Introduction
Porcine reproductive and respiratory syndrome (PRRS) is one of the most severe swine diseases worldwide. It is characterized by reproductive failures in sows and respiratory illness in growing pigs, which has caused an enormous economic burden on the swine industry globally. The etiological agent, Porcine reproductive and respiratory syndrome virus (PRRSV), is an enveloped, positive single-stranded RNA virus that belongs to the family Arteriviridae of the order Nidovirales [
21]. The genome size of PRRSV varies from 14.9 to 15.5 kb in length, with 11 open reading frames (ORFs). ORF1a and ORF1b, which occupy about 70% of the viral genome, encode two long polyproteins, pp1a and pp1ab. Subsequently, proteolytic processing of these polyproteins yields at least 14 non-structural proteins (nsps), which include four proteases (NSP1α, NSP1β, NSP2, and NSP4), the RNA-dependent RNA polymerase (NSP9), a helicase (NSP10), and an endonuclease (NSP11). ORFs 2–7 encode structure proteins including, glycosylated membrane proteins GP2-GP5, a non-glycosylated membrane protein (M), and the nucleocapsid (N) protein. [
5].
The virus was initially isolated in Europe and the United States in the early 1990s and categorized into the European PRRSV and the North American PRRSV genotypes [
28]. In 2006, highly pathogenic PRRSV (HP-PRRSV), a novel variant of type 2 PRRSV featured by the 30-aa deletion in the coding region of non-structure protein 2, emerged in China [
12]. The clinical features of HP-PRRSV infections are high fever, high morbidity, and fatality. Since its first outbreak, HP-PRRSV has caused an inestimable economic loss. A novel NADC30-like virus with 133-aa deletion in the nsp2 was isolated in the field in 2013 [
36]. It has been reported that NADC30-like viruses are undergoing recombination with HP-PRRSV, which makes disease control more challenging.
PRRSV infection impairs host innate and adaptive immune responses, leading to ineffective virus clearance and the establishment of chronic infection. The type I interferon response is a critical component of the host innate immune response against virus infection. The receptor for type I IFNs is ubiquitously expressed. Upon IFN binding, transmits the signal through two receptor-associated tyrosine kinases, tyrosine kinase 2 (TYK2) and Janus kinase 1 (JAK1), resulting in the phosphorylation of signal transducers and activators of transcription STAT1 and STAT2. The phosphorylated STATs form heterodimers and bind to IFN-regulatory factor 9 (IRF9) to form the active transcription factor complex IFN-stimulated gene factor 3 (ISGF3). ISGF3 initiates transcription by binding to the promoters of interferon-stimulated genes (ISGs), which contain IFN-stimulated response elements (ISREs) [
26,
27]. Some ISGs encode proteins with direct antiviral activity, such as ISG15, Mx1, Viperin, and interferon-inducible RNA-dependent protein kinase(PKR). Many of the ISGs encode transcription factors that enhance the production of interferons and other cytokines.
PRRSV has developed a set of mechanisms for suppressing interferon signaling. Several viral proteins have been reported to impair IFN production (Nsp1, Nsp2, Nsp4, Nsp11, and N protein) [
22]. Nsp1 translocates to the nucleus, inhibits the binding of transcription factor IRF3 with CBP, and facilities CBP degradation, resulting in down-regulation of IFN promoter activity [
8]. Nsp1β restrains STAT1 phosphorylation and ISGF3 nuclear translocation. Nsp2 inhibits NF-κB activation by interfering with the polyubiquitination process of IκBα, subsequently preventing the degradation of the IκBα protein, which are required for the releasing of NF-κB dimers and their translocation to the nucleus, where they regulate transcription of type I interferons [
23]. Nsp11 induces STAT2 degradation, and it also interacts with IRF9 to impair the nuclear translocation of the ISGF3 complex [
33]. Nsp11 and N protein both inhibits IRF3 phosphorylation and nuclear translocation to prevent IFN transcription [
19]. N protein also interacts with SOCS1 (a negative regulator of JAK-STAT signaling) to inhibit IFN production [
14].
Non-coding RNAs include long ncRNAs (lncRNAs) and short ncRNAs such as microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and small nuclear RNAs (snRNAs) [
6]. By definition, lncRNAs are transcripts lacking protein-coding potential and longer than 200 nt. The majority of lncRNAs are 7-methylguanosine-capped, spliced, and polyadenylated. A number of reports have delineated the critical roles of lncRNAs in a wide variety of biological processes [
15]. Precise regulation of gene expression in the immune system is crucial to protect hosts from pathogen infections by generating effective immune responses while limiting autoimmunity [
20]. Emerging evidence has established that lncRNAs are involved in the regulation of immune cell differentiation and immune responses. LncRNAs modify the virus-host interactions by regulating ISG expression or through ISG independent manner, such as stabilizing virus RNA structure or modulating viral replication [
17,
18,
25‐
27].
The role of host proteins and microRNAs in virus-host interactions of PRRSV has been extensively studied. However, the function of host lncRNAs in regulating PRRSV-induced immune response is largely unknown. Transcriptomic profiles of PRRSV infected host cells have been analyzed either in vitro or in vivo [
1,
29,
31]. These studies revealed that PRRSV infection resulted in alteration of gene expression evolved in innate immune response signaling pathways such as type I IFN signaling, TLR, and RIG-I signaling. MicroRNAs also play critical roles in regulating host innate immunity. For example, miR-24-3p facilitates PRRSV replication by suppression of HO-1 expression [
31]. MiR-373 promotes PRRSV replication through impairing type I IFN production [
4]. However, the functions of lncRNAs in host immune response against PRRSV are unknown. In our previous study, we analyzed expression profiles of HP-PRRSV strain GSWW/2015 and FL-12 infected PAMs by RNA-sequencing to identify lncRNAs regulated by PRRSV infection. We found a lncRNA, TCONS_00054158, induced by different strains of PRRSV and poly (I: C) but not by the heat-inactivated virus. The function of this lncRNA is under study in our lab (Zhang et al., 2017b). Zhen and colleagues analyzed mRNA, lncRNA, and microRNA profiles of PRRSV infected PAMs from two pig breeds [
35]. Another study used co-expression network analysis of differently expressed mRNAs and lncRNAs after PRRSV infection to identify the function of these lncRNAs. They found some of the lncRNAs associated with the interferon-induced genes [
30].
To explore the role of lncRNAs in the virus-host interaction of PRRSV, we sequenced RNA transcripts of PAMs infected with GSWW/2015 (GSWW for short) and vaccine strain VR2332 at 24 h post-infection. Using rigorous methods to evaluate the coding potential of the transcripts, 1,147 novel lncRNAs were characterized, and a total of 293 lncRNAs were differentially expressed. We identified a lncRNA, LNC_000397, which was up-regulated after PRRSV infection and negatively regulated PRRSV replication by inducing ISGs. This research firstly reported the function of lncRNA in regulating PRRSV replication, which might be beneficial for understanding the interaction between PRRSV and the host immune system.
Discussion
PRRSV has caused tremendous economic losses to the world swine industry. The high mutation rate and immune suppression nature of the virus make it challenging for vaccine development. Further understanding of the interaction between PRRSV and the host immune system would be beneficial for the implementation of novel antiviral strategies. One of the main goals of this study was to explore the role of host lncRNAs in regulating PRRSV-induced immune response. In this research, we characterized a total of 293 differently expressed lncRNAs after infection with two PRRSV strains. Inconsistent with our and other groups’ reports, predicted targets of DE lncRNAs were enriched in immune-response related pathways such as NF-κB and RIG-I signaling [
7,
34].
Importantly, we identified a lncRNA, LNC_000397, suppressed PRRSV replication in PAMs. The expression of lnc_000397 was up-regulated in PRRSV GSWW and VR2332 infected cells. It also could be induced by poly (I: C), a synthetic dsRNA analog, in PAM and PK15 cells. Using RNA-seq, we found that knocking down of this lncRNA down-regulated the expression of ISGs such as CXCL10, MX1, ISG15, and RSAD2. Furthermore, we demonstrated that LNC_000397 was induced by type I-IFN treatment. Because of the low transfection efficiency of primary macrophage, we could not detect the effect of LNC_000397 over-expression on PRRSV replication. Our results suggested that LNC_000397 is an IFN-dependent antiviral lncRNA.
Interferon signaling is one of the most critical parts of innate immunity to defense against virus infection. To date, the vast majority of ISGs involved in the antiviral immune response are proteins, such as Mx, IFIT protein family, and OAS protein family. In recent years, the roles of interferon-dependent lncRNAs have emerged. An IFN-β induced lncRNA, lnc-ISG20, inhibits IAV replication by enhancing ISG20 expression [
3]. Lnc-MxA is an interferon-stimulated gene (ISG) functions as a negative regulator of the antiviral immune response [
11]. These reports indicated that lncRNAs represent another set of ISGs that exert important roles in the antiviral immune response. Our studies showed that LNC_000397 was stimulated by PRRSV infection and negatively regulated virus replication as a novel ISG.
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