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

Background 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. Finding 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). Conclusion 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.

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][18][19][20][21][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 [25]. 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.
Full-length cDNA clone of HuN4-F112 was constructed as shown in Figure 1 as according to Zhang et al. [27]. The cDNA clone (pSK-F112) contains the SP6 RNA polymerase promoter at the 5′-end of PRRSV genome, the 3′-end sequence flanked the 32 poly (A), and a MluI restriction enzyme site at nt 14667 which was a genetic marker to differentiate the rescued viruses and parental virus (HuN4-F112).
The nsp2 deletion strains were generated by the Quick Change Multi site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations. The deletion regions of nsp2 were showed in Figure 2. Sequences of deletion primers were listed in Table 1. The construction of the deletion full-length cDNA clone was performed in two steps. First, the deletion fragments ABC (named pSK-ABC-Δ) were created by mutagenesis kit. Then, the deletion full-length infectious clones were assembled by ligating fragments DEF with pSK-ABC-Δ as shown on Figure 1.
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 SwaI, 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).
The successfully recovered recombinant viruses were tested as below. The nsp2 mutant regions of the recombinant viruses were examined by RT-PCR (Reverse Figure 1 Assembly of the full-length cDNA clone of PRRSV strain HuN4-F112. The total genome was amplified in six fragments (A to E). Each of these fragments was cloned to pSK vector with unique restriction enzyme cleavage site, respectively. Then, A-B-C and D-E-F were linked into pSK, respectively. Finally, the full-length clone was assembled from pSK-ABC and pSK-DEF with PacI and ClaI . A SP6 polymerase promoter and genetic marker (MluI enzyme cleavage sites at 14667nt) were added by PCR. Arrowheads and numbers indicate enzyme cleavage sites in PRRSV strain HuN4-F112.
Transcriptase Polymerase Chain Reaction) after 3 passages in Marc-145 cells. Two pairs of primers were used to identify each mutant virus: one (F2436/R3442) targeting the deletion region, and the other (F14320/R14752) targeting the mutant MluI restriction enzyme site. As shown in Figure 3A, all the successfully recovered mutant viruses maintained the respective engineered deletion with the expected size. The mutant MluI restriction   enzyme site of the successfully recovered viruses were tested ( Figure 3B), and these regions were also sequenced (at least 10 clones) (data not shown). Subsequently, the successfully recovered viruses were test by immunofluorescence assay (IFA). Marc-145 cells grown on 6-well plates and infected with virus were fixed in 80% cold ethanol at 4°C for 30 min. The cells were incubated for 60 min with PRRSV nucleocapsid protein monoclone antibody and nsp4 protein monoclone antibody, respectively, and then stained with lexa Fluor W 568 goat anti-mouse lg G (H + L) (Invitrogen, USA) and examined by fluorescence microscopy. The images were collected under an Olympus IX-70 inverted microscope ( Figure 4). The results showed that all the successfully recovered viruses could be detected by antibody of the nsp4 and N protein of PRRSV.
To determine the effect of the nsp2 deletions on PRRSV growth, the ability to form plaques was examined. Confluent monolayers of Marc-145 cells cultured in a 6-well plate were infected with 10-fold serially diluted viruses (10 to 0.001 TCID 50 ). After 1 h incubation, the excessive inoculum was removed and an agar overlay was applied onto the monolayer. Plaques were stained at 37°C with crystal violet 5 days post infection. The results showed that the plaque sizes of the deletion viruses had no significant difference in comparison to that of parental virus HuN4-F112 ( Figure 5).
Viral growth assays was finished as the papers reported. The titers of PRRSV were all expressed as TCID 50 . Marc-145 cell monolayer in T-25 flasks was infected with either parental or mutant viruses at 100TCID 50 . After 1 h of attachment at 37°C in 5% CO 2  with gentle mixing every 15 minutes, the inocula were removed and the monolayer was washed three times with serum-free DMEM. After washing, 5 ml 2% FBS DMEM was added and the flasks were then incubated for up to 4 days at 37°C in 5% CO 2 . Samples were collected from the medium at every 12 hours post infection (hpi) and titrated by the method of Reed-Muench on Marc-145 cells in 96-well plates. The results of the viral growth curve analysis ( Figure 6) showed that nsp2 with small deletions did not affect the viral growth on Marc-145 cells, but the titers of the deletion virus were higher than the parental virus during the early stage of infection (before 36 h). The titers of the rHuN4-F112-Δ480-532, rHuN4-F112-Δ508-532, and rHuN4-F112-Δ480-667 before 36 h were about 1000-fold higher than that of the parental virus HuN4-F112, and the titers of the rHuN4-F112-Δ480-620 before 36 h were about 100-fold higher than that of the parental virus HuN4-F112.
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 [28]. 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][30][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][33][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 [29]. 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.