key: cord-017824-0pinevfc authors: Tekes, Gergely title: Vaccinia Virus-Based Reverse Genetics for Feline Coronaviruses date: 2015-09-10 journal: Animal Coronaviruses DOI: 10.1007/978-1-4939-3414-0_7 sha: doc_id: 17824 cord_uid: 0pinevfc For decades, the genetic modification of coronavirus genomes and the generation of recombinant coronaviruses have been hampered mostly due to the extraordinary large size of the coronaviral genome. The very first reverse genetic system for feline coronaviruses (FCoVs) was established in the early 2000s; the respective approach exclusively enabled the manipulation of the 3′-third of the viral genome. Later on, vaccinia virus- and bacterial artificial chromosome (BAC)-based systems have been developed. Both systems have the advantage that the entire FCoV genome is amenable for mutagenesis. The main focus of this chapter is the vaccinia virus-based reverse genetic system for FCoVs. Here we present protocols for (1) the generation of a full-length cDNA clone, (2) the manipulation of the FCoV genome, and (3) the rescue of recombinant FCoVs. The establishment of a reverse genetic system for feline coronaviruses (FCoVs), which allows to modify the entire coronaviral genome, was successfully achieved for the fi rst time in 2008 [ 1 ] . This system relies on vaccinia virus, which serves as a cloning vector for the full-length FCoV cDNA. The very fi rst vaccinia virusbased reverse genetic system was developed for the human coronavirus (HCoV) 229 E [ 2 ] and since then it has been successfully applied for other coronaviruses, e.g., infectious bronchitis virus (IBV) [ 3 ] or mouse hepatitis virus (MHV) [ 4 ] . The major advantage of the vaccinia virus-based reverse genetic system lies in the stable integration of full-length corona viral cDNAs into the vaccinia virus genome. In contrast, other conventional cloning techniques often are not suitable for the accommodation of large coronaviral cDNA inserts due to instability of the plasmids caused by certain coronaviral sequences. However, reverse genetic systems, which are based on different techniques other than vaccinia virus, have been developed and used successfully for the generation of various recombinant coronaviruses [ 5 -11 ] . Here, the vaccinia virus-based FCoV reverse genetic system is described. In the fi rst part of this chapter, different strategies for the assembly of the full-length FCoV cDNA and its integration into the vaccinia virus genome are presented (Sect. 3.1 ). Second, the manipulation of the FCoV cDNA integrated into the vaccinia virus genome is outlined (Sect. 3.2 ), followed by the recovery of recombinant feline coronaviruses (Sect. 3.3 ). 1. Cloning and ligation. -pGemT TA cloning kit (Promega). -High-concentrated T4 DNA ligase (Fermentas). -Antarctic phosphatase (NEB). -Qiaex II gel extraction kit (Qiagen). -CHEF Mapper-II pulse-fi eld gel electrophoresis System (Bio-Rad). -Vaccinia Virus vNotI/tk [ 12 ] , fowl-pox virus. 2. Vaccinia virus large-scale DNA preparation. -BHK-21 cells. -DMEM cell culture medium (Sigma). -10× buffer A (10 mM Tris-HCl pH 9.0, 1 mM EDTA). -MagNaLyser Green Beads (Roche). -MagNaLyser Instrument (Roche). -Beckman ultracentrifuge with rotor SW28. -Trypsin. -RNase-free DNase with the appropriate 10× buffer (Promega). -Proteinase K (Roche). -2× proteinase K buffer (200 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8.0, 0.5 % SDS, 400 mM NaCl). -RNase-free water. 1. DMEM cell culture medium (Sigma). 2. CV-1 cells [ 13 ] . 3. D980R cells [ 13 ] . 4 . Lipofectamine 2000 (Life Technologies). 5 . Sonication water bath. 6 . Selective medium for GPT positive selection : (1) xanthine (Sigma) 10 mg/ml in 0.1 M NaOH; (2) hypoxanthine (Sigma) 10 mg/ml in 0.1 M NaOH; (3) mycophenolic acid (MPA) (Sigma). For an entire 6-well plate use 12 ml of medium and supplement it with 300 μl xanthine, 18 μl hypoxanthine, and 30 μl MPA from the above described stocks. 7. Selective medium for GPT negative selection : 6-thioguanine (6-TG) (Sigma) 1 mg/ml in 0.1 M NaOH. For an entire 6-well plate use 12 ml of medium and supplement it with 12 μl 6-TG from the above described stock. 1. RiboMax large-scale RNA production system-T7 (Promega). 2. Cap analog 30 mM (7mGpppG) (NEB). 4. RNase-free DNase (Promega). 5. LiCl solution (7.5 M LiCl, 50 mM EDTA pH 7.5). 6. RNase-free water. 1. Crfk-TetOn-N cells (generated in our laboratory). 2. 1× phosphate-buffered saline (PBS). 3. Electroporator (Bio-Rad Gene Pulser). 4. Electroporation cuvette (4 mm gap) (VWR). Two alternative strategies for the assembly of the full-length FCoV infectious clone are described. The fi rst strategy represents a twostep approach (Sect. 3.1.1 ).This method involves (1) the introduction of the major part of the FCoV genome through in vitro ligation into the vaccinia virus genome (section "Integration of the FCoV cDNA into the Vaccinia Virus Genome via In Vitro Ligation") followed by (2) the completion of the full-length FCoV cDNA via vaccinia virus-mediated homologous recombination (section "Second Step"). The reason for this strategy is that certain coronavirus-derived sequences can cause instability in cloning plasmids, which makes it diffi cult to assemble the full-length FCoV cDNA by in vitro ligation of FCoV cDNA fragments originating from plasmids. Such an approach was applied for the establishment of the fi rst FCoV infectious clone [ 1 ] . Since then, this infectious clone has been successfully applied to study different aspects of FCoV biology [ 14 -16 ] described, which simplifi es the procedure for the generation of the FCoV infectious clone (Tekes, unpublished) . This approach omits all of the in vitro ligation steps and it is based exclusively on vaccinia virus-mediated homologous recombination. Regardless of the applied strategy, viral RNA serves as a starting material for the generation of all FCoV-sequence containing plasmids (sections "First Step", 3.1.1.3, "Generation of plasmids suitable for vaccinia virus-mediated homologous recombination"). 1. Coronavirus-derived sequences, which can cause instability in cloning vectors, are often located between nt 5000 and 15,000 of the coronaviral genomic sequence. Therefore, this part of the genome should be introduced into the vaccinia virus genome by vaccinia virus-mediated homologous recombination in the second step as described in section "Completion of the FCoV cDNA Using Vaccinia Virus-Mediated Homologous Recombination". 2. Analyze the remaining parts of the FCoV sequence (1-5000 and 15,000-29,000 nt) and choose restriction enzyme sites encoded by the FCoV genome that will allow the in vitro ligation of cloned cDNA inserts. After the analysis, prepare a set of plasmids (no more than 4-5) covering the FCoV genome from nt 1 to 5000 and from nt 15,000 to 29,000 (Fig. 1a ). 3. Plasmid A (pA) contains upstream of the FCoV sequence a Bsp120I site followed by a T7 promoter and a G nucleotide. The Bsp120I site is required for the ligation of the cDNA insert derived from pA with the NotI digested vaccinia virus DNA (section "Large-Scale Vaccinia Virus DNA Preparation").The T7 promoter for the T7 RNA polymerase enables the generation of full-length FCoV RNA via in vitro transcription (IVT) (Sect. 3.3.1 ). The presence of the G nucleotide is recommended for the proper initiation of the T7 polymerase. Furthermore, plasmid D (pD) should contain downstream of the FCoV 3′UTR 20-30 A nucleotides, which will serve as a synthetic poly-A tail after IVT. These A nucleotides should be followed by a unique cleavage site (e.g., ClaI ), the hepatitis delta ribozyme (HDR) sequence, and a Bsp120I cleavage site (Fig. 1a ) . 6. Collect the supernatant from the three tubes and combine it into a new tube. 8. Take one of the aliquots, pellet the cells and perform vaccinia virus DNA preparation using proteinase K digestion followed by phenol-chloroform extraction as described (section "Large-Scale Vaccinia Virus DNA Preparation", steps 12-18). 9. Analyze the presence of the FCoV sequence in the vaccinia virus genome by PCR . 10. If the FCoV sequence was successfully integrated into the vaccinia virus genome, proceed with the second step (section "Generation of Plasmids Suitable for Vaccinia Virus-Mediated Homologous Recombination"). The resulting recombinant vaccinia virus is called vrecFCoV-ABCD. In order to achieve the integration of the full-length FCoV cDNA into the vaccinia virus genome, the remaining part of the FCoV genome (nt 5000-15,000) is introduced by vaccinia virus-mediated homologous recombination. 1. Generate DNA fragments AB1, AB2, and AB3 by RT-PCR . Clone fragments AB1 and AB3 corresponding approximately to the FCoV sequence nt 4500-8500 and 115,000-15,500, respectively, upstream and downstream of the phosphoribosyltransferase (GPT) gene in the plasmid pGPT-1 [ 13 ] in order to generate pGPT-AB-1/3 (Fig. 2a ) . The fragment AB-1 contains a 500 nt long overlapping piece at its 5′ terminal with fragment A. Fragment AB-3 possesses a 500 nt long overlapping piece at its 3′ terminal with fragment B. These overlapping sequences are required for the vaccinia virus-mediated homologous recombination. Clone fragment AB2 corresponding approximately to the Second Step FCoV sequence nt 8000-1200 into a suitable plasmid backbone (Fig. 2a ) . This fragment contains at both ends 500 nt long overlapping fragments with AB-1 and AB-3, respectively. The overlapping sequences are prerequisites for the vaccinia virus-mediated homologous recombination. The required cleavage sites of AB1, AB2, and AB3 for the cloning reactions must be included through the primer sequences designed for the RT-PCR . The missing part of the FCoV genome (nt 5000-15,000) is introduced by two rounds of vaccinia virus-mediated homologous recombination using GPT as a positive and negative selection marker. The overall strategy is presented in Fig. 2b ; the technical details are described in Sect. 3.2 . If no cloning diffi culties are observed with large (4-5 kb) fragments covering the entire FCoV genome, the use of the following method is recommended. One major advantage of this approach is that the time consuming and complicated in vitro ligation steps 1. Divide the FCoV genome into eight segments. Accordingly, prepare eight fragments by RT-PCR with a size of 3.5-4 kb each covering the entire FCoV genome (Fig. 3 ).Clone these RT-PCR fragments into a commercial available TA cloning vector in order to generate plasmids 1-8 (p1, p2, p3, p4, p5, p6, p7, and p8). Generation of Plasmids Suitable for Vaccinia Virus-Mediated Homologous Recombination Fig. 3 Generation of plasmids required for the introduction of the full-length FCoV genome into the vaccinia virus genome by vaccinia virus-mediated homologous recombination. For the assembly of the cDNA clone, the approximately 30 kb long FCoV genome is divided into eight overlapping fragments (1) (2) (3) (4) (5) (6) (7) (8) . The fragments are color-coded. Eight plasmids (p1, p2, p3, p4, p5, p6, p7, and p8) corresponding to these fragments are depicted. Out of these eight plasmids, the generation of four fi nal plasmids (pA, pB, pC, and pD), which are required for the introduction of the entire FCoV cDNA into the vaccinia virus (VV) genome, are shown. The overlapping parts of the various fragments are required for the vaccinia virus-mediated homologous recombination. GPT guanosine phosphoribosyl transferase promoter and a G nucleotide. Plasmid 8 (p8) corresponds to the 3′ end of the FCoV genome sequence followed by 20-30 A nucleotides (synthetic poly-A tail), a unique cleavage site, the hepatitis delta ribozyme (HDR) sequence and a 500 nt long piece of the vaccinia virus genome. Plasmid 2 (p2) contains an approximately 500 nt long overlapping part with p1. Plasmid 7 (p7) has an approximately 500 nt long overlapping part with p8. Plasmid 3 (p3) has an approximately 500 nt long overlapping part with p2. Plasmid 6 (p6) contains an approximately 500 nt long overlapping part with p7. Plasmid 4 (p4) has an approximately 500 nt long overlapping part with p3. Plasmid 5 (p5) comprises an approximately 500 nt long overlapping part with p6. These overlapping parts are required for the vaccinia virusmediated homologous recombination. In order to fi nalize the plasmids needed for the vaccinia virus-mediated homologous recombination, additional cloning steps are required (steps 3-6). 3. Clone fragments 1 and 8 originating from p1 and p8, respectively, upstream and downstream of the GPT gene in the multiple cloning sites of the pGPT-1. The specifi c restriction enzyme cleavage sites required for the subcloning originate from the primer sequences used for the generation of the RT-PCR products. The resulting plasmid is called plasmid A (pA) (Fig. 3 ). 4. Clone fragment 2 originating from p2 in the p7 in order to obtain plasmid B (pB). The specifi c restriction enzyme cleavage sites required for the subcloning originate from the primer sequences used for the generation of the RT-PCR products. 5. Clone fragments 3 and 6 originating from p3 and p6, respectively, upstream and downstream of the GPT gene in the pGPT-1. The specifi c restriction enzyme cleavage sites required for the subcloning originate from the primer sequences used for the generation of the RT-PCR products. The resulting plasmid is called plasmid C (pC). 6. Clone fragment 4 originating from p4 in the p5 in order to obtain plasmid D (pD). The cleavage site between fragments 4 and 5 is present in the original FCoV sequence. The other restriction enzyme cleavage sites required for the subcloning originate from the primer sequences used for the generation of the RT-PCR products. The entire FCoV genome is introduced by four rounds of vaccinia virus-mediated homologous recombination using GPT as a positive and negative selection marker. The overall strategy is presented in Fig. 4 ; the technical details are described in Sect. 3.2 . For the manipulation of the FCoV sequence integrated in the vaccinia virus genome, specifi cally designed plasmids in combination with vaccinia virus-mediated homologous recombination are required. This approach allows the replacement of defi ned parts of the FCoV genome. In an initial step, the GPT gene is introduced into the vaccinia virus genome. For this purpose a specifi c plasmid is required, which contains upstream and downstream of the GPT gene homologous sequences (500 nt) with the targeted FCoV region. These homologous sequences in the plasmid enable the insertion of the GPT gene (preceded by a vaccinia virus promoter) by vaccinia virus-mediated double homologous recombination in the vaccinia virus genome. The resulting recombinant vaccinia virus with GPT gene can be selected using the GPT as a positive selection marker (Sect. 3.2.2 ). In the following step, the GPT gene is replaced by the FCoV sequence with the desired mutations. The required specifi c plasmid Fig. 4 Strategy for introduction of the full-length FCoV cDNA into the vaccinia virus genome by vaccinia virusmediated homologous recombination. In order to integrate the full-length FCoV cDNA, fragments 1-8 are introduced via four rounds of vaccinia virus-mediated homologous recombination using GPT as a positive and a negative selection marker. The required plasmids (pA, pB, pC, and pD) and the resulting recombinant vaccinia viruses are shown. GPT guanine phosphoribosyl transferase contains two homologous regions (500 nt) with the targeted FCoV sequence upstream and downstream of the FCoV sequence to be introduced. Vaccinia virus -mediated homologous recombination leads to replacement of the GPT gene by the FCoV sequence with the desired mutations. In the absence of the GPT gene, the resulting recombinant vaccinia virus can be selected using GPT as a negative selection marker (Sect. 3.2.3 ) . In order to generate the GPT positive/negative recombinant vaccinia viruses, cells are infected with the appropriate vaccinia virus and transfected with the specifi cally designed plasmids. During incubation of infected/transfected cells vaccinia virus-mediated homologous recombination takes place between the homologous sequences of the vaccinia genome and the plasmid sequence. 6. This material contains the newly generated GPT positive vs. negative recombinant vaccinia viruses, which will be selected using GPT as positive (Sect. 3 8. Transfer the cells into a 10 cm dish and fi ll up the dish with 10 ml of fresh medium. 9. Optionally, change the medium 2-3 h after the electroporation. Harvest the supernatant containing the recombinant FCoVs and use it for further analysis. 1. Avoid the application of UV-light to excise the bands from the agarose gel. We experienced that exposing the DNA to UVlight can hamper the ligation of these fragments into the vaccinia virus genome. To assess the exact position of a DNA band without damaging it with UV-light, load DNA marker on both sides of your digested fragment on the agarose gel. After electrophoresis, separate the lanes with the marker from the remaining part (contains the digested fragments) of the gel. Using UV-light, cut out a small piece in each of the marker lanes at the expected height of the digested product. Position these lanes back together with the part containing the digested fragment. Cut the agarose gel between the two indicated positions in order to remove the agarose slice with the digested fragment. 2. Do not pipette vaccinia virus DNA with regular tips after proteinase K digestion. These tips can damage the DNA due to their narrow opening. Cut the end of the tips with scissors to enlarge the opening. 3. In order to keep the DNA intact, do not vortex the tubes containing vaccinia virus DNA after proteinase K digestion. Vortexing can break the large vaccinia virus DNA. 4 . Do not dry the vaccinia virus pellet but resuspend it immediately in water. 5 . In our laboratory CHEF Mapper-II pulse-fi eld gel electrophoresis System (Bio-Rad) is used. For the optimal separation of the bands a 1 % agarose gel should be prepared. The gel runs for approximately 18 h (switch time 3-30 s, angle 120°, voltage 6 V/cm). The set up can vary depending on the system used for PFGE . 7. The conditions for the electroporation depend on the cell line and device used for the electroporation. It is recommended to test a given electroporator with the cells to achieve optimal effi ciency. 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