Porcine 2′,5′-oligoadenylate synthetase-like protein inhibits replication of porcine reproductive and respiratory syndrome virus

Background : Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious pathogen that causes $664 million losses per year to the swine industry. There are few useful vaccines that can provide protection against PRRSV infection. 2′, 5′-oligoadenylate synthetase-like protein (OASL) has antiviral activity, this has not been shown for PRRSV and the mechanism is unknown. Methods : Expression of OASL in porcine alveolar macrophages induced by interferon (IFN)-b stimulation and PRRSV infection was examined by real-time polymerase chain reaction. Exogenous expression and knockdown of OASL were used to determine the role of OASL in the PRRSV replication cycle. The type I IFN signaling pathway was evaluated after OASL overexpression. Results : In this study, we found that the expression of OASL in porcine alveolar macrophages was significantly increased by IFN-b stimulation and PRRSV infection. Porcine-OASL-specific small interfering RNA (siRNA) promoted PRRSV replication, whereas exogenous expression of porcine OASL inhibited replication of the virus. The anti-PRRSV activity of porcine OASL was lost after knockdown of retinoic acid-inducible gene I ( DDX58 , also known as RIG-I ). Conclusions : Porcine OASL suppresses PRRSV replication.


Background
Porcine reproductive and respiratory syndrome (PRRS) has been the most aggressive disease affecting the swine industry worldwide, resulting in economic losses of $664 million per year in the US [1,2]. In 2006, the swine industry in China was greatly impacted by the disease, which resulted in substantial economic losses 3 [3]. Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, belongs to the Arteriviridae family. The entire PRRSV genome contains 10 open reading frames (ORFs), namely, ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5, ORF5a, ORF6, and ORF7. ORF1a and ORF1b make up four-fifths of the genome, encoding the main nonstructural proteins involved in viral replication and transcription, whereas the viral structural proteins GP2-GP5, M, N, E, and GP5a are encoded by ORF2-5, ORF6, ORF7, ORF2b, and ORF5a, respectively [4][5][6]. PRRSV strains are mainly subdivided into types 1 and 2 according to their antigenicity. The majority of China's epidemic strains are type 2 [7]. Current vaccines hardly provide protection against this virus, finding new ways to control this virus is in urgent need. The host intrinsic restriction factors usually inhibit virus infection by direct interaction with viral proteins, and they are more promising because they are less likely to mutant under drug-mediated selective pressure.
Previous transcriptomics showed that after PRRSV infection, OAS expression increased [31,32]. The anti-PRRSV effects of porcine OAS1 and OAS2 have been proven [33,34], but those of porcine OASL and the relationship between OASL and IFN are not clear. Moreover, OASL has a different sequence at its C terminus (relative to other OAS proteins), it remains to be determined whether this feature has a different inhibitory effect on viral replication as compared with the other three OAS subtypes. Therefore, in this study, we evaluated the effect of porcine OASL on PRRSV replication in vitro and attempted to elucidate the mechanisms underlying its antiviral activity.  Table 1. Table 1 Primers used in the research

Statistical analysis
All experiments were biologically repeated three times, data represent means ± standard deviations of three independent experiments, and all assays were conducted in triplicate. The results were analyzed by the Student's t-test. Data with a P value < 0.05 were considered statistically significant.

Porcine OASL mRNA expression was increased by IFN-β and PRRSV infection
After 6 h of stimulation with 1000 IU/mL IFN-β, porcine OASL mRNA expression in the PAMs increased quickly to a peak of 125 times than that in the untreated control cells (Fig. 1A). The OASL mRNA expression peak occurred at 12 h post-stimulation.
The OASL mRNA expression level in the PRRSV-infected PAMs peaked at 36 h postinfection (Fig. 1B), showing a 15-fold increase compared to the untreated cells.
Furthermore, OASL exerted no cytotoxic activity toward this cell line after transfection for 48 h (Fig. 2B).
After OASL transfection for 24 h, PRRSV infected the cells, and 24 post-transfection, the PRRSV N mRNA level and viral titer were tested. In comparison with empty 11 vector-transfected cells, the PRRSV N mRNA level decreased significantly in the CRL-2843-CD163 cells transfected with p3xFLAG-CMV™-7.1-OASL (Fig. 2C). PRRSV TCID 50 for the p3xFLAG-CMV™-7.1-OASL group was also lower (Fig. 2D). The phosphorylation level of IRF3 was higher in the OASL-overexpressing cells than in the empty vector-transfected cells (Fig. 3C). After poly (I:C) treatment for 6 h and PRRSV infection for 24 h, IFN-β promoter activity was tested, and the results show that OASL also increased the IFN-β promoter activity (Fig. 3D).

OASL siRNA increased PRRSV replication
si-OASL (60 nM) was transfected into PAMs for 48 h, resulting in efficient reduction of OASL expression ( Fig. 4A and 4B). si-OASL itself was not cytotoxic to the cells (Fig. 4C). After 60 nM si-OASL was transfected into PAMs, PRRSV infected the cells for 48 h, and the results showed that in the presence of si-OASL, the PRRSV N mRNA level was higher than that in cells treated with si-Ctrl (Fig. 4D). The TCID 50 results were in agreement with the mRNA results (Fig. 4E). After 60 nM si-OASL was transfected into PAMs, the cells were subjected to Poly(I:C) treatment for 6 h; the results showed that IFN-β and IFN-α mRNA levels in the OASL knockdown group were also significantly lower ( Fig. 4F and 4G).
3.4. Anti-PRRSV activity was dependent on RIG-I but not RNase L and there were no decreases in either the PRRSV N mRNA expression level or virus titers relative to the levels in the control group ( Fig. 6C and 6D).
In the case of MDA5, 60 nM si-MDA5 was transfected into CRL-2843-CD163 cells for 48 h. mRNA results showed that MDA5 mRNA expression in CRL-2843-CD163 cells was decreased by approximately 72% (Fig. 6E); the western blot confirmed this result (Fig. 6F). In the CRL-2843-CD163 cells cotransfected with 800 ng OASL and 60 nM si-MDA5 and then infected with PRRSV (MOI 1.0), there were significant decreases in both the PRRSV N mRNA expression level and virus titers relative to the control levels ( Fig. 6G and 6H).

Discussion
In this study, we showed that exogenous expression of porcine OASL restricts PRRSV replication, the schematic representation of the signaling pathway is presented in 13 Fig. 7.
Reports have revealed that human OASL interacts with RIG-I and exerts an antiviral effect. Even though it has no enzymatic activity, OASL is usually maintained at low expression levels in cells. When viruses infect human cells, human OASL is notably upregulated by the double-stranded RNA, and IFN is induced by IRF3 [27,40,41]. In the present study, after PRRSV infection of the cells, porcine OASL was produced, which then induced the phosphorylation of IRF3 to enhance IFN production, whereupon viral replication was inhibited. In stark contrast to our results, Lee demonstrated that murine OASL1 downregulates IFN via IRF7 to impede its expression and therefore aids in viral replication [28]. The discrepancies in the results could be explained as follows: effects of porcine OASL are mediated by IRF3, and different OASL isoforms might have different regulatory mechanisms in the signaling pathway.
Since porcine OASL had a nucleotidyltransferase region (data not shown), we surmised that its antiviral activity is dependent on RNase L. Nevertheless, our results indicated that this was not the case; porcine OASL did not exert its action via the OASL-RNase L pathway, and there may be another critical factor influencing the antiviral effect. Similarly, another study showed that porcine OASL also inhibits JEV replication but not through the OASL-RNase L signaling pathway [30]. Thus, our finding for PRRSV is the same as that for JEV. A limitation of this study is that testing of other virulent strains was not done. The NADC-30 strains responsible for the most recent epidemics are presumed to follow the same trends as PRRSV, largely because of their ability to induce IFN. Therefore, future studies should include these viruses.
In conclusion, we demonstrate that pOASL is an anti-PRRSV factor. Upregulation of pOASL activity may boost host immunity to limit PRRSV infection. Knockout of pOASL may increase the PRRSV titer during the virus production. Future investigation of pOASL activity might provide the insight and opportunities needed for the therapeutic development and improved vaccine candidate. 15

Conclusions
Porcine OASL was proven to inhibit PRRSV replication in vitro through a RIG-Idependent process. These data may

Ethics approval and consent to participate
This study was approved by the Institutional Animal Care and Use Committee of Henan Academy of Agricultural Sciences. All processes were followed the "Guidelines for the care and use of laboratory animals in biomedical research".

Consent for publication
Not applicable.

Author Contributions
MZ, RW conceived and designed the experiments. MZ, RW, YK, HL, WW performed the experiments and wrote the manuscript. MZ analysed the data; all authors read and approved the final manuscript.

Availability of data and materials
All datasets are available in the main manuscript.

Competing interests
The authors declare that they have no competing interests. The porcine OASL knockdown increases virus replication in macrophages. (A) qRT-PCR quanti Schematic representation of the signaling pathways involved in OASL-induced interferon (IFN