Cellular microRNAs and Picornaviral Infections

Abstract

microRNAs (miRNAs) are a subtype of short, endogenous, and non-coding RNAs, which post-transcriptionally regulate gene expression. The miRNA-mediated gene silencing mechanism is involved in a wide spectrum of biological processes, such as cellular proliferation, differentiation, and immune responses. Picornaviridae is a large family of RNA viruses, which includes a number of causative agents of many human and animal diseases viz., poliovirus, foot-and-mouth disease virus (FMDV), and coxsackievirus B3 (CVB3). Accumulated evidences have demonstrated that replication of picornaviruses can be regulated by miRNAs and picornaviral infections can alter the expression of cellular miRNAs. Herein, we outline the intricate interactions between miRNAs and picornaviral infections.

Introduction

miRNAs are ~22 nucleotides (nt) long, non-coding regulatory RNAs that are involved in the regulation of gene expression at post-transcriptional level. The first miRNA, lin-4, was discovered in the nematode, Caenorhabditis elegans (C. elegans) by genetic screens.^1,2 With greater progress in research, miRNAs have been identified in many viruses, plants, and animals, such as Epstein-Barr virus, Kaposi's sarcoma-associated herpes virus, Arabidopsis, mouse, and humans.^3-6 miRNAs regulate gene expression through combination of miRNA induced silencing complex (miRISC) to induce translational repression or degradation of targeted mRNAs. Individual miRNA can directly or indirectly repress hundreds or even thousands of genes. Likewise, individual gene can be targeted by different types of miRNAs simultaneously.⁷ The miRNA-mediated regulatory network is essential for a wide spectrum of biological processes, including cellular proliferation, differentiation, apoptosis, and immune responses.^8-10

Picornaviridae is a family of single-stranded and positive sense RNA viruses and currently comprises 37 species distributed among 17 genera.¹¹ Members of the picornavirus family, including poliovirus, hepatitis A virus, and foot-and-mouth disease virus (FMDV), are widespread pathogens of humans and domestic animals. The genome of picornavirus, which is encapsulated in a non-enveloped icosahedral virion, varies between 7000–8500 nt in length and shares a conserved organization across the family.¹² The 3′ end of the genome is polyadenylated, and the 5′ terminus is linked to a small virus-encoded peptide, VPg, which is rapidly lost after the translocation of viral genome into cytoplasm.¹³ The genome of picornavirus contains a single open reading frame (ORF) flanked by untranslated regions (UTRs) at both terminuses. The 5′ UTR possesses a number of cis-acting structural elements, such as internal ribosomal entry site (IRES) and a ‘clover-leaf’ domain that are involved in picornaviral translation and replication, respectively.^14,15 The viral RNA encodes a large polyprotein, which is subsequently processed to produce mature viral proteins by virus-encoded proteases.¹⁶ The structural proteins (‘1A-1D’ or ‘VP4-VP1’) are located within the N-terminal portion of the polyprotein, followed by the nonstructural proteins (namely, ‘2A-2C’ and ‘3A-3C’). The structural proteins, VP1-VP3, together form the icosahedral shell of the virion, while VP4 is located on the inner surface of the capsid. The nonstructural proteins directly or indirectly facilitate the picornaviral replication.¹²

The key component of miRNA machinery is the small RNA induced silencing complex (RISC), which directs the small regulatory RNAs to impact the stability of picornaviral genomes. Similarly, the other component, Dicer2, can also indirectly repress picornaviral infections. These observations indicate that miRNA machinery is essential in picornaviral infections. The present article outlines the current understanding of the relationship between miRNAs and picornaviral infections.

Biogenesis of miRNAs in animal cells

As shown in Figure 1, in the conventional pathway of miRNA biogenesis, the miRNA genes are transcribed typically by RNA polymerase П (pol П) to produce long primary mRNAs (pri-miRNAs); although a minor proportion of miRNAs that are associated with Alu repeats can be transcribed by RNA polymerse Ш (pol Ш).^17,18 The pri-miRNAs possess a 5′ cap and a 3′ polyA tail, and can form two or more hairpins, each containing a distinct mature miRNA species as part of the double stranded (ds) stem connected by a terminal loop.^19,20 Subsequently, these pri-miRNAs are specifically recognized and spliced by the multiprotein complex, the Microprocessor, which consists of Drosha and its cofactor, DiGeorge syndrome critical region gene 8 (DGCR8) in mammals or Pasha in Drosophila melanogaster and C. elegans.^21,22 DGCR8 or Pasha, which serves as a dsRNA binding protein, directs the Drosha (a nuclear RNase Ш-type protein) to cleave the pri-miRNA at the base of the hairpin and release the precursor miRNA (pre-miRNA) of ~70 nt in length.²³ After nuclear processing, pre-miRNAs are transported into the cytoplasm; export of pre-miRNAs is mediated by exprotin-5, which recognizes the short overhang at the 3′ end of pre-miRNA, via a Ran-GTP dependent mechanism.^24,25 Within the cytoplasm, pre-miRNA is recognized and spliced by another RNase Ш protein, known as Dicer, which works in cooperation with the dsRNA-binding protein, TRBP (human immunodeficiency virus trans-activating response RNA binding protein) or Loquacious, releasing a ~22 nt miRNA duplex.^26-28 Following Dicer cleavage, shortened double-stranded miRNA duplex is loaded onto an Argonaute (AGO) protein to generate miRISC.^29-31 The mature miRNA is retained, whereas the passenger strand (referred to as miRNA*) is excluded from the miRISC and is degraded in most cases. However, recent publications have revealed that miRNAs* are also present at a relatively high level and have the ability to repress targets.³² Once loaded onto miRISC, miRNAs guide AGO proteins to target mRNAs via imperfect sequence complementarities with sites in the 5′ UTRs, coding regions, or 3′ UTRs, to repress their translation.^33-35

Figure 1. Biogenesis of miRNAs in animal cells. In the standard pathway, the transcription of miRNA genes are mainly mediated by Pol П. The long primary transcripts (pri-miRNAs) are recognized and cleaved by Dorsha-DGCR8 complex, generating the precursors of miRNAs (pre-miRNAs). Then, pre-miRNAs are transported by exportin-5 from nucleus to cytoplasm. Once arrival at cytoplasm, pre-miRNAs are recognized and spliced by Dicer-TRBP complex to generate miRNA/miRNA* duplexes. Finally, mature miRNAs are loaded onto Argonaute proteins, resulting in cleavage or degradation of the targeted mRNAs. In the mirtron pathway, the mirtrons, derived from miRNA-containing introns, are processed by splicesome and debranching enzymes to generate pre-miRNAs. Afterwards, the pre-miRNAs access the standard miRNA biogenesis pathway. In the Argonaute dependent pathway, transcripts of miRNA genes are spliced by Drosha and then transported to cytoplasm. Afterwards, pre-miRNAs are directly loaded onto AGO proteins that bypass the ‘Dicer-processing’ step, and the catalytic center of Argonaute protein helps to catalyze the maturation of the miRNAs.

The canonical miRNA biogenesis pathway uses Drosha and Dicer RNase Ш enzymes to govern the maturation of miRNAs; the two steps of ‘RNase Ш-processing’ are critical for the canonical pathway of miRNA biogenesis. However, deviations from this pathway have been observed: the biogenesis of several classes of miRNAs bypasses a key ‘RNase Ш-processing’ (Drosha or Dicer) step involved in the canonical miRNA biogenesis pathway; and these alternative pathways are classified as Drosha-independent and Dicer-independent pathways.³⁶

Approximately 40% of animal miRNAs, termed as mirtron miRNAs, are derived from short introns of protein-coding genes.^20,37 These miRNAs genes are transcribed by RNA pol П, and the short intron-derived pri-miRNAs are subsequently spliced into intron-removed mRNAs and intron lariats by the spliceosome.³⁸ The intron lariats, in which the 3′ branchpoints are ligated to the 5′ end of the intron, are then debranched into pre-miRNA hairpins that are suitable for Dicer cleavage with the help of a debranching enzyme, thus bypassing the ‘Drosha processing center’^39,40. Subsequently, translocation of the intron-derived pre-miRNAs from nucleus to cytoplasm is mediated by Exportin-5. Once encountering Dicer, pre-miRNAs are cleaved to generate miRNA duplexes, which are then loaded onto miRISC to perform the silencing activity. Furthermore, there are several other Drosha-independent miRNA pathways, such as tRNA-derived miRNA pathway, small nucleolar (sno) RNA-derived pathway, and short hairpin (sh) RNA-derived pathway.^41-43

In addition to Drosha-independent pathways, there is also a Dicer-independent pathway called the AGO dependent pathway. AGO proteins are the key components of miRISC that regulate gene expression. These proteins possess the pre-miRNA binding domains, namely the PAZ domain and Mid domain, which can respectively bind 3′ end and 5′ end of the pre-miRNA; and a splicing domain, known as the PIWI domain, which forms an RNaseH-like fold in the tertiary structure.^44,45 In this pathway, transcripts of miRNA genes are spliced by Drosha and then transported to cytoplasm. Eventually, the pre-miRNAs are indirectly loaded onto AGO proteins, bypassing the ‘Dicer-processing’ step, and the catalytic center of AGO helps to catalyze the maturation of the miRNAs.⁴⁶

Apart from the biogenesis pathways of miRNA mentioned above, recent studies have discovered novel tRNA-derived miRNAs that are produced by the RNase Ш-independent biogenesis pathway; neither Drosha nor Dicer is involved in generation of this subclass of miRNAs.⁴⁷ Such alternative pathways of miRNA biogenesis provide an additional level of complexity to miRNA-dependent regulation of gene expression.

miRNAs machinery regulate the picornavirus life cycle

miRNAs can directly or indirectly regulate various stages of the life cycle to impact picornaviral replication. Picornaviral infection is initiated when virus encounters its cognate receptor. The presence or absence of a particular receptor on the cell surface determines the viral tropism for that particular cell type.⁴⁸ Receptors used by different picornaviruses include the immunoglobulin superfamily (IgSF), low density lipoprotein receptor family (LDLR), the integrin family of cell adhesion molecules, and the T cell immunoglobulin domain mucin-like domain receptors.⁴⁹ In addition, miRNAs can indirectly determine the viral tropism by silencing certain types of receptors. Ouda et al. reported that in rhinovirus 1B (RV1B) infected cells, miR-23b targeted the mRNA of the picornaviral receptor, very low density lipoprotein receptor (VLDLR), thus leading to repression of viral replication.⁵⁰ At physiological temperature, the binding of receptors induces irreversible conformational changes in the native virion that initiates the formation of endocytosed vesicles. The clathrin-mediated endocytosis is best characterized among several endocytic pathways for the genome translocation of picornaviruses. The clathrin-coated pits are internalized to form clathrin-coated vesicles and are subsequently uncoated, following which the viral genome is released. All picornavirus genomes contain a single ORF flanked by lengthy UTRs. IRES, which resides in the 5′ UTR, consists of cis-acting RNA structures that usually require specific RNA-binding proteins for translational machinery recruitment.⁵¹ miRNAs can affect the replication of picornaviruses by regulating the IRES activity. Chang et al. reported the presence of potential target sites of miR-153, miR-220, miR-242, and miR-276 in the IRES of FMDV, and also observed that the overexpression of these four miRNAs resulted in the inhibition of FMDV infection in vitro and in vivo.⁵² The majority of miRNAs binding sites reside in 3′ UTR of the targeted mRNAs. It is, therefore, not surprising that miRNAs repress the translation of picornavirus genome through binding to the 3′ UTR of the RNA genome. For instance, Karaa et al. observed that miR-16 inhibited the IRES-B activity by targeting the 3′ UTR of genome of encephalomyocarditis virus (EMCV), resulting in repression of IRES-driven translation of the virus.⁵³ In a another study, Kelly et al. reported that four miRNAs were incorporated in the 3′ UTR of coxsackievirus A21 (CVA21), and miR-142–3p provided the most complete and most consistent protection against CVA21 replication.⁵⁴ In addition to UTRs, cellular miRNAs can also target the protein coding region to regulate viral infections. The protein coding regions of picornaviruses can be divided into structural and nonstructural protein coding regions. In the structural protein coding region of enterovirus 71 (EV71), there are two target sites of miR-296–5p located in the VP1 and VP3 coding regions, respectively. miR-296–5p can inhibit EV71 replication by targeting these two viral sites.⁵⁵ In the nonstructural coding region of coxsackievirus B3 (CVB3), two target sites are located in 2C and 3D coding regions for miR-342–5p and miR-10*, respectively. While miR-342–5p shows the ability to repress CVB3 replication, miR-10* promotes viral replication.^56,57 In addition to directly targeting the picornaviral genomes, miRNAs can also target the mRNAs of relevant proteins to regulate the process of viral infections. As identified for cytokines, the miR-548 family (including miR-548b-5p, miR-548c-5p, miR-548i, miR-548j, and miR-548n) targeted the 3′ UTR of IFN-λ1, resulting in suppression of EV71 infection.⁵⁸ For transcription factors, miR-203 was found to repress ZFB-148, which resulted in downregulation of genes involved in cell cycle arrest and upregulation of genes responsible for cell growth; finally leading to cell growth that benefited picornaviral replication.⁵⁹ In case of signaling molecules, miR-126 had three specific targets, SPRED1, LRP6, and WRCH1, distributed in the ERK1/2 and Wnt/β-catenin signal pathways; the collaboration of these two signal pathways coordinated by miR-126 maintained a dynamic balance in the formation and release of viral particles to initiate new infections.⁶⁰ Collectively, these findings illustrate the intricate miRNA-mediated positive and negative regulatory events that influence picornaviral infections (Fig. 2, Table 1).

Figure 2. miRNAs involved in regulating different stage of picornaviral life cycle.

Table 1. miRNAs involved in regulating different stages of picornaviral life cycle.

miRNAs	Target	Function	Ref
miR-23b	VLDLR	Repression of viral replication	50
miR-153	IRES	Inhibition of FMDV infection	52
miR-220	IRES	Inhibition of FMDV infection	52
miR-242	IRES	Inhibition of FMDV infection	52
miR-276	IRES	Inhibition of FMDV infection	52
miR-16	3′ UTR of viral genome	Repression of the IRES driven translation	53
miR-142–3p	3′ UTR of viral genome	Repression of viral replication	54
mir-296–5p	VP1 and VP3	Repression of viral replication	55
miR-10*	3D	Repression of viral replication	56
miR-342–5p	2C	Repression of viral replication	57
miR-548	IFN-λ1	Inhibition of FMDV infection	58
miR-203	ZFB-148	Repression of viral replication	59
miR-126	SPRED1, LRP6, WRCH1	Balance the viral particle formation	60

Picornaviral infections alter cellular miRNAs profile

As noted before, miRNAs can affect picornaviral replication; and conversely, the infections by picornaviruses can change the expression of miRNA in the cell. For picornaviruses, the first study to this effect was performed by Cui et al. using the deep sequencing approach. In human epidermoid carcinoma (Hep2) cells, 64 miRNAs were found to be differentially expressed between infected and non-infected cells. 42 of the 64 miRNAs were upregulated, and the remaining miRNAs were reported to be downregulated.⁶¹ Furthermore, it was predicted that majority of the target genes of these aberrantly expressed miRNAs were responsible for neurological processes, apoptosis, and immune response, indicating that EV71 infection can cause lethal encephalitis of myocarditis.⁶¹ In a subsequent study, Ho et al. verified that miR-141, induced by EV71 infection, targeted the cap-dependent translation initiation factor, eIF4E, to shutoff host protein synthesis; this in turn facilitated viral propagation.⁶² In a separate investigation, Xu et al. found that miR-1 was upregulated upon CVB3 infection and was involved in viral myocarditis by post-transcriptional repression of its target gene, Cx43.⁶³ In a similar study, Corsten et al. reported that CVB3 infection significantly upregulated the expression of miR-155, miR-146b, and miR-21 and further identified that miR-155 contributed to the adverse inflammatory response of the heart to picornaviral infection.⁶⁴ In a more recent study, Liu et al. demonstrated that in CVB3-infected heart and spleen tissues, miR-21 and miR-124b were upregulated, while miR-451 was downregulated. Subsequently, they revealed that inhibition of expression of miR-21 and miR-124b can decrease the expression levels of Th17 and ROPγt; further suggesting that these two miRNAs serve as a regulator of TH-17 differentiation by affecting the expression of the TH-17 transcription factor, ROPγt.⁶⁵ Although previous studies have verified the presence of many aberrantly expressed miRNAs in picornavirus-infected cells, the function of majority of such miRNAs have not been completely understood in picornaviral infections. However, there lies a possibility of studying these miRNAs in infections by other invading pathogens. We hereby summarize the target genes and the functions of the miRNAs, which is significantly changed in abundance during picornaviral infections, in Table 2, which can provide greater insights into the roles of miRNAs in various biological mechanisms during picornaviral infections.

Table 2. The verified targets and functions of aberrantly expressed miRNAs in picornaviral infections.

miRNAs	Expression	Verified Targets	Verified Functions	Ref
miR-21	Up	MyD88, IRAK1, PREN, SMAD7, PDCD4, IL-12p35	Innate immunity	61 , 66 - 70
miR-421	Up	PAI1, FOXO4, FXR	Inflammatory responses; cell proliferation	61 , 71 - 73
miR-1226	Up	MUC1	Cell death	61 , 74
miR-140	Up	Mitofusin1	Apoptosis	61 , 75
MiR-627	Up	JMJD1A	Cell proliferation	61 , 76
miR-27	Down	?	Viral replication	61 , 77
miR-26a	Down	TLR3	Repression of innate immunity; Cytokines expression	61 , 78 , 79
miR-19	Down	TLR2,CYLD	Innate immunity	61 , 80 , 81
miR-30a	Down	Lyn	Immune cell activity	61 , 82
Let-7e	Down	IL-23R	Cytokines expression	61 , 83
miR-155	Up	MyD88, TAB2, IKKε	Innate immunity	84 - 87
miR-223	Up	TLR3,TLR4,STAT3, IKKα	Innate immunity	84 , 88 - 92
miR-146b	Up	TRAF6,IRAK1, MyD88,TLR4,STAT3	Innate immunity	84 , 93 - 98
miR-106a	Up	PDCD4, STAT3, IL10	Innate immunity; Cytokines expression	84 , 99 - 102
miR-130b	Up	STAT3	Cellular proliferation	84 , 103
miR-499	Down	PDCD4,SOX6	Innate immunity; Cellular proliferation	84 , 104 , 105
miR-148a	Up	P27, Bcl-2,CaMKIIα	Innate immunity; Cellular proliferation; Apoptosis	106 - 109
miR-143	Up	COX2	Apoptosis; Inflammasome formation	106 , 110 , 111
miR-324–3p	Up	Rel	Innate immunity	106 , 112
miR-206	Up	?	Immune cells differentiation	113
miR-545	Up	CyclinD1,CDK4	Cell proliferation	106 , 114
miR-362–3p	Up	E2F1,USF2,PTPN1	Cell proliferation	106 , 115

‘?’, unclear

Conclusions

During viral infections, miRNAs serve as post-transcriptional regulators of gene expression in a variety of biological process, including viral replication, cellular proliferation, and immune responses. A number of miRNAs are known to be involved in the regulation of picornaviral infections though multiple steps, thus affecting the outcomes of these infections. Conversely, picornaviral infections can also alter the profile of cellular miRNAs. Some of these miRNAs have been well characterized in other pathogenic diseases, such as miR-21 in HCV and HIV infections. Although it is possible that miR-21 may influence the picornaviral infections by regulating the innate immunity as has been verified in HCV and HIV infections, the precise mechanism of miR-21 in picornaviral infections is not yet clear. Future studies are, therefore, required to investigate the regulatory network of these miRNAs, in order to increase our understanding of the interactions of picornavirus with the miRNA machinery of the host.

10.4161/rna.29357