key: cord-1010804-cv47h083
authors: Johansen, I. Elisabeth; Lund, Ole Søgaard
title: Insertion of Introns: A Strategy to Facilitate Assembly of Infectious Full Length Clones
date: 2008
journal: Plant Virology Protocols
DOI: 10.1007/978-1-59745-102-4_36
sha: 049a3686399336c189e23f0b0ea953097f19f482
doc_id: 1010804
cord_uid: cv47h083

Some DNA fragments are difficult to clone in Escherichia coli by standard methods. It has been speculated that unintended transcription and translation result in expression of proteins that are toxic to the bacteria. This problem is frequently observed during assembly of infectious full-length virus clones. If the clone is constructed for transcription in vivo, interrupting the virus sequence with an intron can solve the toxicity problem. The AU-rich introns generally contain many stop codons, which interrupt translation in E. coli, while the intron sequence is precisely eliminated from the virus sequence in the plant nucleus. The resulting RNA, which enters the cytoplasm, is identical to the virus sequence and can initiate infection.

Infectious cDNA clones are useful for analysis of various properties of plant viruses. The cDNA clone allows introduction of specific mutations and exchange, deletion, or insertion of genes or gene fragments. Unfortunately, full-length cDNA clones of many RNA viruses have proved difficult or even impossible to clone and amplify in Escherichia coli. Full-length clones have frequently turned out to be noninfectious, possibly due to spontaneous mutations or deletions, which reduces toxicity to E. coli. The cause for the toxicity to E. coli is not known, but it has been observed that artificially introduced deletions; insertions or frame shifts can alleviate the cloning problems (1, 2) .

Insertion of an intron into the virus sequence has proven to be an efficient way to facilitate maintenance of the cDNA in E. coli (1, 3, 4, 5) . Upon inoculation of the cDNA to the host cell, the intron is precisely spliced from the precursor nuclear messenger RNA (pre-mRNA) in the nucleus, and a true copy of the viral RNA is transported to the cytoplasm where infection can start. The function of clones with introns requires cloning of the viral cDNA between promoter and terminator From sequences, which are active in the host cell. The 35S promoter and NOS terminator are frequently used in plants (see Chaps. 32 and 33 for a description of construction of infectious clones of RNA viruses). Cloning of the viral cDNA in this context also allows inoculation by agroinfiltration, if the transcription cassette is moved to a binary vector (see Chap. 38 for a description of agroinoculation).

Construction of infectious clones with introns has been reported for viruses belonging to the genera Potyvirus (1, 4, 6) Tobravirus (7, 8) , Coronavirus (3), and Flavivirus (5) . In theory, intron insertion should be applicable for clones of viruses, which are replicated from an RNA template in the cytoplasm.

The sequences required for pre-mRNA intron splicing lie mainly within the intron. These include consensus sequences at the 5′ (GU) and 3′ (AG) ends of the intron; the branchpoint located 18-40 nucleotides upstream of the 3′ splice site and AU-rich sequence elements (9) . Furthermore, introns are characterized by high U or AU content compared to more GC-rich exons. In the sequence next to the intron, there is a high representation of A and G at the two positions next to the 5′ end of the intron and G at the first positions 3′ to the intron (10). This is shown as a consensus sequence in Fig. 1 . Therefore, it is probably safest to insert introns at positions in the virus sequence matching the AG/G consensus. There are minor differences between monocot and dicot introns (10) , and therefore, it is recommended to select introns from the class in which the infectious clone is going to be analyzed. Furthermore, it should be noted that the monocot sequences included in the analysis by (10) almost exclusively represent introns from the order Poalis. Some differences have been observed between species within the dicots (9) . However, at present there is no evidence that differences in dicot introns are important for their use in virus clones. For example, Arabidopsis introns were used in Tobacco mosaic virus, which efficiently infected two species of Nicotiana (11) , and an intron from Solanum tuberosum is spliced in Pisum sativum and Capsicum annuum (1, 6) .

Introns can also be inserted into native PstI restrictions sites as described in detail by (1) . Here, we describe a more flexible method based on overlap extension PCR (12 1 . Commercial DNA marker to estimate DNA concentration of the purified PCR fragments 2. Heat stable DNA polymerase, buffer, and 5 mM dNTP mix for PCR 3. Kit for cloning of PCR fragments

1. Add mercaptoethanol to the 2X CTAB buffer 2. Grind 100 mg plant material in a 1.5-ml microfuge tube immersed in liquid nitrogen. Use safety glasses 3. Add 500 µL 2X CTAB buffer, mix, and incubate at 65°C for 30-60 min 4. Centrifuge at 5000g for 10 min at room temperature 5. Transfer the supernatant to a clean microfuge tube 6. Add an equal volume of chloroform/isoamylalcohol, shake the tube for 10 min 7. Centrifuge at 5000g for 2 min at room temperature 8. Repeat steps 5-7 9. Add 0.8 volume cold isopropanol and 0.1 volume 3 M sodium acetate 

1. If introns are inserted into the virus sequence in order to reduce toxicity, the intron insertion site should be placed in a region, which has been difficult to clone or amplify in E. coli. In addition the insertion site should match the AG/G exon border sequences as indicated in Fig. 1 and Fig. 2a (see note 1) 2. Paste in the intron sequence and run the virus sequence with the intron through a program, which predicts intron splice sites (e.g., NetPlantGene Server). 3. The output will display potential donor sites, acceptor sites, and branchpoints.

Only the transcribed virus sense strand is relevant. 4. Check that the intron is recognized in the virus sequence. If the intron is not recognized, then try another insertion site and/or another intron sequence. 5. Also inspect the output for cryptic introns in the virus sequence, which may interfere with correct processing of the virus sequence (see note 1).

1. Identify restriction sites, SiteA and SiteB, flanking the intron insertion site, which are suitable for reintroduction of the intron containing subclone into the full-length clone or a larger subclone (see note 7) Choose restriction sites that are no more than 2 kb apart to avoid problems, which may arise with generation of long PCR products. 2. Six primers are needed as shown in Fig. 2b. a. Primers 1 FW and 6 RV are forward and reverse primers annealing to the regions with the restriction sites SiteA and SiteB (see note 2). Primers 1 FW and 6 RV are designed like normal PCR primers and it is advisable to check them for primer dimer formation and proper annealing the target sequence using for example vector NTI, which is available at http://www.invitrogen.com b. Primers 3 FW and 2 RV 3. Make a 10 µM dilution of each primer in H 2 O, mix well, and store at −20°C.

Flanking the Introns 1 . Three separate PCR reactions are set up to amplify overlapping fragments: The virus region 5′ to the intron, the intron and the region 3′ to the intron (Fig. 2c) . The virus regions 5′ and 3′ to the intron are amplified from a virus plasmid cDNA clone with primer pairs 1 FW /2 RV and 5 FW /6 RV , respectively. The intron is amplified with primers 3 FW and 4 RV from purified plant DNA. Optimized heatstable high-fidelity DNA polymerases with proofreading are recommended for the PCR. For example, Expand or Phusion, whereas pure Pwo or Pfu are less efficient. 2. The following two protocols are based on the instructions provided by the suppliers. Please refer to these instructions for further information and troubleshooting. Note also that other products can be used. (Finnzymes) . Thaw, mix, and centrifuge all solutions except the enzyme before use. Place on ice and assemble reactions on ice. Add in the following order: H 2 O to a total reaction volume of 50 µL; 10 µL 5X Phusion HF buffer; 2 µL 5 mM dNTPs; 2.5 µL 10 µM primer 1 FW ; 2.5 µL 10 µM primer 6 RV ; approximately 50 ng of each PCR product from the initial reactions; 1.5 µL DMSO (optional); 0.5 µL Phusion DNA polymerase, mix, and centrifuge. Run the following PCR scheme: Initial denaturation 98°C for 30 s; 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 30 s per 1,000 bases; final extension at 72°C for 5-10 min; 4°C. Note that Phusion ™ produces blunt end PCR products. Go to step 2. 2. A small sample (1-5 µL) is analyzed by agarose gel electrophoresis to check that a PCR product of the expected size has been amplified. 3. The PCR product of the expected size is gel purified and the concentration determined. It is recommended to clone the fragment cloned using a T/A PCR cloning cloning kit for products with 3′dA overhangs (Expand) or blunt ends (Phusion ™ ) (see note 6). The cloned fragment should be sequenced to check for errors before it is reinserted into the virus cDNA using restriction sites SiteA and SiteB (see note 7).

1. It is likely that sequences surrounding the intron may affect splicing efficiency, and the virus may contain cryptic 5′ and or 3′ splice sites, which can interfere with splicing of the true intron. The presence of cryptic splice sites in the virus sequence can be identified using, for example, the 'NetPlantGene Server' at http://www.cbs.dtu.dk/services/NetPGene/. To prevent cryptic splicing it may be necessary to eliminate either the 5′ or the 3′ splice site. This may be accomplished by silent mutations (Marillonnet et al., 2005) . In our experience, cryptic sites do not seem to be a major problem if the goal is to generate an infectious clone because only a fraction of the transcripts need to be correctly spliced to initiate infection. However, insertion of true introns can improve the specific infectivity as demonstrated by Marillonnet et al. (2005) . It is also known that sequences in the exon, known as exon splicing enhancers (ESE), are likely to play an important role in plant intron splicing. However, no splicing event in Arabidopsis has yet been shown to be enhancer dependent. For more information see http://www.tigr.org/ software/SeeEse/background.html 2. Primers 1 FW and 6 RV should contain the sequence of the restriction sites SiteA and SiteB, or flank the sites so the restriction sites are contained within the PCR fragments. If the primers contain the restriction sites, the restriction site consensus should be placed at least three nucleotides from the 5′ end of the primer. 3. The length of the annealing sequence will depend on the GC content. It is advisable to use a longer primer for regions with low GC content. 4. Optimal annealing temperature depends on the melting temperature of the primers and the system used. Annealing at 55°C is usually a good starting point unless one of the primers anneal to a short and very AT-rich region. In this case, the annealing temperature should be lower.

5. The optimal annealing temperature will depend on the primer sequences, but in our experience 55°C is a good starting point. 6. PCR fragments are most conveniently cloned using PCR cloning kits adapted for cloning PCR products with dA overhangs or blunt ends. Although the PCR products described here contain restriction sites at least three nucleotides from the ends of the fragment, it is sometimes difficult to clone PCR fragments with the aid of such sites. 7. Multiple fragment ligations can facilitate reinsertion of the fragment containing the intron if the restriction sites SiteA and SiteB are not unique in the full-length clone. We have assembled potyvirus clones from up to five fragments with sticky ends in a single cloning step. The amounts of DNA of each fragment are adjusted when the fragments are cut from the agarose gel and can be purified together on a single spin column

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