloned upstream of the IRES region in a bicistronic construct.
In a transient expression system ( The expression of GFP is detected in transfected cells by viewing under ultraviolet light. The cells are harvested and western blot detection is performed using the expression of β-tubulin gene as a control to normalize the densitometry readings. The expression of both GFP and reporter gene positioned after the IRES indicates that the IRES element s active in mammalian cells. The IRES activity in Cos-7 cells is further analyzed by expression of deletion constructs.
Construction of a translation reporter vector. The vector can be a pBluescript® or any suitable plasmid that contains a T7-RNA polymerase promoter so that a full-length sequence containing a putative IRES element can be cloned into it for transcription. The transcribed RNA template can then be used for in vitro translation. A reporter gene (such as green fluorescent protein, GFP) at the 5′ end is used as an internal control for the internal initiation event and the stability of RNA templates during translation. The region containing the IRES element is inserted into a bicistronic construct containing GFP gene as the 5′ cistron and the envelope (E) protein gene of coronavirus A series of sequential deletions of the full-length sequence containing a putative IRES element is carried out to delineate the region responsible for internal initiation. When the expression of the reporter gene is observed to be reduced by several folds, it indicates that the IRES element has been deleted. Construct pGFP-E, containing the GFP gene and the E protein gene in different reading frames, are used as controls ( Expression of pGFP-E resulted in the detection of GFP. With the insertion of the putative IRES element inserted between the 5′ and 3′ cistrons, the GFP encoded by the 5′ cistron and the E protein encoded by the 3′ cistron were expressed. To avoid repeated freezing and thawing of the coupled transcription and translation kit. It is advisable to aliquot the wheat germ extracts after the first usage. Constructs containing deletions under the control of a T7 promoter are expressed transiently in semiconfluent monolayers of vTF7–3-infected Cos-7 cells. To handle RNA with care so that it will not be degraded by RNase and affect the results.
The authors would like to thank the National University of Singapore for financial support of our research on Hibiscus latent Singapore virus (RP 154–000–295–112) and Hibiscus chlorotic ringspot virus (RP 154–000–252–112); and A*Star for research support in Institute of Molecular and Cell Biology.