Relationship of Noncoding RNA and Swine Viruses Open Access

Most of the large multicellular eukaryote genome encodes RNA that does not code for protein [1]. These noncoding RNA (ncRNA) molecules differ from each other in size, abundance, and protein coding capability [2] and have long been regarded as genome noise [3, 4].

NcRNAs can be divided into housekeeping RNAs and regulatory RNAs based on their expression patterns and functions [5]. The former includes ribosomal RNAs and transport RNAs [6], which are common in eukaryotic cells, while the regulatory RNAs can be split into small RNAs (such as small interfering RNAs and microRNAs [miRNAs]), long ncRNAs (lncRNAs), and circular RNAs [7, 8].

Increased attention has been paid in recent years to the biological significance of RNA interactions on cellular processes and interactions between pathogens and host. Our knowledge of ncRNAs-medicated gene expression regulation has been greatly expanded due to these novel methodologies [9]. Increasing evidence reveals such mechanisms play a central role in all aspects of regulating cellular response to invading viruses [10‒15].

When getting infected by virus, the host cells generate a variety of ncRNAs in order to counteract infection; at the same time, viruses themselves also express many ncRNAs to resist cellular antiviral activity, so as to promote and establish their infection [16]. Besides, evidence has shown that viral ncRNAs can also regulate the replication of virus itself [17]. Thus, an in-depth understanding of the complex networks of interactions coordinated by ncRNAs is needed to provide a unique opportunity to antiviral approaches. In this study, we focus on the relationship of ncRNA and swine viruses.

Mammalian cells express a large amount of regulatory ncRNAs, some of which are essential for regulating the immune system whereas others are exploited by viruses [9]. Upon virus infection, a great variety of cellular ncRNAs, including lncRNAs and miRNAs, are manipulated, as a cellular response to infection or as a viral evasion strategy, and the impact of outcome can be either proviral or antiviral.

Porcine circovirus type 2 (PCV2) is the major cause of post-weaning multi-systemic wasting syndrome and other PCV-related diseases [18]. PCV2 is a small virus with a circular single-stranded DNA genome [19] and has long been held to a known viral pathogen of immense economic value in swine industry [20].

Host miRNAs play an important role in the regulation of inflammation caused by PCV2 [21]. miRNA is a small ncRNA (sncRNA) which participates in RNA silencing [22] and post-transcriptional regulation of gene expression [23], functioning together with mRNA molecules, and it plays a prominent role in a wide range of biological processes [24‒27]. Several researches have shown that miRNA can contribute to the host-virus interactions [28‒31]. They are believed to play a role in the host immune response to PCV2-modulated infection [32]. Tens of miRNAs were induced by the expression of PCV2-encoded proteins [33]. Eight differentially expressed miRNAs (five upregulated, 3 downregulated) were identified in the mediastinal lymph node of infected pigs [32]. Among them, ssc-miR-122 could indirectly inhibit the DNA replication process of PCV2 and repress the protein synthesis in cells.

Besides, host miRNAs also exert effects on PCV2. For instance, ssc-miR-30a-5p in 3D4/21 cells promotes autophagy to enhance replication of PCV2 through targeting 14-3-3 [34].

lncRNAs constitute a diverse type of nonprotein-coding RNA molecules [35], regulating gene expression at multiple levels by interacting with mRNA, DNA, protein, and miRNA [36]. For example, lncRNAs could regulate gene expression by binding to transcription factors and other proteins, promoting or inhibiting gene expression [37]. After PCV2 infection, differentially expressed lncRNAs were identified in an intestinal porcine epithelial cell line (IPEC-J2) [18]. A total of 199 differentially expressed lncRNAs were found in PCV2-infected cells [18]. Trans analysis further revealed lncRNA-regulated target genes were mostly responsible for binding DNA or RNA, performing transcription factor activity. Most of the predicted target genes of expressed lncRNAs are related to infectious diseases, such as influenza A virus infection [38] and arthritogenic alphavirus infection [39]. Therefore, PCV2 infection-related lncRNAs have a huge potential in modulating viral infection by regulating targeted genes.

Porcine epidemic diarrhea (PED) is a kind of intestinal disease that seriously endangers pigs. It is characterized by acute watery diarrhea, vomiting, dehydration, and severe enteritis [40]. PED virus (PEDV), a member in the Coronaviridae family, is the main etiological agent of PED [41]. It is a single-stranded, positive sense RNA virus with a genome of approximately 30 kb [42]. The virus infects pigs of all ages, causing severe economic losses to pig industry around the world, especially in Europe and Asia [43].

A number of studies have implicated that cellular miRNA is participated in virus infection [44‒46]. MiRNAs regulate host antiviral immune response through multiple ways. Mature miRNA forms part of an RNA-induced silencing complex, which can bind to viral RNA for helping cell to recognize it and further degrade it [47]. 214 miRNAs were found differentially expressed in PK-15 cells (porcine epithelial cells) infected by PEDV [48], and the regulatory relationship between PEDV infection and miRNA-221-5p was investigated [49]. The result showed that overexpression of miR-221-5p inhibited the replication of PEDV in a dose-dependent manner. Further study revealed that miR-221-5p inhibits the replication of virus by directly binding to 3′ untranslated region (UTR) of PEDV RNA. Overexpression of miR-221-5p upregulated interferon-stimulated gene 15, interferon-B, and MX1 expression by activating nuclear factor NF-kB signal pathway. NF-κB-inhibitor α and suppressor of cytokine signaling 1 were found to be targets of miR-221-5p. Together, miR-221-5p targets the viral 3′ UTR and activates the NF-κB signaling, therefore inhibiting the replication of PEDV [49].

Conversely, host miRNAs regulate PEDV as well. A recent study has revealed that exosomal ssc-miR-328-3p-derived PEDV-infected Vero E6 cells suppress PEDV infection in recipient cells by targeting tight junction protein 3 [50].

lncRNA expression was regulated by porcine endemic diarrhea virus infection. During PEDV infection, the lncRNA expression profiles were investigated in intestinal porcine epithelial cell-jejunum 2 cell lines [40]. 6,188 novel lncRNAs were identified by next-generation sequencing, and these lncRNAs were likely to be associated with genes related to immune signaling pathways, proving that in IPEC-J2 cell line, lncRNA expression patterns were influenced by PEDV infection.

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major important swine diseases worldwide [51]. It affects swine of any age with reproductive failure and a complex respiratory syndrome, causing an enormous economic loss [52]. Therefore, it has been considered as a devastatingly factor to the global swine industry.

A wide variety of sncRNAs were found in PRRSV-infected pigs to be variably expressed. The altered expression of the sncRNA molecules can provide explanations about how PRRSV could significantly interfere, modulate, or inhibit the host’s innate and adaptive immunity development. Moreover, the class membership of each host noncoding molecule expressed during PRRSV infection could determine the classification of the sncRNAs profiled [53].

miRNAs have been regarded as vital regulators that play a major role during PRRSV replication [54]. The miRNAs which are involved in modulating the replication of PRRSV can be divided into three different categories according to their targets [55]. The first category targets the PRRSV genome directly; for example, miR-181 [56] and miR-23 [57] directly targeted the genomic RNA of PRRSV to inhibit viral replication; the second one can target the signal pathways which are involved in PRRSV replication; for example, miR-27b, miR-29b, miR-30a-3p, miR-132, miR-146a, and miR-9-2 were found to participate in multiple cell signaling pathways involved in immune modulation and cytokine and transcription factor production [58]; MiR-125b inhibited the replication of PRRSV by regulating the NF-kB pathway negatively [59]. The last category mainly targets the host factors associated with PRRSV replication.

PRRSV modulates the host immune responses and induces host gene expression. Study on the HPPRRSV GSWW15 and the North American strain FL-12 in infected porcine alveolar macrophages (PAMs) found 12,867 novel lncRNAs [60], and 299 lncRNAs from them were expressed differentially after the infection of PRRSV. The study on microRNAomes inside PRRSV-infected swine lungs and PAMs revealed that ssc-miR-30d-R_1 and miR-147 play regulatory roles in PRRSV infection and host antiviral responses [58, 61].

Transmissible gastroenteritis virus (TGEV) is a member of Coronaviridae family [62], and TGEV infection can activate NF-κB pathway in porcine intestinal epithelial cells and cause severe inflammatory response [63]. Ssc_circ_009380 was found to promote the activation of NF-κB pathway in the inflammatory response to TGEV by binding miR-22 [63]. Differentially expressed lncRNAs might be involved in inflammatory responses induced by TGEV via acting as miRNA sponges, regulating their upstream and downstream genes [64]. Data analysis based on enrichment of Gene Ontology of host target genes demonstrated that the differentially expressed miRNAs participated in regulatory networks, including cellular process, metabolic process, and immune system response [65]. The important role of miR-222 in the regulation of mitochondrial dysfunction caused by TGEV infection had been confirmed [62]. Mir-4331 aggravated mitochondrial damage induced by TGEV by inhibiting RB1 expression, promoting IL1RAP, and activating p38 MAPK pathway [66]. TGEV selectively regulated the expression of miRNAs in some cells in order to regulate its subgenomic transcription [67].

In the process of virus infection, host cell produces a variety of ncRNAs to counteract infection. Similarly, swine viruses also encode their own ncRNAs. Proteins associated with viral ncRNAs that are critical to the viral stability, function, or both of them. Besides, viral ncRNAs have many biological functions, including regulating virus replication, virus persistence, immune evasion, pathogenesis, and cellular transformation. However, the knowledge of viral ncRNAs in PCV2, PEDV, and TGEV is unknown. Therefore, this section will focus on viral ncRNAs in other swine viruses except PCV2, PEDV, and TGEV.

PRRSV also encodes miRNA-like viral small RNAs (vsRNAs) contributing to PRRSV replication. To date, four PRRSV vsRNAs are found, which are named PRRSV-vsRNA1-4 [68]. Among these PRRSV vsRNAs, PRRSV-vsRNA1 inhibits PRRSV replication through directly suppressing viral nonstructural protein 2 expression [68].

African swine fever (ASF) is a highly contagious swine viral disease with a high mortality rate of nearly 100% in domestic pigs. ASF is caused by the ASF fever virus (ASFV), a relatively large double-stranded DNA virus [69].

Replication of ASFV occurs predominantly in the cytoplasm of cells and has complex interactions with the host cell components, including sncRNA [69]. The host and virus miRNAs were extracted from primary macrophages (PAMs) infected by ASFV for analyzation, and the result suggested that only 6 miRNAs had different expressions in the process of infection which means ASFV infection had only a modest effect on host miRNAs [70].

A number of DNA viruses are known to manipulate sncRNA by encoding their own. In order to investigate the interplay between ASFV and sncRNAs, study of host and vsRNAs extracted from ASFV-infected primary porcine macrophages (PAMs) was undertaken. The data revealed 3 potential novel small RNAs encoded by ASFV as ASFV small RNA 1, 2, and 3 (ASFVsRNA1, ASFVsRNA2, ASFVsRNA3), from which ASFVsRNA2 had the highest mean abundance. Overexpression of ASFVsRNA2 led to up to a 1 log reduction in ASFV growth indicating that ASFV utilized a virally encoded small RNA to disrupt its own replication. Further study showed that ASFVsRNA2 had a variable number of 3′ U residues and it did not fit the classic miRNA biogenesis pathway, so called the canonical miRNA biogenesis pathway; therefore, ASFVsRNA2 cannot be viewed as miRNA [64].

Classical swine fever (CSF) is a contagious and transboundary viral disease of pigs worldwide and causing significant economic losses. It is characterized by acute symptoms like high fever, lethargy, vomiting, and diarrhea [71, 72].

CSF is caused by CSF virus (CSFV) which replicates efficiently in cell lines and monocytes (including macrophages) [73]. CSFV, border disease virus, and bovine viral diarrhea virus form the genus Pestivirus of the family Flaviviridae [74]. It carries an RNA genome with a 3′ UTR, a single large open reading frame, and a 5′ UTR functioning as the cap-independent translation initiation entry site [75]. The UTRs of CSFV RNA were identified with the function of triggering apoptosis in transfected cells. This has been stated in detail by Hsu et al. [76]. The research also discovered that the 5′ and 3′ UTR RNAs work together in cis in order to perform this function. Follow-up studies showed that translation shutoff was involved in the UTR-mediated apoptosis.

As a porcine alpha-herpes virus, pseudorabies virus (PRV) is considered as the etiologic pathogen of Aujeszky’s disease, which induces respiratory system ailments, neurological disorders, and reproductive disease in pig [77, 78]. To date, at least 20 viral miRNAs encoded by PRV are confirmed [79, 80]. Among these viral miRNAs, prv-miR-LLT11a might repress PRV replication in PK15 cells through targeting swine leukocyte antigen-1 [81].

Besides, three viral lncRNAs encoded by PRV are identified by Northern blotting and real-time PCR, including LUI, LDI, and LEULR [82]. Moreover, LDI might regulate PRV gene expression by upregulating IE180, which is a transcriptional activator of viral genes [82].

It is well known that short interfering RNAs play a temporary role in transfected animal cells, and small RNA expression vectors have been developed to induce RNA silencing with lasting effects in mammalian cells [83]. RNAi refers to the process in which cells or viruses produce a single-strand RNA fragment called miRNA, which serves as a guiding template for recognizing specific mRNA. Soon after RNAi was discovered, it was considered by virologists to be a promising method to inhibit virus replication, which opened the door for a novel RNA virus treatment [84].

So far, RNAi is being increasingly used to inhibit the replication of swine virus such as porcine TGEV [85], foot-and-mouth disease virus [86], and PRRSV [87‒89]. As the therapy is considered as simpler and easier to implement, an ideal RNAi treatment for viruses will exhibit a more competitive advantage than the use of antiviral drugs [84]. Moreover, since the ncRNAs play important roles in swine virus, RNAi targeting ncRNAs can also be a promising approach. On the other hand, ncRNA half-lives vary over a wide range, and mutations can lead to RNAi escaping [84]. Therefore, RNAi together with antiviral drugs and other antiviral treatments might make it more difficult for the viruses to evade.

It is well known that ncRNAs play an important role in many cellular processes and they are capable to modulate the responses of RNA-mediated host-virus infection in multiple ways [9]. Functional studies in knockout animal models have provided insights and confirm that ncRNAs are not transcriptional noise or evolutionary junk, but are clearly needed for normal development [2].

NcRNAs are key regulators of antiviral responses which play various roles in different aspects during the process of virus infection, including virus persistence, pathogenesis, and replication. Both DNA and RNA viruses generate regulatory ncRNAs aside from host ncRNAs that are influenced and exploited during infection. These ncRNAs coordinating with various DNA, RNA, and protein molecules perform modulating functions in gene expression; for example, viral lncRNAs can affect RNA stability and many DNA viruses. A few RNA viruses encode miRNAs to evade host immune responses [17, 90]. Many more such cases of functional lncRNAs and miRNAs await to be explored during viral infections. Combinatorial therapeutic approaches with antiviral drugs and ncRNA targeting RNAi provide a promising effective antiviral strategy.

Ethics approval was not required. Written informed consent was not required.

The authors declare that they have no competing interests.

This work was supported by the Hunan Provincial Natural Science Foundation of China (2018JJ2177), the Science and Technology Project of Jiangxi Provincial Department of Education (GJJ211632), and the Initial Scientific Research Fund of Yichun University (3360119046).

Conceptualization: Zhibang Deng and Manxin Fang. Data curation, formal analysis, and manuscript: Manxin Fang and Wei Hu. Funding acquisition, supervision, and approval: Zhibang Deng.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.