MicroRNAs as mediators of insect host-pathogen interactions and immunity.

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Contents lists available at ScienceDirect

Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys

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Review

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MicroRNAs as mediators of insect host–pathogen interactions and immunity

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Australian Infectious Disease Research Centre, School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia

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a r t i c l e

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Mazhar Hussain, Sassan Asgari ⇑

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Article history: Received 20 June 2014 Received in revised form 13 August 2014 Accepted 14 August 2014 Available online xxxx

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Keywords: MicroRNA Insect Pathogen Virus Immunity Interactions

a b s t r a c t Insects are the most successful group of animals on earth, owing this partly to their very effective immune responses to microbial invasion. These responses mainly include cellular and humoral responses as well as RNA interference (RNAi). Small non-coding RNAs (snRNAs) produced through RNAi are important molecules in the regulation of gene expression in almost all living organisms; contributing to important processes such as development, differentiation, immunity as well as host–microorganism interactions. The main snRNAs produced by the RNAi response include short interfering RNAs, microRNAs and piwi-interacting RNAs. In addition to the host snRNAs, some microorganisms encode snRNAs that affect the dynamics of host–pathogen interactions. In this review, we will discuss the latest developments in regards to the role of microRNA in insect host–pathogen interactions and provide some insights into this rapidly developing area of research. Ó 2014 Published by Elsevier Ltd.

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Contents 1.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Unfolding the multiple layers of insect responses to microbial infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. RNA interference an effective anti-viral defence against viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . miRNA biogenesis at a glance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of microRNAs as moderators of host–pathogen interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Host–virus interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Impact of viral infection on the host miRNA profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Cellular miRNAs target viral genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Virus-encoded miRNAs can target host genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. Virus-encoded miRNAs target viral own genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Bacterial infection can modulate host miRNAs in insects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of miRNAs in regulation of insect immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

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1.1. Unfolding the multiple layers of insect responses to microbial infection

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Multiple mechanisms of attack and counter-attack or host defences have evolved during the natural process of the host–

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⇑ Corresponding author. Address: Australian Infectious Disease Research Centre, School of Biological Sciences, The University of Queensland, St Lucia, QLD 4072, Australia. Tel.: +61 (617) 3365 2043; fax: +61 (617) 3365 1655. E-mail address: [email protected] (S. Asgari). http://dx.doi.org/10.1016/j.jinsphys.2014.08.003 0022-1910/Ó 2014 Published by Elsevier Ltd.

Please cite this article in press as: Hussain, M., Asgari, S. MicroRNAs as mediators of insect host–pathogen interactions and immunity. Journal of Insect Physiology (2014), http://dx.doi.org/10.1016/j.jinsphys.2014.08.003

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pathogen arms race. Animals defend themselves against microorganisms in two major ways: innate and/or acquired immune systems. The main difference between the two systems is the involvement of germline encoded factors for recognizing and destroying pathogens in innate immunity, while in acquired immunity effector molecules are produced to recognize antigens leading to the development of immunological memory. In the insect world, the immune system is based on innate immune responses that can be further categorised into three main parts: (1) pathogen immobilization, (2) core immune signalling pathways (Toll, Imd and JAK-STAT) and (3) the RNAi pathway. Pathogen immobilization is a cellular response towards fungal and bacterial infections in the form of phagocytosis, engulfment, melanisation, coagulation and autophagy (Dushay, 2009; Jiravanichpaisal et al., 2006). These responses help in immobilizing pathogens, which are then destroyed extracellularly by antimicrobial peptides (AMPs) or eliminated intracellularly. In response to systemic infection, AMPs are produced and secreted in the hemolymph that directly attack microbes (Bulet et al., 1999). Upon pathogen invasion, activation of immune signalling pathways usually ensues. In insects, these are mainly the Toll, Imd and JAK/STAT pathways. The Toll pathway is activated through an endogenous ligand namely the Nerve Growth Factor-related cytokine Spätzle (Spz) (Weber et al., 2003). Binding of Spz triggers a conformational change in the Toll receptor which activates the downstream signalling pathway (Gangloff et al., 2008). The Toll– Spz interaction is the convergence point for the activation of three pathogen recognition pathways; one triggered by fungal or bacterial proteases, one induced by recognition of fungal cell wall and one activated by Lysine-type bacterial peptidoglycan (El Chamy et al., 2008; Gottar et al., 2006; Ligoxygakis et al., 2002). Following receptor activation, MyD88 proteins interact with Toll through their respective Toll/Interleukin-1 receptor domains and recruit the Drosophila melanogaster IRAK homologue, the kinase Pelle that phosphorylates Cactus for degradation. Upon Cactus degradation, the NF-jB homologues, Dorsal or Dif, are freed to move to the nucleus and regulate hundreds of target genes (Tauszig-Delamasure et al., 2002; Towb et al., 2001). The Immune deficiency (Imd) pathway is activated by mesodiaminopimelic acid (DAP)-type bacterial peptidoglycans that form the cell wall of Gram-negative bacteria and some Gram-positive Bacilli. Pathogen recognition in Imd occurs through the transmembrane receptor PGRP-LC and the intracellular receptor PGRP-LE that are critical for recognizing extracellular bacteria. Flies deficient in both of these proteins are unable to induce AMPs in response to Gram-negative bacteria and are therefore highly susceptible to these infections (Kaneko et al., 2006). The JAK/STAT signalling pathway has been shown to be involved in insect immunity, in particular anti-viral responses and hemocyte development (Agaisse and Perrimon, 2004; Dostert et al., 2005); although the mechanism of activation of the pathway by viruses is not known. The signal transduction pathway consists of unpaired 1–3 ligands, the receptor domeless, the kinase JAK and the transcription factor STAT. Upon activation of the pathway, STAT is phosphorylated and translocated into the nucleus, thus, activating several target genes.

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1.2. RNA interference an effective anti-viral defence against viruses

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RNA interference (RNAi) is another arm of the multilayered defence system in insects involved in gene regulation, genome protection and anti-viral responses. Short interfering RNAs (siRNAs), microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs) are the three main types of small non-coding RNAs (snRNAs) central to the RNAi response pathway. Recently, a connection between the JAK/STAT signalling transduction and the RNAi pathway has been

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established in mosquitoes as part of the anti-viral immunity through the secretion of the signalling molecule, vago (Paradkar et al., 2012). This suggests the effective communications between different pathways associated with the innate immune responses in insects. Likewise, involvement of several miRNAs generated by host cells or encoded by viral genomes in host–pathogen interactions have recently opened up new research avenues in insect– microbe interactions. As the field of RNAi is rapidly expanding, in this review article, we will mainly focus on the role of miRNAs in insect host–pathogen crosstalk and immunity with the majority of examples from viruses as more relevant literature is available. For other types of small RNAs and their role in anti-viral responses, readers are referred to recent reviews (e.g., Sabin and Cherry, 2013; Bronkhorst and van Rij, 2014).

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2. miRNA biogenesis at a glance

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Since the discovery of the first miRNA from Caenorhabditis elegans just over two decades ago (Lee et al., 1993) our understanding of miRNA biogenesis and its interaction with targets has tremendously increased and is continuously evolving. In addition to the canonical pathway of miRNA biogenesis, several non-canonical pathways have been described. Based on the canonical pathway, a miRNA gene is transcribed in the nucleus by RNA polymerase II producing the primary miRNA (pri-miRNA) transcript that could be variable in length but contains one or several stem-loop structures (Fig. 1). A nuclear residing ribonuclease, Drosha, in association with Pasha (in insects) cleaves the stem-loop producing a 70 nt hairpin precursor miRNA (pre-miRNA) that is translocated into the cytoplasm by Exportin-5. In the cytoplasm, the hairpin head is cleaved by another ribonuclease enzyme, Dicer, producing a short duplex RNA molecule. In insects, two dicer proteins have been identified: Dicer-1 that is mainly involved in the miRNA biogenesis, and Dicer-2 that produces siRNAs. The miRNA duplex triggers the formation of the RNA induced silencing complex (RISC) in which an Argonaut (Ago) protein is the main component. Most miRNAs are loaded into RISC that includes Ago1 and siRNAs into Ago2 complexes; however, recent research indicates that miRNAs can also be loaded into Ago2 complexes (Ghildiyal et al., 2010; Rubio and Belles, 2013), especially as the insect ages (Abe et al., 2014). Once the miRNA duplex becomes associated with the RISC, one of the strands is degraded (referred to as miRNA⁄ strand) and the other one forms the mature miR-RISC complex. In some instances, the miRNA⁄ strand is retained and loaded into Ago2 and can be functional (Ghildiyal et al., 2010). Finally, the mature miRNA guides the complex to target mRNA(s) in a sequence specific manner, leading to post-transcriptional gene regulation, or the complex might be transported into the nucleus where the miRNA normally binds to the promoter regions of target genes regulating gene expression at the transcriptional level. Notably, each miRNA may have multiple mRNA targets and each mRNA may be targeted by multiple miRNAs.

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3. Role of microRNAs as moderators of host–pathogen interactions

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While there is a lot known about the role of miRNAs in insect development, less is understood about their function in host–pathogen interactions. Nevertheless, accumulating evidence in recent years has suggested their effects on host–microorganisms interactions and cross-kingdom communications. For example, several cellular as well as viral miRNAs have been found to be involved in assisting or limiting virus replication (see below). What is commonly observed in various host–pathogen systems is the differential abundance of host miRNAs following an infection, which varies at different stages of infection and type of pathogen involved. Con-

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nucleus

TGR RNA Pol II

5' cap

AAAA

pri-miRNA

Drosha Pasha

pre-miRNA

cytoplasm

Exportin-5

Dicer-1

Ago2

TRBP

miRNA duplex

miR-RISC AAAAA miR*-RISC AAAAA

5’ cap

5’ cap

Fig. 1. Canonical pathway of miRNA biogenesis. miRNA genes are transcribed in the nucleus mostly by RNA polymerase II in the form of primary miRNA (pri-miRNA) that contains one or more stem-loop structures, and similar to mRNAs it is 50 capped and polyadenylated. The stem-loop is cleaved from pri-miRNA by Drosha in association with Pasha to form the precursor miRNA (pre-miRNA which is then transported into the cytoplasm facilitated by Exportin-5/Ran. The hairpin head is removed either by Dicer-1 or Ago2 resulting in a miRNA duplex which initiates the formation of the RNA induced silencing complex (RISC). One of the strands in usually degraded (miRNA⁄) and the other strand guides the miR-RISC complex to target mRNA (posttranscriptional regulation) or transported into the nucleus where it binds to a promoter region and regulate gene transcription (transcriptional gene regulation, TGR). The miRNA⁄ may also be loaded into one of the Ago proteins and perform a function by interacting with target sequences. The outcome of miRNA-target interaction may vary from mRNA degradation, translational repression to upregulation of target transcript levels.

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currently, a large number of host genes are found down- or upregulated that could be regulated by miRNAs or other types of snRNAs. These changes in miRNA/mRNA profiles could be part of the host anti-pathogen response or controlled by pathogen manipulation, including miRNAs expressed by viruses. Below, we will examine several relevant examples of the role miRNAs play in mediating host–pathogen interactions.

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3.1. Host–virus interactions

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3.1.1. Impact of viral infection on the host miRNA profile In recent years, based on deep sequencing or microarray analysis, several publications have appeared reporting changes in the expression levels of cellular miRNAs in insects in response to pathogen infection, although in the majority of these cases functional analyses are still lacking. For example, the abundance of a large

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number of miRNAs was changed in Spodoptera frugiperda (Sf9) cells following infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). These included several upregulated miRNAs (e.g., miR-184, miR-998 and miR-10) at 24 h post-infection (hpi), with expression of some of those downregulated at 72 hpi (Mehrabadi et al., 2013). In addition, three of the novel S. frugiperda miRNAs identified in the study showed upregulation upon virus infection. Relevant to baculoviruses, Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) was shown to cause changes in the abundance of host miRNAs (Jayachandran et al., 2013). Among those, miR-14 that positively regulates H. armigera ecdysone receptor (Ha-EcR) in this insect was downregulated upon infection providing an example of virus-medicated manipulation of host physiology. In Bombyx mori larvae infected with B. mori cytoplasmic polyhedrosis virus (BmCPV), 58 miRNAs were found significantly up or


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downregulated in the midgut as compared with that of noninfected larvae at 72 and 96 hpi (Wu et al., 2013). Based on gene ontology (GO) analysis, the putative target genes of six of the differentially expressed miRNAs (bmo-miR-275, miR-14, miR-1a, N-50, N-46 and N-45) were associated with response to stimulus and immune system process. The authors speculated that significant increases in miR-275 levels following BmCPV infection could explain the stunted growth phenotype in virus-infected B. mori, since the miRNA has been shown to regulate development during metamorphosis (Liu et al., 2010). In another study, microarray analysis showed differential expression of at least 20 miRNAs in a Heliothis zea fat body (HzFB) cell line upon infection with Heliothis virescens ascovirus (HvAV3e) with three of them (Hz-miR-24, -22, -7) significantly downregulated (Hussain and Asgari, 2010). Arthropod-borne viruses (arboviruses) have also been demonstrated to alter the host miRNA profile. For example, in Culex quinquefasciatus infected with West Nile virus (WNV) downregulation of miR-989 and upregulation of miR-92 was noted (Skalsky et al., 2010). Deep sequencing analysis of Aedes aegypti mosquitoes infected with dengue virus (DENV) at 2, 4 and 9 days post-infection revealed differential abundance of 35 miRNAs. Among those miR5119-5p, miR-34-3p, miR-87-5p and miR-988-5p were upregulated (Campbell et al., 2013). As more of these studies emerge, it will become possible to carry out comparative studies to find out which miRNAs are commonly involved in host–pathogen responses in different host insects and whether particular miRNAs could be specifically regulated in response to particular groups of pathogens (e.g., viruses, bacteria, etc). 3.1.2. Cellular miRNAs target viral genes Given alteration of the abundance of host miRNAs following infection, it is likely that some of those could directly target viral genes. For instance, in H. zea HzFB cells, miR-24 downregulates DNA dependent RNA polymerase II RPC2 (DdRP; ORF64) and DdRP b subunits (DdRP; ORF82) of HvAV3e (Hussain and Asgari, 2010). Interestingly, this miRNA is selectively downregulated early in infection allowing transcription of viral genes required for virus replication suggesting that the virus may actively suppress host miRNAs that directly target its genes. Relevant to this, in B. mori, several targets of the cellular bmo-miR-8 were predicted in B. mori nucleopolyhedrovirus (BmNPV) genes (see below). Inhibition of this miRNA led to 8-fold increase in viral titre in the fat bodies of infected larvae (Singh et al., 2012). A recent paper highlighted the possibility of utilizing cellular miRNAs as a tool to target virus genes as a control strategy (Zhang et al., 2014a). This work was conducted in B. mori against BmNPV using three cellular miRNAs; bmo-miR-279, bmomiR-92b, and bmo-miR-2764. Accordingly, the cellular pre-miRNA sequences were used as backbone to replace the natural miRNA duplex sequences with artificial siRNA duplex sequences targeting the viral lef-11 gene, which is involved in virus replication (Zhang et al., 2014a). For this, a virus inducible artificial miRNA (amiRNAs) expression cassette was constructed targeting the viral gene. Infection with the virus stimulates the baculovirus-induced promoter 39 K included in the expression cassette leading to production of pre-amiRNA and eventually formation of siRNA duplexes. The mature amiRNAs successfully inhibited BmNPV proliferation by downregulating the lef-11 gene in silkworm BmN-SWU1 cells. It will be interesting to see if the approach could be utilized in whole insects to inhibit BmNPV replication without compromising the insect’s fitness. Viruses might also subvert host miRNAs to their own benefit. For example, in American eastern equine encephalitis virus, binding of the mammalian host miR-142-3p to the 30 UTR of the virus genome in myeloid-lineage cells restricts replication of the virus in this particular cell type leading to minimal interferon induction

and hence suppression of the host innate immune system (Trobaugh et al., 2013). Interestingly, this study showed that these same binding sites in the virus 30 UTR are also required for efficient infection of the mosquito vector Aedes taeniorhynchus. However, it is not known whether a mosquito miRNA might interact with the sequences facilitating its replication in the insect host similar to the mammalian host.

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3.1.3. Virus-encoded miRNAs can target host genes Recent investigations have revealed that insect virus miRNAs, similar to those reported from human viruses, might target and regulate host genes to establish infection and successfully replicate. However, the information is very limited in respect to insect viruses. The impact of the miRNA might be via regulation of a specific gene leading to the modification of a particular pathway or might cause a global reduction in the host miRNA depending on the target genes. For example, baculovirus encoded BmNPV-miR1 downregulates the nuclear Exportin-5 cofactor Ran resulting in the blockage of the export of cellular pre-miRNAs into the cytoplasm generally affecting the abundance of cellular mature miRNAs in infected cells (Singh et al., 2012) (Table 1). This may affect regulation of cellular as well as viral genes. For instance, predicted targets of four selected host miRNAs affected by downregulation of Ran via BmNPV-miR-1 were mostly related to immunity. In addition, targets of these miRNAs were also predicted in the viral genome. For example, bmo-miR-8, which is downregulated following infection, targets the immediate early-1 (ie1) gene (Singh et al., 2012). Downregulation of the miRNA is obviously to the benefit of the virus. However, it remains to be elucidated how viral miRNAs are produced while the host miRNA biogenesis pathway is interrupted. In addition, a WNV (Kunjin strain, WNVKUN) encoded miRNA, KUN-miR-1, derived from the 30 SL stem-loop located at the end of the 30 UTR of the virus in mosquito cells positively regulates the host GATA-binding protein 4 (GATA-4) (Hussain et al., 2012) (Table 1). RNAi-mediated silencing of GATA-4 led to a significant reduction in virus RNA replication, suggesting that GATA-4-induction via KUN-miR-1 plays an important role in virus replication. However, the mechanism remains to be explored. Members of the GATA family are transcription factors that regulate various biological processes in insects and vertebrates (Shapira et al., 2006). In particular, GATA-4 has been shown to be involved in regulation of egg development, vitellogenesis, and lipid metabolism in mosquitoes (Cheon et al., 2006; Park et al., 2006).

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3.1.4. Virus-encoded miRNAs target viral own genes Targeting virus genes by virus-encoded miRNAs appears to be common among different groups of viruses to regulate their own replication, enter latency or suppress host anti-viral responses. The first insect virus-encoded miRNA, HvAV-miR-1, was reported from the ascovirus HvAV-3e originating from the major capsid protein (MCP) gene of the virus (Hussain et al., 2008). The virusencoded miRNA was shown to autoregulate virus replication late in infection by downregulating the virus’s own DNA polymerase (Table 1). In a recent report, it was revealed that BmNPV expresses a miRNA, bmnpv-miR-3, early in infection, which targets and regulates expression of the viral DNA binding protein (P6.9) as well as other late genes (Singh et al., 2014) (Table 1). This may confer several benefits to the virus: (1) autoregulation of virus proliferation to avoid over replication, (2) regulation of the expression levels of the late genes to suppress their expression early in infection, (3) modulation of the host immune response by regulating virus proliferation. In regards to the latter, the investigators showed that virus load correlates with expression levels of the AMP gloverin, which has been shown to be upregulated upon baculovirus infection and inactivates budded viruses (Moreno-Habel et al., 2012).

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Hussain and Asgari (2014) Hussain et al. (2012) Targets and induces transcript level of cellular GATA-4 transcription factor

Autoregulates viral replication late in infection Enhances viral RNA replication NS1 gene region

West Nile virus (Kunjin strain, WNVKUN)

DENV-vsRNA-5 is produced from a stem-loop in the 30 UTR of DENV-2 KUN-miR-1 is derived from the 30 SL located at the end of the 30 UTR

AcMNPV-miR-1 is located on the negative strand of the core gene ODVE25 Autographa californica nucleopolyhedrovirus (AcMNPV)

RNA virus Dengue virus-2 (DENV-2)

Zhu et al. (2013) Results in a reduction of infectious budded virions and accelerates the formation of occlusion-derived virions

Singh et al. (2014)

Bombyx mori nucleopolyhedrovirus (BmNPV) BmNPV

bmnpv-miR-3 is derived from the plus strand early in infection

Downregulates Exportin-5 cofactor Ran resulting in the blockage of pre-miRNA export into the cytoplasm

Both miRNAs target hhi1 transcript

hv-miR-246 and -2959 from pag-1 gene BmNPV-miR-1

Targets 30 UTR of BmNPV DNA binding protein (P6.9) mRNA that transcribes from the opposite strand at the same locus Downregulates ODV-E25 mRNA on positive strand

Helps virus to escape the early immune response from the host

Singh et al. (2012)

Wu et al. (2011)

Hussain et al. (2008)

Limits viral DNA replication during late infection Help virus to enter latent phase Downregulates DNA polymerase 1 HvAV-miR-1 is derived from MCP

Effect on virus infection Target in viral/host genome miRNA and its origin Virus

Table 1 miRNAs encoded in viral genomes that target viral own or host transcripts.

DNA virus Heliothis virescens ascovirus (HvAV3e) Heliothis zea nudivirus (HzNV-1)

References


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A miRNA from the baculovirus AcMNPV, AcMNPV-miR-1, was found to be expressed from the negative strand of the viral genome and target ODV-E25 transcripts on the opposite strand with 100% complementarity (Zhu et al., 2013) (Table 1). ODV-E25 is a late gene encoding a 25 kDa structural protein of occlusion derived virus (ODV) and also found on the budded virus (BV) envelope (Russell and Rohrmann, 1993; Wang et al., 2010). The protein is conserved in baculoviruses that infect lepidopterans and important in ODV formation and BV infectivity (Chen et al., 2012). AcMNPVmiR-1 was expressed late in infection starting from 12 hpi. Bioinformatics analysis identified eight potential targets for AcMNPVmiR-1 in the virus genome with ODV-E25 showing 100% complementarity since it is expressed on the complementary strand of the miRNA. Validation experiments confirmed that AcMNPVmiR-1 causes cleavage of ODV-E25 mRNA and its higher expression levels coincided with reduced levels of the target mRNA levels. Notably, using AcMNPV-miR-1 mimic, it was found that the small RNA had no effect on budded virus production but affected their infectivity by reducing production of ODV-E25 (Chen et al., 2012). Furthermore, the mimic increased ODV formation, and inhibition of AcMNPV-miR-1 dramatically reduced the number of occluded ODVs. This implied that the miRNA promotes ODV formation. In another example, a functional viral small RNA (DENV-vsRNA5) produced from a stem-loop of DENV-2 30 UTR was found in virusinfected A. aegypti mosquitoes and cell lines. The vsRNA was shown to target the virus genome at NS1 gene region (Hussain and Asgari, 2014) (Table 1). Synthetic RNA inhibitor or mimic of DENV-vsRNA5 resulted in significant increases or decrease in viral replication, respectively. The autoregulation of virus replication may facilitate low but transmissible level of virus infection in the mosquito vector. In a number of vertebrate herpesviruses, it has been shown that virus-encoded miRNAs are important in switching between lytic and latent phases (reviewed in Cullen, 2011). In insect viruses, H. zea nudivirus (HzNV-1) expresses only one non-protein coding gene, the persistency-associated gene 1 (pag1), during the latency phase (Wu et al., 2011) (Table 1). Two miRNAs encoded by pag1 were shown to downregulate the viral early gene hhi1, which allows the virus to enter the latent infection stage in cell culture. In the absence of the pag1 gene, the virus does not enter latency phase. It will be interesting to find out whether virus-encoded miRNAs play any role in latency or persistent infection observed in other insect viruses.

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3.2. Bacterial infection can modulate host miRNAs in insects

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A number of studies have revealed that similar to viruses bacterial infections can lead to changes in cellular miRNAs in different insect species, with information again being limited. For example, association of the endosymbiotic bacterium Wolbachia (wMelPop strain) with alterations in cellular miRNAs and miRNA pathway proteins in A. aegypti has been documented (Hussain et al., 2011, 2013a; Mayoral et al., 2014; Osei-Amo et al., 2012; Zhang et al., 2013). Of note, this strain causes life-shortening in the mosquito as well as inhibit replication of several vector-borne pathogens especially RNA viruses such as DENV, WNV and Chikungunya virus (Moreira et al., 2009a,b). Initially, microarray analysis revealed that a number of mosquito miRNAs were differentially expressed with two of them, aae-miR-2940 and -309a, showing high abundance in Wolbachia-infected mosquitoes as compared with non-infected ones (Hussain et al., 2011). A target sequence of aae-miR-2940 was shown to reside in the 30 UTR of the mosquito’s metalloprotease m41 ftsh transcripts; however, the miRNA was shown to positively regulate the metalloprotease transcript levels. miRNA inhibition and silencing of the target gene revealed that the metal-

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loprotease gene and aae-miR-2940 are important in the maintenance of Wolbachia density (Hussain et al., 2011). In a follow-up study, aae-miR-2940 was found to negatively regulate the transcript levels of its other target, the DNA methyltransferase (AaDnmt2) (Zhang et al., 2013). Overexpression of AaDnmt2 in Wolbachia-infected mosquito cells led to significant reductions in Wolbachia density, but significantly enhanced replication of DENV. This suggested that downregulation of AaDnmt2 by aae-miR-2940 may contribute to inhibition of DENV replication in Wolbachiainfected mosquitoes. Notably, significant hypomethylation of A. aegypti genome was recognized in wMelPop infected mosquitoes (Ye et al., 2013). Given that AaDnmt2 is the only DNA methyltransferase gene in the mosquito’s genome, Wolbachia may epigenetically regulate expression of many host genes by suppression of AaDnmt2 via aae-miR-2940 induction. Further, the instances above provide an example of a miRNA regulating more than one gene in opposite ways, both benefiting the endosymbiotic bacterium. Function of another Wolbachia induced cellular miRNA, aaemiR-12, was confirmed in regulation of two target genes, DNA replication licensing factor MCM6 and monocarboxylate transporter MCT1, and shown to be important for the maintenance of Wolbachia density in mosquito cells (Osei-Amo et al., 2012). Recently, it was uncovered that Wolbachia blocks shuttling of Ago1 to the nucleus through upregulation of aae-miR-981 (Hussain et al., 2013a) and that leads to changes in the distribution of miRNAs in infected cells (Mayoral et al., 2014). Interestingly, this miRNA downregulates expression of importin b-4 that facilitates Ago1 distribution to the nucleus (Hussain et al., 2013a). A study on honeybees (Apis mellifera) showed activation of AMPs and regulation of a large number of immune-related genes in response to infection by the Gram-negative bacterium Serratia marcescens (Lourenco et al., 2013). Analysis of miRNAs from the infected bees also revealed differential expression of cellular miRNAs upon bacterial infection. Among the downregulated miRNAs were ame-bantam, miR-12, -34, -184, -278, -375, -989 and 1175, while some were upregulated such as let-7, miR-2, -13a, and -92a. Predicted interactions revealed that the miRNA could potentially regulate genes involved in Imd, Toll, JNK, and JAK/STAT pathways, regulation of AMPs and melanization. However, functional analysis and validation of these interactions remain to be investigated.

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4. Role of miRNAs in regulation of insect immunity

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Despite many reports demonstrating the role of miRNAs in vertebrate immunity, the information pertinent to their role in insect immunity is scant and mostly based on bioinformatics predictions or correlating miRNA abundance with that of mRNAs. For example, Fullaondo et al. identified over 70 miRNAs that could be involved in regulating immune related pathways such as Toll, Imd and JAK/STAT, melanization and JNK pathways using bioinformatics approaches (Fullaond and Lee, 2012). Similarly, a computational approach was used to identify Anopheles gambiae miRNAs that may be involved in regulating immune responses. Two miRNAs, aga-miR-2304 and aga-miR-2390, were predicted to target the genes encoding suppression and prophenoloxidase (Thirugnanasambantham et al., 2013). However, these predictions await experimental confirmation of miRNA-target interactions. In the flour beetle Tribolium castaneum, microbial challenge and stress led to alteration of the beetle’s miRNA profile (Freitak et al., 2012). Induction of immunity by injection of peptidoglycan into the beetles resulted in differential expression of 59 miRNAs from which 21 were upregulated. Similarly, changes in the abundance of Manduca sexta miRNAs were observed following immune challenge involved in pattern recognition, activation of prophenoloxidase, synthesis of

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AMPs and conserved immune signal transduction pathways (Zhang et al., 2014b). In A. gambiae, the possibility of involvement of miRNAs in defence against Plasmodium berghei invasion was demonstrated as Dicer-1 and Ago1 depletion in the mosquitoes led to increased sensitivity of the mosquitoes to P. berghei infection (Winter et al., 2007). However, no specific miRNA was identified responsible for the effect observed. One of the miRNAs whose role in insect immunity has experimentally been established is miR-8 which negatively regulates expression of the AMPs, such as Drosomycin and Diptericin, in D. melanogaster (Choi and Hyun, 2012). This allows maintenance of low level of AMPs expression under non-infection conditions, enabling the homeostasis of immunity. However, in miR-8 null Drosophila, the transcript levels of the AMPs significantly increased; although it was not shown how miR-8 regulates AMPs. It was predicted that the miRNA could potentially target the transcripts of Gram-negative binding protein 3 (GNBP3), an inducer of the Toll pathway upon fungal infection, and Pvf, a modulator of the JNK pathway. Negative regulation of AMPs by miR-8 was also demonstrated in the larvae of Plutella xylostella (Etebari and Asgari, 2013). This study further showed that miR-8 positively regulates the transcript levels of serpin-27, inhibiting activation of the Toll pathway and hence low level of production of AMPs when there is no infection/parasitization. Upon infection, miR-8 levels are reduced leading to a decrease in serpin-27 levels and consequently activation of the Toll pathway. In Aedes albopictus (C6/36) cells, the abundance of the mosquito-specific aae-miR-2940 was shown to be selectively reduced following WNVKUN infection, a flavivirus (Slonchak et al., 2014). As indicated above, aae-miR-2940 positively regulates the metalloprotease ftsh in mosquitoes (Hussain et al., 2011), and this protein was found to enhance WNVKUN replication (Slonchak et al., 2014). Reduced aae-miR-2940 levels led to lower metalloprotease levels and consequently reduced virus titers. This suggested that reduction in the miRNA levels might be an anti-viral response to WNV infection. Similarly, reduced levels of aae-miR-2940 were detected in A. albopictus Singh’s cell line infected with Chikungunya virus, an alphavirus (Shrinet et al., 2014). These results suggest that reduced levels of aae-miR-2940 might be a common response to viral infection. In A. aegypti, two immune related genes, cactus and REL1, were found to be the target of a blood meal induced miRNA, aae-miR375 (Hussain et al., 2013b). Interestingly, cactus, which is an inhibitor of the Toll pathway, was positively regulated by aae-miR-375, whereas REL1, which is an activator of AMPs, was downregulated by the same miRNA. The increase in cactus and decrease in REL1 was found to have a positive effect on DENV replication since AMPs have been shown to negatively affect DENV (Luplertlop et al., 2012; Xia et al., 2008). Coincidence of negative regulation of AMPs upon blood feeding may provide a fitness advantage for the virus. Finally, it is well-known that immune related genes evolve faster than non-immune genes due to constant evolutionarily pressures on hosts to neutralize pathogen novelties. Interestingly, when analysing 50 genes involved in the Drosophila Toll and Imd signalling pathways, Han et al. realized a strong negative correlation between the rate of evolution of the immune proteins and the number of their regulatory miRNAs, suggesting that genes regulated by more miRNAs evolve slower due to stronger functional constraints (Han et al., 2013). This suggests the role of miRNAs in the evolution of immune related genes in insects.

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In the last decade, we have witnessed major developments in our understanding of the biogenesis and the function of miRNAs

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in various aspects of insect biology. Most of these efforts have focused on the role of miRNAs in development with the main focus on D. melanogaster. Recently, there have been a number of publications on the role of these small RNAs in host–pathogen interactions. As an initial step towards understanding their role in the interactions, the majority of investigators have focused on the effect of infection on the host miRNA profile with several being identified as differentially abundant miRNAs. It is of great interest to see these efforts to be followed up by empirical approaches to demonstrate the functional role of these differentially abundant miRNAs in host–pathogen interactions and insect immunity. In addition, a number of virus-encoded miRNAs have been characterized that either regulate virus replication or modulate host genes/ miRNAs to facilitate their replication. Extending research on miRNAs beyond scientific curiosity and basic research to utilize them in applied insect biology with the aim to reduce crop damage by agricultural pests, inhibit mortality in beneficial insects and inhibit/limit transmission of vector-borne pathogens should be considered as future prospects. However, these efforts may encounter regulatory limitations when their applications are to extend to the field arena since genetic manipulations of insects or plants are most likely inevitable. Some attempts have already been made in this area with promising results either in cell culture or at the laboratory scale. miRNA research is certainly a very fast moving area of research and we shall see a strong progress in this field in the near future.

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Acknowledgements

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The authors would like to acknowledge Dr. Karyn Johnson from the University of Queensland for critically reading this review. The Q3 authors would like to acknowledge the Australian Research Council Q4 (DP110102112, DE120101512) and National Health and Medical Research (APP1062983, APP1027110) for providing funding that contributed to some of the findings reported in this review from our lab.

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MicroRNAs as Mediators of the Ageing Process.

MicroRNAs as regulators and mediators of c-MYC function.

MicroRNAs as mediators of cardiovascular disease: Targets to be manipulated.

Antimicrobial Peptides as Mediators of Innate Immunity in Teleosts.

Mediators of gut mucosal immunity and inflammation.

Growth factors as mediators of testicular cell-cell interactions.

MicroRNAs in Rice Innate Immunity.

Evolutionary genetics of insect innate immunity.

Duality at the gate: Skin dendritic cells as mediators of vaccine immunity and tolerance.

MicroRNAs as Important Players in Host-hepatitis B Virus Interactions.

Extracellular microRNAs from the epididymis as potential mediators of cell-to-cell communication.

MicroRNAs transported by exosomes in body fluids as mediators of intercellular communication in cancer.

Soluble mediators regulating immunity in early life.

Regulation of immunity and inflammation by mediators from macrophages.

The rise of the undead: pseudokinases as mediators of effector-triggered immunity.

Frugivory, seed predation, and insect-vertebrate interactions.

Insect prophenoloxidase: the view beyond immunity.

Genome scale transcriptomics of baculovirus-insect interactions.

The Role of microRNAs in Bovine Infection and Immunity.

vector interactions.

The diversity of insect antiviral immunity: insights from viruses.

Exosomes as mediators of neuroinflammation.

Interactions between Innate Immunity, Microbiota, and Probiotics.

Intramolecular hydrophobic interactions are critical mediators of STAT5 dimerization.