Identification of 5' and 3' cis-Acting Elements of
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病菌学杂志 2006年第2期
Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Korea
Research Institute of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea
ABSTRACT
We here demonstrate the successful engineering of the RNA genome of porcine reproductive and respiratory syndrome virus (PRRSV) by using an infectious cDNA as a bacterial artificial chromosome. Runoff transcription from this cDNA by SP6 polymerase resulted in capped synthetic RNAs bearing authentic 5' and 3' ends of the viral genome that had specific infectivities of >5 x 105 PFU/μg of RNA. The synthetic viruses recovered from the transfected cells were genotypically and phenotypically indistinguishable from the parental virus. Using our system, a series of genomic RNAs with nucleotide deletions in their 5' ends produced viruses with decreased or no infectivity. Various pseudorevertants were isolated, and acquisition of novel 5' sequences of various sizes, composed predominantly of A and U bases, restored their infectivities, providing a novel insight into functional elements of the 5' end of the PRRSV genome. In addition, our system was further engineered to generate a panel of self-replicating, self-limiting, luciferase-expressing PRRSV viral replicons bearing various deletions. Analysis of these replicons revealed the presence and location of a 3' cis-acting element in the genome that was required for replication. Moreover, we produced enhanced green fluorescent protein-expressing infectious viruses, which indicates that the PRRSV cDNA/viral replicon/recombinant virus can be developed as a vector for the expression of a variety of heterologous genes. Thus, our PRRSV reverse genetics system not only offers a means of directly investigating the molecular mechanisms of PRRSV replication and pathogenesis but also can be used to generate new heterologous gene expression vectors and genetically defined antiviral vaccines.
INTRODUCTION
Porcine reproductive and respiratory syndrome (PRRS) was first recognized in 1987 in North America (21) and shortly thereafter in Europe (35). It has since become one of the most common and economically significant infectious diseases in the swine industry worldwide (1, 15). It is characterized by mild to severe reproductive failure in sows and gilts and respiratory problems in piglets (9, 10, 17). The PRRS virus (PRRSV) was first isolated almost simultaneously in Europe and North America; the strains are designated Lelystad (53) and VR-2332 (6, 10), respectively. Although these strains induce phenotypically indistinguishable disease symptoms (19), they are genetically (2, 25, 32) and serologically (33, 54, 58) distinct. They currently constitute the two distinct genotypes of known PRRSV species.
PRRSV belongs to the family Arteriviridae in the order Nidovirales together with equine arteritis virus (EAV), simian hemorrhagic fever virus, and the lactate dehydrogenase-elevating virus of mice (8, 45). Like other arteriviruses, PRRSV is a small-envelope virus with a positive-sense, single-stranded RNA genome of 15 kb in length. The genome has a cap structure at its 5' end and a poly(A) tail at its 3' end. The genome contains at least nine open reading frames (ORFs) flanked by 5' and 3' noncoding regions (NCRs) (13, 29, 45, 59). Two overlapping ORFs, ORF1a and ORF1b, are expressed from the genomic RNA. These two ORFs are predicted to be processed into 13 mature nonstructural proteins that are believed to be involved in viral replication (5, 43, 44, 45, 49, 57). ORF2a, ORF2b, and ORF3 to ORF7 are translated from the 5' ends of a coterminal nested set of subgenomic mRNAs. The small ORF2b is completely embedded within the larger ORF2a (59). These ORFs encode the viral structural proteins (14, 27, 29, 59).
To analyze positive-sense RNA viruses such as PRRSV at molecular and genetic levels, a number of reverse genetics systems that allow the determination of the functions of the genes and gene products of these RNA viruses through their manipulation and genetic analysis have been developed (7). With regard to arteriviruses, two "RNA-launched" reverse genetics systems for EAV have been independently developed by two research groups (16, 50). Although both functional EAV cDNAs were constructed using the same virus isolate, different bacteriophage promoters, either the SP6 or T7 RNA polymerase promoter, were placed immediately upstream of the viral genome for runoff transcription in vitro. The first infectious EAV cDNA was constructed in a high-copy-number plasmid, pUC18, and did not show any genetic instability during its construction and propagation in Escherichia coli, in contrast to the infectious cDNAs of several other viruses (50).
For PRRSV, the first RNA-launched reverse genetics system was constructed for the European Lelystad strain by assembling its full-length cDNA under the T7 promoter in the low-copy-number plasmid pOK12 (28). Identical strategies were also previously used in the construction of an infectious cDNA for the North American strain VR-2332 (34). Recently, two additional T7 promoter-driven RNA-launched reverse genetics systems were developed independently for the highly virulent American isolate NVSL 97-7895 (47) and for the highly virulent "atypical" North American isolate P129 (23) by using the low-copy-number plasmid pBR322 and the high-copy-number plasmid pCR2.1, respectively. An additional "DNA-launched" system has also been reported for the P129 isolate and employed full-length cDNA under the human cytomegalovirus immediate early promoter (23).
The present report describes the construction of not only a full-length infectious PRRSV cDNA in a bacterial artificial chromosome (BAC) designed to produce synthetic RNAs bearing authentic 5' and 3' ends of the viral genome but also a panel of self-replicating viral replicons. We have used our system to define 5'- and 3'-terminal nucleotide sequences that are essential for RNA replication. In addition, we have also demonstrated the potential of our system to generate vectors that express heterologous genes in eukaryotic cells in a variety of forms, including recombinant infectious PRRSV cDNA, viral replicons, and synthetic infectious viruses. Thus, the system we have constructed not only provides an important platform from which the basic biology of PRRSV can be investigated but also is a useful tool for designing new heterologous gene expression vectors and for generating genetically defined antiviral vaccines.
MATERIALS AND METHODS
Cells and viruses. MARC-145 (20) and BHK-21 cells (61) were maintained as described previously. All reagents used in cell culture were purchased from Life Technologies, Inc., Gaithersburg, MD. The parental PRRSV used in this study is the first Korean PRRSV strain, PL97-1, which was isolated in 1997 from the serum of an infected pig (20). High-titer virus stocks were obtained by cultivation in MARC-145 cells at a low multiplicity of infection (MOI) of 0.1 for 72 h. The viruses were then clarified by centrifugation (2,000 rpm for 10 min), aliquoted, and stored at –80°C until use.
Virus titration. Virus titers were determined by plaque assay using MARC-145 cells. For this, cells were preseeded in a six-well plate at a density of 3 x 105 per well for 12 to 18 h and then infected with serial 10-fold dilutions of virus for 1 h at 37°C with frequent agitation. The cell monolayers were then overlaid with minimal essential medium containing 0.5% SeaKem LE agarose (FMC BioProducts, Rockland, Maine) and 5% fetal bovine serum and incubated for 4 days at 37°C with 5% CO2. The resulting plaques were visualized by fixation with 7% formaldehyde followed by staining with crystal violet (1% [wt/vol] in 5% ethanol).
Oligonucleotides. All oligonucleotides used for cDNA synthesis, PCR amplification, and mutagenesis in this study are listed in the supplemental material (see Table S1 in the supplemental material). Their sequences were designed according to the complete nucleotide sequence of PL97-1 (GenBank accession number AY585241) (20).
Construction and mutagenesis of the full-length PRRSV cDNA as a BAC. Because of space limitations, only the salient features of plasmids are described here. A detailed description of plasmid construction is provided in the supplemental material, and computer-readable sequence files are also available upon request. All plasmids were constructed using standard molecular biology procedures (38).
pBAC/PRRSV/FL contained the full-length PRRSV PL97-1/LP1 cDNA flanked by the SP6 RNA polymerase promoter and three unique restriction sites (AclI, NotI, and SdaI) in a row for in vitro runoff transcription. Eight constructs (pBAC/PRRSV/FL/nt1 to -15) harbored deletions of 1, 3, 5, 7, 9, 11, 13, and 15 nucleotides (nt) from the utmost 5' end of the viral genome, respectively. pBAC/PRRSV/FL/nt3/Rev2 and pBAC/PRRSV/FL/nt3/Rev3 were reconstructed by the addition of 4 (TATG) and 3 (AAG) nucleotides at the beginning of the viral genome in pBAC/PRRSV/FL/nt3, respectively. Similarly, six additional constructs of pBAC/PRRSV/FL/nt7/Rev1 to -6 were reconstructed by the addition of 7 (ATTATAT), 8 (TATTATAT), 8 (TATCATAT), 10 (ATATATATAT), 12 (ATATATATATAT), or 8 (ATTTATAT) nucleotides, respectively, at the beginning of the viral genome in pBAC/PRRSV/FL/nt7. pBAC/PRRSV/FL/IRES-EGFP was constructed by the insertion of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-driven enhanced green fluorescent protein (EGFP) expression cassette immediately downstream of the first 33 nucleotides of the ORF7 coding region, followed by the 3' 911 nucleotides of the viral genome. pBAC/PRRSV/FL/Npro-EGFP is identical to pBAC/PRRSV/FL/IRES-EGFP, except that the EMCV IRES-driven EGFP expression cassette was replaced by the coding sequence of the Npro-EGFP fusion protein in such a way that expression of the fused Npro-EGFP was driven by its own subgenomic promoter. The autoprotease Npro gene of bovine viral diarrhea virus was fused adjacent to the N terminus of the EGFP gene so that the correct N terminus of the EGFP protein was created by Npro cleavage.
We constructed a panel of 11 PRRSV viral replicon vectors that express the luciferase (LUC) gene as a reporter. One set of three constructs, namely, pBAC/PRRSV/RepLuc MB, pBAC/PRRSV/RepLuc ME, and pBAC/PRRSV/RepLuc DI, were first constructed to contain internal deletions of nt 12714 to 14194, nt 12163 to 14194, and nt 12163 to 15252 in the viral genome, respectively, and also the insertion of the EMCV IRES-driven LUC expression cassette at the site of each deletion to facilitate the monitoring of viral replication. The other set of eight constructs, namely, pBAC/PRRSV/RepLuc S1 to S8, are identical to pBAC/PRRSV/RepLuc ME, except that the internal deletion was further extended to nt 15200, nt 15150, nt 15100, nt 15050, nt 15000, nt 14950, nt 14900, and nt 14500, respectively.
In vitro synthesis of RNA transcripts. The full-length PRRSV cDNA (2 μg) was digested with the appropriate restriction enzyme (AclI, NotI, or SdaI) and/or treated with mung bean nuclease (MBN). With regard to the PRRSV viral replicons, all the recombinant cDNAs were linearized by NotI digestion and extracted with phenol-chloroform and precipitated with ethanol. In vitro transcription from these template cDNAs, subsequent removal of the template cDNAs from transcription reaction, and extraction and quantitation of synthetic RNA transcripts were performed as previously described (61).
Transfection of cells with RNA transcripts. MARC-145 or BHK-21 cells were preseeded at a density of 2 x 106 or 3 x 106 cells per p150 culture dish for 24 h at 37°C with 5% CO2, respectively. The subconfluent cells were then trypsinized and washed three times with ice-cold RNase-free phosphate-buffered saline. After resuspension at a density of 2 x 107 cells/ml in phosphate-buffered saline, 400 μl of the cells was mixed with 2 μg synthetic RNA in a cuvette with a gap width of 0.2 cm. The MARC-145 and BHK-21 cells were electroporated with 10 or 5 pulses of current, respectively, by using an ECM 830 electroporator (BTX Inc., San Diego, CA) set at 900 V and a 99-μs pulse length. The cells were then transferred to 10 ml of fresh medium. Infectious-center assays were used to quantitate the specific infectivity of synthetic RNA transcripts. The electroporated cells were then serially diluted 10-fold and plated onto monolayers of nontransfected MARC-145 cells (3 x 105) in a six-well plate. After 6 h, they were overlaid with agarose-containing minimal essential medium, incubated for 4 or 5 days, and stained with crystal violet.
Real-time quantitative reverse transcription-PCR. Total RNA was extracted from duplicate wells with TRIzol reagent (Invitrogen Co.); 100 ng of total cellular RNA was used for the reverse transcription reaction with primers specific for the PRRSV ORF1a region as well as for the BHK-21 -actin RNA to normalize total RNA levels. PRRSV and BHK-21 -actin cDNAs were generated by reverse transcription at 45°C for 30 min, followed by inactivation of the reverse transcriptase at 95°C for 10 min. PRRSV-specific and BHK-21 -actin-specific cDNAs were amplified with an iQ Supermix quantitative PCR system (Bio-Rad Laboratories, Hercules, CA) and detected with an iCycler iQ multicolor real-time PCR detection system (Bio-Rad Laboratories). One-tenth of the reverse transcription reaction mixture was used for PCR amplification with 45 cycles of 95°C for 15 s and 60°C for 1 min. The PRRSV forward and reverse primers were 5'-CATGTGAGTGATAAACCTTTCCCG and 5'-TCATAGACAGTAGCCATAGCACAC, respectively. The probe sequence (nt 659 to 682) was 5'-6FAM-ACGTGTTGACCAACCTGCCGCTCC-BHQ1 (where 6FAM is 6-carboxyfluorescein and BHQ is black hole quencher; Integrated DNA Technologies Inc., Coralville, IA). The forward and reverse primers for BHK-21 -actin were 5'-ACTGGCATTGTGATGGACTC and 5'-CATGAGGTAGTCTGTCAGGTC, respectively. The probe sequence was 5'-HEX-CCAGCCAGGTCCAGACGCAGG-BHQ2 (where HEX is hexachlorofluorescein; Integrated DNA Technologies Inc.). The 2–CT method was used to analyze relative changes in PRRSV RNA levels from real-time, quantitative PCR experiments (41, 56).
Direct immunofluorescence and analysis of reporter gene expression. PRRSV ORF7 proteins were visualized on the surface of formaldehyde-fixed cells by incubation with mouse anti-ORF7 monoclonal antibody (MAb; 6D7/D2) followed by secondary labeling with either fluorescein isothiocyanate-conjugated or Cy3-conjugated goat anti-mouse immunoglobulin G (IgG; Jackson ImmunoResearch Labs Inc., West Grove, PA), using a procedure described elsewhere (61). LUC activity was estimated in cell lysates, as previously described (61). EGFP expression was visualized under a confocal microscope fitted with a fluorescein filter, as described previously (61).
RESULTS
Assembly of the PRRSV full-length cDNA as a BAC. The full-length PRRSV cDNA BAC was constructed by sequentially assembling six overlapping cDNA fragments of the PRRSV PL97-1/LP1 isolate (GenBank accession number AY612613), a clone of a genetically homogeneous population of large-plaque-forming PRRSV, plaque purified from the first PRRSV Korean isolate PL97-1 (Fig. 1A) (see the supplemental material for a detailed description). During the assembly, we took advantage of natural preexisting restriction sites in the viral genome rather than introducing intentional mutations to generate new restriction sites that would then be used to assemble the cDNAs, since such mutations, even silent mutations, may impair viral replication. To enable in vitro runoff transcription, the SP6 RNA polymerase promoter sequence was placed precisely at the beginning of the PL97-1/LP1 viral sequence in such a way that synthetic RNA transcripts with an authentic 5' end would be generated (Fig. 1A and B). To generate an artificial runoff site, three unique restriction endonuclease recognition sites (AclI, NotI, and SdaI) were placed in a row immediately downstream of the viral poly(A)54 stretch of PL97-1/LP1 (Fig. 1A and B). The genetic integrity of the cloned cDNAs in the BACs was verified throughout the subcloning and assembly process by extensive restriction analyses and sequencing. The cloned inserts did not exhibit genetic instability, as deletions, rearrangements, or nonsense mutations were never detected (data not shown). As a result, the full-length PRRSV cDNA clone encoding the entire PL97-1/LP1 viral genome downstream of the SP6 promoter was assembled. It was designated pBAC/PRRSV/FL, and its complete genome sequence was confirmed (Fig. 1A and B).
In vitro generation of infectious synthetic PRRSV RNAs transcribed from pBAC/PRRSV/FL. We estimated the specific infectivities of the synthetic RNAs that were transcribed in vitro from pBAC/PRRSV/FL. First, pBAC/PRRSV/FL was linearized by digestion with AclI, NotI, or SdaI to prepare three different cDNA templates. These were designated pBAC/PRRSV/FL/AclI, pBAC/PRRSV/FL/NotI, and pBAC/PRRSV/FL/SdaI, respectively (Fig. 1B), and served as templates for SP6 polymerase runoff transcription in the presence of the m7G(5')ppp(5')A cap structure analog. As summarized in Fig. 1B, all transcription reactions produced capped synthetic RNA transcripts with authentic 5' ends (11). However, the 3' ends varied depending on the template used in the transcription reaction. Transcription of the pBAC/PRRSV/FL/AclI template yielded synthetic RNAs containing two extra nucleotides (CG) of nonviral sequence at their 3' ends as a result of copying the 5' overhang left by the AclI digestion (Fig. 1B). Transcription of the pBAC/PRRSV/FL/NotI and pBAC/PRRSV/FL/SdaI templates also produced synthetic RNAs containing 10 (CGUUGCGGCC) and 14 (CGUUGCGGCCGCCC) extra nucleotides of nonviral sequence at their 3' ends, respectively (Fig. 1B). In some flaviviruses, unrelated sequences at the 3' ends of synthetic RNAs transcribed from an infectious cDNA have been reported to diminish their specific infectivities (60, 61). Thus, we sought to generate a synthetic RNA bearing the authentic 3' end of the PRRSV genome. This was achieved by linearizing pBAC/PRRSV/FL with AclI followed by MBN treatment to remove the 5' overhang left by the AclI digestion. The resulting construct was designated pBAC/PRRSV/FL/AclIMBN (Fig. 1B).
Transfection of the synthetic RNA transcripts into susceptible MARC-145 cells by using our optimized electroporation conditions (5% transfection efficiency) indicated that the specific infectivities of the transcripts produced from pBAC/PRRSV/FL/AclIMBN, pBAC/PRRSV/FL/AclI, and pBAC/PRRSV/FL/NotI were similar, at 3.3 x 104, 2.3 x 104, and 2.2 x 104 PFU/μg, respectively (Fig. 1C). On the other hand, the specific infectivities of the transcripts from pBAC/PRRSV/FL/SdaI were 20-fold lower, at 1.3 x 103 PFU/μg (Fig. 1C). Thus, reconstitution of the authentic 3' end of the PRRSV genome is important for the generation of highly infectious synthetic PRRSV RNA transcripts. Furthermore, transfection of the synthetic RNA transcripts into BHK-21 cells (>90% transfection efficiency) always produced >20-fold-higher infectivity than that produced by MARC-145 cells (Fig. 1C). This indicates that a system employing BHK-21 cells may be superior to MARC-145 cells for the purpose of characterizing viral mutants whose infectivities have been lost or reduced and which, therefore, can be studied only immediately following transfection.
The specific infectivities of all four RNA transcripts reflected the virus titers in the culture supernatants of the transfected MARC-145 cells (Fig. 1D). Cells transfected with synthetic RNA transcripts containing the authentic 3' end rapidly produced 105 PFU/ml of PRRSV virus, although 2 to 10 extra nucleotides (but not 14) were tolerated within the nonviral sequence at the 3' end (Fig. 1D). Additionally, we also confirmed that specific infectivity requires the transcription of RNA from the full-length PRRSV cDNA template (see the supplemental material).
Synthetic PRRSVs generated from the infectious cDNAs are identical to the parental virus. We compared the synthetic PRRSVs recovered from the four infectious cDNA templates with the parental virus PL97-1/LP1. Infection of MARC-145 cells with the synthetic viruses or with PL97-1/LP1 resulted in homogeneous large plaques (Fig. 2A). The growth properties on MARC-145 cells at an MOI of 1 showed that equivalent virus titers accumulated over time for all four synthetic viruses and the parental virus (Fig. 2B). Similar growth kinetics were also obtained with low (0.1) and high (10) MOIs (data not shown). Western blot analyses using a PRRSV-positive pig antiserum (Fig. 2C) and a rabbit antiserum raised against the glutathione S-transferase-fused PRRSV Nsp1a protein (Fig. 2D, top) showed that the levels of PRRSV-specific viral proteins and PRRSV Nsp1a produced in cells infected with any of the four synthetic viruses or the parental virus were similar. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was employed as a loading control (Fig. 2D, bottom). The immunostaining patterns of the cells infected with any of the four synthetic viruses or with PL97-1/LP1 were also identical (data not shown). Thus, the synthetic viruses recovered from the four infectious cDNA templates are phenotypically indistinguishable from the parental virus in terms of plaque morphology, cytopathogenicity, growth kinetics, and protein expression.
We sequenced the 5' and 3' ends of the four synthetic PRRSV viral genomes and found that, as expected, their 5' ends began with 1AUG ACG UAU-, which is identical to the 5' end of the parental virus (data not shown). With regard to the 3' end, we found that the genomic RNAs of all four synthetic viruses terminated with a poly(A) tail and did not retain the 2, 10, or 14 extra nucleotides of nonviral sequence at their 3' ends (data not shown; see Discussion). Thus, the genomes of the viruses recovered from the four infectious cDNA templates bear authentic 5' and 3' ends. Furthermore, a single point mutation acting as a genetic marker was introduced into the infectious cDNA and was found in the genome of the recovered synthetic virus, indicating that the recovered virus originates from the cDNA template (see the supplemental material). Therefore, these results validate the use of the infectious PRRSV cDNA for direct molecular genetic analyses.
Serial nucleotide deletions of the PRRSV 5' end in genomic RNAs decrease or completely abolish their infectivity, which appears to be restored by acquisition of novel 5' sequences. We noticed previously that significant nucleotide sequence differences in the PRRSV 5' NCR exist between the North American and European genotypes (20). However, a nucleotide sequence alignment using 10 fully sequenced PRRSV isolates showed that about 30 consecutive nucleotides proximal to the 5' end are highly conserved among nine isolates with the North American genotype (Fig. 3A). Moreover, 10 out of 12 nucleotides proximal to the PRRSV 5' end were absolutely conserved between the North American and European genotypes (Fig. 3A), despite their otherwise low nucleotide identity of 55 to 70% (2, 6, 10, 20, 25, 32, 53). To examine the role of the PRRSV-conserved 5'-terminal nucleotides in viral replication, a set of eight truncated mutants was constructed by serial deletion of an odd number of nucleotides from the 5' end (Fig. 3B). We first determined the specific infectivities of the synthetic RNA transcripts derived from each truncated mutant cDNA template. As shown in Fig. 3C, cDNAs of mutants with deletions of 1 (pBAC/PRRSV/FL/nt1) and 3 (pBAC/PRRSV/FL/nt3) nucleotides at the utmost 5' end were equally decreased in their infectivities by 15-fold, to 1.2 x 104 to 3.1 x 104 PFU/μg, compared to that of the wild-type infectious pBAC/PRRSV/FL cDNA (4.4 x 105 to 5.2 x 105 PFU/μg). Moreover, deletions of 5 (pBAC/PRRSV/FL/nt5) and 7 (pBAC/PRRSV/FL/nt7) nucleotides drastically decreased the infectivity to 1.0 x 102 to 1.8 x 102 PFU/μg and 8.6 x 102 to 9.3 x 102 PFU/μg, 3,500-fold and 550-fold lower than that of the wild type, respectively (Fig. 3C). Interestingly, the infectivities of the synthetic RNAs containing the 7-nucleotide deletion was generally approximately sixfold higher than that containing the 5-nucleotide deletion. Furthermore, in the cells transfected with the synthetic RNAs derived from pBAC/PRRSV/FL/nt1 and pBAC/PRRSV/FL/nt3, we observed a relatively homogeneous population of large plaques and foci visualized by staining with a mouse anti-ORF7 MAb, as seen in the cells transfected with the wild-type infectious RNAs (Fig. 3C). In the cells transfected with the synthetic RNAs derived from pBAC/PRRSV/FL/nt5 and pBAC/PRRSV/FL/nt7, on the other hand, a relatively heterogeneous population of smaller plaques and foci was observed (Fig. 3C). Neither infectivity nor foci or plaques were detectable with synthetic RNAs containing more than 9-nucleotide deletions at the utmost 5' end (Fig. 3C).
It should be noted that two mutants harboring deletions of 5 or 7 nucleotides produced plaques of heterogeneous sizes, indicating some instability. This was more evident when supernatants harvested from the transfected cells were passaged once on nave MARC-145 cells. These passaged pseudorevertants derived from the mutant cDNAs containing 1-, 3-, 5-, and 7-nucleotide deletions produced similar amounts of the PRRSV Nsp1a protein upon infection with the same amounts of the viruses as did the wild-type virus (Fig. 4A). Furthermore, their growth kinetics (Fig. 4B) and plaque morphologies (Fig. 4C) were very similar, with a relatively homogeneous population of large plaques observed in the cells infected with each of these pseudorevertants, including the 5- and 7-nucleotide deletion mutants.
The nucleotide sequences at the utmost 5'-end regions of all these pseudorevertants were determined by 5' rapid amplification of cDNA end analysis, cloning of reverse transcription-PCR amplicons, and sequencing of a number of the independently picked clones containing the insert. As summarized in Fig. 5A, 33 of 42 independent clones obtained from the pBAC/PRRSV/FL/nt1-derived pseudorevertants appeared to be converted to the wild-type virus by the acquisition of one A nucleotide at the site of the deletion. The remainder of the nine clones had the same sequence as pBAC/PRRSV/FL/nt1 (Fig. 5A). In the case of the pBAC/PRRSV/FL/nt3-derived pseudorevertants, 32 out of 57 independent clones had acquired 3 nucleotides (AUG) at the utmost 5' end, which rendered the sequence identical to that of the wild-type virus (Fig. 5A). Twenty-one clones contained an insertion of 4 nucleotides (UAUG), while four clones appeared to contain an insertion of 3 nucleotides (AAG) at the deletion site (Fig. 5A). Interestingly, the pBAC/PRRSV/FL/nt5-derived pseudorevertants were found to have a deletion of the 5'-end single G nucleotide, the first nucleotide in this mutant construct. Moreover in 39 of 49 independent clones, there was an insertion of 6 novel (AUUAUA) nucleotides at the deletion site, while 10 clones contained a 7-nucleotide insertion (UAUUAUA) (Fig. 5A). For the pBAC/PRRSV/FL/nt7-derived pseudorevertants, the novel 5'-end sequences acquired were found to be more heterogeneous than those of the mutants described above. Specifically, a majority of the sequenced clones (28/48 and 9/48 clones) had insertions of 7 (AUUAUAU) and 8 (UAUUAUAU) nucleotides at the site of the deletion, respectively, and appeared to be identical to two of the pBAC/PRRSV/FL/nt5-derived pseudorevertants (Fig. 5A). In addition, four independent clones had 8 novel nucleotides (UAUCAUAU) inserted at the deletion site, while two further clones had the 8-nucleotide sequence AUUUAUAU inserted at this site (Fig. 5A). In addition, three and two independent clones appeared to have five and six AU repeats at the site of the deletion, respectively (Fig. 5A). Overall, these pseudorevertants appeared to have acquired novel 5' sequences of various sizes, composed mainly of A and U bases.
To address the importance of these novel 5' sequences, we reconstructed eight derivatives of the PRRSV 5'-end truncated mutants with all of these novel sequences and determined the specific infectivities of their RNA transcripts (Fig. 5B). In all reconstructed cases, the specific infectivities were increased to a level similar to that of the wild type (Fig. 5B). Among all of the pseudorevertants, we found three cases that were not reconstructed, since the resulting mutants were identical to either the wild type (PRRSV/FL/nt1/Rev1 and PRRSV/FL/nt3/Rev1) or the original truncated mutant (PRRSV/FL/nt1/Rev2) (Fig. 5A). According to plaque/focus morphology, the cells transfected with four synthetic RNAs (derived from pBAC/PRRSV/FL/nt3/Rev2, pBAC/PRRSV/FL/nt7/Rev1, pBAC/PRRSV/FL/nt7/Rev2, and pBAC/PRRSV/FL/nt7/Rev6) formed a homogeneous population of large plaques/foci, as seen with the wild-type infectious cDNA (Fig. 5B). In addition, we also observed a homogeneous population of medium-sized (pBAC/PRRSV/FL/nt3/Rev3 and pBAC/PRRSV/FL/nt7/Rev3) and small (pBAC/PRRSV/FL/nt7/Rev4 and pBAC/PRRSV/FL/nt7/Rev5) plaques/foci (Fig. 5B). These results demonstrated that the addition of novel AU-rich sequences to the utmost 5' end of the PRRSV 5' deletion mutants, but not changes elsewhere in their genomes, allowed efficient PRRSV replication.
Generation of a panel of self-replicating, self-limiting PRRSV viral replicons expressing the LUC gene as a reporter. We sought to construct a panel of self-replicating, self-limiting PRRSV viral replicons by using pBAC/PRRSV/FL. We initially constructed a set of three viral replicons (pBAC/PRRSV/RepLuc MB, pBAC/PRRSV/RepLuc ME, and pBAC/PRRSV/RepLuc DI) which lacked various portions of the structural protein-coding region of the PRRSV genome (Fig. 6A). At the same time, we also inserted at the site of each deletion the expression cassette containing the EMCV IRES-driven LUC gene to facilitate the monitoring of viral replication (Fig. 6A). LUC was chosen as the reporter since its expression is easy to monitor in a highly quantitative and sensitive manner. pBAC/PRRSV/RepLuc MB had the smallest deletion, of nt 12714 to 14194, and lacked the 3'-terminal 129 nucleotides of ORF2a, the majority of ORF3 except for its 5'-terminal 18 nucleotides, the complete sequence of ORF4, and the 5'-terminal 406 nucleotides of ORF5 (Fig. 6A). pBAC/PRRSV/RepLuc ME was the same as pBAC/PRRSV/RepLuc MB except for an additional 5' deletion (nt 12163 to 14194). It lacked the majority of ORF2a and ORF2b except for their 5'-terminal 90 (ORF2a) and 85 (ORF2b) nucleotides, the complete sequence of ORF3 and ORF4, and the 5'-terminal 406 nucleotides of ORF5 (Fig. 6A). pBAC/PRRSV/RepLuc DI had the largest deletion, of nt 12163 to 15252, which extended towards the 3' end of pBAC/PRRSV/RepLuc ME, and lacked the majority of ORF2a and ORF2b, except for their 5'-terminal 90 (ORF2a) and 85 (ORF2b) nucleotides, and the complete sequence of ORF3 through ORF7, except for the 3'-terminal 8 nucleotides of ORF7 (Fig. 6A). Since none of these viral replicons contained all of the structural genes, they were expected not to produce viral particles, as first established for EAV (31, 55, 62).
Next, we examined whether the viral replicon RNAs derived from the three cDNA templates were replication competent by monitoring the expression of the LUC gene after their transfection in BHK-21 cells. The LUC activities 6 h after transfection with pBAC/PRRSV/RepLuc MB or pBAC/PRRSV/RepLuc ME RNAs were 9.95 x 102 and 9.72 x 102 relative light units (RLU), respectively (Fig. 6B), and were dramatically increased at 18 and 24 h posttransfection to 1.12 x 104 to 1.49 x 104 and 2.12 x 104 to 2.45 x 104 RLU, respectively. These activities were maintained until 48 h posttransfection (Fig. 6B), after which they gradually decreased due to the cell death induced by the replication of these viral replicons and the lack of cell-to-cell spread. In BHK-21 cells transfected with pBAC/PRRSV/RepLuc DI RNA, the initial LUC activity at 6 h posttransfection was similar to that of the other two (9.81 x 102 RLU) but then gradually decreased over time to 3.60 RLU at 72 h posttransfection, which was at the level of the background luminescence from the mock-transfected cells (Fig. 6B). Thus, pBAC/PRRSV/RepLuc MB and pBAC/PRRSV/RepLuc ME viral replicon RNAs were competent in replication, but pBAC/PRRSV/RepLuc DI RNA was not. In addition, no virus particle formation was observed in the two replication-competent viral replicon RNA-transfected cells (data not shown). To determine the location of the minimal cis-acting element required for viral replication at the 3' end of the viral genome, we constructed the eight viral replicons, pBAC/PRRSV/RepLuc S1 to S8, by systematically deleting additional sequences towards the 3' end of pBAC/PRRSV/RepLuc ME (Fig. 6A). Of the eight viral replicons, only pBAC/PRRSV/RepLuc S8 RNAs were replication competent (Fig. 6B).
The production levels of viral replicon RNA itself in BHK-21 cells transfected with each of the 11 viral replicon RNAs (pBAC/PRRSV/RepLuc MB, pBAC/PRRSV/RepLuc ME, pBAC/PRRSV/RepLuc DI, and pBAC/PRRSV/RepLuc S1 to S8) were directly quantitated by real-time quantitative reverse transcription-PCR using a PRRSV-specific probe complementary to a sequence of nt 659 to 682 in ORF1a (Fig. 6C). In agreement with the LUC activity (Fig. 6B), the levels of the replication-competent viral replicon RNAs, i.e., pBAC/PRRSV/RepLuc MB, pBAC/PRRSV/RepLuc ME, and pBAC/PRRSV/RepLuc S8 RNA, were increased approximately 1.1-fold, 6.2-fold, and 16.0-fold at 24 h posttransfection relative to the levels at 6 h posttransfection, and these increases were subsequently reduced over time due to the lack of viral spread to the adjacent cells (Fig. 6C). On the other hand, the levels of all replication-incompetent viral replicon RNAs at 6 h posttransfection were drastically decreased and were below the level of detection at all time points, confirming that these replicon RNAs were indeed incapable of RNA replication (Fig. 6C). Thus, these findings showed that the 911 nucleotides from the 3' end of the viral genome contained the minimal cis-acting element required for viral replication and that the internal deletion in the coding region of the structural genes (including ORF2a to ORF6) altered the level of the ORF1a and ORF1b-containing replicon RNA replication.
Immunostaining of the ORF7 protein showed that it was expressed in BHK-21 cells transfected with the replication-competent pBAC/PRRSV/RepLuc S8, pBAC/PRRSV/RepLuc MB, and pBAC/PRRSV/RepLuc ME RNAs but not in cells transfected with the replication-incompetent viral replicons (data not shown). Mutagenetic analyses with another EAV system have indicated that all of the structural proteins, including ORF7, are dispensable for genomic RNA replication and subgenomic mRNA transcription (31, 55, 62). On the other hand, previous studies with the distantly related coronavirus systems have indicated that expression of the N protein, which is equivalent to the PRRSV ORF7 protein, is required for efficient replication of the genomic RNA (3, 39, 46). To examine the possibility that the replication-incompetent viral replicons that we constructed would be able to replicate when the ORF7 protein was provided in trans, all PRRSV viral replicon RNAs were transiently cotransfected with the infectious PRRSV viral RNAs transcribed from pBAC/PRRSV/FL, and LUC activity was monitored over time. This revealed that the replication-incompetent viral replicon RNAs were still replication incompetent when the ORF7 protein was provided in trans (data not shown).
Expression of the EGFP reporter gene by infectious PRRSV cDNA/recombinant viruses. We sought to produce recombinant infectious PRRSV viruses that express the EGFP reporter gene upon infection. To do this, pBAC/PRRSV/FL was engineered in two ways. First, we inserted the EMCV IRES-driven EGFP expression cassette immediately downstream of the first 33 nucleotides of the ORF7 coding region that include a short PRRSV transcription-regulating sequence and followed it with the cis-acting element in the 3' 911 nucleotides of the viral genome (Fig. 7A). The EGFP gene in the resulting pBAC/PRRSV/FL/IRES-EGFP construct was driven by the EMCV IRES, and its expression was dependent on viral replication. Second, the autoprotease Npro gene of bovine viral diarrhea virus was fused adjacent to the N terminus of the EGFP gene so that the correct N terminus of the EGFP protein was created by Npro cleavage (Fig. 7A). The fused Npro-EGFP gene was then inserted at the same site where the EMCV IRES-driven EGFP expression cassette was inserted. This resulted in the pBAC/PRRSV/FL/Npro-EGFP construct. In both constructs, the EGFP expression cassette was inserted downstream of the first 33 nucleotides of the ORF7 coding region because this region might be involved in the gene expression and regulation of its own subgenomic mRNA. In both cases, the insertions did not alter the infectivities of the synthetic RNA transcripts (data not shown).
EGFP expression was then assessed by transfecting nave BHK-21 cells with the pBAC/PRRSV/FL, pBAC/PRRSV/FL/IRES-EGFP, or pBAC/PRRSV/FL/Npro-EGFP RNAs and examining them by confocal microscopy. No fluorescence was observed in the PRRSV/FL RNA-transfected (Fig. 7B) or mock-transfected (data not shown) cells, but the other two cell types showed green fluorescence (Fig. 7B). The fluorescence was in the nucleus and the cytoplasm because EGFP (30 kDa) is small enough to permit diffusion between the nucleus and the cytoplasm. All three RNA-transfected cell types also expressed the ORF7 protein (Fig. 7B). Similar observations were made when MARC-145 cells were transfected (data not shown).
The production of recombinant EGFP-expressing PRRSV viruses was estimated by measuring (at the indicated time points) the viral titers in the culture supernatants of BHK-21 or MARC-145 cells transfected with the three synthetic RNA transcripts. Production of the EGFP-expressing PRRSV viruses was monitored by infecting nave MARC-145 cells and subsequently counting the green focus-forming units (GFU) per ml. Several experiments showed that transfection of MARC-145 cells with pBAC/PRRSV/FL/IRES-EGFP RNAs generated on average 1.2 x 103, 1.0 x 104, and 9.6 x 104 GFU/ml at 24, 48, and 72 h posttransfection, respectively (Fig. 7C). Similar virus titers were produced by pBAC/PRRSV/FL/Npro-EGFP RNA-transfected MARC-145 cells (Fig. 7C). In addition, at 24 h posttransfection, BHK-21 cells transfected with the EGFP-expressing PRRSV RNAs produced slightly higher virus titers (4.1 x 104 to 5.5 x 104 GFU/ml) than the MARC-145 cells did, due to their higher transfection efficiency (data not shown). Moreover, unlike MARC-145 cells, the BHK-21-generated virus titers gradually decreased to 6.4 x 102 to 7.8 x 102 GFU/ml at 96 h posttransfection, due to a lack of viral spread and cell death of the synthetic RNA-containing cells (data not shown). Thus, we produced recombinant PRRSVs carrying the EGFP gene as a reporter and thereby showed the possibility of utilizing infectious PRRSV cDNA/recombinant viruses as heterologous gene expression vectors.
DISCUSSION
In this report, we have described the construction and characterization of a reverse genetics system for PRRSV that involves generating a functional PRRSV cDNA as a BAC which is based on E. coli and its single-copy plasmid F factor (42, 52). BACs have been successfully used to clone and maintain large fragments of DNA from a variety of complex genomic sources (including humans and herpesvirus) in bacteria (26, 42). Using this functional cDNA, we not only demonstrated the importance of the PRRSV 5'-end nucleotide sequences for replication but also discovered novel AU-rich sequences of various sizes that can functionally replace the authentic 5'-proximal 7 nucleotides of the viral genome for RNA replication. We also showed the utility of this system by generating a panel of self-replicating, self-limiting PRRSV viral replicons that aided the identification of a 3' cis-acting element required for PRRSV replication. This reverse genetics system will thus permit direct molecular genetic studies of the PRRSV pathogen that will greatly enhance our understanding of the molecular mechanisms behind its replication, transcription, translation, virulence, and pathogenesis. In addition, the results we obtained present the possibility that this system can be used to produce novel heterologous gene expression vectors that can express a foreign gene of interest. Thus, this system will also be useful in developing new, safe, and genetically defined antiviral vaccines.
With the aim of generating an infectious cDNA that has an unaltered native sequence, we also sought to ensure that the in vitro transcription of the infectious PRRSV cDNA would generate RNA transcripts possessing the authentic 5'- and 3'-terminal nucleotide sequences of the viral genome. These sequences are usually essential for the replication of viral RNA. For example, it has been shown that the 5'- and 3'-terminal regions of flavivirus RNA are required for the initiation of replication in vitro and in vivo (24). To ensure the generation of authentic 5'- and 3'-terminal sequences, we adapted the approaches that were used previously to design flavivirus infectious cDNAs (37, 61). The cap structure at the 5' end of the PRRSV viral genome was followed by the nucleotide sequence 1AUG ACG6, which is highly conserved in most PRRSV strains, including PL97-1/LP1 (20). We achieved the authentic 5' end of the viral genome by engineering the SP6 promoter transcription start immediately upstream of the viral genome. The in vitro runoff transcription reaction in the presence of the m7G(5')ppp(5')A cap structure analog then allowed us to synthesize capped RNA transcripts with the authentic 5' ends (11). These transcripts were highly infectious upon transfection into susceptible MARC-145 cells and nonpermissive BHK-21 cells. Previous studies with PRRSV cDNAs used the m7G(5')ppp(5')G cap structure analog instead in the in vitro T7 polymerase-driven transcription reaction (23, 28, 34, 47). This would result in capped RNA transcripts containing one (23, 28, 34) or two (47) extra nonviral G nucleotides upstream of the 1AUG ACG6 sequence. When we used the m7G(5')ppp(5')G cap structure analog in our own transcription reactions, we found that the extra G did not affect the infectivities of the RNA transcripts (data not shown). We also found that the uncapped RNA transcripts were not infectious, which indicates that the cap structure is essential for viral replication (data not shown), in agreement with a previous report (28).
The 3' end of the PRRSV viral genome terminates with the poly(A) tail (8, 29, 45). The poly(A) tail in PL97-1/LP1 is 54 nucleotides long. To ensure that the infectious PPRSV cDNA that we constructed would produce RNAs bearing the 54 nucleotides of the poly(A) tail, we followed the tail sequence with a unique restriction recognition site that would linearize the cDNA for runoff transcription. The resulting RNA transcripts contained the 54-nucleotide poly(A) tail and gave a specific infectivity of >105 PFU/μg of RNA that produces synthetic viruses of >5 x 104 PFU/ml upon transfection. That the 54-nucleotide poly(A) tail is important was then clearly demonstrated by the fact that unpolyadenylated RNA transcripts were not infectious (data not shown). With regard to the other PRRSV cDNAs published previously, poly(A) tails of various lengths, such as 109 (28), 43 (47), 40 (34), or 21 (23) A's, have been included during construction of each cDNA. It is not clear whether poly(A) tails of such variable lengths may affect viral replication.
Several studies have shown that it is important that the synthetic RNA transcripts that are generated from functional cDNAs for RNA viruses carry the authentic 3' end, as this promotes their infectivity and the establishment of a productive infection (60, 61). To ensure this for the PRRSV RNAs, we designed the infectious PRRSV cDNA template so that AclI digestion followed by MBN treatment would generate a linearized template lacking the 5' CG overhang left by the AclI digestion. This then served as the template in SP6 polymerase runoff transcription and was found to produce highly infectious RNA transcripts bearing the authentic 5' and 3' termini of the viral genome. We also observed similar levels of infectivity with RNA transcripts that terminated with 2 and 10 extra nonviral nucleotides at their 3' ends. However, 14 extra nonviral 3' nucleotides slightly decreased the specific infectivity and delayed virus production. Interestingly, the 2 to 14 extra nonviral nucleotides were not found in the genomic RNAs of the synthetic viruses produced from the cDNA in cells. This could be because these extra nonviral nucleotides are lost during propagation in the transfected cells. Alternatively, it is also possible that the SP6 RNA polymerase in the in vitro transcription reaction may not efficiently synthesize the entire poly(A) tail each time, which would sometimes result in synthetic RNA transcripts with poly(A) tails of different sizes that lack these extra 3' nonviral nucleotides. Thus, replication-competent RNA transcripts could be selected over replication-incompetent RNAs containing these extra nonviral nucleotides. Further investigation will be needed to distinguish between these two possibilities. With regard to the other PRRSV cDNAs published previously, linearization was induced by a unique restriction site, such as PvuI (28), AclI (34, 47), or SwaI (23), which was introduced downstream of the poly(A) tail. The digestion of either PvuI or AclI would produce synthetic RNA transcripts bearing two extra nonviral nucleotides (CG) downstream of the poly(A) tail, whereas SwaI digestion would make synthetic RNAs with nine nonviral nucleotides. Our own observations suggest that these extra 2 or 9 nucleotides should not affect the infectivities of their resulting RNA transcripts.
Our work presented here elucidates the importance of the PRRSV-conserved 5'-end nucleotide sequence 1AUGACGU7 in RNA replication. Several novel AU-rich PRRSV 5' sequences detected in pseudorevertants were able to functionally replace the deleted 1AUGACGU7. According to the predicted RNA secondary structure of PRRSV 5' NCRs (48), the presence of these novel AU-rich PRRSV 5' sequences did not seem to significantly alter their stem-loop structures, which might be critical for viral replication. Although the functional role of these novel sequences is not understood at all, the complementary sequence of each of these novel 5' sequences at the utmost 3' end of negative-sense RNA is predicted to be involved in the initiation of positive-sense RNA synthesis. For some positive-sense RNA viruses, only minimal cis-acting sequences at the 3' ends of negative-sense RNAs are essential for positive-sense RNA synthesis (4, 18, 36). It is interesting to speculate on the origin of these novel 5' AU-rich sequences and the molecular mechanism of their insertion at the very beginning of the viral genome. Based on the heterogeneity and size of these novel 5' sequences, it is likely that they are acquired from cellular RNAs in the process of recombination involving template switching, during either negative- or positive-sense RNA synthesis, as has been described for pestivirus (30) and poliovirus (22). It is less likely, although not impossible, that these sequences are derived from the viral genome. Understanding this issue may provide new information on RNA replication of arteriviruses.
Several studies have shown that cis-acting elements within the coding region of the viral genome are essential for RNA virus replication (24). By analyzing a panel of self-replicating, self-limiting, LUC-expressing PRRSV viral replicons with internal deletions starting from the beginning of ORF2a and extending towards the 3' NCR, we found that there was a cis-acting element within the coding region of PRRSV as well. These analyses revealed that the three viral replicons that contained at least 911 nucleotides from the 3' end of the genome were replication competent. In contrast, eight viral replicons that did not contain 911 nucleotides at the 3' end of the genome were all replication incompetent. The LUC-expressing PRRSV viral replicon system is advantageous for monitoring viral replication in a highly quantitative and sensitive manner. On the other hand, the sequence requirements for RNA replication of the LUC-containing PRRSV viral replicon might not necessarily be the same as those for full-length genome replication. Especially, the insertion of a highly structured RNA sequence of EMCV IRES and the 1,653-nucleotide LUC gene could have a deleterious effect on the overall level of RNA structure when this insertion is combined with a specific deletion within the viral genome. Investigations on how the cis-acting element mapped in this study works in RNA replication in the context of the full-length viral genome might be an important step towards a better understanding of PRRSV RNA synthesis. A study using the infectious Lelystad strain cDNA has also shown that a 34-nucleotide stretch within the PRRSV ORF7 is essential for RNA replication (51). In addition to this stretch of 34 nucleotides, the presence of an additional 3' cis-acting element which is essential for viral replication was revealed in our own data. Investigations using our replicon system that aim to finely map this minimal 3' cis-acting element and elucidate the molecular mechanism by which it controls viral replication are currently under way.
Several RNA viruses have been successfully exploited as eukaryotic expression vectors (12, 40). We showed here that the infectious PRRSV cDNA BAC that we constructed could also serve as a novel PRRSV-based virus/vector system in which foreign genes of interest could be expressed. We showed the possibility that this system can be used to express foreign genes in two ways: first, by generating infectious recombinant vector RNAs/viruses that carry a foreign gene, and second, by producing viral replication-competent but propagation-deficient PRRSV viral replicon vector RNA that carries a foreign gene. As a transient-expression system, PRRSV offers several advantages: (i) the recombinant virus is rapidly produced, (ii) PRRSV can replicate in a variety of eukaryotic cells upon transfection of synthetic RNAs, (iii) PRRSV is unable to infect humans, (iv) the genetically stable infectious cDNA is available and readily manipulated, and (v) the cytoplasmic replication of the RNA genome minimizes the possibility of integration and unwanted mutagenic consequences. Recently, a human coronavirus with a positive-strand RNA genome has been developed as a multigene expression vector; the transcription strategy of this vector involves producing a nested set of multiple subgenomic mRNAs (46). PRRSV employs a similar strategy in its gene expression. That the genome of PRRSV is smaller (15 kb) than that of coronaviruses (30 kb) suggests that our PRRSV-based expression system can be even more useful as a multigene expression vector.
In conclusion, our reverse genetics system for PRRSV, which involves an infectious cDNA BAC and viral replicons, may be useful in a variety of research fields. First, this system enables us not only to investigate the molecular mechanisms of viral replication, transcription, and translation but also to identify the viral genetic elements involved in virulence and pathogenesis. Second, it can serve as an invaluable genetic tool for heterologous gene expression in a wide variety of eukaryotic cells; this has many applications in biological research. Finally, targeted manipulation of the infectious PRRSV cDNA may permit the production of effective genetically modified antiviral vaccines against this pathogen that may abrogate the ongoing worldwide spread of PRRSV.
ACKNOWLEDGMENTS
We are grateful to Jae-Young Song, Animal Disease Research Department, National Veterinary Research & Quarantine Service, for sharing a PRRSV-positive pig antiserum.
This work was supported by grant R01-2001-000-00249-0 from the Korea Science & Engineering Foundation.
Supplemental material for this article may be found at http://jvi.asm.org/.
These authors contributed equally to this work.
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Research Institute of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea
ABSTRACT
We here demonstrate the successful engineering of the RNA genome of porcine reproductive and respiratory syndrome virus (PRRSV) by using an infectious cDNA as a bacterial artificial chromosome. Runoff transcription from this cDNA by SP6 polymerase resulted in capped synthetic RNAs bearing authentic 5' and 3' ends of the viral genome that had specific infectivities of >5 x 105 PFU/μg of RNA. The synthetic viruses recovered from the transfected cells were genotypically and phenotypically indistinguishable from the parental virus. Using our system, a series of genomic RNAs with nucleotide deletions in their 5' ends produced viruses with decreased or no infectivity. Various pseudorevertants were isolated, and acquisition of novel 5' sequences of various sizes, composed predominantly of A and U bases, restored their infectivities, providing a novel insight into functional elements of the 5' end of the PRRSV genome. In addition, our system was further engineered to generate a panel of self-replicating, self-limiting, luciferase-expressing PRRSV viral replicons bearing various deletions. Analysis of these replicons revealed the presence and location of a 3' cis-acting element in the genome that was required for replication. Moreover, we produced enhanced green fluorescent protein-expressing infectious viruses, which indicates that the PRRSV cDNA/viral replicon/recombinant virus can be developed as a vector for the expression of a variety of heterologous genes. Thus, our PRRSV reverse genetics system not only offers a means of directly investigating the molecular mechanisms of PRRSV replication and pathogenesis but also can be used to generate new heterologous gene expression vectors and genetically defined antiviral vaccines.
INTRODUCTION
Porcine reproductive and respiratory syndrome (PRRS) was first recognized in 1987 in North America (21) and shortly thereafter in Europe (35). It has since become one of the most common and economically significant infectious diseases in the swine industry worldwide (1, 15). It is characterized by mild to severe reproductive failure in sows and gilts and respiratory problems in piglets (9, 10, 17). The PRRS virus (PRRSV) was first isolated almost simultaneously in Europe and North America; the strains are designated Lelystad (53) and VR-2332 (6, 10), respectively. Although these strains induce phenotypically indistinguishable disease symptoms (19), they are genetically (2, 25, 32) and serologically (33, 54, 58) distinct. They currently constitute the two distinct genotypes of known PRRSV species.
PRRSV belongs to the family Arteriviridae in the order Nidovirales together with equine arteritis virus (EAV), simian hemorrhagic fever virus, and the lactate dehydrogenase-elevating virus of mice (8, 45). Like other arteriviruses, PRRSV is a small-envelope virus with a positive-sense, single-stranded RNA genome of 15 kb in length. The genome has a cap structure at its 5' end and a poly(A) tail at its 3' end. The genome contains at least nine open reading frames (ORFs) flanked by 5' and 3' noncoding regions (NCRs) (13, 29, 45, 59). Two overlapping ORFs, ORF1a and ORF1b, are expressed from the genomic RNA. These two ORFs are predicted to be processed into 13 mature nonstructural proteins that are believed to be involved in viral replication (5, 43, 44, 45, 49, 57). ORF2a, ORF2b, and ORF3 to ORF7 are translated from the 5' ends of a coterminal nested set of subgenomic mRNAs. The small ORF2b is completely embedded within the larger ORF2a (59). These ORFs encode the viral structural proteins (14, 27, 29, 59).
To analyze positive-sense RNA viruses such as PRRSV at molecular and genetic levels, a number of reverse genetics systems that allow the determination of the functions of the genes and gene products of these RNA viruses through their manipulation and genetic analysis have been developed (7). With regard to arteriviruses, two "RNA-launched" reverse genetics systems for EAV have been independently developed by two research groups (16, 50). Although both functional EAV cDNAs were constructed using the same virus isolate, different bacteriophage promoters, either the SP6 or T7 RNA polymerase promoter, were placed immediately upstream of the viral genome for runoff transcription in vitro. The first infectious EAV cDNA was constructed in a high-copy-number plasmid, pUC18, and did not show any genetic instability during its construction and propagation in Escherichia coli, in contrast to the infectious cDNAs of several other viruses (50).
For PRRSV, the first RNA-launched reverse genetics system was constructed for the European Lelystad strain by assembling its full-length cDNA under the T7 promoter in the low-copy-number plasmid pOK12 (28). Identical strategies were also previously used in the construction of an infectious cDNA for the North American strain VR-2332 (34). Recently, two additional T7 promoter-driven RNA-launched reverse genetics systems were developed independently for the highly virulent American isolate NVSL 97-7895 (47) and for the highly virulent "atypical" North American isolate P129 (23) by using the low-copy-number plasmid pBR322 and the high-copy-number plasmid pCR2.1, respectively. An additional "DNA-launched" system has also been reported for the P129 isolate and employed full-length cDNA under the human cytomegalovirus immediate early promoter (23).
The present report describes the construction of not only a full-length infectious PRRSV cDNA in a bacterial artificial chromosome (BAC) designed to produce synthetic RNAs bearing authentic 5' and 3' ends of the viral genome but also a panel of self-replicating viral replicons. We have used our system to define 5'- and 3'-terminal nucleotide sequences that are essential for RNA replication. In addition, we have also demonstrated the potential of our system to generate vectors that express heterologous genes in eukaryotic cells in a variety of forms, including recombinant infectious PRRSV cDNA, viral replicons, and synthetic infectious viruses. Thus, the system we have constructed not only provides an important platform from which the basic biology of PRRSV can be investigated but also is a useful tool for designing new heterologous gene expression vectors and for generating genetically defined antiviral vaccines.
MATERIALS AND METHODS
Cells and viruses. MARC-145 (20) and BHK-21 cells (61) were maintained as described previously. All reagents used in cell culture were purchased from Life Technologies, Inc., Gaithersburg, MD. The parental PRRSV used in this study is the first Korean PRRSV strain, PL97-1, which was isolated in 1997 from the serum of an infected pig (20). High-titer virus stocks were obtained by cultivation in MARC-145 cells at a low multiplicity of infection (MOI) of 0.1 for 72 h. The viruses were then clarified by centrifugation (2,000 rpm for 10 min), aliquoted, and stored at –80°C until use.
Virus titration. Virus titers were determined by plaque assay using MARC-145 cells. For this, cells were preseeded in a six-well plate at a density of 3 x 105 per well for 12 to 18 h and then infected with serial 10-fold dilutions of virus for 1 h at 37°C with frequent agitation. The cell monolayers were then overlaid with minimal essential medium containing 0.5% SeaKem LE agarose (FMC BioProducts, Rockland, Maine) and 5% fetal bovine serum and incubated for 4 days at 37°C with 5% CO2. The resulting plaques were visualized by fixation with 7% formaldehyde followed by staining with crystal violet (1% [wt/vol] in 5% ethanol).
Oligonucleotides. All oligonucleotides used for cDNA synthesis, PCR amplification, and mutagenesis in this study are listed in the supplemental material (see Table S1 in the supplemental material). Their sequences were designed according to the complete nucleotide sequence of PL97-1 (GenBank accession number AY585241) (20).
Construction and mutagenesis of the full-length PRRSV cDNA as a BAC. Because of space limitations, only the salient features of plasmids are described here. A detailed description of plasmid construction is provided in the supplemental material, and computer-readable sequence files are also available upon request. All plasmids were constructed using standard molecular biology procedures (38).
pBAC/PRRSV/FL contained the full-length PRRSV PL97-1/LP1 cDNA flanked by the SP6 RNA polymerase promoter and three unique restriction sites (AclI, NotI, and SdaI) in a row for in vitro runoff transcription. Eight constructs (pBAC/PRRSV/FL/nt1 to -15) harbored deletions of 1, 3, 5, 7, 9, 11, 13, and 15 nucleotides (nt) from the utmost 5' end of the viral genome, respectively. pBAC/PRRSV/FL/nt3/Rev2 and pBAC/PRRSV/FL/nt3/Rev3 were reconstructed by the addition of 4 (TATG) and 3 (AAG) nucleotides at the beginning of the viral genome in pBAC/PRRSV/FL/nt3, respectively. Similarly, six additional constructs of pBAC/PRRSV/FL/nt7/Rev1 to -6 were reconstructed by the addition of 7 (ATTATAT), 8 (TATTATAT), 8 (TATCATAT), 10 (ATATATATAT), 12 (ATATATATATAT), or 8 (ATTTATAT) nucleotides, respectively, at the beginning of the viral genome in pBAC/PRRSV/FL/nt7. pBAC/PRRSV/FL/IRES-EGFP was constructed by the insertion of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-driven enhanced green fluorescent protein (EGFP) expression cassette immediately downstream of the first 33 nucleotides of the ORF7 coding region, followed by the 3' 911 nucleotides of the viral genome. pBAC/PRRSV/FL/Npro-EGFP is identical to pBAC/PRRSV/FL/IRES-EGFP, except that the EMCV IRES-driven EGFP expression cassette was replaced by the coding sequence of the Npro-EGFP fusion protein in such a way that expression of the fused Npro-EGFP was driven by its own subgenomic promoter. The autoprotease Npro gene of bovine viral diarrhea virus was fused adjacent to the N terminus of the EGFP gene so that the correct N terminus of the EGFP protein was created by Npro cleavage.
We constructed a panel of 11 PRRSV viral replicon vectors that express the luciferase (LUC) gene as a reporter. One set of three constructs, namely, pBAC/PRRSV/RepLuc MB, pBAC/PRRSV/RepLuc ME, and pBAC/PRRSV/RepLuc DI, were first constructed to contain internal deletions of nt 12714 to 14194, nt 12163 to 14194, and nt 12163 to 15252 in the viral genome, respectively, and also the insertion of the EMCV IRES-driven LUC expression cassette at the site of each deletion to facilitate the monitoring of viral replication. The other set of eight constructs, namely, pBAC/PRRSV/RepLuc S1 to S8, are identical to pBAC/PRRSV/RepLuc ME, except that the internal deletion was further extended to nt 15200, nt 15150, nt 15100, nt 15050, nt 15000, nt 14950, nt 14900, and nt 14500, respectively.
In vitro synthesis of RNA transcripts. The full-length PRRSV cDNA (2 μg) was digested with the appropriate restriction enzyme (AclI, NotI, or SdaI) and/or treated with mung bean nuclease (MBN). With regard to the PRRSV viral replicons, all the recombinant cDNAs were linearized by NotI digestion and extracted with phenol-chloroform and precipitated with ethanol. In vitro transcription from these template cDNAs, subsequent removal of the template cDNAs from transcription reaction, and extraction and quantitation of synthetic RNA transcripts were performed as previously described (61).
Transfection of cells with RNA transcripts. MARC-145 or BHK-21 cells were preseeded at a density of 2 x 106 or 3 x 106 cells per p150 culture dish for 24 h at 37°C with 5% CO2, respectively. The subconfluent cells were then trypsinized and washed three times with ice-cold RNase-free phosphate-buffered saline. After resuspension at a density of 2 x 107 cells/ml in phosphate-buffered saline, 400 μl of the cells was mixed with 2 μg synthetic RNA in a cuvette with a gap width of 0.2 cm. The MARC-145 and BHK-21 cells were electroporated with 10 or 5 pulses of current, respectively, by using an ECM 830 electroporator (BTX Inc., San Diego, CA) set at 900 V and a 99-μs pulse length. The cells were then transferred to 10 ml of fresh medium. Infectious-center assays were used to quantitate the specific infectivity of synthetic RNA transcripts. The electroporated cells were then serially diluted 10-fold and plated onto monolayers of nontransfected MARC-145 cells (3 x 105) in a six-well plate. After 6 h, they were overlaid with agarose-containing minimal essential medium, incubated for 4 or 5 days, and stained with crystal violet.
Real-time quantitative reverse transcription-PCR. Total RNA was extracted from duplicate wells with TRIzol reagent (Invitrogen Co.); 100 ng of total cellular RNA was used for the reverse transcription reaction with primers specific for the PRRSV ORF1a region as well as for the BHK-21 -actin RNA to normalize total RNA levels. PRRSV and BHK-21 -actin cDNAs were generated by reverse transcription at 45°C for 30 min, followed by inactivation of the reverse transcriptase at 95°C for 10 min. PRRSV-specific and BHK-21 -actin-specific cDNAs were amplified with an iQ Supermix quantitative PCR system (Bio-Rad Laboratories, Hercules, CA) and detected with an iCycler iQ multicolor real-time PCR detection system (Bio-Rad Laboratories). One-tenth of the reverse transcription reaction mixture was used for PCR amplification with 45 cycles of 95°C for 15 s and 60°C for 1 min. The PRRSV forward and reverse primers were 5'-CATGTGAGTGATAAACCTTTCCCG and 5'-TCATAGACAGTAGCCATAGCACAC, respectively. The probe sequence (nt 659 to 682) was 5'-6FAM-ACGTGTTGACCAACCTGCCGCTCC-BHQ1 (where 6FAM is 6-carboxyfluorescein and BHQ is black hole quencher; Integrated DNA Technologies Inc., Coralville, IA). The forward and reverse primers for BHK-21 -actin were 5'-ACTGGCATTGTGATGGACTC and 5'-CATGAGGTAGTCTGTCAGGTC, respectively. The probe sequence was 5'-HEX-CCAGCCAGGTCCAGACGCAGG-BHQ2 (where HEX is hexachlorofluorescein; Integrated DNA Technologies Inc.). The 2–CT method was used to analyze relative changes in PRRSV RNA levels from real-time, quantitative PCR experiments (41, 56).
Direct immunofluorescence and analysis of reporter gene expression. PRRSV ORF7 proteins were visualized on the surface of formaldehyde-fixed cells by incubation with mouse anti-ORF7 monoclonal antibody (MAb; 6D7/D2) followed by secondary labeling with either fluorescein isothiocyanate-conjugated or Cy3-conjugated goat anti-mouse immunoglobulin G (IgG; Jackson ImmunoResearch Labs Inc., West Grove, PA), using a procedure described elsewhere (61). LUC activity was estimated in cell lysates, as previously described (61). EGFP expression was visualized under a confocal microscope fitted with a fluorescein filter, as described previously (61).
RESULTS
Assembly of the PRRSV full-length cDNA as a BAC. The full-length PRRSV cDNA BAC was constructed by sequentially assembling six overlapping cDNA fragments of the PRRSV PL97-1/LP1 isolate (GenBank accession number AY612613), a clone of a genetically homogeneous population of large-plaque-forming PRRSV, plaque purified from the first PRRSV Korean isolate PL97-1 (Fig. 1A) (see the supplemental material for a detailed description). During the assembly, we took advantage of natural preexisting restriction sites in the viral genome rather than introducing intentional mutations to generate new restriction sites that would then be used to assemble the cDNAs, since such mutations, even silent mutations, may impair viral replication. To enable in vitro runoff transcription, the SP6 RNA polymerase promoter sequence was placed precisely at the beginning of the PL97-1/LP1 viral sequence in such a way that synthetic RNA transcripts with an authentic 5' end would be generated (Fig. 1A and B). To generate an artificial runoff site, three unique restriction endonuclease recognition sites (AclI, NotI, and SdaI) were placed in a row immediately downstream of the viral poly(A)54 stretch of PL97-1/LP1 (Fig. 1A and B). The genetic integrity of the cloned cDNAs in the BACs was verified throughout the subcloning and assembly process by extensive restriction analyses and sequencing. The cloned inserts did not exhibit genetic instability, as deletions, rearrangements, or nonsense mutations were never detected (data not shown). As a result, the full-length PRRSV cDNA clone encoding the entire PL97-1/LP1 viral genome downstream of the SP6 promoter was assembled. It was designated pBAC/PRRSV/FL, and its complete genome sequence was confirmed (Fig. 1A and B).
In vitro generation of infectious synthetic PRRSV RNAs transcribed from pBAC/PRRSV/FL. We estimated the specific infectivities of the synthetic RNAs that were transcribed in vitro from pBAC/PRRSV/FL. First, pBAC/PRRSV/FL was linearized by digestion with AclI, NotI, or SdaI to prepare three different cDNA templates. These were designated pBAC/PRRSV/FL/AclI, pBAC/PRRSV/FL/NotI, and pBAC/PRRSV/FL/SdaI, respectively (Fig. 1B), and served as templates for SP6 polymerase runoff transcription in the presence of the m7G(5')ppp(5')A cap structure analog. As summarized in Fig. 1B, all transcription reactions produced capped synthetic RNA transcripts with authentic 5' ends (11). However, the 3' ends varied depending on the template used in the transcription reaction. Transcription of the pBAC/PRRSV/FL/AclI template yielded synthetic RNAs containing two extra nucleotides (CG) of nonviral sequence at their 3' ends as a result of copying the 5' overhang left by the AclI digestion (Fig. 1B). Transcription of the pBAC/PRRSV/FL/NotI and pBAC/PRRSV/FL/SdaI templates also produced synthetic RNAs containing 10 (CGUUGCGGCC) and 14 (CGUUGCGGCCGCCC) extra nucleotides of nonviral sequence at their 3' ends, respectively (Fig. 1B). In some flaviviruses, unrelated sequences at the 3' ends of synthetic RNAs transcribed from an infectious cDNA have been reported to diminish their specific infectivities (60, 61). Thus, we sought to generate a synthetic RNA bearing the authentic 3' end of the PRRSV genome. This was achieved by linearizing pBAC/PRRSV/FL with AclI followed by MBN treatment to remove the 5' overhang left by the AclI digestion. The resulting construct was designated pBAC/PRRSV/FL/AclIMBN (Fig. 1B).
Transfection of the synthetic RNA transcripts into susceptible MARC-145 cells by using our optimized electroporation conditions (5% transfection efficiency) indicated that the specific infectivities of the transcripts produced from pBAC/PRRSV/FL/AclIMBN, pBAC/PRRSV/FL/AclI, and pBAC/PRRSV/FL/NotI were similar, at 3.3 x 104, 2.3 x 104, and 2.2 x 104 PFU/μg, respectively (Fig. 1C). On the other hand, the specific infectivities of the transcripts from pBAC/PRRSV/FL/SdaI were 20-fold lower, at 1.3 x 103 PFU/μg (Fig. 1C). Thus, reconstitution of the authentic 3' end of the PRRSV genome is important for the generation of highly infectious synthetic PRRSV RNA transcripts. Furthermore, transfection of the synthetic RNA transcripts into BHK-21 cells (>90% transfection efficiency) always produced >20-fold-higher infectivity than that produced by MARC-145 cells (Fig. 1C). This indicates that a system employing BHK-21 cells may be superior to MARC-145 cells for the purpose of characterizing viral mutants whose infectivities have been lost or reduced and which, therefore, can be studied only immediately following transfection.
The specific infectivities of all four RNA transcripts reflected the virus titers in the culture supernatants of the transfected MARC-145 cells (Fig. 1D). Cells transfected with synthetic RNA transcripts containing the authentic 3' end rapidly produced 105 PFU/ml of PRRSV virus, although 2 to 10 extra nucleotides (but not 14) were tolerated within the nonviral sequence at the 3' end (Fig. 1D). Additionally, we also confirmed that specific infectivity requires the transcription of RNA from the full-length PRRSV cDNA template (see the supplemental material).
Synthetic PRRSVs generated from the infectious cDNAs are identical to the parental virus. We compared the synthetic PRRSVs recovered from the four infectious cDNA templates with the parental virus PL97-1/LP1. Infection of MARC-145 cells with the synthetic viruses or with PL97-1/LP1 resulted in homogeneous large plaques (Fig. 2A). The growth properties on MARC-145 cells at an MOI of 1 showed that equivalent virus titers accumulated over time for all four synthetic viruses and the parental virus (Fig. 2B). Similar growth kinetics were also obtained with low (0.1) and high (10) MOIs (data not shown). Western blot analyses using a PRRSV-positive pig antiserum (Fig. 2C) and a rabbit antiserum raised against the glutathione S-transferase-fused PRRSV Nsp1a protein (Fig. 2D, top) showed that the levels of PRRSV-specific viral proteins and PRRSV Nsp1a produced in cells infected with any of the four synthetic viruses or the parental virus were similar. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was employed as a loading control (Fig. 2D, bottom). The immunostaining patterns of the cells infected with any of the four synthetic viruses or with PL97-1/LP1 were also identical (data not shown). Thus, the synthetic viruses recovered from the four infectious cDNA templates are phenotypically indistinguishable from the parental virus in terms of plaque morphology, cytopathogenicity, growth kinetics, and protein expression.
We sequenced the 5' and 3' ends of the four synthetic PRRSV viral genomes and found that, as expected, their 5' ends began with 1AUG ACG UAU-, which is identical to the 5' end of the parental virus (data not shown). With regard to the 3' end, we found that the genomic RNAs of all four synthetic viruses terminated with a poly(A) tail and did not retain the 2, 10, or 14 extra nucleotides of nonviral sequence at their 3' ends (data not shown; see Discussion). Thus, the genomes of the viruses recovered from the four infectious cDNA templates bear authentic 5' and 3' ends. Furthermore, a single point mutation acting as a genetic marker was introduced into the infectious cDNA and was found in the genome of the recovered synthetic virus, indicating that the recovered virus originates from the cDNA template (see the supplemental material). Therefore, these results validate the use of the infectious PRRSV cDNA for direct molecular genetic analyses.
Serial nucleotide deletions of the PRRSV 5' end in genomic RNAs decrease or completely abolish their infectivity, which appears to be restored by acquisition of novel 5' sequences. We noticed previously that significant nucleotide sequence differences in the PRRSV 5' NCR exist between the North American and European genotypes (20). However, a nucleotide sequence alignment using 10 fully sequenced PRRSV isolates showed that about 30 consecutive nucleotides proximal to the 5' end are highly conserved among nine isolates with the North American genotype (Fig. 3A). Moreover, 10 out of 12 nucleotides proximal to the PRRSV 5' end were absolutely conserved between the North American and European genotypes (Fig. 3A), despite their otherwise low nucleotide identity of 55 to 70% (2, 6, 10, 20, 25, 32, 53). To examine the role of the PRRSV-conserved 5'-terminal nucleotides in viral replication, a set of eight truncated mutants was constructed by serial deletion of an odd number of nucleotides from the 5' end (Fig. 3B). We first determined the specific infectivities of the synthetic RNA transcripts derived from each truncated mutant cDNA template. As shown in Fig. 3C, cDNAs of mutants with deletions of 1 (pBAC/PRRSV/FL/nt1) and 3 (pBAC/PRRSV/FL/nt3) nucleotides at the utmost 5' end were equally decreased in their infectivities by 15-fold, to 1.2 x 104 to 3.1 x 104 PFU/μg, compared to that of the wild-type infectious pBAC/PRRSV/FL cDNA (4.4 x 105 to 5.2 x 105 PFU/μg). Moreover, deletions of 5 (pBAC/PRRSV/FL/nt5) and 7 (pBAC/PRRSV/FL/nt7) nucleotides drastically decreased the infectivity to 1.0 x 102 to 1.8 x 102 PFU/μg and 8.6 x 102 to 9.3 x 102 PFU/μg, 3,500-fold and 550-fold lower than that of the wild type, respectively (Fig. 3C). Interestingly, the infectivities of the synthetic RNAs containing the 7-nucleotide deletion was generally approximately sixfold higher than that containing the 5-nucleotide deletion. Furthermore, in the cells transfected with the synthetic RNAs derived from pBAC/PRRSV/FL/nt1 and pBAC/PRRSV/FL/nt3, we observed a relatively homogeneous population of large plaques and foci visualized by staining with a mouse anti-ORF7 MAb, as seen in the cells transfected with the wild-type infectious RNAs (Fig. 3C). In the cells transfected with the synthetic RNAs derived from pBAC/PRRSV/FL/nt5 and pBAC/PRRSV/FL/nt7, on the other hand, a relatively heterogeneous population of smaller plaques and foci was observed (Fig. 3C). Neither infectivity nor foci or plaques were detectable with synthetic RNAs containing more than 9-nucleotide deletions at the utmost 5' end (Fig. 3C).
It should be noted that two mutants harboring deletions of 5 or 7 nucleotides produced plaques of heterogeneous sizes, indicating some instability. This was more evident when supernatants harvested from the transfected cells were passaged once on nave MARC-145 cells. These passaged pseudorevertants derived from the mutant cDNAs containing 1-, 3-, 5-, and 7-nucleotide deletions produced similar amounts of the PRRSV Nsp1a protein upon infection with the same amounts of the viruses as did the wild-type virus (Fig. 4A). Furthermore, their growth kinetics (Fig. 4B) and plaque morphologies (Fig. 4C) were very similar, with a relatively homogeneous population of large plaques observed in the cells infected with each of these pseudorevertants, including the 5- and 7-nucleotide deletion mutants.
The nucleotide sequences at the utmost 5'-end regions of all these pseudorevertants were determined by 5' rapid amplification of cDNA end analysis, cloning of reverse transcription-PCR amplicons, and sequencing of a number of the independently picked clones containing the insert. As summarized in Fig. 5A, 33 of 42 independent clones obtained from the pBAC/PRRSV/FL/nt1-derived pseudorevertants appeared to be converted to the wild-type virus by the acquisition of one A nucleotide at the site of the deletion. The remainder of the nine clones had the same sequence as pBAC/PRRSV/FL/nt1 (Fig. 5A). In the case of the pBAC/PRRSV/FL/nt3-derived pseudorevertants, 32 out of 57 independent clones had acquired 3 nucleotides (AUG) at the utmost 5' end, which rendered the sequence identical to that of the wild-type virus (Fig. 5A). Twenty-one clones contained an insertion of 4 nucleotides (UAUG), while four clones appeared to contain an insertion of 3 nucleotides (AAG) at the deletion site (Fig. 5A). Interestingly, the pBAC/PRRSV/FL/nt5-derived pseudorevertants were found to have a deletion of the 5'-end single G nucleotide, the first nucleotide in this mutant construct. Moreover in 39 of 49 independent clones, there was an insertion of 6 novel (AUUAUA) nucleotides at the deletion site, while 10 clones contained a 7-nucleotide insertion (UAUUAUA) (Fig. 5A). For the pBAC/PRRSV/FL/nt7-derived pseudorevertants, the novel 5'-end sequences acquired were found to be more heterogeneous than those of the mutants described above. Specifically, a majority of the sequenced clones (28/48 and 9/48 clones) had insertions of 7 (AUUAUAU) and 8 (UAUUAUAU) nucleotides at the site of the deletion, respectively, and appeared to be identical to two of the pBAC/PRRSV/FL/nt5-derived pseudorevertants (Fig. 5A). In addition, four independent clones had 8 novel nucleotides (UAUCAUAU) inserted at the deletion site, while two further clones had the 8-nucleotide sequence AUUUAUAU inserted at this site (Fig. 5A). In addition, three and two independent clones appeared to have five and six AU repeats at the site of the deletion, respectively (Fig. 5A). Overall, these pseudorevertants appeared to have acquired novel 5' sequences of various sizes, composed mainly of A and U bases.
To address the importance of these novel 5' sequences, we reconstructed eight derivatives of the PRRSV 5'-end truncated mutants with all of these novel sequences and determined the specific infectivities of their RNA transcripts (Fig. 5B). In all reconstructed cases, the specific infectivities were increased to a level similar to that of the wild type (Fig. 5B). Among all of the pseudorevertants, we found three cases that were not reconstructed, since the resulting mutants were identical to either the wild type (PRRSV/FL/nt1/Rev1 and PRRSV/FL/nt3/Rev1) or the original truncated mutant (PRRSV/FL/nt1/Rev2) (Fig. 5A). According to plaque/focus morphology, the cells transfected with four synthetic RNAs (derived from pBAC/PRRSV/FL/nt3/Rev2, pBAC/PRRSV/FL/nt7/Rev1, pBAC/PRRSV/FL/nt7/Rev2, and pBAC/PRRSV/FL/nt7/Rev6) formed a homogeneous population of large plaques/foci, as seen with the wild-type infectious cDNA (Fig. 5B). In addition, we also observed a homogeneous population of medium-sized (pBAC/PRRSV/FL/nt3/Rev3 and pBAC/PRRSV/FL/nt7/Rev3) and small (pBAC/PRRSV/FL/nt7/Rev4 and pBAC/PRRSV/FL/nt7/Rev5) plaques/foci (Fig. 5B). These results demonstrated that the addition of novel AU-rich sequences to the utmost 5' end of the PRRSV 5' deletion mutants, but not changes elsewhere in their genomes, allowed efficient PRRSV replication.
Generation of a panel of self-replicating, self-limiting PRRSV viral replicons expressing the LUC gene as a reporter. We sought to construct a panel of self-replicating, self-limiting PRRSV viral replicons by using pBAC/PRRSV/FL. We initially constructed a set of three viral replicons (pBAC/PRRSV/RepLuc MB, pBAC/PRRSV/RepLuc ME, and pBAC/PRRSV/RepLuc DI) which lacked various portions of the structural protein-coding region of the PRRSV genome (Fig. 6A). At the same time, we also inserted at the site of each deletion the expression cassette containing the EMCV IRES-driven LUC gene to facilitate the monitoring of viral replication (Fig. 6A). LUC was chosen as the reporter since its expression is easy to monitor in a highly quantitative and sensitive manner. pBAC/PRRSV/RepLuc MB had the smallest deletion, of nt 12714 to 14194, and lacked the 3'-terminal 129 nucleotides of ORF2a, the majority of ORF3 except for its 5'-terminal 18 nucleotides, the complete sequence of ORF4, and the 5'-terminal 406 nucleotides of ORF5 (Fig. 6A). pBAC/PRRSV/RepLuc ME was the same as pBAC/PRRSV/RepLuc MB except for an additional 5' deletion (nt 12163 to 14194). It lacked the majority of ORF2a and ORF2b except for their 5'-terminal 90 (ORF2a) and 85 (ORF2b) nucleotides, the complete sequence of ORF3 and ORF4, and the 5'-terminal 406 nucleotides of ORF5 (Fig. 6A). pBAC/PRRSV/RepLuc DI had the largest deletion, of nt 12163 to 15252, which extended towards the 3' end of pBAC/PRRSV/RepLuc ME, and lacked the majority of ORF2a and ORF2b, except for their 5'-terminal 90 (ORF2a) and 85 (ORF2b) nucleotides, and the complete sequence of ORF3 through ORF7, except for the 3'-terminal 8 nucleotides of ORF7 (Fig. 6A). Since none of these viral replicons contained all of the structural genes, they were expected not to produce viral particles, as first established for EAV (31, 55, 62).
Next, we examined whether the viral replicon RNAs derived from the three cDNA templates were replication competent by monitoring the expression of the LUC gene after their transfection in BHK-21 cells. The LUC activities 6 h after transfection with pBAC/PRRSV/RepLuc MB or pBAC/PRRSV/RepLuc ME RNAs were 9.95 x 102 and 9.72 x 102 relative light units (RLU), respectively (Fig. 6B), and were dramatically increased at 18 and 24 h posttransfection to 1.12 x 104 to 1.49 x 104 and 2.12 x 104 to 2.45 x 104 RLU, respectively. These activities were maintained until 48 h posttransfection (Fig. 6B), after which they gradually decreased due to the cell death induced by the replication of these viral replicons and the lack of cell-to-cell spread. In BHK-21 cells transfected with pBAC/PRRSV/RepLuc DI RNA, the initial LUC activity at 6 h posttransfection was similar to that of the other two (9.81 x 102 RLU) but then gradually decreased over time to 3.60 RLU at 72 h posttransfection, which was at the level of the background luminescence from the mock-transfected cells (Fig. 6B). Thus, pBAC/PRRSV/RepLuc MB and pBAC/PRRSV/RepLuc ME viral replicon RNAs were competent in replication, but pBAC/PRRSV/RepLuc DI RNA was not. In addition, no virus particle formation was observed in the two replication-competent viral replicon RNA-transfected cells (data not shown). To determine the location of the minimal cis-acting element required for viral replication at the 3' end of the viral genome, we constructed the eight viral replicons, pBAC/PRRSV/RepLuc S1 to S8, by systematically deleting additional sequences towards the 3' end of pBAC/PRRSV/RepLuc ME (Fig. 6A). Of the eight viral replicons, only pBAC/PRRSV/RepLuc S8 RNAs were replication competent (Fig. 6B).
The production levels of viral replicon RNA itself in BHK-21 cells transfected with each of the 11 viral replicon RNAs (pBAC/PRRSV/RepLuc MB, pBAC/PRRSV/RepLuc ME, pBAC/PRRSV/RepLuc DI, and pBAC/PRRSV/RepLuc S1 to S8) were directly quantitated by real-time quantitative reverse transcription-PCR using a PRRSV-specific probe complementary to a sequence of nt 659 to 682 in ORF1a (Fig. 6C). In agreement with the LUC activity (Fig. 6B), the levels of the replication-competent viral replicon RNAs, i.e., pBAC/PRRSV/RepLuc MB, pBAC/PRRSV/RepLuc ME, and pBAC/PRRSV/RepLuc S8 RNA, were increased approximately 1.1-fold, 6.2-fold, and 16.0-fold at 24 h posttransfection relative to the levels at 6 h posttransfection, and these increases were subsequently reduced over time due to the lack of viral spread to the adjacent cells (Fig. 6C). On the other hand, the levels of all replication-incompetent viral replicon RNAs at 6 h posttransfection were drastically decreased and were below the level of detection at all time points, confirming that these replicon RNAs were indeed incapable of RNA replication (Fig. 6C). Thus, these findings showed that the 911 nucleotides from the 3' end of the viral genome contained the minimal cis-acting element required for viral replication and that the internal deletion in the coding region of the structural genes (including ORF2a to ORF6) altered the level of the ORF1a and ORF1b-containing replicon RNA replication.
Immunostaining of the ORF7 protein showed that it was expressed in BHK-21 cells transfected with the replication-competent pBAC/PRRSV/RepLuc S8, pBAC/PRRSV/RepLuc MB, and pBAC/PRRSV/RepLuc ME RNAs but not in cells transfected with the replication-incompetent viral replicons (data not shown). Mutagenetic analyses with another EAV system have indicated that all of the structural proteins, including ORF7, are dispensable for genomic RNA replication and subgenomic mRNA transcription (31, 55, 62). On the other hand, previous studies with the distantly related coronavirus systems have indicated that expression of the N protein, which is equivalent to the PRRSV ORF7 protein, is required for efficient replication of the genomic RNA (3, 39, 46). To examine the possibility that the replication-incompetent viral replicons that we constructed would be able to replicate when the ORF7 protein was provided in trans, all PRRSV viral replicon RNAs were transiently cotransfected with the infectious PRRSV viral RNAs transcribed from pBAC/PRRSV/FL, and LUC activity was monitored over time. This revealed that the replication-incompetent viral replicon RNAs were still replication incompetent when the ORF7 protein was provided in trans (data not shown).
Expression of the EGFP reporter gene by infectious PRRSV cDNA/recombinant viruses. We sought to produce recombinant infectious PRRSV viruses that express the EGFP reporter gene upon infection. To do this, pBAC/PRRSV/FL was engineered in two ways. First, we inserted the EMCV IRES-driven EGFP expression cassette immediately downstream of the first 33 nucleotides of the ORF7 coding region that include a short PRRSV transcription-regulating sequence and followed it with the cis-acting element in the 3' 911 nucleotides of the viral genome (Fig. 7A). The EGFP gene in the resulting pBAC/PRRSV/FL/IRES-EGFP construct was driven by the EMCV IRES, and its expression was dependent on viral replication. Second, the autoprotease Npro gene of bovine viral diarrhea virus was fused adjacent to the N terminus of the EGFP gene so that the correct N terminus of the EGFP protein was created by Npro cleavage (Fig. 7A). The fused Npro-EGFP gene was then inserted at the same site where the EMCV IRES-driven EGFP expression cassette was inserted. This resulted in the pBAC/PRRSV/FL/Npro-EGFP construct. In both constructs, the EGFP expression cassette was inserted downstream of the first 33 nucleotides of the ORF7 coding region because this region might be involved in the gene expression and regulation of its own subgenomic mRNA. In both cases, the insertions did not alter the infectivities of the synthetic RNA transcripts (data not shown).
EGFP expression was then assessed by transfecting nave BHK-21 cells with the pBAC/PRRSV/FL, pBAC/PRRSV/FL/IRES-EGFP, or pBAC/PRRSV/FL/Npro-EGFP RNAs and examining them by confocal microscopy. No fluorescence was observed in the PRRSV/FL RNA-transfected (Fig. 7B) or mock-transfected (data not shown) cells, but the other two cell types showed green fluorescence (Fig. 7B). The fluorescence was in the nucleus and the cytoplasm because EGFP (30 kDa) is small enough to permit diffusion between the nucleus and the cytoplasm. All three RNA-transfected cell types also expressed the ORF7 protein (Fig. 7B). Similar observations were made when MARC-145 cells were transfected (data not shown).
The production of recombinant EGFP-expressing PRRSV viruses was estimated by measuring (at the indicated time points) the viral titers in the culture supernatants of BHK-21 or MARC-145 cells transfected with the three synthetic RNA transcripts. Production of the EGFP-expressing PRRSV viruses was monitored by infecting nave MARC-145 cells and subsequently counting the green focus-forming units (GFU) per ml. Several experiments showed that transfection of MARC-145 cells with pBAC/PRRSV/FL/IRES-EGFP RNAs generated on average 1.2 x 103, 1.0 x 104, and 9.6 x 104 GFU/ml at 24, 48, and 72 h posttransfection, respectively (Fig. 7C). Similar virus titers were produced by pBAC/PRRSV/FL/Npro-EGFP RNA-transfected MARC-145 cells (Fig. 7C). In addition, at 24 h posttransfection, BHK-21 cells transfected with the EGFP-expressing PRRSV RNAs produced slightly higher virus titers (4.1 x 104 to 5.5 x 104 GFU/ml) than the MARC-145 cells did, due to their higher transfection efficiency (data not shown). Moreover, unlike MARC-145 cells, the BHK-21-generated virus titers gradually decreased to 6.4 x 102 to 7.8 x 102 GFU/ml at 96 h posttransfection, due to a lack of viral spread and cell death of the synthetic RNA-containing cells (data not shown). Thus, we produced recombinant PRRSVs carrying the EGFP gene as a reporter and thereby showed the possibility of utilizing infectious PRRSV cDNA/recombinant viruses as heterologous gene expression vectors.
DISCUSSION
In this report, we have described the construction and characterization of a reverse genetics system for PRRSV that involves generating a functional PRRSV cDNA as a BAC which is based on E. coli and its single-copy plasmid F factor (42, 52). BACs have been successfully used to clone and maintain large fragments of DNA from a variety of complex genomic sources (including humans and herpesvirus) in bacteria (26, 42). Using this functional cDNA, we not only demonstrated the importance of the PRRSV 5'-end nucleotide sequences for replication but also discovered novel AU-rich sequences of various sizes that can functionally replace the authentic 5'-proximal 7 nucleotides of the viral genome for RNA replication. We also showed the utility of this system by generating a panel of self-replicating, self-limiting PRRSV viral replicons that aided the identification of a 3' cis-acting element required for PRRSV replication. This reverse genetics system will thus permit direct molecular genetic studies of the PRRSV pathogen that will greatly enhance our understanding of the molecular mechanisms behind its replication, transcription, translation, virulence, and pathogenesis. In addition, the results we obtained present the possibility that this system can be used to produce novel heterologous gene expression vectors that can express a foreign gene of interest. Thus, this system will also be useful in developing new, safe, and genetically defined antiviral vaccines.
With the aim of generating an infectious cDNA that has an unaltered native sequence, we also sought to ensure that the in vitro transcription of the infectious PRRSV cDNA would generate RNA transcripts possessing the authentic 5'- and 3'-terminal nucleotide sequences of the viral genome. These sequences are usually essential for the replication of viral RNA. For example, it has been shown that the 5'- and 3'-terminal regions of flavivirus RNA are required for the initiation of replication in vitro and in vivo (24). To ensure the generation of authentic 5'- and 3'-terminal sequences, we adapted the approaches that were used previously to design flavivirus infectious cDNAs (37, 61). The cap structure at the 5' end of the PRRSV viral genome was followed by the nucleotide sequence 1AUG ACG6, which is highly conserved in most PRRSV strains, including PL97-1/LP1 (20). We achieved the authentic 5' end of the viral genome by engineering the SP6 promoter transcription start immediately upstream of the viral genome. The in vitro runoff transcription reaction in the presence of the m7G(5')ppp(5')A cap structure analog then allowed us to synthesize capped RNA transcripts with the authentic 5' ends (11). These transcripts were highly infectious upon transfection into susceptible MARC-145 cells and nonpermissive BHK-21 cells. Previous studies with PRRSV cDNAs used the m7G(5')ppp(5')G cap structure analog instead in the in vitro T7 polymerase-driven transcription reaction (23, 28, 34, 47). This would result in capped RNA transcripts containing one (23, 28, 34) or two (47) extra nonviral G nucleotides upstream of the 1AUG ACG6 sequence. When we used the m7G(5')ppp(5')G cap structure analog in our own transcription reactions, we found that the extra G did not affect the infectivities of the RNA transcripts (data not shown). We also found that the uncapped RNA transcripts were not infectious, which indicates that the cap structure is essential for viral replication (data not shown), in agreement with a previous report (28).
The 3' end of the PRRSV viral genome terminates with the poly(A) tail (8, 29, 45). The poly(A) tail in PL97-1/LP1 is 54 nucleotides long. To ensure that the infectious PPRSV cDNA that we constructed would produce RNAs bearing the 54 nucleotides of the poly(A) tail, we followed the tail sequence with a unique restriction recognition site that would linearize the cDNA for runoff transcription. The resulting RNA transcripts contained the 54-nucleotide poly(A) tail and gave a specific infectivity of >105 PFU/μg of RNA that produces synthetic viruses of >5 x 104 PFU/ml upon transfection. That the 54-nucleotide poly(A) tail is important was then clearly demonstrated by the fact that unpolyadenylated RNA transcripts were not infectious (data not shown). With regard to the other PRRSV cDNAs published previously, poly(A) tails of various lengths, such as 109 (28), 43 (47), 40 (34), or 21 (23) A's, have been included during construction of each cDNA. It is not clear whether poly(A) tails of such variable lengths may affect viral replication.
Several studies have shown that it is important that the synthetic RNA transcripts that are generated from functional cDNAs for RNA viruses carry the authentic 3' end, as this promotes their infectivity and the establishment of a productive infection (60, 61). To ensure this for the PRRSV RNAs, we designed the infectious PRRSV cDNA template so that AclI digestion followed by MBN treatment would generate a linearized template lacking the 5' CG overhang left by the AclI digestion. This then served as the template in SP6 polymerase runoff transcription and was found to produce highly infectious RNA transcripts bearing the authentic 5' and 3' termini of the viral genome. We also observed similar levels of infectivity with RNA transcripts that terminated with 2 and 10 extra nonviral nucleotides at their 3' ends. However, 14 extra nonviral 3' nucleotides slightly decreased the specific infectivity and delayed virus production. Interestingly, the 2 to 14 extra nonviral nucleotides were not found in the genomic RNAs of the synthetic viruses produced from the cDNA in cells. This could be because these extra nonviral nucleotides are lost during propagation in the transfected cells. Alternatively, it is also possible that the SP6 RNA polymerase in the in vitro transcription reaction may not efficiently synthesize the entire poly(A) tail each time, which would sometimes result in synthetic RNA transcripts with poly(A) tails of different sizes that lack these extra 3' nonviral nucleotides. Thus, replication-competent RNA transcripts could be selected over replication-incompetent RNAs containing these extra nonviral nucleotides. Further investigation will be needed to distinguish between these two possibilities. With regard to the other PRRSV cDNAs published previously, linearization was induced by a unique restriction site, such as PvuI (28), AclI (34, 47), or SwaI (23), which was introduced downstream of the poly(A) tail. The digestion of either PvuI or AclI would produce synthetic RNA transcripts bearing two extra nonviral nucleotides (CG) downstream of the poly(A) tail, whereas SwaI digestion would make synthetic RNAs with nine nonviral nucleotides. Our own observations suggest that these extra 2 or 9 nucleotides should not affect the infectivities of their resulting RNA transcripts.
Our work presented here elucidates the importance of the PRRSV-conserved 5'-end nucleotide sequence 1AUGACGU7 in RNA replication. Several novel AU-rich PRRSV 5' sequences detected in pseudorevertants were able to functionally replace the deleted 1AUGACGU7. According to the predicted RNA secondary structure of PRRSV 5' NCRs (48), the presence of these novel AU-rich PRRSV 5' sequences did not seem to significantly alter their stem-loop structures, which might be critical for viral replication. Although the functional role of these novel sequences is not understood at all, the complementary sequence of each of these novel 5' sequences at the utmost 3' end of negative-sense RNA is predicted to be involved in the initiation of positive-sense RNA synthesis. For some positive-sense RNA viruses, only minimal cis-acting sequences at the 3' ends of negative-sense RNAs are essential for positive-sense RNA synthesis (4, 18, 36). It is interesting to speculate on the origin of these novel 5' AU-rich sequences and the molecular mechanism of their insertion at the very beginning of the viral genome. Based on the heterogeneity and size of these novel 5' sequences, it is likely that they are acquired from cellular RNAs in the process of recombination involving template switching, during either negative- or positive-sense RNA synthesis, as has been described for pestivirus (30) and poliovirus (22). It is less likely, although not impossible, that these sequences are derived from the viral genome. Understanding this issue may provide new information on RNA replication of arteriviruses.
Several studies have shown that cis-acting elements within the coding region of the viral genome are essential for RNA virus replication (24). By analyzing a panel of self-replicating, self-limiting, LUC-expressing PRRSV viral replicons with internal deletions starting from the beginning of ORF2a and extending towards the 3' NCR, we found that there was a cis-acting element within the coding region of PRRSV as well. These analyses revealed that the three viral replicons that contained at least 911 nucleotides from the 3' end of the genome were replication competent. In contrast, eight viral replicons that did not contain 911 nucleotides at the 3' end of the genome were all replication incompetent. The LUC-expressing PRRSV viral replicon system is advantageous for monitoring viral replication in a highly quantitative and sensitive manner. On the other hand, the sequence requirements for RNA replication of the LUC-containing PRRSV viral replicon might not necessarily be the same as those for full-length genome replication. Especially, the insertion of a highly structured RNA sequence of EMCV IRES and the 1,653-nucleotide LUC gene could have a deleterious effect on the overall level of RNA structure when this insertion is combined with a specific deletion within the viral genome. Investigations on how the cis-acting element mapped in this study works in RNA replication in the context of the full-length viral genome might be an important step towards a better understanding of PRRSV RNA synthesis. A study using the infectious Lelystad strain cDNA has also shown that a 34-nucleotide stretch within the PRRSV ORF7 is essential for RNA replication (51). In addition to this stretch of 34 nucleotides, the presence of an additional 3' cis-acting element which is essential for viral replication was revealed in our own data. Investigations using our replicon system that aim to finely map this minimal 3' cis-acting element and elucidate the molecular mechanism by which it controls viral replication are currently under way.
Several RNA viruses have been successfully exploited as eukaryotic expression vectors (12, 40). We showed here that the infectious PRRSV cDNA BAC that we constructed could also serve as a novel PRRSV-based virus/vector system in which foreign genes of interest could be expressed. We showed the possibility that this system can be used to express foreign genes in two ways: first, by generating infectious recombinant vector RNAs/viruses that carry a foreign gene, and second, by producing viral replication-competent but propagation-deficient PRRSV viral replicon vector RNA that carries a foreign gene. As a transient-expression system, PRRSV offers several advantages: (i) the recombinant virus is rapidly produced, (ii) PRRSV can replicate in a variety of eukaryotic cells upon transfection of synthetic RNAs, (iii) PRRSV is unable to infect humans, (iv) the genetically stable infectious cDNA is available and readily manipulated, and (v) the cytoplasmic replication of the RNA genome minimizes the possibility of integration and unwanted mutagenic consequences. Recently, a human coronavirus with a positive-strand RNA genome has been developed as a multigene expression vector; the transcription strategy of this vector involves producing a nested set of multiple subgenomic mRNAs (46). PRRSV employs a similar strategy in its gene expression. That the genome of PRRSV is smaller (15 kb) than that of coronaviruses (30 kb) suggests that our PRRSV-based expression system can be even more useful as a multigene expression vector.
In conclusion, our reverse genetics system for PRRSV, which involves an infectious cDNA BAC and viral replicons, may be useful in a variety of research fields. First, this system enables us not only to investigate the molecular mechanisms of viral replication, transcription, and translation but also to identify the viral genetic elements involved in virulence and pathogenesis. Second, it can serve as an invaluable genetic tool for heterologous gene expression in a wide variety of eukaryotic cells; this has many applications in biological research. Finally, targeted manipulation of the infectious PRRSV cDNA may permit the production of effective genetically modified antiviral vaccines against this pathogen that may abrogate the ongoing worldwide spread of PRRSV.
ACKNOWLEDGMENTS
We are grateful to Jae-Young Song, Animal Disease Research Department, National Veterinary Research & Quarantine Service, for sharing a PRRSV-positive pig antiserum.
This work was supported by grant R01-2001-000-00249-0 from the Korea Science & Engineering Foundation.
Supplemental material for this article may be found at http://jvi.asm.org/.
These authors contributed equally to this work.
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