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1 National Influenza Center, Department of Virology and Postgraduate School of Molecular Medicine, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands
2 Solvay Pharmaceuticals BV, Weesp, The Netherlands
Correspondence
Ron A. M. Fouchier
r.fouchier{at}erasmusmc.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Luytjes et al., 1989), important improvements have been made. First of all, reverse-genetics systems were introduced that required only plasmid DNA (Fodor et al., 1999; Neumann et al., 1999), making the use of helper virus for the generation of recombinant virus obsolete. Transfection of eight plasmids under the control of an RNA polymerase I promoter for the production of viral RNA (vRNA) and four expression plasmids encoding the three polymerase proteins and NP under the control of an RNA polymerase II promoter resulted in the rescue of infectious virus. Next, a bidirectional reverse-genetics system was developed based on transfection of eight plasmids instead of 12 (Hoffmann et al., 2000b). In this reverse-genetics system, cDNA encoding negative-sense vRNA is inserted between the RNA polymerase I promoter and terminator sequences. This entire complex is inserted in the positive-sense orientation between an RNA polymerase II promoter and polyadenylation site. Transfection of this set of eight plasmids results in the production of mRNA and negative-sense vRNA and does not require additional plasmids.
A recent improvement to the unidirectional transcription system was the generation of plasmids containing up to eight RNA polymerase I transcription cassettes, thereby reducing the number of plasmids to be transfected and thus enabling the use of cell lines with low transfectability (Neumann et al., 2005).
Although both the unidirectional and bidirectional reverse-genetics systems are sufficient for generating recombinant virus and have been used for various research purposes and generation of vaccine seed strains in experimental settings, these reverse-genetics systems have one major disadvantage. Expression of vRNA in these systems is under control of the RNA polymerase I promoter. Unlike RNA polymerase II, RNA polymerase I transcription exhibits stringent, although not absolute, species specificity. A chicken RNA polymerase I promoter was described recently (Massin et al., 2005), but influenza virus reverse genetics is still limited to cells of primate or avian origin. Because different reverse-genetics vectors must be used in different cell types, separate sets of eight or more plasmids must be constructed for each cell type. For research purposes and for the preparation of vaccine seed virus strains, it would be an advantage if there was one, universally applicable reverse-genetics system for use in any given cell type.
Reverse-genetics systems for negative-strand viruses other than influenza virus are often based on transcription of vRNA by the T7 RNA polymerase. Schnell et al. (1994) were the first to rescue a non-segmented negative-strand virus solely from cloned cDNA, using T7 RNA polymerase promoter and terminator sequences for the production of rabies virus vRNA. Since then, similar systems have been described for numerous viruses representing the families Paramyxoviridae, Rhabdoviridae, Filoviridae, Bunyaviridae and Arenaviridae of non-segmented and segmented negative-strand viruses (Blakqori & Weber, 2005; Conzelmann, 2004; Ikegami et al., 2006; Sanchez & de la Torre, 2006). Although T7 RNA polymerase has been used for transcription of single genes in influenza A virus minigenome systems (Massin et al., 2001; Perez & Donis, 1998), rescue of recombinant influenza virus by using T7 RNA polymerase has never been described.
Here, we describe a reverse-genetics system for the rescue of recombinant Influenza A virus based on transcription of vRNA under the control of a T7 RNA polymerase promoter. As the T7 RNA polymerase is provided in the form of plasmid DNA that is cotransfected, this system is not dependent on expression of a species-specific RNA polymerase promoter and can be used in cell lines originating from different species. We developed a unidirectional reverse-genetics system for the production of recombinant Influenza A virus and were able to produce recombinant virus in cell lines of such diverse origin as human, avian and canine cells by using a single set of plasmids.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were cultured in Medium 199 (Cambrex) supplemented with 5 % FCS, 1 % chicken serum, 5 % tryptose phosphate broth, 100 IU penicillin ml–1, 100 µg streptomycin ml–1, 2 mM glutamine, 0.75 mg sodium bicarbonate ml–1 and non-essential amino acids.
Viruses.
Influenza virus A/PR/8/34 was kindly provided by Dr J. Wood, National Institute for Biological Standards and Control, Potters Bar, UK, and was described previously (de Wit et al., 2004). Influenza virus A/NL/219/03 was isolated from a fatal case of H7N7 infection and was passaged twice in embryonated chicken eggs (Fouchier et al., 2004).
Plasmids.
The expression plasmids encoding nuclear and cytoplasmic versions of T7 RNA polymerase under the control of a simian virus 40 promoter (pAR3126 and pAR3132) were kindly provided by Dr J. Dunn (Brookhaven National Laboratory, Upton, NY, USA) (Dunn et al., 1988). Plasmids pHMG-PB1, pHMG-PB2, pHMG-PA, pHMG-NP and pCAGGS were a kind gift from Dr A. García-Sastre and Dr P. Palese (Mount Sinai School of Medicine, New York, USA).
The hepatitis delta virus ribozyme sequence (HDVR) was cloned in the XbaI–BamHI sites of pSP72, thus constructing pSP72-HDVR. A BamHI–EcoRV fragment from a pET vector containing a T7 RNA polymerase terminator (TT7) was cloned in the BamHI–HpaI sites of pSP72-HDVR, resulting in pSP72-HDVR-TT7. An oligonucleotide encoding the T7 RNA polymerase promoter (PT7; 5'-TATTGTAATACGACTCACTATAGGGTCTT) was ligated in the NdeI–XbaI sites of pSP72-HDVR-TT7 in the appropriate context to the BbsI sites. The resulting vector was named pSP72-PT7-HDVR-TT7.
Green fluorescent protein (GFP), flanked by non-coding regions (NCRs) from segment 5 of influenza virus A/PR/8/34, was cloned in the BbsI sites of pSP72-PT7-HDVR-TT7, using pSP-Hu-GFP-Mu (de Wit et al., 2004) as a template. GFP was cloned in the sense and antisense orientations, with no, two or three additional G residues directly after the T7 RNA polymerase promoter.
The gene segments of influenza viruses A/PR/8/34 and A/NL/219/03 were cloned in the BbsI restriction sites of PT7-pSP72-HDVR-TT7 in the antisense orientation, containing two additional G residues after the T7 RNA polymerase promoter.
Plasmids pCAGGS-PB2, pCAGGS-PB1, pCAGGS-PA and pCAGGS-NP were constructed by cloning the appropriate gene segments of A/NL/219/03 in the KpnI–NheI sites of pCAGGS.
All plasmids were sequenced by using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and a 3100 Genetic Analyzer (Applied Biosystems), according to the instructions of the manufacturer.
Transfection of cells.
Transient calcium phosphate-mediated transfections of 293T and QT6 cells were performed essentially as described by Pear et al. (1993). Cells were plated the day before transfection in gelatinized 100 mm diameter culture dishes to obtain 50 % confluent monolayers. After overnight transfection, the transfection medium was replaced with fresh medium supplemented with 2 % FCS for virus production or 10 % FCS for all other transfections. Cells were incubated for 30–72 h, after which supernatants were harvested and cells were analysed for fluorescence if appropriate.
Transient transfection of MDCK cells was performed essentially as described previously (Basler et al., 2000). Briefly, 1344 µl Optimem I medium (Gibco BRL) was added to 56 µl Lipofectamine 2000 (Invitrogen) and incubated at room temperature for 5 min. This mixture was added to the intended amount of DNA, adjusted to a volume of 280 µl by using Optimem I medium. This mixture was incubated at room temperature for 20 min. After incubation, 5.6 ml MDCK culture medium without penicillin and streptomycin was added and this mixture was added to 5.6x106 MDCK cells in suspension in a 100 mm diameter culture dish. After 4–5 h incubation, cells were washed twice with PBS and cultured in 10 ml MDCK culture medium without penicillin and streptomycin. This medium was replaced with MDCK culture medium containing 2 % FCS after overnight incubation.
For production of recombinant viruses, cells were transfected with 5 µg of the eight genomic plasmids and the four plasmids expressing the polymerase proteins and NP, and 15 µg of the plasmid expressing T7 RNA polymerase. Plasmid pEGFP-N1 (Clontech) was transfected in parallel and the percentage of fluorescent cells was measured in a FACSCalibur (Becton Dickinson) flow cytometer, confirming that the transfection efficiency ranged from 95 to 100 % for 293T, QT6 and MDCK cells in all experiments. In the minigenome assays, relative GFP expression was calculated as GFP-expressing cells (%)xGFP expression in these cells. Virus-containing supernatants were cleared by centrifugation for 10 min at 300 g. Virus titres in the supernatant were determined either directly, or after storage at 4 °C for less than 1 week or at –80 °C for longer than 1 week.
Virus titrations.
Virus titrations were performed as described previously (Rimmelzwaan et al., 1998). Infectious titres were calculated from five replicates according to the method of Spearman–Kärber.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Several possible methods for optimization of expression from T7 RNA polymerase promoter-regulated genes have been described. Although the described RNA polymerase I-based reverse-genetics systems all produce negative-sense vRNA, for successful recovery of non-segmented negative-strand viruses using the T7 RNA polymerase promoter, positive-sense antigenomic RNA is often used. Furthermore, it has been described that transcription by T7 RNA polymerase can be enhanced by inserting two or three additional G residues at the start of transcription (Pattnaik et al., 1992). Whilst this leads to genomic terminal sequences that are different from those of wild-type viruses, most negative-strand viruses can correct this problem. Therefore, a GFP open reading frame (ORF) flanked by the NCRs of segment 5 of influenza virus A/PR/8/34 was cloned in pSP72-PT7-HDVR-TT7 in the sense (S) and antisense (AS) orientations with no, two or three additional G residues (Fig. 1a). These constructs were named S, S-GG, S-GGG, AS, AS-GG and AS-GGG, respectively. Each of these plasmids was cotransfected into 293T cells with a T7 RNA polymerase expression plasmid (pAR3132) and pHMG-PB2, pHMG-PB1, pHMG-PA, pHMG-NP, expressing the PB2, PB1, PA and NP proteins of influenza virus A/PR/8/34. At 30 h after transfection, the cells were analysed for fluorescence in a FACSCalibur.
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). In the third experiment, S-GGG led to the highest relative GFP expression, but the difference from AS-GG was small. Therefore, we used pSP72-PT7-HDVR-TT7 with viral sequences inserted in the negative-sense orientation and two additional G residues downstream of PT7 for further experiments.
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).
The results of this experiment are shown in Fig. 2(b)
. When a wild-type, predominantly cytoplasmic T7 RNA polymerase expression plasmid was used, relative GFP expression [GFP-positive cells (%)xGFP expression in these cells] was 8440. This was enhanced significantly by using a T7 RNA polymerase that contained an NLS (relative GFP expression, 20 571). When both the T7 RNA polymerase constructs with and without the NLS were combined (1 : 1 ratio, keeping the total amount of transfected plasmid unchanged), an intermediate level of GFP expression was observed. In subsequent experiments, we have made use of the T7 RNA polymerase containing an NLS.
GFP minigenome assays in different host cells
Next, we tested the efficiency of the T7 RNA polymerase reverse-genetics system in different host cells. 293T, MDCK and QT6 cells were transfected with the antisense GFP minigenome with two additional G residues, HMG-PB2, HMG-PB1, HMG-PA, HMG-NP and the nuclear T7 RNA polymerase expression plasmid. Fluorescence of the cells was analysed 30 h after transfection. As can be seen in Fig. 3, the T7 RNA polymerase minigenome system is functional in all three cell types. Despite the fact that the transfection efficiency of 293T, MDCK and QT6 cells is similar (95–100 %), relative GFP production is four to five times higher in 293T cells than in MDCK and QT6 cells. This difference in relative GFP production is probably caused by the higher expression of the polymerase complex expression plasmids in 293T cells compared with MDCK and QT6 cells (data not shown).
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We transfected 293T cells with the eight constructs encoding the gene segments of influenza virus A/PR/8/34, pHMG-PB1, pHMG-PB2, pHMG-PA and pHMG-NP and the nuclear T7 RNA polymerase expression plasmid. After transfection, trypsin was added to the medium to allow replication of the produced viruses. At 72 h after transfection, supernatants were harvested and used to inoculate MDCK cells. At 3 days after inoculation, a haemagglutination assay was performed on the supernatant of these MDCK cells as an indication of virus replication, and virus titres of 293T and MDCK supernatant were determined. For comparison, we also transfected the constructs of the previously described RNA polymerase I-based unidirectional and bidirectional reverse-genetics systems encoding A/PR/8/34 (de Wit et al., 2004). In line with previous observations, the unidirectional RNA polymerase I-based reverse-genetics system was very inefficient for influenza virus A/PR/8/34 and we were able to produce recombinant virus from only two out of three transfections (de Wit et al., 2004). The T7 RNA polymerase-based system yielded virus in every transfection, but with very low titres (Fig. 4). The T7 RNA polymerase-based reverse-genetics system is thus more reliable than the unidirectional RNA polymerase I-based system, but less efficient than the bidirectional RNA polymerase I-based system, which yields titres between 104 and 105 TCID50 ml–1 routinely. MDCK passage of these three rescue experiments yielded equal virus titres (Fig. 4), indicating that the low virus titres in the 293T supernatant of cells transfected with the plasmids of the unidirectional RNA polymerase I and T7 RNA polymerase-based systems are not due to a replication deficiency of the produced viruses.
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). These 12 plasmids and the nuclear T7 RNA polymerase expression plasmid were cotransfected into 293T cells. After transfection, virus was produced in the supernatant of these cells with a mean titre of 6.8x102 TCID50 ml–1 (Fig. 5). Passaging of these supernatants in MDCK cells yielded high virus titres. To show that the T7 RNA polymerase-based reverse-genetics system is applicable in different cell types, we next tried to rescue the same virus in MDCK and QT6 cells. Virus was produced in the supernatant of MDCK and QT6 cells at titres of 2.0x105 and 9.3x105 TCID50 ml–1, respectively (Fig. 5). Thus, rescue of A/NL/219/03 in MDCK and QT6 cells yields higher virus titres than in 293T cells. Passaging of the transfection supernatant in MDCK cells resulted in virus titres equal to those of the 293T transfections, indicating that replication-competent virus was produced in all three cell types. There seems to be a contradiction between expression of a reporter in a minigenome assay, which is highest in 293T cells (Fig. 3), and the production of recombinant virus, which is most efficient in QT6 cells (Fig. 5). This can be explained by the fact that influenza virus A/NL/219/03, which has a multibasic cleavage site in the haemagglutinin, replicates more efficiently in QT6 cells than in 293T cells (data not shown). We conclude that the T7 RNA polymerase system can be used to produce recombinant virus from different host cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The system that we present here offers the possibility to produce recombinant Influenza A virus in a cell type of their original host, whether they are of avian or mammalian origin. Influenza A viruses are prevalent in many different species and are known to adapt to their host. Mutations in human and avian viruses upon passaging in embryonated chicken eggs and mammalian cell culture, respectively, have been described (Rogers et al., 1985; Schild et al., 1983). The T7 RNA polymerase reverse-genetics system creates the possibility to avoid host adaptation when generating recombinant viruses, but also offers the opportunity to study host adaptation, as it increases the range of cell types in which viruses can be produced. The T7 RNA polymerase vector that is used for production of recombinant virus can, for instance, also be used in minigenome assays. This creates the possibility to compare polymerase activity of a virus in different host cells. Especially in the light of recent zoonotic events, with avian influenza viruses being transmitted to humans, there is increasing focus on determinants of these zoonotic events and subsequent adaptation of the virus to the new host. Mutations in polymerase genes have been described upon transmission to a new host species (Fouchier et al., 2004; Hatta et al., 2001). By using a T7 RNA polymerase minigenome assay, it would be possible to compare the effect of mutations in polymerase genes on replication in different cell types.
Apart from research purposes, the T7 RNA polymerase-based reverse-genetics system can also be used for production of vaccine seed strains. Currently, there are only few cell lines registered for the production of vaccine seed virus, including MDCK cells. However, a canine RNA polymerase I promoter has not been described thus far. The T7 RNA polymerase-based system can be used to produce vaccine seed virus in MDCK cells and in other cells that might be registered for vaccine production in the future.
Although we were able to generate recombinant virus in different host cells using the T7 RNA polymerase system, and rescue of virus is more efficient than with the RNA polymerase I unidirectional reverse-genetics system, production of recombinant influenza virus A/PR/8/34 is not as efficient as with the bidirectional RNA polymerase I system. Therefore, it may be possible to enhance rescue efficiency of the T7 RNA polymerase system by developing a bidirectional T7 RNA polymerase system. We have attempted to produce a bidirectional T7 RNA polymerase-based reverse-genetics system. When a cytomegalovirus promoter was cloned in front of TT7 in the unidirectional T7 RNA polymerase vector, this did not result in production of recombinant influenza virus A/PR/8/34 (data not shown). For all gene segments except PB1, we were able to produce recombinant A/PR/8/34 when seven constructs of the polymerase I bidirectional reverse-genetics system were used together with one bidirectional T7 RNA polymerase construct, indicating that PB1 is responsible for the lack of virus production when a complete bidirectional T7 RNA polymerase reverse-genetics system is used (data not shown). We have described previously that PB1 of influenza virus A/PR/8/34 limits its rescue in a bidirectional polymerase I-based reverse-genetics system (de Wit et al., 2004). When we tried to produce a tandem-promoter vector (Hoffmann et al., 2000a) based on T7 RNA polymerase, this also did not result in production of recombinant virus. However, this does not mean that it will be impossible to develop a functional bidirectional T7 RNA polymerase reverse-genetics system that is more efficient than the unidirectional reverse-genetics system.
In recent years, the use of reverse-genetics systems for research purposes has increased tremendously. Although the currently available RNA polymerase I-based reverse-genetics systems suffice to produce recombinant virus and no reports have yet appeared of viruses that could not be rescued by using these systems, the species specificity of RNA polymerase I is a major disadvantage. Following the example of reverse-genetics systems for other negative-strand RNA viruses, we have developed a T7 RNA polymerase-based reverse-genetics system for Influenza A virus that can be used to rescue virus in cells from different hosts. By using one set of plasmids, virus can be generated in any cell type, without the need for recloning gene segments in vectors with different RNA polymerase I promoters.
It is likely that the T7 RNA polymerase-based reverse-genetics system described here can be used for other members of the family Orthomyxoviridae, e.g. Influenza B virus, Influenza C virus and Infectious salmon anemia virus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 8 August 2006;
accepted 1 December 2006.
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