Identification of nonessential regions of the nsp2 protein of an attenuated vaccine strain (HuN4-F112) of highly pathogenic porcine reproductive and respiratory syndrome virus for replication in marc-145 cell
© Xu et al.; licensee BioMed Central Ltd. 2012
Received: 7 November 2011
Accepted: 25 July 2012
Published: 2 August 2012
The regions in the middle of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) have been shown to be nonessential for PRRSV replication, and these nonessential regions are different in various viral strains.
In this study, the nonessential regions of the nsp2 of an attenuated vaccine strain (HuN4-F112) of highly pathogenic porcine reproductive and respiratory syndrome virus were identified based on an infectious cDNA clone of HuN4-F112. The results demonstrated that the segments of nsp2 [amino acids (aa) 480 to 667] tolerated deletions. Characterization of the mutants demonstrated that those with small deletions did not affect the viral growth on Marc-145 cells, but deletion of these regions led to earlier PRRSV replication increased (before 36 h after infectious in vitro).
The functional roles of nsp2 variable middle region for PRRSV HuN4-F112 replication have been identified. Our results also suggested that none-essential region might be an ideal insertion region to express foreign gene in PRRSV genome.
Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Based on a difference in nucleotide sequence, two PRRSV genotypes have been identified including European genotype (type 1) and North American genotype (type 2) represented by prototype viruses Lelystad and VR-2332, respectively [2–5]. The genome of PRRSV is approximately 15 kb in size, and it contains ten open reading frames (ORF), designated as ORF1a, ORF1b, ORF2a, ORF2b, and ORF 3 to ORF5, ORF5a, ORF6 and ORF7 [3, 6–10]. ORF1a and ORF1b encode viral nonstructural proteins which are directly translated upon viral entry. Then the polyproteins are cleaved into 14 nonstructural protein (nsp) (nsp1α, nsp1β, nsp2 to nsp6, nsp7α, nsp7β, and nsp8 to nsp12) that participate viral replication and transcription. Among the 14 nonstructural proteins, the nsp2 protein is the largest PRRSV replicative protein [2, 11, 12]. Through the alignment of arterivirus nsp2 proteins, nsp2 could be recognized containing three major domains: a N-terminal cysteine proteinase domain (PL2), a middle region, and a hydrophobic transmembrane (TM) region close to the C terminus [11, 13–15]. Nsp2 has been shown to be highly heterogeneous and variable, and become a key factor in distinguishing between PRRSV type 1 and type 2 strains due to their length difference and less than 40% similarity of their amino acid sequences [2, 4, 16].
It was reported that natural mutations, insertions, or deletions always occurred in the middle region or near to the N-terminal region of the nsp2, while the putative PL2 domain and TM domain remains well conserved [2, 4, 13, 17–22]. In 2006, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) (a virulent form of PRRSV, i.e., HP-PRRSV HuN4) was identified in China [21, 23, 24]. HuN4-F112 was an attenuated strain by passaging the HP-PRRSV HuN4 on Marc-145 cell in our laboratory . One character of the virulent PRRSV strain was two discontinuous deletions of 1 aa and 29 aa at positions 482 and 534–562, respectively, relative VR-2332 [21, 26]. So in our study, we wanted to explore the genetic flexibility of the virulent form of PRRSV nsp2 protein and identified the nonessential regions in the middle of nsp2 of HP-PRRSV Strain HuN4-F112.
Oligonucleotide primers used in this study
5′- TCCCTAACGGTTCGGAAGAAACAACGCTGACGCACCAGGA -3′
5′- TCCTGGTGCGTCAGCGTTGTTTCTTCCGAACCGTTAGGGA -3′
5′- AGCCCGTACTTATGCCCGCGACAACGCTGACGCACCAGGA -3′
5′- TCCTGGTGCGTCAGCGTTGTCGCGGGCATAAGTACGGGCT -3′
To rescue these infectious cDNA clones in vitro, transcription and transfection of the full length viral cDNA clones were performed according to the manufacturer’s instructions. Briefly, the full-length cDNA clone was linearized by cleavage with restriction enzyme Swa I, which cuts downstream of the poly (A) tail. Then, the capped RNA transcripts from cDNA were performed using the mMESSAGE MACHINE SP6 kit (Ambion, USA). Subsequently, the in vitro transcribed RNA was transfected into BHK-21 cells using DMRIE-C reagent (Invitrogen, USA). To rescue the virus, cell culture supernatant obtained 24 h post-transfection was serially passaged on Marc-145 cells. The infected Marc-145 cells were monitored daily for the formation of CPE (cytopathic effect).
In this study, eight deletion constructs were produced as shown in Figure 2: rHuN4-F112-Δ480-532, rHuN4-F112-Δ508-532, rHuN4-F112-Δ480-620, rHuN4- F112-Δ480-667, rHuN4-F112-Δ420-532, rHuN4-F112-Δ400-532, rHuN4-F112- Δ444-667, and rHuN4-F112-Δ400-700. The transfection results suggested that four mutants (rHuN4-F112-Δ480-532, rHuN4-F112-Δ508-532, rHuN4-F112-Δ480-620, and rHuN4-F112-Δ480-667) resulted in viable viruses. All others produced nonreplicating PRRSV genomes, and the deletion cDNA clones appeared to be lethal to the virus, which have been rescued at least five clones. Recently, Han J et al. reported that deletion aa 324 to 746 of nsp2 of PRRSV VR-2332 strain did not affect the viral replication, and they also found that the mutant virus displayed decreased cytolytic activity on Marc-145 cell and did not develop visible plaques . The combined data showed that HuN4-F112 nsp2 harbors smaller nonessential region for viral replication than VR-2332 strain in cell culture. Nsp2 between the HuN4-F112 and VR-2332 have different length and share less than 70% similarity of their amino acid, and this may be the reason why the nonessential regions is different among the different PRRSV strains.
Although deletions of the segments of nsp2 [amino acids (aa) 480 to 667] did not affect the viral growth on Marc-145 cells, deletion of these regions led to earlier PRRSV replication increased (before 36 h after infectious in vitro), which suggested that the sequences of the nonessential regions of nsp2 might play an important role in regulating the viral replication through an unknown mechanism. Recently, nsp1, nsp2, nsp4 and nsp11 were found to have strong ability to moderate inhibitory effects on beta interferon (IFN-β) promoter activation to regulate host innate immune response, but the roles of the nsp4 were not clear . Therefore, the function of these nonessential sequences of nsp2 should be under investigated.
PRRSV as a potential viral vector has been investigated previously. Most of the concern was focused on the region between the ORFs and the 3′ end of the genome [29–31], while recently some researchers were interested in using nsp2 to express foreign gene since the nonessential regions of nsp2 for replication had been identified [15, 32–34]. When viral structural proteins were used to express the foreign genes, the size of the insertion was limited to less than 10 amino acids . Inserting the fragment into the internal ORFs would affect the PRRSV replication. Therefore, the nonessential region of PRRSV nsp2 suggested the likelihood of expressing a large foreign gene as a fusion protein in the viral replicase region. Therefore, these identified nonessential regions suggested they might be the potential inserted sites to express the foreign gene in PRRSV genome.
The study was supported by grants from National High Tech Plan (863 Plan) (2011AA10A208), the Ministry of Agriculture of China (No. 2009ZX08010-022B), NSFC-Guangdong Joint Foundation (U0931003), International Sci & Tech Cooperation Program (2010DFB33920), National Nonprofit Institute Research Grant of CATAS-ITBB (2011JB03) and the Excellent Scientist Program of Shanghai (09XD1405400).
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