The Emerging Role of Non-Coding RNAs in the Regulation of Virus Replication and Resultant Cellular Pathologies

Non-coding RNAs, particularly lncRNAs and miRNAs, have recently been shown to regulate different steps in viral infections and induction of immune responses against viruses. Expressions of several host and viral lncRNAs have been found to be altered during viral infection. These lncRNAs can exert antiviral function via inhibition of viral infection or stimulation of antiviral immune response. Some other lncRNAs can promote viral replication or suppress antiviral responses. The current review summarizes the interaction between ncRNAs and herpes simplex virus, cytomegalovirus, and Epstein–Barr infections. The data presented in this review helps identify viral-related regulators and proposes novel strategies for the prevention and treatment of viral infection.


Introduction
The human genome consists of a variety of non-protein-coding DNA. A proportion of these genomic regions are transcribed into RNA. These non-coding RNAs (ncRNAs) are believed to have diverse roles in cellular functions. In addition to those with characterized functions, further functional ncRNAs certainly need to be discovered and categorized [1]. Since the first evidence of biological functions of transfer and ribosomal RNAs in the 1950s, several other classes of ncRNAs have been identified. Two classes of these transcripts, namely, long ncRNAs (lncRNAs) and microRNAs (miRNAs), have gained special attention because of their regulatory roles on gene expression. While lncRNAs regulate gene expression at different levels, miRNAs mainly act at a post-transcriptional level. LncRNAs and miRNAs have another distinctive feature arising from their size. While the former group is longer than 200 nt, the latter are approximately 22 nt in length. LncRNAs interplay with other RNA species, particularly miRNAs, in a way that they sequester them and decrease their bioavailability [2]. Besides this, lncRNAs have biological functions through serving as scaffolds and enhancer RNAs [3].
The regulatory role of miRNAs on gene expression is mediated through miRNAs pairing with the miRNA recognition elements in the mRNAs. These elements are found particularly in the 3' untranslated region (UTR) of transcripts; however, they are also present in 5'UTRs and coding regions. When the RNA-induced silencing complex is recruited to miRNA recognition elements, the target mRNA is destabilized, or its expression is repressed [4]. Based on an individual miRNA's ability to target numerous miRNA recognition elements and suppression of hundreds of mRNAs, it is estimated that more than 60% of human protein-coding genes can be targeted by miRNAs [2].
NcRNAs, particularly lncRNAs, have recently been shown to regulate different steps in viral infections and induce immune responses against viruses [5]. Expressions of several host and viral lncRNAs have been found to be altered during viral infection [2,6]. These lncRNAs can exert antiviral function via inhibition of viral infection or stimulation of antiviral immune response. Some other lncRNAs can promote viral replication or suppress antiviral responses [2].
In the current review, we summarize the interaction between ncRNAs and herpes simplex virus (HSV), cytomegalovirus (CMV), and Epstein-Barr (EBV) infections.

ncRNAs and HSV Infection
Kaposi's sarcoma-associated HSV-encoded miRNAs have been shown to affect the expression of host lncRNAs such as Maternally Expressed 3 (MEG3), antisense non-coding RNA in the INK4 locus (ANRIL), and Urothelial Cancer Associated 1 (UCA1) in favor of cancer development. More than 120 host lncRNAs have been identified as putative targets for viral miRNAs. Notably, in addition to the miRNA-dependent route, this type of HSV can affect the expression of host lncRNAs through direct interactions between lncRNAs and latency-related proteins. The impact of HSV on UCA1 expression has pro-proliferative and pro-migratory effects on endothelial cells [7].
HSV-1 has also been shown to increase the expression of Nuclear Enriched Abundant Transcript 1 (NEAT1) and the establishment of paraspeckles through influencing Signal Transducer And Activator Of Transcription 3 (STAT3). NEAT1 and other paraspeckle constituents, namely, P54nrb and PSPC1, interact with HSV-1 genomic DNA. Paraspeckle Component 1 (PSPC1) binds with STAT3 to facilitate its recruitment to paraspeckles and increase its interplay with viral gene promoters. This interaction increases the expression of viral genes and the replication of viruses. Suppression of NEAT1 or STAT3 has improved the healing of HSV-1-related skin lesions in animal models [8]. Another study has shown that HSV infection can result in the construction of higher numbers of paraspeckles via increasing expression of NEAT1 [9]. NEAT1 has also been shown to cooperate with HEXIM1 to construct a multi-subunit complex that participates in the regulation of DNA-associated innate responses of the immune system. This complex encompasses DNA-PK subunits as well as paraspeckle proteins. In fact, binding of HEXIM1 to NEAT1 has an essential role in the assembly of this complex. This complex has a vital participation in induction of innate immune responses against foreign DNA via induction of cGAS-STING-IRF3 pathway [10].
A high throughput RNA sequencing experiment has shown over-expression of lncR-NAs in murine 661 W cells following HSV-1 infection. U90926 RNA has been identified as the most over-expressed lncRNA after infection with this virus. Being located in the nucleus, U90926 enhanced replication of HSV-1 DNA and increased proliferation of this virus. U90926-silenced cells have exhibited higher survival rates [11].
During the latent phase of HSV infection, one region of its genome which encodes the latency-associated transcript (LAT) is not silenced. This lncRNA has been first identified as an enhancer of HSV-1 reactivation. Yet, subsequent studies have shown this lncRNA's role in the inhibition of cell apoptosis and enhancement of the formation of latency. Experiments in a rabbit model have revealed that reduction in LAT levels in neurons after the establishment of the latent phase decreases the capacity of HSV to reactivate. Thus, the HSV-1 LAT transcript is involved in the reactivation in an independent manner from its role in establishing latency [12].
These two miRNAs are encoded by Kaposi's sarcoma herpes virus and could activate the mTORC1 signaling pathway and improve cell proliferation and colony formation.

ncRNAs and CMV Infection
RNA1.2 has been recognized as one of four principal lncRNAs which are expressed by human CMV. This lncRNA has an essential function in manipulating the cellular NF-κB-dependent pathways of cytokine and chemokine production in the course of CMV infection. Thus, this lncRNA can affect host immune responses [29]. Another study has shown that human CMV encodes lncRNA4.9, which is localized to the viral replication compartment in the nucleus. Depletion of this lncRNA decreases CMVV DNA replication and its growth. Notably, CRISPR-Cas9-mediated targeting the RNA4.9 promoter results in the reduction of viral ssDBP levels implying the relation between ssDBP levels and oriLyt activity [30].
An in vitro study has shown the ability of human CMV in infecting primary human mammary epithelial cells. This infection leads to inactivation of Rb and p53 proteins, enhancement of telomerase activity, and activation of c-Myc and Ras as well as Akt and STAT3 signaling pathways. CMV-transformed cells exhibited a CMV signature associated with the lncRNA4.9 gene. The sequence of this lncRNA has also been detected in xenograft tumors originated from CMV-transformed cells. Most notably, similar lncRNA4.9 genomic sequences have been found in tumor samples of breast cancer patients [31].
Persistent CMV infection in elderlies has been associated with down-regulation of Non-Coding Repressor Of NFAT (NRON) lncRNA, while up-regulation of its immunityassociated target gene NFAT, in both CD28nullCD8+ T cells and CMVpp65CD8+ T cells ( Figure 1). Thus, NRON has been suggested to contribute to CMV-induced CD28nullCD8+ T cell aging through affecting IL-4-associated NFAT signals [32].
Another study has shown that human CMV encodes several ncRNAs such as miR-US5-1 and miR-UL112-3p that protect CD34+ hematopoietic progenitors from apoptosis through inactivation of Forkhead box class O 3a (FOXO3a) [34]. miR-UL112-3p has an established role in the enhancement of the progression of glioblastoma [35]. Tables 3 and 4 show the role of host and viral-encoded ncRNAs in CMV infection.

ncRNAs and EBV Infection
Nasopharyngeal carcinoma is one of the EBV-associated cancers. EBV has been shown to express very few viral proteins in nasopharyngeal carcinoma cells, probably in order to evade induction of immune responses. Yet, it expresses high amounts of EBV BamHI-A region rightward transcript (BART) miRNAs and lncRNAs (Figure 1). These ncRNAs are implicated in the pathogenesis of EBV-related disorders. The expression of BARTs has been shown to be regulated by the NF-κB pathway. In fact, EBV LMP1 as an effective activator of the NF-κB pathway can increase the expression of BARTs via this pathway. Meanwhile, BART miRNAs can decrease the expression of LMP1. NF-κB pathway and expression of BARTs construct an autoregulatory circuit to preserve EBV latency in nasopharyngeal carcinoma cells [46]. BHLF1 gene of EBV has been shown to encode several lncRNAs in linear and circular forms that participate in viral replication. However, an open reading frame has been detected in this gene in a proportion of EBV isolates. BHLF1 transcripts have also been detected during the latent phase. In fact, this lncRNA participates in important features of EBV latency, such as its capacity to induce the constant proliferation of B lymphocytes and their malignant transformation [47]. On the other hand, the EBV-encoded miRNA miR-BART6-3p has been shown to act as a tumor suppressor. This miRNA could inhibit metastasis and invasion processes and suppress the proliferation of EBV-related neoplasms via decreasing the expression of LOC553103. LOC553103 has been found to directly bind with the 3'UTR of STMN1 and increase its stability. Cumulatively, miR-BART6-3p/LOC553103/STMN1 molecular route can modulate levels of cell cycle-related proteins, which subsequently suppress the EBV-related proliferation of tumor cells [48].
Over-expression of EBV-miR-BART5-3p has been shown to promote the growth of nasopharyngeal and gastric cancer cells. This miRNA can directly target 3'UTR of TP53 and subsequently decrease Cyclin Dependent Kinase Inhibitor 1A (CDKN1A), BCL2 Associated X, Apoptosis Regulator (BAX), and Fas Cell Surface Death Receptor expression levels. Thus, this miRNA accelerates cell cycle progression and inhibits cell apoptosis. Most notably, BART5-3p participates in chemo/radioresistance. Furthermore, it increases p53 protein degradation [49].
Huang et al. have sequenced more than 11,000 lncRNAs and 144,000 protein-coding transcripts from four EBV-associated and EBV-negative gastric cancer samples in addition to their adjacent unaffected tissues. They have shown specific expression of SNHG8 in EBVassociated gastric cancer. This lncRNA has been demonstrated to influence the activity of numerous gastric cancer-specific signaling pathways and genes being affected by EBV [50]. Tables 5 and 6 show the role of host and viral-encoded ncRNAs in EBV infection.   In Burkitt lymphoma and diffuse large B-cell lymphomas, miR-34a acts as a tumor suppressor due to downregulating PD-L1 and activating T cells. [54] In gastric cancer cell lines, miR-34a downregulation leads to increased cell viability and reduced apoptosis rate. [55]

Impact of Drugs on the Expression of ncRNAs in Infected Patients
EBV-miR-BART22 has been shown to promote stemness properties, metastatic abilities of cancer cells, and increase their resistance to cisplatin. This viral-encoded miRNA directly targets the MAP2K4 and up-regulates MYH9 levels through PI3K/AKT/c-Jun signaling. Notably, cinobufotalin has been found to suppress miR-BART22-associated cisplatin resistance through enhancing expression of MAP2K4 to inhibit MYH9/GSK3β/β-catenin cascade and EMT process in nasopharyngeal carcinoma [71]. Another study has shown that RAS G12V and irinotecan up-regulate BART3-3p expression to hamper gastric cancer cells' senescence and lower NK cells and macrophages infiltration. In fact, BART3-3p reduces TP53, TP21, and inflammatory cytokines such as IL-1A, IL-1B, IL-6, and IL-8 [72]. Table 7 shows the impact of drugs on the expression of ncRNAs in infected patients.

Diagnostic Value of Non-Coding RNAs in EBV-Infected Individuals
Expression levels of EBV-encoded miRNAs such as miR-BART2-5p, miR-BART7-3p, miR-BART9-3p, and miR-BART13-3p have been shown to distinguish patients with nasopharyngeal cancer from healthy controls with diagnostic values ranging from 0.87 to 0.97 (Table 8).

Discussion
The interactions between ncRNAs and viral genes result in different pathophysiological consequences, thus affecting the clinical course of infection. In the current review, we have focused on the role of lncRNAs and miRNAs in HSV, CMV, and EBV infections and the induction of antiviral response. Most of the conducted studies have summarized the expression pattern of host transcripts. However, virus-derived ncRNAs have also been studied.
A possible mechanism for the oncogenic function of HSV is hijacking some cellular elements such as lncRNAs and miRNAs. Interference with this process possibly has therapeutic effects. Moreover, interference with viral-encoded ncRNAs might affect pathogenic processes in the course of viral infections. For instance, LAT-targeting ribozymes have been suggested as a possible strategy for treating recurrent HSV-related diseases such as herpes stromal keratitis [12].
A number of viral-encoded lncRNAs and miRNAs regulate the establishment of permanent latency, which might be a preceding phase of tumor development. Since this phase might be continued for a long time, it provides a window for therapeutic interventions in order to reverse the carcinogenic process. These viral transcripts have been shown to affect the expression of both protein-coding genes and ncRNAs encoded by the host cells. Thus, the interactions between cellular and viral ncRNAs are entirely complicated, necessitating the conduction of high throughput sequencing experiments and integrative bioinformatics analyses.
A number of cellular signaling pathways, such as NF-κB signaling, can affect viral latency. Additional investigations about the mechanisms by which NF-κB signaling can regulate the latency of viral particles might reveal novel therapeutic options for the treatment of viral-associated cancers.
Since most viral-encoded miRNAs and lncRNAs do not have similar sequences in the human genome, they can be considered appropriate diagnostic biomarkers, particularly for viral-associated malignancies. This type of application has been studies for EBV-encoded miRNAs such as miR-BART2-5p, miR-BART7-3p, miR-BART9-3p, and miR-BART13-3p, showing promising results in EBV-associated nasopharyngeal carcinoma. Further research in other types of viral-associated cancers is needed to expand the diagnostic application of these ncRNAs.
The data presented in this review helps identify viral-related regulators and proposes novel strategies for the prevention and treatment of viral infection.