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Comparison of RNA and cDNA transfection methods for rescue of infectious bursal disease virus
Journal of Virological Methods 97 (2001) 67 – 76 www.elsevier.com/locate/jviromet
Comparison of RNA and cDNA transfection methods for rescue of infectious bursal disease virus Hein J. Boot *, Kristina Dokic, Ben P.H. Peeters Department of A6ian Virology, Institute for Animal Science and Health, ID-Lelystad, PO Box 65, NL-8200 AB Lelystad, The Netherlands Received 8 November 2000; received in revised form 21 May 2001; accepted 23 May 2001
Abstract Specific alterations in the genetic material of RNA viruses rely on a technique known as reverse genetics. Transfection of cells with the altered generic material is a critical step of this procedure. In this report we have compared RNA and cDNA transfection methods for the efficiency of transient protein expression and rescue of (recombinant) infectious bursal disease virus (IBDV). Quantitative expression analysis of the secreted alkaline phosphatase reporter protein, and qualitative expression levels of an IBDV protein showed both that cDNA transfection results in a much higher level of protein expression than RNA transfection. Because the rescue of a crippled variant of IBDV was achieved consistently using the cDNA transfection method, but failed when we used the RNA transfection method, we favor the cDNA transfection method for the rescue of (recombinant) IBDV from cloned cDNA. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Reverse genetics; mRNA transfection; cDNA transfection; IBDV; SEAP
1. Introduction Infectious bursal disease virus (IBDV), a member of the family of Birnaviridae, genus a6ibirna6irus, is the causative agent of an avian restricted disease known as Gumboro. The first outbreak of IBDV was reported in commercial chicken flocks in Delaware, USA (Cosgrove, 1962). The IBDV strains, which were isolated during this outbreak, are now referred to as classi* Corresponding author. Tel.: + 31-320-238-238; fax: + 31320-238-668. E-mail address: [email protected] (H.J. Boot).
cal serotype I isolates. Later on, a second serotype (serotype II) of IBDV was identified (McNulty and Saif, 1988). Serotype II IBDV isolates are apathogenic and are recovered mainly from turkeys (Ismail et al., 1988). Antigenic variants of serotype I IBDV isolates were recovered from infected flocks in the USA starting in 1985, probably resulting from the selection pressure of field vaccination against classical IBDV serotype I (Snyder, 1990). Although being antigenic variant these isolates have only minor amino acid changes and do not form a separate serotype. In 1991, IBDV isolates, which were able to break through levels of maternal antibodies that normally were
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protective, were reported in Europe (Chettle et al., 1989). These isolates, the so-called very virulent IBDV (vvIBDV), are causing more severe clinical signs during an outbreak, and are now found almost world-wide (VandenBerg, 2000). The genome of IBDV consists of a double stranded RNA (dsRNA) genome divide over two segments, the A- and B-segment (Dobos et al., 1979). The A-segment (3.3 kbp) contains two partly overlapping open reading frames (ORFs). The first, smallest ORF encodes the non-structural Viral Protein 5 (VP5, 17 kDa, non-essential). The second ORF encodes a polyprotein (110 kDa), which is cleaved autocatalytically into pVP2 (48 kDa, capsid protein), VP4 (28 kDa, protease) and VP3 (32 kDa, capsid protein). During virus release pVP2 is further processed into VP2 (38 kDa). The conformation dependent neutralizing epitopes are found exclusively in a specific region of the VP2 protein, the so-called hypervariable region (amino acids 224– 314). The B-segment (2.9 kbp) contains one large ORF, encoding the 91 kDa VP1 protein, the RNA dependent RNA polymerase (Bruenn, 1991). VP1 is, furthermore, covalently linked (VPg) to the 5%-ends of the genomic RNA segments (Muller and Nitschke, 1987). In 1996, the first report of rescue of IBDV from cloned cDNA was published (Mundt and Vakharia, 1996), opening the way to manipulate the IBDV genome, and analyzing the effect of the introduced mutations in rescued IBDV (rIBDV). The introduction of mutations and the ability to generate chimeric IBDV strains is particularly important to get a better understanding of virulence factors. The ability to manipulate the IBDV genome in a rational way will also enhance greatly vaccine development. Variations on the original IBDV mRNA transfection method (Mundt and Vakharia, 1996) have been published recently by us (Boot et al., 1999) and others (Lim et al., 1999). These methods rely on transfection of cloned cDNA instead of RNA. RNA transfections have the drawback that in vitro production of RNA is expensive and laborious in comparison to DNA transfections. Furthermore, protein expression levels from transfected RNA are generally low in comparison to cDNA transfections
(Boyer and Haenni, 1994). Mutations introduced in the IBDV genome might hamper the assembly and/or replication of the virus. A highly efficient and reproducible transfection method will increase the chances of rescuing such crippled viruses. Here we show that cDNA transfections give rise to a much higher transient expression levels than RNA transfections. Furthermore, a particular mutation in the B-segment prevented the rescue of recombinant IBDV when RNA transfections were carried out, while this recombinant IBDV could be rescued consistently when the cDNA transfection method was used.
2. Materials and methods
2.1. Virus, plasmids, and cells The IBDV strain CEF94 is a cell culture adapted classical serotype I isolate (Boot et al., 1999), which is derived from the PV1 isolate (Italia, 1973). The full-length cDNA of this strain has been cloned (GeneBank: AF194428 and AF194429) in a pUC18 derived vector, which contains a hepatitis delta virus ribozym and a T7 promoter and terminator (see Fig. 1), yielding plasmids pHB-36W (A-segment cDNA) and pHB34Z (B-segment cDNA) (Boot et al., 1999). For transfection of cDNA and mRNA, and for propagation of wild-type or rescued CEF94 we have used the differentiation incompetent quail myogenic cell line QM5 (Antin and Ordahl, 1991), which was maintained in QT35 medium (GibcoBRL Life Technologies) supplemented with 5% fetal calf serum and 2% antibiotics solution ABII (1000 U/ml Penicillin (Yamanouchi), 1 mg/ml Streptomycin (Radiumfarma, Italy), 20 mg/ml Fungizone, 500 mg/ml; Polymixin B, and 10 mg/ ml Kanamycin). IBDV infected cells were detected using a VP3 specific monoclonal antibody Mab 9.7 (Boot et al., 2001).
2.2. Replacement of the polyprotein ORF by the secreted alkaline phosphatase gene The polyprotein encoding region (nt 131– 3166) of pHB-36W was replaced by the secreted alkaline
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phosphatase (SEAP) gene (Clontech). First, two independent polymerase chain reactions (PCRs) were carried out. In PCR-1, we used primers T7ACO (Boot et al., 2000) and ASSC (TCATCGATGA TGCATGCCTG CGATCGTTTG TCTGATC) and pHB-36W as template (resulting in a 171-bp fragment), while in PCR-2 we used primer ANC1 (Boot et al., 2000) and ATSC (CAGGCATGCA TCATCGATGA GGCTCCTGGG AGTC) and pHB-36W as template (resulting in a 113-bp fragment). In a subsequent fusion PCR (Higuchi, 1990), we used the purified (Qiaex II Gel Extraction Kit, Qiagen) PCR-1 and PCR-2 fragments as template and T7ACO an ANCO (Boot et al., 2000) as primers. The resulting PCR product (PCR-3, 264 bp) was cloned in pUC18-Ribo, which contains the cis-acting antigenomic hepatitis delta virus ribozym (Boot et al., 1999), yielding plasmid pHB-3. The integrity of the introduced nucleotide sequence was checked by sequence analysis. The two unique restriction sites of pHB-3 (SphI and ClaI), which were introduced in the fusion PCR, were used to insert the SEAP gene from plasmid pSEAP-Basic (Clontech), resulting in plasmid pHB-32. To generate a reporter plasmid, which has exactly the 5%-untranslated regions (UTR) of wild type IBDV we restored the nucleotides (GC) at position 129–
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130 to the wild type (CG) sequence. These changes were introduced by performing a PCR using primers ATG3 (CATCGCTGCGA TCGTTTGTCT GATCTCTAC) and T7EcoRI (GGAATTCTAA TACGACTCAC TATAGG), pHB-32 as template, and Pwo polymerase (Boeringher Mannheim) as enzyme (PCR-4, 159 bp). Plasmid pHB-32 was digested with SphI and the 3%-overhangs were trimmed by using Klenow polymerase (Pharmacia). After heat inactivation of Klenow the plasmid was digested with EcoRI, agarose gel purified and used in a ligation reaction with the EcoRI digested PCR-4 fragment, yielding plasmid pKD-ATG3R (Fig. 1).
2.3. RNA synthesis Plasmid DNA was isolated from 50 ml overnight Escherichia coli (DH5a) cultures using the Qiagen midiprep procedure (Qiagen, Germany). The isolated plasmids were treated with Proteinase K (Amresco, 1.0 mg/ml) in the presence of 0.1% sodium dodecyl sulphate (SDS) at 50 °C for 60 min to remove all RNase contamination. The plasmids were purified subsequently by phenol/chloroform/isoamylacohol treatment (two times) and ethanol precipitation (Sambrook et al., 1989), and dissolved (0.2 mg/ml) in diethyl
Fig. 1. Schematic representation of the plasmids containing a full-length IBDV A-segment (pHB-36W), B-segment (pHB-34Z), or a derivative of pHB-36W in which the coding region of the polyprotein has been replaced by SEAP gene (pKD-ATG3R). Boxes indicate open reading frames, and several functional genetic elements like hepatitis delta ribozyme (HDR) and T7 RNA polymerase promoter and terminator sequences are indicated. The fusion sites between the SEAP reporter gene and the 5%- and 3%-UTR of the IBDV A-segment is shown for pKD-ATG3R. The nucleotides originating from the 5%- and 3%- UTR are underlined.
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pyrocarbonate (DEPC) treated H2O. Capped RNA was synthesized in 15 ml DEPC treated H2O containing 1 mg DNA, 40 mM Tris (pH 8.0), 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 1 mM ATP, CTP and UTP, 0.2 mM GTP, 0.5 mM CAP analog (New England Biolabs), 1 U T7 polymerase (United States Biochemicals), 15 U RNasin (Promega) and 10 mM dithiothreitol (DTT). After incubation for 30 min at 37 °C, DNase (2 U, Amersham Pharmacia Biotech) was added and incubation at 37 °C was prolonged for 15 min.
once with PBS (3 ml) and twice with Optimem (3 ml). The monolayer was incubated for 10 min at 37 °C between the different washing steps. Transfection was carried out by mixing 1 mg DNA with 250 ml Optimem and 12.5 ml Lipofectamine (GibcoBRL Life technologies). This mixture was kept at room temperature for 30 min and added subsequently to the QM5 monolayer, which had been covered with 2 ml fresh Optimem. After overnight incubation (18 h, 37 °C, 5.0% CO2) the monolayer was rinsed once with PBS and covered with 2.5 ml QT35 medium. Each cDNA transfection was undertaken in three- or four-fold.
2.4. Transfection of mRNA Confluent monolayer (80%) of QM5 cells (10 cm2 per transfection) was washed once with phosphare buffer saline (PBS; 3 ml), twice with Optimem (3 ml) prior to transfection, and covered with 1 ml Optimem. The monolayer was incubated for 10 min at 37 °C between each washing step. In a polystyrene tube 125 ml Optimem 1 (GibcoBRL Life Technologies) was mixed with 12.5 ml lipofectin (GibcoBRL Life Technologies), and incubated at room temperature for 15 min. The RNA synthesis mixture (see Section 2.3) was added subsequently and incubation at room temperature was prolonged for 5 min. The mRNA/ Lypofectine/Optimem mixture was mixed with the supernatant of the QM5 monolayer. Transfection was allowed to proceed at 37 °C (5.0% CO2) for 3 h, after which time the monolayer was washed once with PBS and covered with 2.5 ml QT35 medium. Each RNA transfection was carried out in three- or four-fold with independent mRNA preparations.
2.5. Infection with fowlpox-T7 6irus and transfection of cDNA The plasmid preparation, which was used for the synthesis of mRNA was also used to transfect an 80% confluent monolayer of QM5 cells (10 cm2 per transfection) directly. The QM5 monolayer was washed once with PBS and covered with PBS contained 107 pfu fowlpox-T7 virus. Infection with fowlpox-T7 virus was allowed for 1 h at 37 °C (5% CO2), and the monolayer was washed
2.6. Quantitation of the SEAP reporter protein after transfection Plasmid pKD-ATG3R (see Fig. 1) was used to transfect QM5 cells directly (cDNA transfection), or was used for in vitro synthesis of RNA, which was transfected subsequently (RNA transfection). The last washing step at the end of each transfection procedure was taken as T=0 h. Samples (200 ml) were taken from the supernatant of transfected monolayers at different time points after T= 0 and stored at −20 °C. The SEAP activity (relative light units, RLU) in each sample was determined by using a chemoluminescense assay (Phospha-Light detecion kit (Tropix)), and a 1450 Microbeta luminometer (Wallac), as described by the supplier. The average RLU of the four independent transfections with either cDNA or RNA was determined for each time point.
2.7. Detection of IBDV antigen after transfection Plasmid pHB-36W (see Fig. 1) was used either directly (cDNA transfection) or after conversion into RNA (RNA transfection) to transfect QM5 cells. Transfected cells were incubated for 24 h in 2.5 ml QT35 medium (37 °C, in the presence of 5% CO2) after the last washing step in each procedure. The monolayer was washed once with PBS, air-dried, and frozen at − 70 °C. The cells were fixed subsequently by incubation for 10 min in PBS supplemented with 4% para-formaldehyde (4 °C). After washing the fixed cells twice with PBS (supplemented with 0.05% Tween-80) the
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first antibody (Mab 9.7 in PBS supplemented with 30 g/ml NaCl and 2% horse serum) was allowed to bind to viral antigens. Three washing steps were performed (PBS supplemented with 0.05% Tween-80) and the second antibody (rabbit anti mouse coupled to horseradish peroxidase (Dako, 1000-fold diluted,) in PBS supplemented with 30 g/ml NaCl and 2% horse serum) was allowed to bind. Detection of the second antibody was carried out after three washing steps (PBS supplemented with 0.05% Tween-80) by addition of a solution of 50 mM NaAc (pH 5.0), 0.2 mg/ml 3-amino-9-ethyl-carbazole (AEC, Sigma) and 0.01% H2O2.
2.8. Quantitation of rescued IBDV after transfection Plasmid pHB-36W was co-transfected with pHB-34Z, either directly (cDNA co-transfection), or after conversion into RNA (RNA co-transfection). QM5 cells transfected with either mRNA or cDNA were incubated for 24 h in 2.5 ml QT35 medium (37 °C, in the presence of 5% CO2) after the last washing step in each transfection procedure. The cells and supernatant were freeze/ thawed subsequently, filtered through a 200-nm-pore-size filter (Acrodisc, Gelman Sciences) and stored at −20 °C. The amount of IBDV (50% tissue culture infectious dose (TCID50)) was determined by infecting fresh QM5 cells with 10-fold dilutions of the filtered transfection lysates. IBDV infected wells were detected in an immunoperoxidase monolayer assay (IPMA) using the IBDV specific Mab 9.7 (see Section 2.6).
2.9. Mutagenesis of the B-segment cDNA A point mutation at position 2803 in the 3%UTR region of the B-segment was introduced by carrying out a PCR using the primers MB-10 (GGGGGCCCCC GCAGGCGAAG GCCCGGGAT) and BC10 (GCTCTAGATC AAGAACCCAC AGACCG), pHB-52 as template, and Pwo polymerase (Boehringer Mannheim) as enzyme (PCR-5, 277 bp). The pHB-52 plasmid is a derivative of pHB-34Z that contains a NgoMIV restriction site at nt 2741. This PCR fragment was
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purified subsequently, digested with NgoMIV and used to replace the corresponding part of plasmid pHB-52 (digested with NgoMIV and SmaI), yielding pMB-10. The presence of the point mutation at position (nt 2803) in pMB-10 was confirmed by sequence analysis. Secondary structure prediction of the 3%-UTR region of the codingstrand B-segment RNA was carried out using the Mfold program (version 3.0).
3. Results
3.1. Construction of a SEAP reporter plasmid To follow accurately protein expression originating from mRNA or cDNA after transfection of eukaryotic cells, the polyprotein encoding ORF of IBDV was replaced by the secreted form of the human placental alkaline phosphatase (SEAP). This enzyme is very stable and is efficiently secreted from expressing cells (Berger et al., 1988). The SEAP activity can easily be determined using the CSPD reagent, and the measured activity in the culture medium is directly proportional to changes in intracellular concentrations of SEAP (Berger et al., 1988). An additional advantage of this reporter protein is that it is secreted by the producing cells, making it possible to follow the expression kinetics by just removing a sample of the culture medium at different time points. The mechanism of translation initiation of the IBDV ORFs is unclear at present (see also Section 4). We chose to replace the polyprotein ORF, because the polyprotein is the precursor of the two capsid proteins and most likely the highest expressed IBDV protein. The ORF for the polyprotein was replaced in such a way that the same startcodon was used for the initiation of the SEAP and polyprotein, and no nucleotide differences were present in the 5%-UTR of the A-segment and reporter plasmid (Fig. 1).
3.2. Transfection of QM5 cells with reporter mRNA and cDNA To evaluate expression of the SEAP reporter protein a quail derived cell line (QM5) was used.
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mRNA, as pox viruses are capable of producing capped mRNA cytoplasmatically. After transfection of FPT7 infected QM5 cells with the reporter plasmid, we found a high level of SEAP activity in the supernatant (Fig. 3). SEAP activity was already detectable 5 h after transfection, and continued to increase for at least 2 days, indicating ongoing transcription and translation of the transfected plasmid DNA.
3.3. Transfection of QM5 cells with full-length IBDV RNA and cDNA
Fig. 2. Agarose gel electrophoresis (1.0%, 0.5* TBE) of linearized plasmid DNA (0.2 mg, lanes 1 – 3) or in vitro produced mRNA (lanes 4 – 6). Lane 1, pHB-36W (full length A-segment plasmid, 6175 bp); lane 2, pHB-34Z (full length B-segment plasmid, 5742 bp); lane 3, pKD-ATG3R (SEAP reporter plasmid, 4724 bp); lane 4, mRNA derived from pHB-36W (full length coding strand A-segment, 3260 nt); lane 5, mRNA derived from pHB-34Z (full length coding strand B-segment, 2827 nt); lane 6, mRNA derived from pKD-ATG3R (1808 nt); lane M, size marker DNA (Smartladder, Eurogentech).
About thirty percent of the fibroblast cells present in the monolayer can be transfected by plasmid DNA using established transfection protocols. Furthermore, several avian viruses, including IBDV and fowlpox virus are able to replicate on this cell line. We started to evaluate the relative efficiency of reporter protein expression by transfecting mRNA of the SEAP reporter gene. This mRNA was produced in vitro by using T7 RNA polymerase and the SEAP encoding reporter plasmid pKD-ATG3R in the presence of cap analogue. Although we were able to produce this capped mRNA reproducible (see Fig. 2) no detectable SEAP expression levels were obtained (Fig. 3). Alternatively we used an in vivo based T7 expression system. QM5 cells were infected with a recombinant fowlpox virus containing the T7 polymerase gene under control of the vaccinia virus early/late P7.5 promoter (Britton et al., 1996), before transfection with the reporter plasmid. Artificially introduced cDNA containing a T7 promoter will be transcribed into capped
Transfection of the reporter plasmid showed that the introduction of cDNA in FPT7 infected QM5 cells resulted in high levels of reporter protein expression, in contrast to mRNA transfection. To investigate whether IBDV proteins can be detected after transfection of full-length A- or B-segment RNA we transfected A-segment RNA alone or in combination with B-segment RNA. Both cDNA and the derived RNA (see Fig. 2) were used to transfect QM5 cells. Expression of VP3 (located at the C-terminal end of the polyprotein) was detected at 24 h post transfection by an IPMA. No VP3 expressing cells could be detected in case of RNA transfection (Fig. 4). This is in contrast to transfection with cDNA A-segment in which about 25% of the cells were expressing VP3 (Fig. 4). When A-segment RNA
Fig. 3. SEAP activity (RLU) in the supernatant of the QM5 monolayer after transfection with either plasmid pKDATG3R or in vitro produced mRNA using pKD-ATG3R as template. Aliquots of the supernatant were removed at different time points after transfection and stored at −20 °C, until the SEAP activity was determined. Each value is the average of four independent transfection experiments, error bars represent standard deviation.
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made, as the high level of transient expression of the VP3 protein hampered the identification of a group of cells (plaques) resulting from IBDV infection/replication after cDNA co-transfection.
3.4. Rescue of infectious IBDV from transfected QM5 cells
Fig. 4. An immunoperoxidase monolayer assay (IPMA) was performed using a VP3 specific monoclonal antibody (Mab 9.7). Nearly confluent monolayers of QM5 cells were either transfected with A-segment RNA or cDNA, or co-transfected with A- and B-segment RNA or cDNA. VP3 producing cells were visualized (dark cells), 24 h after the end of each transfection procedure.
was co-transfected in combination with B-segment RNA, several clustered cells (plaques) were found to produce VP3 at 24-h post-infection (Fig. 4). In case of co-transfection of the A- and B-segment cDNA we found about 25% of the cells expressing VP3 at 24-h post-infection (Fig. 4), resembling the result of the transfection of the A-segment alone. No comparison of the number of plaques of the mRNA and cDNA co-transfections could be
To determine the rescue efficiency of IBDV after co-transfection of A- and B-segment cDNA or RNA in a different way, we collected the supernatant 24 h after the end of each transfection method. Transfected monolayers, including the supernatant, were freeze-thawed once and the IBDV titer (50% TCID50) was determined after filtration of these lysates. All cDNA and RNA co-transfections yielded infectious virus with about equal efficiencies (TCID50 ] 5, Table 1). Subsequently, we compared both transfection methods by using a B-segment plasmid in which a single point mutation was present in the 3%-UTR at position nt 2803 (cytosine guanine). The stem–loop structure, which is present in the 3%UTR of the B-segment coding strand mRNA (Mundt and Muller, 1995; Boot et al., 1999) is extended by this single point mutation (Fig. 5). Using this altered B-segment cDNA we were not able to rescue IBDV after co-transfection of RNA. If on the other hand, the cDNA transfection method was used we were able to rescue recombinant IBDV in all four independent experiments, although the observed IBDV titer was
Table 1 Efficiency of the rescue of IBDV after cDNA and RNA transfections log10 TCID50a Experiment 1
Experiment 2
Experiment 3
Experiment 4
Average
cDNA pHB-36W+pHB-34Z PHB-36W+pMB-10
5.8 0.7
5.9 0.5
4.8 0.8
4.9 0.5
5.4 0.6
RNA pHB-36W+pHB-34Zb PHB-36W+pHB-34Z pHB-36W+pMB-10
0.0 4.7 0.0
0.0 5.0 0.0
0.0 4.7 0.0
– 5.4 –
0.0 5.0 0.0
a b
TCID (50%) 24 h after each transfection. In vitro synthesis of RNA in the absence of CAP analogue.
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Fig. 5. Prediction of the secondary structure of the 3%-terminus of the coding strand of the IBDV B-segment, using the Mfold program (version 3.0). Both the stem –loop structure of the wild-type B-segment and mutant MB-10 are shown. The point mutation of MB-10 at position nt 2803 (corresponding with nt-24) is indicated with an arrow.
reduced severely compared with co-transfection of the wild-type cDNA (Table 1).
4. Discussion Specific alterations in the genetic material of RNA viruses rely on a technique known as reverse genetics. First, the complete genomic RNA of the virus is reversed transcribed into cDNA, this cDNA is then altered, and transcribed into RNA. The recombinant RNA is used subsequently to rescue virus by transfection into cells that support the replication of the virus in question. Several methods to generate infectious RNA viruses have been developed during recent years (Boyer and Haenni, 1994) and issue 53 of Adv. Virus Res. (1999). In this study, we compared two transfection methods to rescue IBDV. The mechanism of translation initiation of birnaviruses is unknown currently. At least one unused start codon is present in the 5%-UTR of the A- and B-segment coding strand RNA, and the RNA also contains a large viral protein genome linked molecule (VPg, see Section 1). These two properties exclude a simple CAP-scanning mechanism of translation initiation. Furthermore, the AUG codon at position 131– 133 is used to initiate
translation of the polyprotein, making the 5%-UTR much smaller than the known 5%-UTR’s, which harbor an Internal Ribosome Entry Site (Le et al., 1996). It seems most likely that VPg is involved in translation initiation, and has a function comparable with a CAP-structure. Despite the lack of knowledge concerning translation initiation, it has been shown that the substitution of the VPg by a CAP-analogue is sufficient to initiate translation initiation of the IBDV RNA. Co-transfection of in vitro synthesized RNA of the IBDV A- and B-segment gives rise to infectious viral particles (Mundt and Vakharia, 1996). Also co-transfection of the Aand B-segment cDNA (preceded by a T7 promoter) into fowlpox-T7 virus infected cells, which express T7 polymerase leads to the formation of infectious viral particles (Boot et al., 1999). To assess, which of these two methods is the most efficient to recover infectious IBDV, we replaced firstly the ORF of the polyprotein for the SEAP reporter gene. Using this plasmid and the corresponding, in vitro synthesized, capped mRNA a marked difference was observed in the levels of reporter protein expression. There was no detectable SEAP expression in case of mRNA transfection, while a good and time dependent expression of SEAP occurred in case of cDNA transfection (Fig. 3). Also the transient expression of the VP3 capsid protein after transfection with either A-segment mRNA or the corresponding cDNA shows that only cDNA transfection lead to detectable protein expression (Fig. 4). Despite the marked difference between the protein expression levels, we observed no difference between IBDV recovery efficiencies between the two transfection methods. Both methods gave rise to 105 rIBDV particles per ml, 24 h after each transfection method. Although the final rIBDV titers are the same, it might still be that the efficiencies of the two transfection methods are quite different. IBDV replicates quite fast, as virus release is already occurring 8 h after the initial infection (Petek et al., 1973). Thus samples taken at 24 h post co-transfection do not only represent virus from initially virus producing cells, but also from secondary infected cells. Furthermore, the time of the mRNA (3 h) and cDNA (18 h)
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transfection procedures are quite different, and an additional transcription step (cDNA mRNA) has to occur in the cDNA transfection method. All these factors make direct comparison between the two transfection methods impossible. An efficient transfection method is needed mostly when mutations in the viral genome are introduced, which interfere with replication of the virus. Only with a very efficient transfection system, mutations can be studied that lead to viruses that replicate extremely slowly, or mutations which need to be reverted, or suppressed by a second-site mutation. To determine whether there is indeed a difference between the rescue efficiencies of the two transfection methods we have used a mutated B-segment plasmid. In this plasmids (pMB-10) we introduced a single point mutation, which leads to an extended stem– loop structure at the 3%-terminus of the coding strand. Although no data have been published on the function of this stem–loop structure we believe that this stem –loop structure might be involved in replication or translation of the viral genome. The introduction of the single point mutation leads to a more stable stem–loop structure, as the stem region is extended by one GC basepair (Fig. 5). Transfections using this mutated pMB-10 plasmid showed that the replication of rIBDV is hampered by this mutation. Rescue of the rIBDV is reduced severely when we used the cDNA transfection method (less then ten infections virus particles per ml, Table 1), whereas it was impossible to rescue this virus using the RNA transfection method. Although we have no information on how replication of the mutated virus is impaired by the stem –loop mutation, we can conclude from this transfection experiment that the cDNA transfection method is indeed more efficient for rescuing IBDV, than the RNA transfection method.
References Antin, P.B., Ordahl, C.P., 1991. Isolation and characterization of an avian myogenic cell line. Dev. Biol. 143 (1), 111 – 121. Berger, J., Hauber, J., Hauber, R., Geiger, R., Cullen, B.R., 1988. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukary-
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otic cells. Gene 66 (1), 1 – 10. Boot, H.J., Ter Huurne, A.A.H.M., Peeters, B.P., Gielkens, A.L., 1999. Efficient rescue of infectious bursal disease virus from cloned cDNA: evidence for involvement of the 3%-terminal sequence in genome replication. Virology 265 (2), 330 – 341. Boot, H.J., Ter Huurne, A.A.H.M., Peeters, B.P., 2000. Generation of full-length cDNA of the two genomic dsRNA segments of infectious bursal disease virus. J. Virol. Methods 84 (1), 49 – 58. Boot, H.J., ter Huurne, A.A.H.M., Vastenhouw, S.A., Kant, A., Peeters, B.P.H., Gielkens, A.L.J., 2001. Rescue of Infectious Bursal Disease Virus from mosaic full-length clones composed of serotype I and II cDNA, Archives of Virology, in press. Boyer, J.C., Haenni, A.L., 1994. Infectious transcripts and cDNA clones of RNA viruses. Virology 198 (2), 415 – 426. Britton, P., Green, P., Kottier, S., Mawditt, K.L., Penzes, Z., Cavanagh, D., Skinner, M.A., 1996. Expression of bacteriophage T7 RNA polymerase in avian and mammalian cells by a recombinant fowlpox virus. J. Gen. Virol. 77 (Pt 5), 963 – 967. Bruenn, J.A., 1991. Relationships among the positive strand and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases. Nucleic Acids Res. 19 (2), 217 – 226. Chettle, N.J., Stuart, J.C., Wyeth, P.J., 1989. Outbreak of virulent infectious bursal disease in East Anglia. Vet. Rec. 125 (10), 271 – 272. Cosgrove, A.S., 1962. An apparently new disease of chickens: avian nephrosis. Avian Dis. 6, 385 – 389. Dobos, P., Hill, B.J., Hallett, R., Kells, D.T.C., Becht, H., Teninges, D., 1979. Biophysical and biochemical characterization of five animal viruses with bisegmented doublestranded RNA genomes. J. Virol. 32, 593 – 605. Higuchi, R., 1990. Recombinant PCR. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols. Academic Press, San Diego, CA, pp. 177 – 183. Ismail, Y.M., Saif, Y.M., Moorhead, P.D., 1988. Lack of pathogenicity of five serotype 2 infectious bursal disease viruses in chickens. Avian Dis. 32 (4), 757 – 759. Le, S.Y., Siddiqui, A., Maizel, J.V. Jr, 1996. A common structural core in the internal ribosome entry sites of picornavirus, hepatitis C virus, and pestivirus. Virus Genes 12 (2), 135 – 147. Lim, B.L., Cao, Y., Yu, T., Mo, C.W., 1999. Adaptation of very virulent infectious bursal disease virus to chicken embryonic fibroblasts by site-directed mutagenesis of residues 279 and 284 of viral coat protein VP2. J. Virol. 73 (4), 2854 – 2862. McNulty, M.S., Saif, Y.M., 1988. Antigenic relationship of non-serotype 1 turkey infectious bursal disease viruses from the United States and United Kingdom. Avian Dis. 32 (2), 374 – 375.
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Muller, H., Nitschke, R., 1987. The two segments of the infectious bursal disease virus genome are circularized by a 90 000-Da protein. Virology 159, 174 –177. Mundt, E., Muller, H., 1995. Complete nucleotide sequences of 5%- and 3%-noncoding regions of both genome segments of different strains of infectious bursal disease virus. Virology 209 (1), 10 – 18. Mundt, E., Vakharia, V.N., 1996. Synthetic transcripts of double-stranded Birnavirus genome are infectious. Proc. Natl. Acad. Sci. USA 93, 11131 –11136.
Petek, M., D%Aprile, P.N., Cancellotti, F., 1973. Biological and physico-chemical properties of the infectious bursal disease virus (IBDV). Avian Pathol. 2, 135 – 152. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Snyder, D.B., 1990. Changes in the field status of infectious bursal disease virus. Avian Pathol. 19 (3), 419 – 423. VandenBerg, T.P., 2000. Acute infectious bursal disease virus in poultry: a review. Avian Pathol. 29, 175 – 194.
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Comparison of RNA and cDNA transfection methods for rescue of infectious bursal disease virus Hein J. Boot *, Kristina Dokic, Ben P.H. Peeters Department of A6ian Virology, Institute for Animal Science and Health, ID-Lelystad, PO Box 65, NL-8200 AB Lelystad, The Netherlands Received 8 November 2000; received in revised form 21 May 2001; accepted 23 May 2001
Abstract Specific alterations in the genetic material of RNA viruses rely on a technique known as reverse genetics. Transfection of cells with the altered generic material is a critical step of this procedure. In this report we have compared RNA and cDNA transfection methods for the efficiency of transient protein expression and rescue of (recombinant) infectious bursal disease virus (IBDV). Quantitative expression analysis of the secreted alkaline phosphatase reporter protein, and qualitative expression levels of an IBDV protein showed both that cDNA transfection results in a much higher level of protein expression than RNA transfection. Because the rescue of a crippled variant of IBDV was achieved consistently using the cDNA transfection method, but failed when we used the RNA transfection method, we favor the cDNA transfection method for the rescue of (recombinant) IBDV from cloned cDNA. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Reverse genetics; mRNA transfection; cDNA transfection; IBDV; SEAP
1. Introduction Infectious bursal disease virus (IBDV), a member of the family of Birnaviridae, genus a6ibirna6irus, is the causative agent of an avian restricted disease known as Gumboro. The first outbreak of IBDV was reported in commercial chicken flocks in Delaware, USA (Cosgrove, 1962). The IBDV strains, which were isolated during this outbreak, are now referred to as classi* Corresponding author. Tel.: + 31-320-238-238; fax: + 31320-238-668. E-mail address: [email protected] (H.J. Boot).
cal serotype I isolates. Later on, a second serotype (serotype II) of IBDV was identified (McNulty and Saif, 1988). Serotype II IBDV isolates are apathogenic and are recovered mainly from turkeys (Ismail et al., 1988). Antigenic variants of serotype I IBDV isolates were recovered from infected flocks in the USA starting in 1985, probably resulting from the selection pressure of field vaccination against classical IBDV serotype I (Snyder, 1990). Although being antigenic variant these isolates have only minor amino acid changes and do not form a separate serotype. In 1991, IBDV isolates, which were able to break through levels of maternal antibodies that normally were
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protective, were reported in Europe (Chettle et al., 1989). These isolates, the so-called very virulent IBDV (vvIBDV), are causing more severe clinical signs during an outbreak, and are now found almost world-wide (VandenBerg, 2000). The genome of IBDV consists of a double stranded RNA (dsRNA) genome divide over two segments, the A- and B-segment (Dobos et al., 1979). The A-segment (3.3 kbp) contains two partly overlapping open reading frames (ORFs). The first, smallest ORF encodes the non-structural Viral Protein 5 (VP5, 17 kDa, non-essential). The second ORF encodes a polyprotein (110 kDa), which is cleaved autocatalytically into pVP2 (48 kDa, capsid protein), VP4 (28 kDa, protease) and VP3 (32 kDa, capsid protein). During virus release pVP2 is further processed into VP2 (38 kDa). The conformation dependent neutralizing epitopes are found exclusively in a specific region of the VP2 protein, the so-called hypervariable region (amino acids 224– 314). The B-segment (2.9 kbp) contains one large ORF, encoding the 91 kDa VP1 protein, the RNA dependent RNA polymerase (Bruenn, 1991). VP1 is, furthermore, covalently linked (VPg) to the 5%-ends of the genomic RNA segments (Muller and Nitschke, 1987). In 1996, the first report of rescue of IBDV from cloned cDNA was published (Mundt and Vakharia, 1996), opening the way to manipulate the IBDV genome, and analyzing the effect of the introduced mutations in rescued IBDV (rIBDV). The introduction of mutations and the ability to generate chimeric IBDV strains is particularly important to get a better understanding of virulence factors. The ability to manipulate the IBDV genome in a rational way will also enhance greatly vaccine development. Variations on the original IBDV mRNA transfection method (Mundt and Vakharia, 1996) have been published recently by us (Boot et al., 1999) and others (Lim et al., 1999). These methods rely on transfection of cloned cDNA instead of RNA. RNA transfections have the drawback that in vitro production of RNA is expensive and laborious in comparison to DNA transfections. Furthermore, protein expression levels from transfected RNA are generally low in comparison to cDNA transfections
(Boyer and Haenni, 1994). Mutations introduced in the IBDV genome might hamper the assembly and/or replication of the virus. A highly efficient and reproducible transfection method will increase the chances of rescuing such crippled viruses. Here we show that cDNA transfections give rise to a much higher transient expression levels than RNA transfections. Furthermore, a particular mutation in the B-segment prevented the rescue of recombinant IBDV when RNA transfections were carried out, while this recombinant IBDV could be rescued consistently when the cDNA transfection method was used.
2. Materials and methods
2.1. Virus, plasmids, and cells The IBDV strain CEF94 is a cell culture adapted classical serotype I isolate (Boot et al., 1999), which is derived from the PV1 isolate (Italia, 1973). The full-length cDNA of this strain has been cloned (GeneBank: AF194428 and AF194429) in a pUC18 derived vector, which contains a hepatitis delta virus ribozym and a T7 promoter and terminator (see Fig. 1), yielding plasmids pHB-36W (A-segment cDNA) and pHB34Z (B-segment cDNA) (Boot et al., 1999). For transfection of cDNA and mRNA, and for propagation of wild-type or rescued CEF94 we have used the differentiation incompetent quail myogenic cell line QM5 (Antin and Ordahl, 1991), which was maintained in QT35 medium (GibcoBRL Life Technologies) supplemented with 5% fetal calf serum and 2% antibiotics solution ABII (1000 U/ml Penicillin (Yamanouchi), 1 mg/ml Streptomycin (Radiumfarma, Italy), 20 mg/ml Fungizone, 500 mg/ml; Polymixin B, and 10 mg/ ml Kanamycin). IBDV infected cells were detected using a VP3 specific monoclonal antibody Mab 9.7 (Boot et al., 2001).
2.2. Replacement of the polyprotein ORF by the secreted alkaline phosphatase gene The polyprotein encoding region (nt 131– 3166) of pHB-36W was replaced by the secreted alkaline
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phosphatase (SEAP) gene (Clontech). First, two independent polymerase chain reactions (PCRs) were carried out. In PCR-1, we used primers T7ACO (Boot et al., 2000) and ASSC (TCATCGATGA TGCATGCCTG CGATCGTTTG TCTGATC) and pHB-36W as template (resulting in a 171-bp fragment), while in PCR-2 we used primer ANC1 (Boot et al., 2000) and ATSC (CAGGCATGCA TCATCGATGA GGCTCCTGGG AGTC) and pHB-36W as template (resulting in a 113-bp fragment). In a subsequent fusion PCR (Higuchi, 1990), we used the purified (Qiaex II Gel Extraction Kit, Qiagen) PCR-1 and PCR-2 fragments as template and T7ACO an ANCO (Boot et al., 2000) as primers. The resulting PCR product (PCR-3, 264 bp) was cloned in pUC18-Ribo, which contains the cis-acting antigenomic hepatitis delta virus ribozym (Boot et al., 1999), yielding plasmid pHB-3. The integrity of the introduced nucleotide sequence was checked by sequence analysis. The two unique restriction sites of pHB-3 (SphI and ClaI), which were introduced in the fusion PCR, were used to insert the SEAP gene from plasmid pSEAP-Basic (Clontech), resulting in plasmid pHB-32. To generate a reporter plasmid, which has exactly the 5%-untranslated regions (UTR) of wild type IBDV we restored the nucleotides (GC) at position 129–
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130 to the wild type (CG) sequence. These changes were introduced by performing a PCR using primers ATG3 (CATCGCTGCGA TCGTTTGTCT GATCTCTAC) and T7EcoRI (GGAATTCTAA TACGACTCAC TATAGG), pHB-32 as template, and Pwo polymerase (Boeringher Mannheim) as enzyme (PCR-4, 159 bp). Plasmid pHB-32 was digested with SphI and the 3%-overhangs were trimmed by using Klenow polymerase (Pharmacia). After heat inactivation of Klenow the plasmid was digested with EcoRI, agarose gel purified and used in a ligation reaction with the EcoRI digested PCR-4 fragment, yielding plasmid pKD-ATG3R (Fig. 1).
2.3. RNA synthesis Plasmid DNA was isolated from 50 ml overnight Escherichia coli (DH5a) cultures using the Qiagen midiprep procedure (Qiagen, Germany). The isolated plasmids were treated with Proteinase K (Amresco, 1.0 mg/ml) in the presence of 0.1% sodium dodecyl sulphate (SDS) at 50 °C for 60 min to remove all RNase contamination. The plasmids were purified subsequently by phenol/chloroform/isoamylacohol treatment (two times) and ethanol precipitation (Sambrook et al., 1989), and dissolved (0.2 mg/ml) in diethyl
Fig. 1. Schematic representation of the plasmids containing a full-length IBDV A-segment (pHB-36W), B-segment (pHB-34Z), or a derivative of pHB-36W in which the coding region of the polyprotein has been replaced by SEAP gene (pKD-ATG3R). Boxes indicate open reading frames, and several functional genetic elements like hepatitis delta ribozyme (HDR) and T7 RNA polymerase promoter and terminator sequences are indicated. The fusion sites between the SEAP reporter gene and the 5%- and 3%-UTR of the IBDV A-segment is shown for pKD-ATG3R. The nucleotides originating from the 5%- and 3%- UTR are underlined.
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pyrocarbonate (DEPC) treated H2O. Capped RNA was synthesized in 15 ml DEPC treated H2O containing 1 mg DNA, 40 mM Tris (pH 8.0), 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 1 mM ATP, CTP and UTP, 0.2 mM GTP, 0.5 mM CAP analog (New England Biolabs), 1 U T7 polymerase (United States Biochemicals), 15 U RNasin (Promega) and 10 mM dithiothreitol (DTT). After incubation for 30 min at 37 °C, DNase (2 U, Amersham Pharmacia Biotech) was added and incubation at 37 °C was prolonged for 15 min.
once with PBS (3 ml) and twice with Optimem (3 ml). The monolayer was incubated for 10 min at 37 °C between the different washing steps. Transfection was carried out by mixing 1 mg DNA with 250 ml Optimem and 12.5 ml Lipofectamine (GibcoBRL Life technologies). This mixture was kept at room temperature for 30 min and added subsequently to the QM5 monolayer, which had been covered with 2 ml fresh Optimem. After overnight incubation (18 h, 37 °C, 5.0% CO2) the monolayer was rinsed once with PBS and covered with 2.5 ml QT35 medium. Each cDNA transfection was undertaken in three- or four-fold.
2.4. Transfection of mRNA Confluent monolayer (80%) of QM5 cells (10 cm2 per transfection) was washed once with phosphare buffer saline (PBS; 3 ml), twice with Optimem (3 ml) prior to transfection, and covered with 1 ml Optimem. The monolayer was incubated for 10 min at 37 °C between each washing step. In a polystyrene tube 125 ml Optimem 1 (GibcoBRL Life Technologies) was mixed with 12.5 ml lipofectin (GibcoBRL Life Technologies), and incubated at room temperature for 15 min. The RNA synthesis mixture (see Section 2.3) was added subsequently and incubation at room temperature was prolonged for 5 min. The mRNA/ Lypofectine/Optimem mixture was mixed with the supernatant of the QM5 monolayer. Transfection was allowed to proceed at 37 °C (5.0% CO2) for 3 h, after which time the monolayer was washed once with PBS and covered with 2.5 ml QT35 medium. Each RNA transfection was carried out in three- or four-fold with independent mRNA preparations.
2.5. Infection with fowlpox-T7 6irus and transfection of cDNA The plasmid preparation, which was used for the synthesis of mRNA was also used to transfect an 80% confluent monolayer of QM5 cells (10 cm2 per transfection) directly. The QM5 monolayer was washed once with PBS and covered with PBS contained 107 pfu fowlpox-T7 virus. Infection with fowlpox-T7 virus was allowed for 1 h at 37 °C (5% CO2), and the monolayer was washed
2.6. Quantitation of the SEAP reporter protein after transfection Plasmid pKD-ATG3R (see Fig. 1) was used to transfect QM5 cells directly (cDNA transfection), or was used for in vitro synthesis of RNA, which was transfected subsequently (RNA transfection). The last washing step at the end of each transfection procedure was taken as T=0 h. Samples (200 ml) were taken from the supernatant of transfected monolayers at different time points after T= 0 and stored at −20 °C. The SEAP activity (relative light units, RLU) in each sample was determined by using a chemoluminescense assay (Phospha-Light detecion kit (Tropix)), and a 1450 Microbeta luminometer (Wallac), as described by the supplier. The average RLU of the four independent transfections with either cDNA or RNA was determined for each time point.
2.7. Detection of IBDV antigen after transfection Plasmid pHB-36W (see Fig. 1) was used either directly (cDNA transfection) or after conversion into RNA (RNA transfection) to transfect QM5 cells. Transfected cells were incubated for 24 h in 2.5 ml QT35 medium (37 °C, in the presence of 5% CO2) after the last washing step in each procedure. The monolayer was washed once with PBS, air-dried, and frozen at − 70 °C. The cells were fixed subsequently by incubation for 10 min in PBS supplemented with 4% para-formaldehyde (4 °C). After washing the fixed cells twice with PBS (supplemented with 0.05% Tween-80) the
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first antibody (Mab 9.7 in PBS supplemented with 30 g/ml NaCl and 2% horse serum) was allowed to bind to viral antigens. Three washing steps were performed (PBS supplemented with 0.05% Tween-80) and the second antibody (rabbit anti mouse coupled to horseradish peroxidase (Dako, 1000-fold diluted,) in PBS supplemented with 30 g/ml NaCl and 2% horse serum) was allowed to bind. Detection of the second antibody was carried out after three washing steps (PBS supplemented with 0.05% Tween-80) by addition of a solution of 50 mM NaAc (pH 5.0), 0.2 mg/ml 3-amino-9-ethyl-carbazole (AEC, Sigma) and 0.01% H2O2.
2.8. Quantitation of rescued IBDV after transfection Plasmid pHB-36W was co-transfected with pHB-34Z, either directly (cDNA co-transfection), or after conversion into RNA (RNA co-transfection). QM5 cells transfected with either mRNA or cDNA were incubated for 24 h in 2.5 ml QT35 medium (37 °C, in the presence of 5% CO2) after the last washing step in each transfection procedure. The cells and supernatant were freeze/ thawed subsequently, filtered through a 200-nm-pore-size filter (Acrodisc, Gelman Sciences) and stored at −20 °C. The amount of IBDV (50% tissue culture infectious dose (TCID50)) was determined by infecting fresh QM5 cells with 10-fold dilutions of the filtered transfection lysates. IBDV infected wells were detected in an immunoperoxidase monolayer assay (IPMA) using the IBDV specific Mab 9.7 (see Section 2.6).
2.9. Mutagenesis of the B-segment cDNA A point mutation at position 2803 in the 3%UTR region of the B-segment was introduced by carrying out a PCR using the primers MB-10 (GGGGGCCCCC GCAGGCGAAG GCCCGGGAT) and BC10 (GCTCTAGATC AAGAACCCAC AGACCG), pHB-52 as template, and Pwo polymerase (Boehringer Mannheim) as enzyme (PCR-5, 277 bp). The pHB-52 plasmid is a derivative of pHB-34Z that contains a NgoMIV restriction site at nt 2741. This PCR fragment was
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purified subsequently, digested with NgoMIV and used to replace the corresponding part of plasmid pHB-52 (digested with NgoMIV and SmaI), yielding pMB-10. The presence of the point mutation at position (nt 2803) in pMB-10 was confirmed by sequence analysis. Secondary structure prediction of the 3%-UTR region of the codingstrand B-segment RNA was carried out using the Mfold program (version 3.0).
3. Results
3.1. Construction of a SEAP reporter plasmid To follow accurately protein expression originating from mRNA or cDNA after transfection of eukaryotic cells, the polyprotein encoding ORF of IBDV was replaced by the secreted form of the human placental alkaline phosphatase (SEAP). This enzyme is very stable and is efficiently secreted from expressing cells (Berger et al., 1988). The SEAP activity can easily be determined using the CSPD reagent, and the measured activity in the culture medium is directly proportional to changes in intracellular concentrations of SEAP (Berger et al., 1988). An additional advantage of this reporter protein is that it is secreted by the producing cells, making it possible to follow the expression kinetics by just removing a sample of the culture medium at different time points. The mechanism of translation initiation of the IBDV ORFs is unclear at present (see also Section 4). We chose to replace the polyprotein ORF, because the polyprotein is the precursor of the two capsid proteins and most likely the highest expressed IBDV protein. The ORF for the polyprotein was replaced in such a way that the same startcodon was used for the initiation of the SEAP and polyprotein, and no nucleotide differences were present in the 5%-UTR of the A-segment and reporter plasmid (Fig. 1).
3.2. Transfection of QM5 cells with reporter mRNA and cDNA To evaluate expression of the SEAP reporter protein a quail derived cell line (QM5) was used.
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mRNA, as pox viruses are capable of producing capped mRNA cytoplasmatically. After transfection of FPT7 infected QM5 cells with the reporter plasmid, we found a high level of SEAP activity in the supernatant (Fig. 3). SEAP activity was already detectable 5 h after transfection, and continued to increase for at least 2 days, indicating ongoing transcription and translation of the transfected plasmid DNA.
3.3. Transfection of QM5 cells with full-length IBDV RNA and cDNA
Fig. 2. Agarose gel electrophoresis (1.0%, 0.5* TBE) of linearized plasmid DNA (0.2 mg, lanes 1 – 3) or in vitro produced mRNA (lanes 4 – 6). Lane 1, pHB-36W (full length A-segment plasmid, 6175 bp); lane 2, pHB-34Z (full length B-segment plasmid, 5742 bp); lane 3, pKD-ATG3R (SEAP reporter plasmid, 4724 bp); lane 4, mRNA derived from pHB-36W (full length coding strand A-segment, 3260 nt); lane 5, mRNA derived from pHB-34Z (full length coding strand B-segment, 2827 nt); lane 6, mRNA derived from pKD-ATG3R (1808 nt); lane M, size marker DNA (Smartladder, Eurogentech).
About thirty percent of the fibroblast cells present in the monolayer can be transfected by plasmid DNA using established transfection protocols. Furthermore, several avian viruses, including IBDV and fowlpox virus are able to replicate on this cell line. We started to evaluate the relative efficiency of reporter protein expression by transfecting mRNA of the SEAP reporter gene. This mRNA was produced in vitro by using T7 RNA polymerase and the SEAP encoding reporter plasmid pKD-ATG3R in the presence of cap analogue. Although we were able to produce this capped mRNA reproducible (see Fig. 2) no detectable SEAP expression levels were obtained (Fig. 3). Alternatively we used an in vivo based T7 expression system. QM5 cells were infected with a recombinant fowlpox virus containing the T7 polymerase gene under control of the vaccinia virus early/late P7.5 promoter (Britton et al., 1996), before transfection with the reporter plasmid. Artificially introduced cDNA containing a T7 promoter will be transcribed into capped
Transfection of the reporter plasmid showed that the introduction of cDNA in FPT7 infected QM5 cells resulted in high levels of reporter protein expression, in contrast to mRNA transfection. To investigate whether IBDV proteins can be detected after transfection of full-length A- or B-segment RNA we transfected A-segment RNA alone or in combination with B-segment RNA. Both cDNA and the derived RNA (see Fig. 2) were used to transfect QM5 cells. Expression of VP3 (located at the C-terminal end of the polyprotein) was detected at 24 h post transfection by an IPMA. No VP3 expressing cells could be detected in case of RNA transfection (Fig. 4). This is in contrast to transfection with cDNA A-segment in which about 25% of the cells were expressing VP3 (Fig. 4). When A-segment RNA
Fig. 3. SEAP activity (RLU) in the supernatant of the QM5 monolayer after transfection with either plasmid pKDATG3R or in vitro produced mRNA using pKD-ATG3R as template. Aliquots of the supernatant were removed at different time points after transfection and stored at −20 °C, until the SEAP activity was determined. Each value is the average of four independent transfection experiments, error bars represent standard deviation.
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made, as the high level of transient expression of the VP3 protein hampered the identification of a group of cells (plaques) resulting from IBDV infection/replication after cDNA co-transfection.
3.4. Rescue of infectious IBDV from transfected QM5 cells
Fig. 4. An immunoperoxidase monolayer assay (IPMA) was performed using a VP3 specific monoclonal antibody (Mab 9.7). Nearly confluent monolayers of QM5 cells were either transfected with A-segment RNA or cDNA, or co-transfected with A- and B-segment RNA or cDNA. VP3 producing cells were visualized (dark cells), 24 h after the end of each transfection procedure.
was co-transfected in combination with B-segment RNA, several clustered cells (plaques) were found to produce VP3 at 24-h post-infection (Fig. 4). In case of co-transfection of the A- and B-segment cDNA we found about 25% of the cells expressing VP3 at 24-h post-infection (Fig. 4), resembling the result of the transfection of the A-segment alone. No comparison of the number of plaques of the mRNA and cDNA co-transfections could be
To determine the rescue efficiency of IBDV after co-transfection of A- and B-segment cDNA or RNA in a different way, we collected the supernatant 24 h after the end of each transfection method. Transfected monolayers, including the supernatant, were freeze-thawed once and the IBDV titer (50% TCID50) was determined after filtration of these lysates. All cDNA and RNA co-transfections yielded infectious virus with about equal efficiencies (TCID50 ] 5, Table 1). Subsequently, we compared both transfection methods by using a B-segment plasmid in which a single point mutation was present in the 3%-UTR at position nt 2803 (cytosine guanine). The stem–loop structure, which is present in the 3%UTR of the B-segment coding strand mRNA (Mundt and Muller, 1995; Boot et al., 1999) is extended by this single point mutation (Fig. 5). Using this altered B-segment cDNA we were not able to rescue IBDV after co-transfection of RNA. If on the other hand, the cDNA transfection method was used we were able to rescue recombinant IBDV in all four independent experiments, although the observed IBDV titer was
Table 1 Efficiency of the rescue of IBDV after cDNA and RNA transfections log10 TCID50a Experiment 1
Experiment 2
Experiment 3
Experiment 4
Average
cDNA pHB-36W+pHB-34Z PHB-36W+pMB-10
5.8 0.7
5.9 0.5
4.8 0.8
4.9 0.5
5.4 0.6
RNA pHB-36W+pHB-34Zb PHB-36W+pHB-34Z pHB-36W+pMB-10
0.0 4.7 0.0
0.0 5.0 0.0
0.0 4.7 0.0
– 5.4 –
0.0 5.0 0.0
a b
TCID (50%) 24 h after each transfection. In vitro synthesis of RNA in the absence of CAP analogue.
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Fig. 5. Prediction of the secondary structure of the 3%-terminus of the coding strand of the IBDV B-segment, using the Mfold program (version 3.0). Both the stem –loop structure of the wild-type B-segment and mutant MB-10 are shown. The point mutation of MB-10 at position nt 2803 (corresponding with nt-24) is indicated with an arrow.
reduced severely compared with co-transfection of the wild-type cDNA (Table 1).
4. Discussion Specific alterations in the genetic material of RNA viruses rely on a technique known as reverse genetics. First, the complete genomic RNA of the virus is reversed transcribed into cDNA, this cDNA is then altered, and transcribed into RNA. The recombinant RNA is used subsequently to rescue virus by transfection into cells that support the replication of the virus in question. Several methods to generate infectious RNA viruses have been developed during recent years (Boyer and Haenni, 1994) and issue 53 of Adv. Virus Res. (1999). In this study, we compared two transfection methods to rescue IBDV. The mechanism of translation initiation of birnaviruses is unknown currently. At least one unused start codon is present in the 5%-UTR of the A- and B-segment coding strand RNA, and the RNA also contains a large viral protein genome linked molecule (VPg, see Section 1). These two properties exclude a simple CAP-scanning mechanism of translation initiation. Furthermore, the AUG codon at position 131– 133 is used to initiate
translation of the polyprotein, making the 5%-UTR much smaller than the known 5%-UTR’s, which harbor an Internal Ribosome Entry Site (Le et al., 1996). It seems most likely that VPg is involved in translation initiation, and has a function comparable with a CAP-structure. Despite the lack of knowledge concerning translation initiation, it has been shown that the substitution of the VPg by a CAP-analogue is sufficient to initiate translation initiation of the IBDV RNA. Co-transfection of in vitro synthesized RNA of the IBDV A- and B-segment gives rise to infectious viral particles (Mundt and Vakharia, 1996). Also co-transfection of the Aand B-segment cDNA (preceded by a T7 promoter) into fowlpox-T7 virus infected cells, which express T7 polymerase leads to the formation of infectious viral particles (Boot et al., 1999). To assess, which of these two methods is the most efficient to recover infectious IBDV, we replaced firstly the ORF of the polyprotein for the SEAP reporter gene. Using this plasmid and the corresponding, in vitro synthesized, capped mRNA a marked difference was observed in the levels of reporter protein expression. There was no detectable SEAP expression in case of mRNA transfection, while a good and time dependent expression of SEAP occurred in case of cDNA transfection (Fig. 3). Also the transient expression of the VP3 capsid protein after transfection with either A-segment mRNA or the corresponding cDNA shows that only cDNA transfection lead to detectable protein expression (Fig. 4). Despite the marked difference between the protein expression levels, we observed no difference between IBDV recovery efficiencies between the two transfection methods. Both methods gave rise to 105 rIBDV particles per ml, 24 h after each transfection method. Although the final rIBDV titers are the same, it might still be that the efficiencies of the two transfection methods are quite different. IBDV replicates quite fast, as virus release is already occurring 8 h after the initial infection (Petek et al., 1973). Thus samples taken at 24 h post co-transfection do not only represent virus from initially virus producing cells, but also from secondary infected cells. Furthermore, the time of the mRNA (3 h) and cDNA (18 h)
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transfection procedures are quite different, and an additional transcription step (cDNA mRNA) has to occur in the cDNA transfection method. All these factors make direct comparison between the two transfection methods impossible. An efficient transfection method is needed mostly when mutations in the viral genome are introduced, which interfere with replication of the virus. Only with a very efficient transfection system, mutations can be studied that lead to viruses that replicate extremely slowly, or mutations which need to be reverted, or suppressed by a second-site mutation. To determine whether there is indeed a difference between the rescue efficiencies of the two transfection methods we have used a mutated B-segment plasmid. In this plasmids (pMB-10) we introduced a single point mutation, which leads to an extended stem– loop structure at the 3%-terminus of the coding strand. Although no data have been published on the function of this stem–loop structure we believe that this stem –loop structure might be involved in replication or translation of the viral genome. The introduction of the single point mutation leads to a more stable stem–loop structure, as the stem region is extended by one GC basepair (Fig. 5). Transfections using this mutated pMB-10 plasmid showed that the replication of rIBDV is hampered by this mutation. Rescue of the rIBDV is reduced severely when we used the cDNA transfection method (less then ten infections virus particles per ml, Table 1), whereas it was impossible to rescue this virus using the RNA transfection method. Although we have no information on how replication of the mutated virus is impaired by the stem –loop mutation, we can conclude from this transfection experiment that the cDNA transfection method is indeed more efficient for rescuing IBDV, than the RNA transfection method.
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