key: cord-332844-2se4d1yp
authors: Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Lee, Young-Min
title: Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses
date: 2015-12-29
journal: J Vis Exp
DOI: 10.3791/53164
sha: 
doc_id: 332844
cord_uid: 2se4d1yp

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

For RNA virologists, the advent of recombinant DNA technology in the late 1970s made it possible to convert viral RNA genomes into cDNA clones, which could then be propagated as plasmids in bacteria for the genetic manipulation of RNA viruses. 1 The first RNA virus to be molecularly cloned was bacteriophage Qβ, a positive-strand RNA virus that infects Escherichia coli. A plasmid containing a complete cDNA copy of the Qβ genomic RNA gave rise to infectious Qβ phages when introduced into E. coli. 2 Shortly thereafter, this technique was applied to poliovirus, a positive-strand RNA virus of humans and animals. A plasmid bearing a full-length cDNA of the poliovirus genomic RNA was infectious when transfected into mammalian cells and capable of producing infectious virions. 3 In this "DNA-launched" approach, the cloned cDNAs should be transcribed intracellularly to initiate viral RNA replication; however, it is unclear how the transcription is initiated and how the transcripts are processed to the correct viral sequence. This concern has led to the development of an alternative "RNA-launched" approach, whereby a complete cDNA copy of the viral RNA genome is cloned under a promoter recognized by an E. coli or phage RNA polymerase for the production of synthetic RNAs in vitro with defined 5' and 3' termini, which undergo the complete viral replication cycle when introduced into host cells. 4, 5 The first success with this approach was reported for brome mosaic virus, 6 ,7 a positive-strand RNA virus of plants. Since then, the RNA-launched approach has been developed for a wide range of positive-strand RNA viruses, including caliciviruses, alphaviruses, flaviviruses, arteriviruses, and coronaviruses. 1, 4, 5, 8 In both the DNA-and RNA-launched reverse genetics systems, the construction of a full-length cDNA clone is the key to generating infectious DNA or RNA of positive-strand RNA viruses, but it becomes a considerable technical challenge as the size of the viral genome increases. [9] [10] [11] [12] [13] [14] [15] [16] [17] In particular, a large RNA genome of ~10-32 kb presents three major obstacles to the cloning of a full-length functional cDNA. 18 The first difficulty is the synthesis of a faithful cDNA copy, since the fidelity of RT-PCR is inversely proportional to the length of the viral RNA. The second hurdle is the presence of potentially toxic sequences, since long RNA molecules are more likely to contain unexpected sequences capable of making the cDNA fragment in plasmids unstable in E. coli. The third and most critical issue is the availability of a suitable vector, since it is difficult to find a cloning vector that can house a viral cDNA insert of >10 kb. Over the past three decades, these barriers have been overcome by several advances in enzymology, methodology, and vectorology. 1, 4, 5, 8 Of these, the most promising and innovative development is the cloning of large positive-strand RNA viruses as infectious bacterial artificial chromosomes (BACs). The BAC vector is a low-copy cloning plasmid (1-2 copies/cell) based on the E. coli fertility factor, with an average DNA insert size of ~120-350 kb. 19 2. Assemble a set of the four modified, overlapping cDNAs into a single full-length SA 14 -14-2 BAC (pBAC/SA 14 -14-2) by joining at three natural restriction sites (BsrG I, BamH I, and Ava I) in a sequential manner using the five-step cloning procedures detailed in Protocols 3.1-3.5 ( Figure 1E) ) in a 6-well plate. 7. After 4-6 hr of incubation, overlay the cells with 0.5% agarose in minimal essential medium containing 10% fetal bovine serum. Incubate the plates for 4 days at 37 °C with 5% CO 2 . 8. Visualize the infectious centers (plaques) by fixation with 7% formaldehyde and staining with 1% crystal violet in 5% ethanol 27 (Figure 6A ). 

For all positive-strand RNA viruses, the reliability and efficiency of a reverse genetics system depend on the genetic stability of a cloned fulllength cDNA, whose sequence is equivalent to the consensus sequence of viral genomic RNA. 27 Figure 1 shows a five-step strategy for the construction of a full-length infectious cDNA as a BAC for JEV SA 14 -14-2 28 :

Step 1, purification of viral RNA from the cell culture supernatant of JEV-infected BHK-21 cells (Figure 1A) ; Step 2, synthesis of four overlapping cDNA amplicons (F1 to F4) spanning the whole viral genome ( Figure 1B); Step 3, subcloning of each of the four contiguous cDNA fragments into a BAC vector, creating pBAC/F1 to pBAC/F4 ( Figure 1C) ;

Step 4, modification of the cloned cDNAs for in vitro run-off transcription with SP6 RNA polymerase, i.e., placing an SP6 promoter sequence immediately upstream of the viral 5'-end (pBAC/F1 Step 5, assembly of a full-length SA 14 -14-2 cDNA BAC, pBAC/SA 14 -14-2 ( Figure 1E) . Table 1 lists the oligonucleotides used in this cloning procedure. 28 For the construction of a functional JEV cDNA, the first important step is the synthesis of the four overlapping cDNA fragments using the purified viral RNA as a template for RT-PCR. Figure 2 provides a representative result for the four RT-PCR products that were electrophoresed on a 0.8% agarose gel. This gel demonstrates clearly that a full-length JEV cDNA is amplified into four overlapping cDNA fragments. Occasionally, RT-PCR reactions might yield one or more additional virus-specific or nonspecific products that are mostly smaller than the expected product, because of the nonspecific annealing of primers during cDNA synthesis/amplification. On the other hand, little or no expected RT-PCR product would be amplified because of accidental RNase contamination during the viral RNA isolation or improper RT-PCR performance.

The next key step is the cloning and modification of a partial-or full-length JEV cDNA in BAC, which is a relatively straightforward procedure that uses standard recombinant DNA techniques. 69 Figure 3 presents a representative outcome for the purification of the BAC clone containing a fulllength cDNA of JEV SA 14 -14-2 by banding in a CsCl-EtBr gradient. In this experiment, after centrifugation for 16 hr at 401,700 × g, two distinct bands, i.e., the E. coli chromosomal DNA above and the supercoiled BAC plasmid DNA below, are visible in the middle of the tube under longwave ultraviolet light. A minimal volume (~400 µl) of the lower BAC DNA band was carefully collected by poking a hole with a syringe on the side of the tube. Subsequently, the EtBr was extracted from the BAC DNA by butanol extraction, and the EtBr-free BAC DNA was concentrated by ethanol precipitation.

The final step is the determination of the specific infectivity of the synthetic RNAs transcribed in vitro from the full-length SA 14 -14-2 BAC (pBAC/ SA 14 -14-2) after RNA transfection into permissive cells (Figure 4 ). This step involves three sequential steps:

Step 1, linearization of the fulllength SA 14 -14-2 cDNA at the 3'-end of the viral genome ( Figure 4A) ;

Step 2, production of synthetic RNAs from the linearized cDNA by runoff transcription ( Figure 4B); and Step 3, rescue of the recombinant viruses in BHK-21 cells transfected with the synthetic RNAs ( Figure 4C) . Experimentally, two independent clones of pBAC/SA 14 -14-2 were linearized with Xba I digestion and treated with MBN to remove the fourbase 5' overhang generated by the Xba I digestion. The linearized BACs were cleaned up by phenol-chloroform extraction, followed by ethanol precipitation. The linearization of the two purified BACs was demonstrated on a 0.8% agarose gel ( Figure 5A) . The phenol-chloroform extraction must be done carefully to ensure that the linearized BACs are RNase-free. Each of the two linearized BACs served as a cDNA template for runoff transcription using SP6 RNA polymerase in the presence of the m 7 G(5')ppp(5')A cap analog. The integrity of the synthetic RNAs was shown by running aliquots of the two transcription reaction mixtures on a 0.6% agarose gel, along with a reference 1 kb DNA ladder ( Figure 5B) . In this simple assay, the major prominent RNA band always migrated just below the 3 kb reference DNA band and appeared to be sharp. However, degraded RNA would have a smeared appearance on the same gel.

An infectious center assay is the gold standard for determining the specific infectivity of the synthetic RNAs. This assay was done by electroporating BHK-21 cells with RNA samples, seeding equal aliquots of the 10-fold serially diluted electroporated cells in 6-well plates containing naïve BHK-21 cells (3 × 10 5 cells/well), and overlaying agarose onto the cell monolayers. After incubation for 4 days, surviving cells were fixed with formaldehyde and stained with a crystal violet solution to quantify the number of infectious centers (plaques), which corresponds to the number of infectious RNA molecules delivered into the cells (Figure 6A ). Since the cDNA template used for in vitro transcription has been proven to be non-infectious, 27 an aliquot of the transcription reaction mixture was directly used for electroporation. Electroporation is the preferred method for RNA transfection; alternatively, RNAs can be transfected by other methods using DEAE-dextran and cationic liposomes. RNA electroporation is very effective, but "arcing" of the electric pulse occurs rarely if salts are present in the electroporation reaction or if the electroporation cuvette is reused. The expression of viral proteins in RNA-transfected cells was examined by immunofluorescence assays using an anti-NS1 rabbit antiserum ( Figure 6B) . The production of viral particles accumulated in the supernatants of RNA-transfected cells was analyzed by plaque assays (Figure 6C) . Nucleotide position refers to the complete genome sequence of JEV SA 14 -14-2 (Genbank accession number JN604986). 

The current protocol has been successfully used to generate full-length infectious cDNA clones for two different strains (CNU/LP2 27 and SA 14 -14-2 28 ) of JEV, a flavivirus whose functional cDNA has proved to be inherently difficult to construct and propagate because of host cell toxicity and the genetic instability of the cloned cDNA. 8, [74] [75] [76] This protocol involves three major components: first, maximizing the synthesis/ amplification of a faithful cDNA copy of the viral RNA using high-fidelity reverse transcriptase/DNA polymerase; second, cloning the viral prM-E coding region containing toxic sequences (unpublished data) 74, 77, 78 in a very low-copy number vector BAC from the initial cDNA subcloning to the final full-length cDNA assembly steps; and third, utilizing a cloning vector BAC that can accommodate a foreign DNA with an average size of 120-350 kb, [19] [20] [21] which apparently tolerates larger DNA inserts than do other cloning vectors. This cloning approach will be generally applicable to many other positive-strand RNA viruses, particularly those with a large RNA genome of ~10 to 32 kb. Generation of an infectious cDNA clone is a key step in developing a reverse genetics system for RNA viruses, especially for positive-strand RNA viruses, because its genome acts as viral mRNA that is translated into proteins by host cell ribosomes. Thus, viral replication can be initiated by the introduction of a cDNA-derived genome-length RNA molecule into a susceptible host cell. The availability of an infectious JEV cDNA clone, when combined with recombinant DNA technology, has increased our understanding of various aspects of the viral life cycle at the molecular level, such as gene expression 73, 79 and genome replication. 63, 64 Also, a full-length JEV cDNA clone has proven to be a valuable tool for the development of antiviral vaccines 28 and gene delivery vectors. 80, 81 As with all positive-strand RNA viruses, there are multiple critical steps in constructing a reliable functional cDNA for JEV from which highly infectious RNAs can be synthesized in vitro. Ideally, the sequence of the synthetic RNAs transcribed from a clone of the full-length cDNA should be identical to that of the viral genomic RNA, particularly the 5'-and 3'-terminal sequences that are required for the initiation of viral RNA replication. [60] [61] [62] In the current protocol, the authentic 5'-and 3'-ends were ensured by placing the SP6 promoter sequence upstream of the first adenine nucleotide of the viral genome and positioning a unique artificial Xba I restriction site downstream of the last thymine nucleotide of the viral genome, respectively. Capped synthetic RNAs with the authentic 5' and 3' ends were produced by run-off transcription of an Xba I-linearized and MBN-treated cDNA template using SP6 RNA polymerase primed with the m 7 G(5')ppp(5')A cap analog. This protocol can be modified in several ways. For in vitro transcription, another bacteriophage RNA polymerase (e.g., T3 or T7) can be used in conjunction with its well-defined promoter sequence. 27 As a run-off site, a different restriction site can be utilized if it is not present in the viral genome and if synthetic RNA from the linearized cDNA ends with the authentic 3' end. The importance of the 3'-end nucleotide sequence has been demonstrated by a ~10-fold decrease in RNA infectivity when a synthetic RNA contains three or four virus-unrelated nucleotides at its 3' end. 27 In an in vitro transcription reaction, both the m 7 G(5')ppp(5')A and m 7 G(5')ppp(5')G cap analog can be used equally well, although the latter places an unrelated extra G nucleotide upstream of the viral 5'-end, but that addition does not alter the infectivity or replication of synthetic RNA. 27 Moreover, removal of the cDNA template from the RNA transcripts by DNase I digestion is not necessary for RNA infectivity tests, because the cDNA template itself is not infectious. 27 The BAC technology has now been applied to constructing infectious cDNA clones for a handful of positive-strand RNA viruses, namely, two JEVs, CNU/LP2 27 and SA 14 -14-2 28 (genome size, ~11 kb); two dengue viruses, BR/90 26 and NGC 29 (~11 kb); the bovine viral diarrhea construction is the high genetic stability of the large, 1-or 2-copy BAC plasmids; however, the intrinsic nature of its extremely low-copy number is also a great disadvantage, because of very low yields of BAC DNA and the consequent reduction in the purity of the BAC DNA with respect to host chromosomal DNA. In the current protocol, the yield of BAC DNA is maximized by growing E. coli DH10B transformed with the infectious BAC pBAC/SA 14 -14-2 in a nutrient-rich medium, 2xYT. Despite this effort, the average yield is only ~15 µg of BAC DNA from 500 ml of 2xYT broth. Also, the purity of the BAC DNA is best achieved by using CsCl-EtBr density gradient centrifugation for purification, rather than the commonly used column-based plasmid isolation. However, it is important to keep in mind that the BAC-transformed E. coli should not overgrow because it might jeopardize the genetic stability of the cloned cDNA, and higher growth does not necessarily lead to greater yields or higherpurity BAC DNA.

The protocol described here is an optimized, efficient, and streamlined method for the construction and propagation of a genetically stable fulllength infectious cDNA clone as a BAC for JEV, a procedure once thought practically impossible. This same cloning strategy may also be applied to many other positive-strand RNA viruses. In general, infectious cDNA clones enable us to introduce a variety of mutations (e.g., deletions, insertions, and point mutations) into a viral RNA genome to study their biological functions in viral replication and pathogenesis. This cDNAbased reverse genetics system makes it possible to develop and test vaccine and therapeutic candidates targeting a virulence factor(s) of a particular positive-strand RNA virus of interest. In addition, this infectious cDNA technology can also be utilized as a viral vector, capable of expressing a foreign gene(s) of interest for many applications in biomedical research.

The authors have declared that they have no competing financial interests.

Digest the vector and insert DNAs with two appropriate restriction endonucleases

30 a derivative of the pBeloBAC11 plasmid (7507 bp, GenBank accession number U51113), with Pme I and Not I in a total volume of 60 µl (containing ~500 ng DNA, 10 U enzyme, 1x digestion buffer, and 1× BSA) at 37 °C for 12-15 hr

Perform a sequential digestion of each of the four cDNA amplicons (insert) with Sma I and Not I in a total volume of 60 µl (containing 1 µg DNA, 20 U enzyme, 1× digestion buffer, and 1× BSA) at 25 °C (Sma I) or 37 °C (Not I) for 12-15 hr

Separate the doubly digested products on a 1% low-melting point agarose gel containing 0.5 µg/ml EtBr. Cut out a band of the desired DNA fragment with a minimal amount of agarose (usually ~200 µl) under long-wave ultraviolet light

Add an equal volume of TEM buffer (10 mM Tris-Cl, 1 mM EDTA, and 10 mM MgCl 2 ) to the excised agarose in a 1.7 ml microtube. Incubate the sample at 72 °C for 10-15 min

Add an equal volume of pre-warmed buffer-saturated phenol, vortex vigorously for 1 min, and centrifuge at 13,400 × g for 10 min at RT. Note: The mixture separates into a lower organic phase and an upper aqueous phase

Add an equal volume of chloroform:isoamyl alcohol (24:1), vortex for 1 min, and centrifuge at 13,400 × g for 10 min at RT. Transfer the upper aqueous phase to a new microtube

Add 0.5 µl of 10 µg/µl yeast tRNA, 1/10 volume of 3 M sodium acetate, and 2.5 volumes of 100% ethanol. Keep the mixture on ice for 20 min

Centrifuge the mixture at 13,400 × g for 10 min at RT. Remove the supernatant and wash the DNA pellet with 1 ml of 70% ethanol by pulse-vortexing three to five times and centrifuging at 13,400 × g for 10 min

Air-dry the DNA pellet for 10 min and dissolve it in 20 µl of TE buffer (10 mM Tris-Cl and 1 mM EDTA

Ligate the desired vector and insert DNA fragments using T4 DNA ligase

Set up a 20 µl ligation reaction containing 50-100 ng of the vector DNA, a ~3-fold molar excess of the insert DNA, 400 U T4 DNA ligase, and 1× ligation buffer. Incubate the ligation reaction at 16 °C for 12-15 hr

Perform four separate ligations, each joining the 15426-bp Pme I-Not I fragment of pBAC/PRRSV/FL with the 2559-bp (for F1), 4157-bp (for F2), 3908-bp (for F3), or 1784-bp (for F4) Sma I-Not I fragment of one of the four cDNA amplicons

Transform the ligated DNA into E. coli DH10B by the CaCl 2 -heat shock method

Take 100 µl aliquots of the CaCl 2 -treated competent DH10B cells 69 stored at -80 °C and thaw them on ice

Note: Use 100 µl of cells per transformation

Add a 10 µl aliquot of the DNA ligation reaction to 100 µl of the thawed cells in a 1.7 ml microtube, mix gently by tapping the tube, and keep on ice for 30 min

Heat-shock the DNA-cell mixture for 45 sec in a 42 °C water bath, place on ice for 2 min, and then add 900 µl of LB broth pre-warmed to RT

Incubate the heat-shocked cells at 35 °C for 1 hr, with shaking at 225-250 rpm

Spread 50 to 200 µl aliquots of the cultured cells on LB agar plates containing 10 µg/ml chloramphenicol (Cml)

Recover the cloned BAC DNA from the host cells by a column-based purification method (Figure 1C)

Pick six to eight bacterial colonies from the LB-Cml agar plates and inoculate them into 3 ml of 2xYT broth containing 10 µg/ml Cml

Perform two analytical restriction enzyme digestions of the isolated BACs for ~6 hr in a total volume of 10 µl, one to identify the presence of the vector with a correct insert using the same enzymes used for cloning (see Protocol 3.1), and the other to test the integrity of the cloned BACs with an appropriate enzyme

Propagate the correctly cloned BACs by inoculating 500 µl of the positive bacterial cultures (from Protocol 3.5.1) in 500 ml of 2xYT-Cml medium and cultivating the inoculum for 6 hr at 35 °C while shaking at 225-250 rpm

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Construction of an infectious cDNA clone for a Brazilian prototype strain of dengue virus type 1: characterization of a temperature-sensitive mutation in NS1

Development and application of a reverse genetics system for Japanese encephalitis virus

A molecularly cloned, live-attenuated Japanese encephalitis vaccine SA 14 -14-2 virus: a conserved single amino acid in the ij hairpin of the viral E glycoprotein determines neurovirulence in mice

Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome

Identification of 5' and 3' cis-acting elements of the porcine reproductive and respiratory syndrome virus: acquisition of novel 5' AU-rich sequences restored replication of a 5'-proximal 7-nucleotide deletion mutant

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Mechanisms of evasion of the type I interferon antiviral response by flaviviruses

Functional RNA elements in the dengue virus genome

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Control of dengue virus translation and replication

3' cis-acting elements that contribute to the competence and efficiency of Japanese encephalitis virus genome replication: functional importance of sequence duplications, deletions, and substitutions

A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication

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The authors would like to acknowledge the Utah Science Technology and Research fund for support of YML and the Korea National Research Foundation grants (2009-0069679 and 2010-0010154) for support of SIY. This research was supported by the Utah Agricultural Experiment Station, Utah State University, and approved as journal paper number UAES #8753. Also, the authors thank Dr. Deborah McClellan for editorial assistance.