Roles and regulation of microRNAs in cytomegalovirus infection

Biochimica et Biophysica Acta 1809 (2011) 613–622

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g r m

Review

Roles and regulation of microRNAs in cytomegalovirus infection☆ Lee Tuddenham, Sébastien Pfeffer ⁎ Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de biologie moléculaire et cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg, France

a r t i c l e

i n f o

Article history: Received 14 March 2011 Received in revised form 12 April 2011 Accepted 14 April 2011 Available online 21 April 2011 Keywords: Cytomegalovirus MiRNA Herpesvirus HCMV MCMV RCMV

a b s t r a c t MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression post-transcriptionally via binding to complementary sites typically located in the 3′ untranslated regions (UTRs) of their target mRNAs. This ancient regulatory system has been conserved in eukaryotes throughout evolution, and it is therefore unsurprising that certain viruses have evolved to express their own miRNAs. Since the initial discovery of Epstein–Barr virus (EBV) derived miRNAs in 2004, over 230 viral miRNAs have been identified, the majority arising from herpesviruses. Although the functions of most viral miRNAs remain to be elucidated, an increasing number of their cellular and viral targets have been experimentally validated. Due to their nonimmunogenic nature, viral miRNAs represent an elegant tool for the virus to evade the host immune system, and likely play a key role in the latent/lytic switch during the viral lifecycle. In this review, we will focus on the interactions of cytomegaloviruses with cellular and viral miRNAs during infection. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation. © 2011 Elsevier B.V. All rights reserved.

1. Introduction MicroRNAs (miRNAs) are ~22 nucleotides (nt) long RNAs that were first identified from forward genetic screens of developmental timing mutants in C. elegans [1–3]. Since these initial reports, miRNAs have been detected throughout the animal kingdom [4–6], as well as in plants [7], and more recently in several DNA viruses [8,9]. Interest in these tiny regulators has increased dramatically over the past decade with more than 15,000 miRNA sequences listed in the latest release of the miRNA registry, miRBase [10]. The biogenesis of miRNAs has been extensively reviewed in the literature [11,12]. Briefly, miRNA biogenesis is a step-wise, compartmentalized process that begins with the transcription of a primary-miRNA (pri-miRNA) mostly by RNA polymerase II to generate 5′-capped, polyadenylated transcripts containing one or more hairpin structures [13]. The RNase III enzyme Drosha cleaves the pri-miRNA in concert with DGCR8 to yield a stemloop precursor miRNA (pre-miRNA) [14], which is subsequently recognized and exported to the cytoplasm by Exportin5 (Exp5) [15,16]. A second RNase III enzyme, Dicer, then cleaves the hairpin into a miRNA/miRNA* duplex [17,18], which is then unwound to incorporate the mature strand into an Argonaute containing RNAinduced silencing complex (RISC) [19]. Mature miRNAs can then either direct translational inhibition or cleavage of their mRNA targets. In plants, miRNAs generally mediate cleavage after they base pair in full with some of their targets, whereas in animal systems, cleavage is exceptional [20]. Typically, nucleotides 2 to 8 of the ☆ This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation. ⁎ Corresponding author. Tel.: + 33 3 88 41 70 60; fax: + 33 3 88 60 22 18. E-mail address: [email protected] (S. Pfeffer). 1874-9399/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagrm.2011.04.002

miRNA, namely the ‘seed’ appear to be crucial in miRNA-target recognition [21]; this partial complementarity (often accompanied by additional 3′ compensatory pairing) leads to translational repression of the target and/or its destabilization. Localization of RISC-bound mRNAs to cytoplasmic processing bodies (P bodies) links them to the decapping and deadenylation complexes associated with mRNA degradation. However, the precise mechanisms of miRNA-mediated downregulation of protein expression, or which factors influence the decision over either translational repression or mRNA decay are unknown [22,23]. Currently, over 1000 miRNAs have been identified in humans, and almost 700 from mouse [10]. As any given miRNA is predicted to regulate tens to several hundreds of mRNAs, the potential for miRNAmediated genome-wide regulation is generally accepted; statistical reports cite that 30–60% of all human mRNAs are direct miRNA targets [24,25]. MiRNAs can often exhibit striking cell/tissue or developmentally specific expression profiles, whereas others display a more ubiquitous pattern [26,27]. Such traits have aided scientists in pinpointing some of their functions, and as such miRNAs have been reported to be involved in diverse cellular and disease processes, including: morphogenesis, hematopoiesis, tumorigenesis and neurogenesis (reviewed in: [28]). 1.1. Viral miRNAs The complexity of an organism and the number/diversity of miRNAs it possesses are generally regarded as evolutionary correlated; complex organisms have typically evolved to express a greater number of miRNAs than their ancient counterparts [29]. MiRNAs that regulate fundamental cellular processes are likely to be the most

614

L. Tuddenham, S. Pfeffer / Biochimica et Biophysica Acta 1809 (2011) 613–622

ancient, and thus conserved throughout bilateral evolution, e.g. let-7 and miR-100. The finding that some DNA viruses express their own miRNAs (although none of which have been reported to encode their own factors essential for miRNA biogenesis [30]), represents a complexity to viruses previously overlooked by virologists. Unlike viral proteins, viral miRNAs, akin to their cellular counterparts are non-immunogenic, and therefore can function undetected by the cellular immune response. As such, miRNAs offer a stealthy tool for viruses to regulate their environment throughout their lifecycle by down-regulating cellular and/or viral transcripts. Such hijacking of a host regulatory mechanism represents yet another ingenious play by certain viruses to thwart host defenses, and opens up potential targets for therapeutics. To date, a total of 235 viral miRNAs are listed in miRBase, almost entirely from members of the herpesvirus family, although a small number of miRNAs have been cloned from adenovirus, an ascovirus, a baculovirus, as well as a handful of polyomaviruses [31–35]. These viruses share in common their nuclear replication, and as such have access to the Drosha/DGCR8 microprocessor complex analogous to cellular miRNAs. Indeed, no miRNAs have as yet been identified from DNA viruses that replicate in the cytoplasm. Although, three miRNAs are purported to derive from HIV-1 [36,37], there is some controversy regarding their validity [9,38]. Despite the counterintuitive nature of a cytoplasmic RNA virus excising part of its own genome to generate pre-miRNAs, insertion of an EBV pre-miRNA into a cytoplasmically replicating flavivirus [39], or of the cellular miR-124 into the Sindbis virus genome [40], yielded functional mature miRNAs without impeding viral replication. Although the exact mechanisms of their maturation are unknown, such reports open up the possibility that many more viruses will be shown to generate non-canonical miRNAlike small RNAs: the debate is still open. 1.2. Herpesvirus miRNAs

picture of HCMV pathogenesis may be drawn to aid researchers in developing tools to counteract the virus. 2. Identification and characteristics of cytomegalovirus miRNAs 2.1. HCMV miRNAs So far, HCMV has been shown to encode for 11 miRNA precursors, which give rise to at least 14 mature miRNAs (see Table 1). Unlike members of the alpha and gamma herpesvirinae, HCMV miRNAs were identified in cells undergoing lytic infection and are distributed throughout the genome either individually or in small clusters on either genomic strand (Fig. 1A) [9,44,45]. Pfeffer et al. used a computational method to predict the likelihood of pre-miRNA structures based on features of known human miRNAs in a range of DNA and RNA viruses, including HCMV. Eleven precursor miRNAs from 9 distinct loci were predicted. Subsequent small RNA cloning from primary human foreskin fibroblasts (HFFs) infected with a clinical isolate of HCMV (VR1814) led to the identification of 9 HCMV pre-miRNAs (see Table 1) [9]. Dunn et al. carried out classical small RNA cloning from HFFs and astrocytoma U373MG cells infected with the HCMV TowneBAC strain, and subsequently cloned and validated 3 miRNAs, miR-UL23-5p, miR-UL23-3p and miR-US24 [44] (later renamed miR-UL22A-5p, miR-UL22A-3p and miR-US25-1 respectively [47]). Grey et al. based their search for HCMV miRNAs on the degree of conservation between the genomes of chimpanzee cytomegalovirus (CCMV) and HCMV, and predicted 13 candidates that matched their strict bioinformatic criteria. Five of these were verified by northern blotting in lytically infected cells, including two novel miRNAs, miR-UL70-1 and miR-US4-1 [45]. The exact number of HCMV miRNAs awaits more extensive studies of HCMV infected cells, preferably by deep sequencing. It is quite likely that HCMV may encode as of yet uncharacterized miRNAs and/or novel small RNA species that may have been missed due to their rarity and/or tissuespecific expression. Of note, miR-US33-1 and miR-UL148D-1 have not been verified by northern but were later verified by real time PCR from clinical isolates of HCMV [48].

MiRNAs have so far been identified in 5 of the 8 human herpesviruses; despite their comparable genome size and related ancestry, computational methods to predict pre-miRNA structures in the genomes of HHV-3, HHV-6 or HHV-7 did not identify any plausible candidates [9]. Further supporting this hypothesis, deep sequencing of varicella zona virus (VZV) latently infected trigeminal ganglia failed to identify any VZV-derived miRNAs [41]. Unlike alpha and gamma herpesviruses, whose miRNAs are expressed in large clusters from latency-associated transcripts (LATs) (reviewed in [30]), cytomegalovirus miRNAs are distributed throughout the genome, and associated with productive lytic infection [9,42–45].

CCMV is the closest relative of HCMV, and although miRNAs have not been experimentally identified in CCMV, 10 out of the 11 HCMV pre-miRNAs share a high degree of sequence homology (62–97%) to predicted CCMV pre-miRNAs; 8 share identical seed regions, and 5 share identical mature miRNA sequences [47]. HCMV miRNAs can be

1.3. Human cytomegalovirus (HCMV): clinical significance

Table 1 Cytomegalovirus miRNAs.

The seroprevalence of HCMV in human populations varies between 45 and 100%, with higher incidence reported in developing countries (geographical factors, socioeconomic status, and race are contributing factors to worldwide differences in seroprevalence) [46]. Following primary infection, HCMV persists for the life of the individual in a latent form, with reactivation accompanied by virus shedding in saliva and other bodily fluids. Although HCMV does not present a significant problem to the immunocompetent, congenital HCMV infection is the leading viral cause of birth defects in industrialized nations. In immunocompromised patients, such as persons with AIDS or those receiving immunosuppressing drugs for allogeneic bone marrow or organ transplants, HCMV presents with a high risk of morbidity and mortality. As such HCMV presents a significant medical and financial burden, warranting the development of a vaccine and antiviral therapeutics. The finding that HCMV encodes miRNAs opens up a new field in HCMV research; through identification of their cellular and viral targets a more complete

2.2. Identification of chimp, mouse, and rat cytomegalovirus miRNAs

Virus

Number of miRNAs

miRNA names

References

HCMV

11

[9,44,45]

MCMV

18

RCMV

24

miR-UL22A-1; miR-UL36-1; miR-UL70-1; miR-UL112-1; miR-UL148D-1; miR-US4-1; miR-US5-1; miR-US5-2; miR-US25-1; miR-US25-2; and miR-US33-1 miR-m01-1; miR-m01-2; miR-m01-3; miRm01-4; miR-m21-1; miR-M23-1; miR-m22-1; miR-M23-2; miR-M44-1; miR-M55-1; miRm59-1; miR-m59-2; miR-M87-1; miR-m88-1; miR-M95-1; miR-m107-1; miR-m108-1; and miRm108-2 miR-r1-1; miR-r1-2; miR-r1-3; miR-r1-4; miR-r6-1; miR-R37-1; miR-r43.1-1; miRr43.1-2; miR-OriLyt-1; miR-OriLyt-2; miRR87-1; miR-R90-1; miR-R91-1; miR-r95.1-1; miR-r111.1-1; miR-r111.1-2; miR-r111.1-3; miR-r111.2-1; miR-r111.2-2; miR-r111.2-3; miR-r111.2-4; miR-r111.2-5; miR-r111.2-6; and miR-r170-1

[42,43]

[50]

L. Tuddenham, S. Pfeffer / Biochimica et Biophysica Acta 1809 (2011) 613–622

A

615

US5-1 UL70-1

UL22A-1

UL112-1

UL148D-1

US4-1

US5-2

HCMV UL 22A

OriLyt

UL36

UL23

UL70

UL114

UL150

US3

US6

US24

US7

US26

US29 US30

US25-2

UL36-1

US33-1

US25-1

B

m21-1

M59-1 M88-1

M59-2 M87-1

m22-1

M95-1

m107-1

MCMV m01

m02 m03

m21

m22

M43

m01-3

M87

M88

M94 M95

7.2kb intron

m107 m108

M44-1

M23-2

m01-1

M59 OriLyt

M23

m01-4

M44

m108-2

M23-1

m108-1

m01-2 OriLyt-1

r6-1

OriLyt-2

r111.1-1

R87-1

r111.1-3 r111.1-2

RCMV r6

r1

R43 R23

r1-1 r1-3

r43.1

OriLyt

r43.1-2

R86

R96

R97

r111.1

r95.1

r111.2 r111.2-1

r1-2

r43.1-1

r1-4

r95.1-1

r111.2-2

r111.2-6 r111.2-4 r111.2-5

r111.2-3

Fig. 1. Schematic representation of cytomegaloviruses miRNAs genomic organization. Open arrows indicate validated and predicted open reading frames, while miRNAs are indicated by black arrows, which indicate the strand from which they are expressed. A. HCMV miRNAs are almost all predicted to be conserved in the chimp CMV (indicated by a gray shadowing of the miRNA name). B. MCMV and RCMV miRNAs are located in conserved positions, as indicated by the alternative gray and white background. In this figure, only the miRNAs located in conserved loci are indicated. OriLyt, lytic origin of replication.

studied in vitro in permissive cell lines; however, appreciation of their function in vivo is clearly not possible. In addition, although CCMV and HCMV share homology in several of their miRNAs, (which are more likely to have kept evolutionary important functions for the virus) chimpanzees are a protected species and as such in vivo work is likely to be limiting. By contrast, although more distantly related to humans than chimpanzees, rhesus monkeys can be infected with rhesus cytomegalovirus (RhCMV), which shares similarity to HCMV and CCMV, and as such is being actively pursued as a non-human primate model for studying CMV biology [49]. It will be of importance for scientists working on such model systems to fully identify the viral miRNAs of these closely related cytomegaloviruses to develop a better understanding of the unique differences between them and their respective host–pathogen interactions. While non-human primate models of infection are highly valuable tools in dissecting the roles of CMV infection, small animal models offer greater flexibility and as such are routinely employed to characterize CMV pathogenesis. MCMV has been routinely used as an in vivo model for the study of cytomegalovirus infection for decades. The finding that MCMV also expresses its own unique miRNAs during lytic infection was reported in 2007 [42,43]. Small RNA cloning identified that MCMV encodes 18 pre-miRNAs (Table 1), producing 21 mature miRNAs (or 27 small RNA species), which are distributed throughout the genome either as individual transcripts or from small clusters generated from either strand (Fig. 1B) [42,43]. Dölken et al. cloned small RNAs from NIH-3T3 fibroblasts infected with MCMV at a multiplicity of infection (MOI) of 10 at 24, 48 and 72 h time points. A striking increase in the contribution of viral miRNA to global miRNAs levels from 35% to 61% was noted [43]. The population of viral miRNAs in MCMV seems to be dependent on cell type and MOI; this may relate to the permissibility of the cells to infection [47]. Infection of bone marrow macrophage (BMM) cells or mouse embryonic fibroblast (MEF) cells with an MOI of 1.0 yielded

0.5% and 30% of viral clones respectively [42]. Clearly, maximizing the infection rate of cells is likely to increase the chances of identifying viral miRNAs, but cells undergoing active infection by viruses maybe more liable to the cloning of RNA degradation products as noted for HCMV [9]. To validate the expression of MCMV miRNAs during the course of in vivo infection, Dölken et al. isolated RNA from liver, lungs, and spleen of infected mice and performed RNase protection assays for the highly expressed miRNAs: miR-m01-4, miR-M23-2 and miRM44-1. Levels of these miRNAs in infected organs correlated with viral load, indicating that miRNA production is an important feature of active viral replication [43]. To expand upon their analysis, deletion mutants for these miRNAs were created using bacterial artificial chromosome (BAC) technology and used to infect murine embryonic fibroblasts (MEFs). The MCMV-ΔmiR-M23-2 mutant is additionally deleted for miR-m21-1, as this miRNA is encoded antisense to miRM23-2. None of these mutants demonstrated significant defects in their ability to replicate on MEFs, although an increase in miR-m01-3 expression, and alterations in the level of the immediate early transcript 1 (IE1) were observed for the ΔmiR-m01-4 mutant [43]. In addition to mice, rats can also be used as small animal models for cytomegalovirus biology. RCMV was recently shown to express its own unique miRNAs [50]. This study is of particular interest, as it is the first to use a deep-scale sequencing approach from both in vitro infected fibroblasts (MOI of 0.1), as well as from in vivo persistently infected salivary glands. The authors identified 24 small RNAs mapping to hairpin structures (Table 1), two of which (miR-r95.1-1 and miR-r170-1) were specific to the salivary gland. However, these two small RNAs are 17-nt in length, and although this is not exceptional, further evidence of their expression is necessary to validate them as bona fide miRNAs. Interestingly, miR-R111.1-2 displayed a clear predominance for salivary gland, and its level increased from 7 to 28 dpi, indicating that this miRNA may play a role in viral persistence [50]. In contrast, miR-R87-1 was highly expressed

616

L. Tuddenham, S. Pfeffer / Biochimica et Biophysica Acta 1809 (2011) 613–622

in most tissues at 7 dpi, but persisted at 28 dpi in salivary glands albeit at levels lower than miR-R112.1-2. In addition, RCMV miRNAs displayed significant 3′, as well as 5′ heterogeneity, with clear differences in isoform usage between fibroblasts and salivary glands [50]. Such 5′ heterogeneity has been previously noted for miR-M23-2 of MCMV [42], and may represent a strategy to broaden the regulatory ability of a miRNA through the use of a ‘shifted-seed’. Although challenging, it will be interesting to understand the functional significance of such miRNA heterogeneity in an in vivo context. HCMV, MCMV and RCMV miRNAs do not share sequence homology, although some are similarly distributed throughout the genome as individual miRNAs or as small clusters (Fig. 1A and B). Unlike HCMV, MCMV and RCMV encode for miRNAs within or proximal to the origin of lytic replication (Fig. 1B) as well as expressing a greater number of miRNAs than HCMV. This feature may reflect their species-specific function, potential redundancy, or simply that HCMV encodes for additional uncharacterized miRNAs. 2.3. Expression kinetics of cytomegalovirus miRNAs Depending on the requirements of viral protein synthesis or viral DNA replication, herpesviral gene expression can be divided into three kinetic classes: immediate early (IE), early, and late classes. IE gene expression is not dependent on de novo protein synthesis, whereas expression of early genes can be blocked with protein synthesis inhibitors such as cycloheximide (CHX). By contrast, late genes are dependent on the synthesis of viral DNA, and can be differentiated from earlier classes by blocking viral DNA replication using Foscarnet (FOS). HCMV miRNAs (except for miR-UL70-1 that displays immediateearly kinetics) display early kinetics [45]. Interestingly, miR-UL36-1 is located within an intron of the immediate-early gene UL36, so if processed by splicing it should naturally be expressed with the same kinetics as its host gene. However, in the presence of CHX pre-miRUL36-1 increased remarkably but was not processed to the mature form. Either the processing of its pre-miRNA is dependent on factors that require the synthesis of early viral proteins (or cellular factors with short half-lives), or this miRNA is transcribed independently from UL36 [45]. As with HCMV, the majority of MCMV miRNAs are early genes. Several MCMV pre-miRNAs were detectable after treatment with CHX, although unlike HCMV-miR-UL36-1, did not accumulate [43]. Differentiating early and immediate-early kinetics for viral miRNAs may be biased by the effects of inhibiting protein synthesis on the miRNA biogenesis machinery, as cellular miRNAs (commonly used as controls) are present prior to drug treatment and may be stable for extended periods [47]. Unlike the majority of cytomegalovirus miRNAs, MCMV miRNAs from the m107/108 cluster display late kinetics [42]. The presence of this cluster located adjacent to a 7.2-kb stable intron important for progress to the persistent phase of infection in vivo [51] warrants further study into the roles of these miRNAs in vivo.

their own transcriptome at each stage of infection [8]. MCMV and RCMV express miRNAs with early or late kinetics (late miRNAs have not yet been identified for HCMV), and some early genes such as miRUL112-1 increase dramatically in their expression during infection, suggesting a situation whereby temporal control of viral infection by miRNAs is plausible. For instance, one simple scenario would dictate that an increase of an early miRNA (or expression of a late miRNA) over a certain threshold could reduce IE genes important for the lytic cascade, permitting the virus to progress to the latent phase. Obvious candidate targets of viral miRNAs are known coding transcripts that are expressed antisense to the miRNA, whereby the miRNA could direct their cleavage. Although such a mechanism may at first seem counterintuitive for the virus, robust mechanisms are likely in place to ensure proper timing of miRNA-mediated regulation by cleavage. Examples of such regulation have been reported for polyomaviruses which all encode a late miRNA down-regulating the early T antigen transcript via cleavage [32,33,35,55,56]; the effects of which in natural in vivo settings are difficult to determine [55]. In addition, herpesviruses have been shown to direct mRNA cleavage (such as for EBV BALF5 by miR-BART2-5p) [57]. Interestingly, the IE transactivator ICP0 of HSV-1 lies directly antisense to the latency-associated miRH2-3p, but in this case the regulation of the transcript is via translation inhibition; cleavage does not seem to occur [58]. In respect to MCMV and RCMV, no viral targets of viral miRNAs are currently known; for HCMV the situation is different (see Table 2). HCMV encodes three miRNAs that lie antisense to coding transcripts (miR-UL112-1 to UL114, miR-US33-1 to US29, and miR-UL148D-1 to UL150) [48]. So far, only miR-UL112-1 has been shown to inhibit its antisense transcript UL114, a uracil DNA glycosylase important in DNA repair [48]. Other viral targets of miR-UL112-1 were found by searching putative binding sites conserved between HCMV and CCMV. After initially generating 3′ UTR datasets for both viruses, Grey et al. searched for putative miRNA binding sites in HCMV. Three out of 14 candidates were subsequently validated: UL123 (IE1, IE72), UL120/ UL121 and UL112/UL113 (Table 2) [59]. Murphy et al. independently confirmed the targeting of IE1 by miR-UL112-1 after applying a bioinformatic approach to predict viral targets of KSHV, EBV, HSV, and HCMV. They observed that all four herpesviruses were likely to use their miRNAs to target transactivators of viral gene transcription [60]. IE1 (immediate-early 1) is one of the first cytomegalovirus genes to be expressed, and although not critical for viral replication in vivo, mutant viruses exhibit severe attenuation and markedly reduced virulence [61]. Targeting of MCMV, or RCMV IE1 by miRNAs has not been verified, although an emerging commonality with herpesvirus miRNAs is that they are likely to be involved in the transition from lytic to latent states and vice versa. In respect to miR-UL112-1, it does not accumulate to significant levels until late in infection once transcription of viral genes is essentially complete [45,59]. As such, targeting of IE1 would inhibit viral transcription and the production of IE genes that may provide the virus with an advantage by reducing the amount of immunogenic material in a given cell until virions are fully

3. Targets of viral miRNAs The identification of miRNA targets remains a challenging aspect of miRNA research. Commonly used algorithms such as TargetScan [25,52], miRanda [53] and PicTar [54] rely heavily on evolutionary conservation between miRNAs and their targets. Such algorithms cannot be easily used to search for viral miRNA targets. Despite this, both viral and cellular targets of viral miRNAs have been identified using bioinformatic, biochemical, and PCR based strategies. 3.1. Viral targets of viral miRNAs The discovery of viral miRNAs raised the possibility that viruses could use them not only to regulate cellular genes, but also to regulate

Table 2 Viral targets of cytomegalovirus miRNAs and their validation methods: WB, western blot; Luc, luciferase reporter assay; I, validated in context of infection; KO, use of knockout/mutant virus for viral miRNA; and SM/del, target site mutation/deletion. miRNA

Target

Description

Validation method

Reference

HCMV-miRUL112-1

UL114

Uracil DNA glycosylase MIE gene, viral transactivator MIE region exons Viral DNA synthesis Maturation of replication compartments

Luc

[48]

Lucdel, WBI LucSM, WBI/KO Luc Luc Luc

[59] [60] [59] [59] [81]

IE72 UL120/1 UL112/3 UL117

L. Tuddenham, S. Pfeffer / Biochimica et Biophysica Acta 1809 (2011) 613–622

formed. In agreement with this hypothesis, expressing miR-UL112-1 prior to infection results in a general inhibition of viral DNA replication [59]. The effect on replication was specific to HCMV, as replication of HSV was unaffected by miR-UL112-1. In contrast, miRUS25-1 and miR-US25-2 were later identified to down-regulate viral replication, but this effect was not specific to HCMV; replication of HSV-1 and adenovirus, but not influenza virus, was also inhibited. Hence, these miRNAs likely target cellular genes important for the lifecycle of DNA viruses [48]. 3.2. Cellular targets of HCMV miRNAs Since the initial identification of viral miRNAs in EBV, researchers have postulated that viral miRNAs could function in interspecies regulation to modulate their environment through regulating both cellular and viral transcripts. Although for the majority of HCMV miRNAs there is no published data on their targets, studies of two highly expressed miRNAs, miR-UL112-1 and miR-US25-1 epitomize why HCMV is ingenious at counteracting host defenses. Table 3 lists known cellular targets of cytomegalovirus miRNAs. HCMV, as with all herpesviruses can persist in a latent state. In order for the virus to achieve latency, it must first of all avoid clearance by the immune system. Paramount to an organism's survival is the ability to recognize cells identifying as ‘self’ or ‘nonself’. Natural killer (NK) cells sense and elicit a robust immune response mediated via activating and inactivating receptors through binding of stress-induced ligands expressed by cells following DNA damage, heat shock, cellular transformation and viral infections [62]. It has been known for some time that both HCMV and MCMV encode multiple proteins to thwart the recognition of infected cell populations by NK cells (reviewed in: [63–65]). In this respect, recent findings that HCMV-miR-UL112-1 is also involved in mediating avoidance of NK cell recognition demonstrates the importance in identifying cellular targets of viral miRNAs. To work around the reliance on evolutionary conservation of most prediction programs, Stern-Ginossar et al. employed an alternative bioinformatic strategy to identify targets of the highly expressed miRUL112-1. The underlying principle of RepTar is that miRNA target sites can repeat within any given UTR. The algorithm therefore scans UTRs for repetitive elements that correspond to potential miRNA target sites. A complementary model (cRepTar) then searches for additional binding sites that may have been missed by the previous analysis (http://reptar.ekmd.huji.ac.il) [66]. The top prediction for miR-UL1121 was the stress-induced ligand MICB (MHC class I polypeptiderelated sequence B). This ligand is upregulated under stress and is recognized by the major activating receptor NKG2D expressed on natural killer cells. Ectopic expression of miR-UL112-1 from lentiviral vectors in a range of cancerous cells endogenously expressing both MICA and MICB resulted in specific down-regulation at the protein level of MICB, but not of MICA. MICB mRNA expression was unaffected, showing that miR-UL112-1 down-regulates MICB via translational inhibition and not mRNA degradation. The relationship Table 3 Cellular targets of cytomegalovirus miRNAs and their validation methods: WB, western blot; Luc, luciferase reporter assay; I, validated in context of infection; KO, use of knockout/mutant virus for viral miRNA; SM, target site mutation. Virus

miRNA

Target

Description

Validation method

References

HCMV

miR-US25-1

CCNE2 H3F3B

G1/S cyclin E2 H3 histone, family 3B Transcriptional corepressor Stress-induced NK cell ligand Chemokine

LucSM, WBI/KO LucSM

[82] [82]

WBI/KO

[82]

TRIM28

MCMV

miR-UL112-1

MICB

miR-M23-2

CXCL16

SM

Luc

, WB

LucI/KO

I/KO

[67] [87]

617

was shown to be of functional significance since NK cell mediated lysis was specifically reduced in cells transduced with miR-UL112-1. Using site-directed mutagenesis, the authors showed that miR-UL112-1 specifically down-regulates MICB by direct binding to its 3′-UTR. The authors also confirmed that this regulation also occurs in the context of infection [67]. This initial report raised interesting questions as to why a viral miRNA would target MICB when it already encodes a protein (UL16) that binds to and sequesters MICB to inhibit surface expression to evade the immune system [68,69]. In addition, how has MICA, which only differs by one nucleotide at the miR-UL112-1 binding site, escaped this issue? 3.3. Viral miRNAs usurp cellular miRNA target sites In a series of insightful reports, a deeper understanding of the relationship between miR-UL112-1 and MICB is developing. Predating the discovery that this particular viral miRNA subverts recognition of NK cells, it was known that in normal tissues MICA and MICB mRNAs can be detected without concomitant expression of protein. In 2008, SternGinossar et al. hypothesized that cellular miRNAs may play a role in targeting the mRNA of these two ligands as to set a threshold for the cell to go undetected by the immune response, while maintaining a pool of mRNA that could be readily translated under certain conditions [70]. Indeed, TargetScan predicted binding sites for several cellular miRNAs in the 3′-UTRs of both MICA and MICB. Cellular miR-20a, miR-93, miR106b, miR-373 and miR-520d were experimentally validated as being able to reduce both MICA and MICB protein, but not mRNA levels. As confirmation of these data, coordinated inhibition of these miRNAs led to the upregulation of MICA and MICB with concomitant increases in specific recognition and lysis by NK cells. To reinforce their hypothesis that cellular miRNA levels set a threshold for these ligands, they demonstrated that all these miRNAs were co-expressed with MICA and MICB mRNAs in a range of tissues and cell lines, and as such have the potential to control their regulation under normal conditions [70]. The authors noted that most of these miRNAs are oncogenic and proposed that their upregulation would inhibit surface expression of one or both of the ligands, enabling the cancerous cell to escape immunosurveillance. Indeed, in tumor-clearance assays, all MICA and MICB targeting miRNAs were shown to inhibit NK cell recognition and lysis in vivo compared to a control miRNA [70]. Although cellular miRNAs mediate both MICB and MICA translational inhibition, MICA has managed to avoid regulation by miRUL112-1. Nachmani et al. linked this phenomena with a shortening of the MICA 3′-UTR when they discovered that both an EBV miRNA (miR-BART2-5p) and a KSHV miRNA (miR-K12-7) also target MICB during authentic viral infection [71]. Unlike HCMV, these miRNAs were identified from latently infected cells and are expressed from transcripts associated with latency [8,9,72,73]. As shown for HCMV, these miRNAs specifically down-regulated MICB protein, and their over-expression resulted in a specific reduction in NK cell recognition and killing. Although MICA contains two potential target sites for the EBV miRNA, no viral miRNA-mediated regulation was observed [71]. With such a plethora of cellular and viral miRNAs targeting MICB, a natural question that arises is: do cellular and viral miRNAs compete for target sites, or do they act together upon MICB to enhance its downregulation? The answer is not straightforward. Cellular and viral miRNAs that possess overlapping target sites such as miR-UL112-1 and miR-373 are bound to compete for the same site, with overexpression of one miRNA effectively increasing overall regulation by binding to MICB transcripts that are not already bound by the other. Nachmani et al. explored this hypothesis in more detail, and observed that in addition to the known miRNAs previously identified to target MICB, miR-376 and miR-433 (which partially overlap sites for miRBART2-5p and miR-K12-7 respectively) also target MICB [74]. These two cellular miRNAs functionally target MICB at sites distinct from the

618

L. Tuddenham, S. Pfeffer / Biochimica et Biophysica Acta 1809 (2011) 613–622

other cellular miRNAs known to regulate MICB. Importantly, they observed that co-expression of these two miRNAs antagonized regulation of a MICB luciferase reporter. However, the surprise finding was that miR-UL112-1 and miR-376 act synergistically within infected cells to down-regulate MICB. Of note, the EBV and KSHV miRNAs only showed an additive effect on down-regulation when expressed with cellular miRNAs, presumably due to the use of distinct sites within the 3′-UTR. The authors identified that for miR-UL112-1 and miR-376a the proximity of their binding sites (24-nt apart) is likely to influence this mechanism [74]. It seems that the usurpation of cellular miRNA target sites by viral miRNAs represents a clever ploy by these three herpesviruses to maintain their regulatory networks of cellular pathways. Whether such synergism or potentially even antagonism is a common trick of viral miRNAs awaits confirmation. 3.4. Additional targets of HCMV miRNAs 3.4.1. Addressing the limitations of bioinformatics The power of bioinformatic tools to determine miRNA-target interactions is improving, but false-positive rates in such predictions are often high. Determining cellular and viral targets of viral miRNAs is further complicated by host specificity, even for closely related viruses such as CCMV and HCMV it can be assumed that important species-specific interactions exist that may be missed with bioinformatic approaches. To overcome these problems, powerful high throughput biochemical techniques have been recently developed that enable the identification of miRNA targets directly from Argonaute-containing RISC complexes. For each technique, a global comparative analysis of Ago2 bound miRNAs/mRNAs is performed following up/down regulation of specific miRNAs. RIP-Chip (Ribonucleoprotein ImmunoPrecipitation-gene Chip) [75–77], HITS-CLIP (High Throughput Sequencing of RNAs isolated by cross-linking immunoprecipitation) [78,79], and PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced CLIP) [80] have all been used to identify miRNA targets bound to Ago2. In addition to Ago2 IP-based approaches, novel hybrid-PCR techniques have recently been developed to identify targets of HCMV miRNAs [81]. The combination of a multitude of methods is likely to aid in the identification of miRNA targets; however, the challenges facing researchers now lie with analyzing and interpreting miRNA-target interactions and their relevance to HCMV pathogenesis during infection. 3.4.2. Biochemical identification of HCMV miRNA targets To determine the cellular targets of exogenously expressed HCMVmiR-US25-1, Grey et al. employed a RIP-CHIP approach using a myctagged Ago2 in parallel with pull-downs of biotinylated miR-US25-1 to generate two data sets to increase confidence in their target predictions. They demonstrated that miR-US25-1 targeted a range of cellular transcripts involved in cell cycle regulation, preferentially via binding to their 5'-UTRs (Table 3). To validate their approach, the authors went on to show that in the context of infection, miR-US25-1 targeted cyclin E2 and TRIM28; protein levels of these two genes increased in the relevant miRNA knockout mutant [82]. The identification of a range of cellular targets important for HCMV biology of a viral miRNA with antiviral properties against DNA viruses [48] highlights the importance to dissect miRNA-based host-pathogen interactions on a genome-wide scale. Recent publications proving that miRNAs can regulate their targets via binding to 5'-UTRs or coding sequences, suggest that the current dogma of 3′-UTR regulation needs to be addressed as bioinformatics tools for miRNA prediction are largely biased in this regard [83,84]. Currently, HCMV-miR-UL112-1 is unique among cytomegalovirus miRNAs in that it has clearly evolved two distinct and important functions for the virus: the targeting of cellular MICB to subvert NK cellrecognition [67], and the targeting of the viral IE1 to impact upon viral replication [60]. Whether, additional miRNAs have evolved to possess

similar multifunctional roles, or whether miR-UL112-1 is an exceptional case of co-evolutionary pressure remains to be determined. For the moment, rodent cytomegaloviruses miRNAs have not been awarded such a status, although it is likely that similar to HCMV, they may use their miRNAs to modulate responses to NK cells, as well as to regulate viral transactivators to properly coordinate their viral lifecycles. Only a determined effort to elucidate and compare the functions of cytomegaloviruses miRNAs will enable such questions to be answered. 4. Roles of viral miRNAs in vivo An advantage of the use of small animal models for the study of CMV is that the native host can be infected with mutant viruses generated by manipulating the viral genome as a bacterial artificial chromosome (BAC) [85,86]. Dölken et al. generated deletion mutants for miR-m01-4, miR-M23-2/m21-1 (located on opposing overlapping strands), and miR-M44-1 [43]. Although these deletion mutants did not impact significantly on viral replication in vitro, in an in vivo context the virus encounters a broad array of cell types and immunoregulatory factors that cannot be addressed in vitro. In order to gain insight into the relevance of MCMV miRNAs in vivo, Dölken et al. infected C57BL/6 and BALB/c mice with either wild type MCMV, a deletion mutant for miR-M23-2 (ΔmiR-M23-2), or a mutant disrupted for pre-miRNA processing (miR-M23-2-mut), and their respective revertant viruses. After 3 days of infection, no effect on viral replication was observed for either mutant in the organs tested, but following 14 days, titers were reduced ~100-fold specifically in the salivary glands of C57BL/6 mice; whereas all viruses replicated to similar levels in the lungs irrespective of miR-M23-2/m21-1 expression [87]. In contrast, only a mild but significant attenuation of replication (2-fold) was observed in BALB/c mice under the same conditions. Lowering the viral dose 20-fold markedly increased the level of miRNA-mediated attenuation, implying that viral load and genetic strain have an impact on the miRNA-specific effect. These features correlate with the ability of different mouse strains to control MCMV infection. Unlike BALB/c, C57BL/6 mice are able to keep MCMV infection in check as they express the Ly49H receptor that recognizes the m157 viral protein to elicit an effective NK response against the virus [88,89]. The specific attenuation of the miR-M23-2-mut held for a range of strains with differing genetic backgrounds, including the 129/SvJ.IFNγR−/−, denoting that the observed effect is independent of IFNγ. Interestingly, this phenotype was reverted only by the combined depletion of NK and CD4+ T cells [87]. Although the precise mechanisms of how miR-M23-2 and/or miRm21-1 act within the salivary glands are unknown, it is clear that they are involved in countering the host immune response by modulating interactions with both NK and CD4+ T cells to favor viral persistence. To further this hypothesis, RepTar [66] was used to identify potential targets of these two miRNAs with a focus on the immune response. The chemokine CXCL16 was identified as a top target of both miRNAs and validated as a target of miR-M23-2 using luciferase reporter assays in the context of infection using mutant viruses (Table 3). However, the regulation of CXCL16 is unlikely to be the sole responsible factor in the attenuation observed, indeed these miRNAs were predicted to target numerous genes involved in the immune response [87]. Elucidation of the precise mechanisms will rely on a comprehensive analysis of their targets in vivo through the use of genome-wide target identification strategies as listed in Section 3.4.1. 5. Interactions of cytomegaloviruses with host cellular miRNAs 5.1. Regulation of viruses by cellular miRNAs In addition to expressing their own miRNAs, viruses have been shown to interact extensively with host miRNAs. Indeed, viruses can be targeted by cellular miRNAs, and the outcome can have both

L. Tuddenham, S. Pfeffer / Biochimica et Biophysica Acta 1809 (2011) 613–622

positive and negative consequences for the virus. However, these interactions are not only occurring unidirectionally, since viral infection can also impact on the expression level of cellular miRNAs. Finally, as we will see, some loops in the relationship between host and pathogen miRNAs have been described. For example, miRNAs may negatively regulate viral replication, but in turn the virus will affect the accumulation of these miRNAs. The first evidence that a cellular miRNA can directly base pair with a viral transcript to affect viral replication came from a study on primate foamy retrovirus (PFV). Voinnet and colleagues showed that the PFV genome contained a putative binding site for miR-32 in a position that would be conserved in all viral transcripts. Blocking miR-32 with antisense oligonucleotides or mutating the binding site, resulted in an increase in the accumulation of PFV mRNA [90]. Huang et al. later reported similar findings for HIV-1. They found that the 3′ end of HIV-1 messenger RNA is targeted by multiple miRNAs, such as miR-28, miR125b, miR-150, miR-223 and miR-382, and that the miRNA-mediated regulation of these viral transcripts played a role in the maintenance of viral latency [91]. The laboratory of J. Han provided another example of miRNAs controlling viral RNA accumulation. They reported that Dicer-deficient mice were hypersusceptible to the negative-strand RNA vesicular stomatitis virus (VSV), and pinpointed the effect to four miRNA binding sites contained within the P and L genes [92]. These findings might indicate that cellular miRNAs represent yet another tool in the cellular arsenal against invading viral pathogens. Nonetheless, being targeted by miRNAs is not always detrimental for the virus. The most striking example of a miRNA-mediated positive impact for a virus is the interaction of the liver-specific miR-122 with hepatitis C virus (HCV). Indeed, two binding sites for this miRNA were found in the 5′ non-coding region of the HCV genomic RNA. Puzzlingly, mutations of these sites, or blocking miR-122 with antisense approaches, resulted in the exact opposite effect as for other viruses, i.e. a drop in HCV RNA accumulation [93]. The importance of miR-122 in the HCV life cycle makes it an interesting therapeutic target, and in this sense encouraging results have already been obtained in the chimp [94]. The exact mechanism by which miR122 mediates its proviral activity is currently unknown, but a recent report from the Sarnow laboratory indicates that it could be through an unconventional mechanism involving the masking of the viral RNA 5′ end to prevent its recognition by innate immunity receptors [95]. 5.2. Regulation of cellular miRNAs during herpesvirus infection To date, no direct targeting of herpesviruses by miRNAs has been reported. However, there are numerous evidences of regulation of miRNA expression during herpesvirus infections. EBV was the first virus shown to alter cellular miRNA expression profiles. More precisely, published results focused mainly on two miRNAs, miR146 and miR-155, which are induced upon EBV infection [96,97]. Interestingly, these miRNAs are induced by the viral protein LMP1 through the NF-κB pathway, but are also involved in regulating the pathway itself [98]. Other miRNAs regulated by EBV include miR-200, miR-429, miR-29b and miR-10b [99–101]. For further details on EBV and cellular miRNAs, we refer the reader to the contribution of F. Grässer in this issue. On the other hand, KSHV does not induce expression of miR-155 and this miRNA is typically expressed at a low-level in infected cells. However, this virus has developed an alternative strategy to maintain miR-155 regulatory networks by expressing an ortholog of miR-155, miR-K12-11 [102,103]. KSHV does nevertheless regulate cellular miRNA expression, among which miR-132, miR-212, miR-146a are all altered upon infection [104]. The case of miR-132 is particularly interesting; it is induced very early (6 h) after infection in a CREB (cyclic AMP response element binding protein) transcription factordependant manner. In respect to KSHV, CREB is expressed minutes

619

after exposure to the virus. One of the identified miR-132 targets is the p300 transcriptional co-activator, which together with CREB-binding protein are essential for the initiation of antiviral immunity through interferon. Interestingly, miR-132 is also induced by viruses such as HSV1 and HCMV (although at later time points of infection for this virus), indicating a more general involvement of this miRNA in the control of antiviral responses. The regulation of miRNAs by herpesviruses to get a grip on host defense systems is not limited to miR-132. Indeed, in an earlier study aimed at identifying cellular miRNA changes induced by HCMV infection, Pellett and collaborators identified a handful of miRNAs showing significant up or down-regulation at various time points following infection. Among the down-regulated miRNAs, two miRNAs, miR-100 and miR-101, were shown to target mTOR (both miRNAs) or raptor (only miR-100) [105]. These proteins are essential components of the mTOR regulatory pathway, which plays important roles in cell death, cell cycle and most importantly HCMV replication (see Fig. 2). As a consequence, over-expressing miR-100 and miR-101 in HCMV-infected cells resulted in a strong inhibitory effect on virus yield [105]. In order to obtain a comprehensive picture of cellular miRNAs with broad antiviral or proviral activity, the Buck laboratory undertook a large-scale approach in 3T3 fibroblasts, consisting of systematically over-expressing or inhibiting all mouse miRNAs and infecting them with GFP-expressing viruses such as MCMV, MHV-68 and HSV-1 [106]. They found that some miRNAs, such as miR-30b, miR-30d and miR-93, had proviral effects for all viruses, whereas others, like miR24, miR-103, miR-214 and miR-199-3p, appeared to have global antiviral effects. The antiviral properties of these four miRNAs were also active for HCMV, and two of them, miR-214 and miR-199-3p, also worked on the Semliki forest RNA virus. In parallel to this analysis, miRNA expression levels were measured upon MCMV or HCMV infection, and both miR-199 and miR-214 were down-regulated in infected cells, indicating that these viruses have evolved ways of counteracting the antiviral properties of specific subsets of miRNAs via modulating their accumulation (Fig. 2). 5.3. Destabilization of cellular miRNAs during viral infection Among the miRNAs possessing antiviral activity against MCMV, miR-27 was one of the most potent identified in the Buck study. Interestingly, it was also one of the most dramatically down-regulated miRNAs following MCMV, but not HCMV infection [106]. This result confirmed earlier observations we made using small RNA cloning and sequencing of MCMV-infected cells. We found that the net effect of MCMV on cellular miRNA profiles was generally modest, with the exception of miR-27a and b, which were completely absent late in infection [107]. Surprisingly, we also noted that miR-23a/b and miR24 (miRNAs located in the same genomic cluster as miR-27a/b) were not affected by MCMV. This observation led us to hypothesize that the regulation of miR-27a was occurring at the posttranscriptional level. Using both northern blot and real-time PCR analysis, we then showed that the levels of pri-miRNA and pre-miRNAs were unaffected upon MCMV infection. Furthermore, the quantity of the passenger form (star) of the miRNA duplex was comparable in both mock and MCMV infected cells. Altogether, our results indicated that miR-27 was regulated at the level of the stability of its mature form. Inhibiting transcription prior to infection prevented this regulation to occur, pointing towards the requirement of a viral transcript for destabilization of miR-27 [107]. Intriguingly, the herpesvirus saimiri (HVS) (a virus that infects monkeys) seems to use a strategy similar to MCMV to regulate the accumulation of miR-27. Cazalla et al. reported that some of the very abundant non-coding HSUR (Herpesvirus Saimiri Urich RNA) RNAs (the function of which had been elusive for years) can bind cellular miRNAs via Ago2 [108]. In particular, HSUR-1 can bind miR-27a and regulate its abundance and decay in a sequence and

620

L. Tuddenham, S. Pfeffer / Biochimica et Biophysica Acta 1809 (2011) 613–622

Cell genomic DNA

Repression: -miR-100, 101 -miR-199a, 214 Activation: -miR-132

pri-miRNA AAA

MCMV HCMV BOTH

pre-miRNA

Degradation: miR-27a/b

mTOR proteins

Antiviral: miR-199a, 214 miR-100, 101 miR-27a/b

mature miRNA

Proviral: miR-132

p300

Fig. 2. Interactions of cytomegaloviruses with cellular miRNAs. Dotted arrows indicate at which steps and in which direction interactions can occur. Colors indicate for which virus a regulation/interaction has been described: MCMV (red), HCMV (green) or both (blue). See text for more details.

binding-specific manner, and this viral RNA can be engineered to regulate another miRNA. As a result of miR-27 down-regulation, the authors also showed that some of its known cellular targets, such as FOXO1 [109] were up-regulated. The mechanism by which the mature form of miR-27 is destabilized upon binding of HSUR-1, or in MCMVinfected cells, is currently not known, but it could involve either 3′ end tailing followed by exonucleolytic degradation of the miRNA [110], or 5′ to 3′ degradation by exoribonucleases such as Xrn-2 [111]. Although two separate viruses down-regulate the same miRNA, it is still unclear as to why, especially since miR-27 was not identified as a miRNA with broad antiviral activity. In that respect, the identification of the cellular targets of this particular miRNA in both mouse and primates should yield important information regarding the biology of these viruses. For now, it can be seen as yet another example of coevolution of herpesviruses with their respective hosts. 6. Conclusions/perspectives Viruses exploit a number of elegant strategies to counteract the host immune response to prevent their subsequent elimination and destruction. This is also the case for HCMV and MCMV, whose exquisite subversion of the host immune system is a remarkable feat of nature and co-evolution. The identification that HCMV, MCMV, and now RCMV express their own unique miRNAs during lytic infection has opened up a new field of cytomegalovirus research, offering promise for the development of antiviral treatments that target these small non-immunogenic regulators. Although the analysis of their targets and roles in active infection is still in their infancy, a handful of well-conducted studies have confirmed that viral miRNAs are important regulators of viral pathogenesis. The use of genome-wide Ago2 IPs has already highlighted that viral miRNAs can regulate cell cycle genes and mediate their regulation through non-classical 5'-UTR interactions [82]. In the future, systematic approaches (preferably using miRNA knockout viruses) will be essential in delineating viral miRNA targetomes and their relevancy to viral pathogenesis. This aspect is particularly difficult to determine in an in vivo context. To this end, small animal models offer advantages in that they can be readily infected with mutant viruses and their roles elucidated throughout infection. Although analysis of miRNA targets of RCMV and MCMV is lagging behind that of HCMV, identification of tissue-specific expressed miRNAs [50], and the first in vivo phenotype of a miRNA knockout

mutant virus of attenuating viral persistence [87], infer that viral miRNAs will open up new avenues into the understanding of cytomegalovirus pathology. Crucially, it is still unknown whether any cytomegalovirus miRNAs are expressed in latently infected cells. If true, one can envision a situation whereby exogenous expression of viral miRNAs may coax the virus out of latency and lead to immune clearance. Alternatively, modulating viral miRNA expression in at-risk patients may subdue the extent of reactivation and associated pathological complications. In addition to the possibility of exploiting viral miRNAs to combat the virus, it is becoming increasingly clear that cellular miRNAs have a broad role to play in antiviral responses, and may represent an ancient arm of the innate immune response. Understanding the precise mechanisms of how viruses modulate cellular miRNA expression, and the implications thereof in respect to their targets (cellular and/or viral) will be essential in devising therapeutic stratagems in the future. Acknowledgments We would like to thank members of the Pfeffer laboratory for critical reading of the manuscript. Work in our laboratory is supported by the European Research Council (ERC Starting Grant ncRNAVIR 260767), by the Agence Nationale de la Recherche (ANR-08-MIEN005), and by an ATIP grant from CNRS. References [1] R.C. Lee, R.L. Feinbaum, V. Ambros, The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14, Cell 75 (1993) 843–854. [2] B. Wightman, I. Ha, G. Ruvkun, Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans, Cell 75 (1993) 855–862. [3] B.J. Reinhart, F.J. Slack, M. Basson, A.E. Pasquinelli, J.C. Bettinger, A.E. Rougvie, H.R. Horvitz, G. Ruvkun, The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans, Nature 403 (2000) 901–906. [4] M. Lagos-Quintana, R. Rauhut, W. Lendeckel, T. Tuschl, Identification of novel genes coding for small expressed RNAs, Science 294 (2001) 853–858. [5] N.C. Lau, L.P. Lim, E.G. Weinstein, D.P. Bartel, An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans, Science 294 (2001) 858–862. [6] R.C. Lee, V. Ambros, An extensive class of small RNAs in Caenorhabditis elegans, Science 294 (2001) 862–864. [7] B.J. Reinhart, E.G. Weinstein, M.W. Rhoades, B. Bartel, D.P. Bartel, MicroRNAs in plants, Genes Dev. 16 (2002) 1616–1626.

L. Tuddenham, S. Pfeffer / Biochimica et Biophysica Acta 1809 (2011) 613–622 [8] S. Pfeffer, M. Zavolan, F.A. Grasser, M. Chien, J.J. Russo, J. Ju, B. John, A.J. Enright, D. Marks, C. Sander, T. Tuschl, Identification of virus-encoded microRNAs, Science 304 (2004) 734–736. [9] S. Pfeffer, A. Sewer, M. Lagos-Quintana, R. Sheridan, C. Sander, F.A. Grasser, L.F. van Dyk, C.K. Ho, S. Shuman, M. Chien, J.J. Russo, J. Ju, G. Randall, B.D. Lindenbach, C.M. Rice, V. Simon, D.D. Ho, M. Zavolan, T. Tuschl, Identification of microRNAs of the herpesvirus family, Nat. Methods 2 (2005) 269–276. [10] S. Griffiths-Jones, H.K. Saini, S. van Dongen, A.J. Enright, miRBase: tools for microRNA genomics, Nucleic Acids Res. 36 (2008) D154–D158. [11] J. Winter, S. Jung, S. Keller, R.I. Gregory, S. Diederichs, Many roads to maturity: microRNA biogenesis pathways and their regulation, Nat. Cell Biol. 11 (2009) 228–234. [12] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116 (2004) 281–297. [13] Y. Lee, C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, V.N. Kim, The nuclear RNase III Drosha initiates microRNA processing, Nature 425 (2003) 415–419. [14] J. Han, Y. Lee, K.H. Yeom, Y.K. Kim, H. Jin, V.N. Kim, The Drosha-DGCR8 complex in primary microRNA processing, Genes Dev. 18 (2004) 3016–3027. [15] R. Yi, Y. Qin, I.G. Macara, B.R. Cullen, Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs, Genes Dev. 17 (2003) 3011–3016. [16] E. Lund, S. Guttinger, A. Calado, J.E. Dahlberg, U. Kutay, Nuclear export of microRNA precursors, Science 303 (2004) 95–98. [17] G. Hutvagner, J. McLachlan, A.E. Pasquinelli, E. Balint, T. Tuschl, P.D. Zamore, A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA, Science 293 (2001) 834–838. [18] R.F. Ketting, S.E. Fischer, E. Bernstein, T. Sijen, G.J. Hannon, R.H. Plasterk, Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans, Genes Dev. 15 (2001) 2654–2659. [19] S.M. Hammond, S. Boettcher, A.A. Caudy, R. Kobayashi, G.J. Hannon, Argonaute2, a link between genetic and biochemical analyses of RNAi, Science 293 (2001) 1146–1150. [20] S. Yekta, I.H. Shih, D.P. Bartel, MicroRNA-directed cleavage of HOXB8 mRNA, Science 304 (2004) 594–596. [21] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell 136 (2009) 215–233. [22] R.S. Pillai, S.N. Bhattacharyya, W. Filipowicz, Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol. 17 (2007) 118–126. [23] J. Krol, I. Loedige, W. Filipowicz, The widespread regulation of microRNA biogenesis, function and decay, Nat. Rev. Genet. 11 (2010) 597–610. [24] R.C. Friedman, K.K. Farh, C.B. Burge, D.P. Bartel, Most mammalian mRNAs are conserved targets of microRNAs, Genome Res. 19 (2009) 92–105. [25] B.P. Lewis, C.B. Burge, D.P. Bartel, Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets, Cell 120 (2005) 15–20. [26] P. Landgraf, M. Rusu, R. Sheridan, A. Sewer, N. Iovino, A. Aravin, S. Pfeffer, A. Rice, A.O. Kamphorst, M. Landthaler, C. Lin, N.D. Socci, L. Hermida, V. Fulci, S. Chiaretti, R. Foa, J. Schliwka, U. Fuchs, A. Novosel, R.U. Muller, B. Schermer, U. Bissels, J. Inman, Q. Phan, M. Chien, D.B. Weir, R. Choksi, G. De Vita, D. Frezzetti, H.I. Trompeter, V. Hornung, G. Teng, G. Hartmann, M. Palkovits, R. Di Lauro, P. Wernet, G. Macino, C.E. Rogler, J.W. Nagle, J. Ju, F.N. Papavasiliou, T. Benzing, P. Lichter, W. Tam, M.J. Brownstein, A. Bosio, A. Borkhardt, J.J. Russo, C. Sander, M. Zavolan, T. Tuschl, A mammalian microRNA expression atlas based on small RNA library sequencing, Cell 129 (2007) 1401–1414. [27] M. Lagos-Quintana, R. Rauhut, A. Yalcin, J. Meyer, W. Lendeckel, T. Tuschl, Identification of tissue-specific microRNAs from mouse, Curr. Biol. 12 (2002) 735–739. [28] N. Bushati, S.M. Cohen, microRNA functions, Annu. Rev. Cell Dev. Biol. 23 (2007) 175–205. [29] A. Grimson, M. Srivastava, B. Fahey, B.J. Woodcroft, H.R. Chiang, N. King, B.M. Degnan, D.S. Rokhsar, D.P. Bartel, Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals, Nature 455 (2008) 1193–1197. [30] R.L. Skalsky, B.R. Cullen, Viruses, microRNAs, and host interactions, Annu. Rev. Microbiol. 64 (2010) 123–141. [31] J. Singh, C.P. Singh, A. Bhavani, J. Nagaraju, Discovering microRNAs from Bombyx mori nucleopolyhedrosis virus, Virology 407 (2010) 120–128. [32] G.J. Seo, L.H. Fink, B. O'Hara, W.J. Atwood, C.S. Sullivan, Evolutionarily conserved function of a viral microRNA, J. Virol. 82 (2008) 9823–9828. [33] G.J. Seo, C.J. Chen, C.S. Sullivan, Merkel cell polyomavirus encodes a microRNA with the ability to autoregulate viral gene expression, Virology 383 (2009) 183–187. [34] M. Hussain, R.J. Taft, S. Asgari, An insect virus-encoded microRNA regulates viral replication, J. Virol. 82 (2008) 9164–9170. [35] C.S. Sullivan, A.T. Grundhoff, S. Tevethia, J.M. Pipas, D. Ganem, SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells, Nature 435 (2005) 682–686. [36] S. Omoto, M. Ito, Y. Tsutsumi, Y. Ichikawa, H. Okuyama, E.A. Brisibe, N.K. Saksena, Y.R. Fujii, HIV-1 nef suppression by virally encoded microRNA, Retrovirology 1 (2004) 44. [37] D.L. Ouellet, I. Plante, P. Landry, C. Barat, M.E. Janelle, L. Flamand, M.J. Tremblay, P. Provost, Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element, Nucleic Acids Res. 36 (2008) 2353–2365. [38] J. Lin, B.R. Cullen, Analysis of the interaction of primate retroviruses with the human RNA interference machinery, J. Virol. 81 (2007) 12218–12226. [39] H. Rouha, C. Thurner, C.W. Mandl, Functional microRNA generated from a cytoplasmic RNA virus, Nucleic Acids Res. 38 (2010) 8328–8337.

621

[40] J.S. Shapiro, A. Varble, A.M. Pham, B.R. Tenoever, Noncanonical cytoplasmic processing of viral microRNAs, RNA 16 (2010) 2068–2074. [41] J.L. Umbach, M.A. Nagel, R.J. Cohrs, D.H. Gilden, B.R. Cullen, Analysis of human alphaherpesvirus microRNA expression in latently infected human trigeminal ganglia, J. Virol. 83 (2009) 10677–10683. [42] A.H. Buck, J. Santoyo-Lopez, K.A. Robertson, D.S. Kumar, M. Reczko, P. Ghazal, Discrete clusters of virus-encoded micrornas are associated with complementary strands of the genome and the 7.2-kilobase stable intron in murine cytomegalovirus, J. Virol. 81 (2007) 13761–13770. [43] L. Dolken, J. Perot, V. Cognat, A. Alioua, M. John, J. Soutschek, Z. Ruzsics, U. Koszinowski, O. Voinnet, S. Pfeffer, Mouse cytomegalovirus microRNAs dominate the cellular small RNA profile during lytic infection and show features of posttranscriptional regulation, J. Virol. 81 (2007) 13771–13782. [44] W. Dunn, P. Trang, Q. Zhong, E. Yang, C. van Belle, F. Liu, Human cytomegalovirus expresses novel microRNAs during productive viral infection, Cell. Microbiol. 7 (2005) 1684–1695. [45] F. Grey, A. Antoniewicz, E. Allen, J. Saugstad, A. McShea, J.C. Carrington, J. Nelson, Identification and characterization of human cytomegalovirus-encoded microRNAs, J. Virol. 79 (2005) 12095–12099. [46] M.J. Cannon, D.S. Schmid, T.B. Hyde, Review of cytomegalovirus seroprevalence and demographic characteristics associated with infection, Rev. Med. Virol. 20 (2010) 202–213. [47] L. Dolken, S. Pfeffer, U.H. Koszinowski, Cytomegalovirus microRNAs, Virus Genes 38 (2009) 355–364. [48] N. Stern-Ginossar, N. Saleh, M.D. Goldberg, M. Prichard, D.G. Wolf, O. Mandelboim, Analysis of human cytomegalovirus-encoded microRNA activity during infection, J. Virol. 83 (2009) 10684–10693. [49] C. Powers, K. Fruh, Rhesus CMV: an emerging animal model for human CMV, Med. Microbiol. Immunol. 197 (2008) 109–115. [50] C. Meyer, F. Grey, C.N. Kreklywich, T.F. Andoh, R.S. Tirabassi, S.L. Orloff, D.N. Streblow, Cytomegalovirus microRNA expression is tissue specific and is associated with persistence, J. Virol. 85 (2011) 378–389. [51] C.A. Kulesza, T. Shenk, Murine cytomegalovirus encodes a stable intron that facilitates persistent replication in the mouse, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 18302–18307. [52] B.P. Lewis, I.H. Shih, M.W. Jones-Rhoades, D.P. Bartel, C.B. Burge, Prediction of mammalian microRNA targets, Cell 115 (2003) 787–798. [53] B. John, A.J. Enright, A. Aravin, T. Tuschl, C. Sander, D.S. Marks, Human MicroRNA targets, PLoS Biol. 2 (2004) e363. [54] A. Krek, D. Grun, M.N. Poy, R. Wolf, L. Rosenberg, E.J. Epstein, P. MacMenamin, I. da Piedade, K.C. Gunsalus, M. Stoffel, N. Rajewsky, Combinatorial microRNA target predictions, Nat. Genet. 37 (2005) 495–500. [55] C.S. Sullivan, C.K. Sung, C.D. Pack, A. Grundhoff, A.E. Lukacher, T.L. Benjamin, D. Ganem, Murine polyomavirus encodes a microRNA that cleaves early RNA transcripts but is not essential for experimental infection, Virology 387 (2009) 157–167. [56] P. Cantalupo, A. Doering, C.S. Sullivan, A. Pal, K.W. Peden, A.M. Lewis, J.M. Pipas, Complete nucleotide sequence of polyomavirus SA12, J. Virol. 79 (2005) 13094–13104. [57] S. Barth, T. Pfuhl, A. Mamiani, C. Ehses, K. Roemer, E. Kremmer, C. Jaker, J. Hock, G. Meister, F.A. Grasser, Epstein–Barr virus-encoded microRNA miR-BART2 downregulates the viral DNA polymerase BALF5, Nucleic Acids Res. 36 (2008) 666–675. [58] J.L. Umbach, M.F. Kramer, I. Jurak, H.W. Karnowski, D.M. Coen, B.R. Cullen, MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs, Nature 454 (2008) 780–783. [59] F. Grey, H. Meyers, E.A. White, D.H. Spector, J. Nelson, A human cytomegalovirusencoded microRNA regulates expression of multiple viral genes involved in replication, PLoS Pathog. 3 (2007) e163. [60] E. Murphy, J. Vanicek, H. Robins, T. Shenk, A.J. Levine, Suppression of immediateearly viral gene expression by herpesvirus-coded microRNAs: implications for latency, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 5453–5458. [61] P. Ghazal, A.E. Visser, M. Gustems, R. Garcia, E.M. Borst, K. Sullivan, M. Messerle, A. Angulo, Elimination of ie1 significantly attenuates murine cytomegalovirus virulence but does not alter replicative capacity in cell culture, J. Virol. 79 (2005) 7182–7194. [62] N. Stern-Ginossar, O. Mandelboim, An integrated view of the regulation of NKG2D ligands, Immunology 128 (2009) 1–6. [63] T. Lenac, J. Arapovic, L. Traven, A. Krmpotic, S. Jonjic, Murine cytomegalovirus regulation of NKG2D ligands, Med. Microbiol. Immunol. 197 (2008) 159–166. [64] C. Powers, V. DeFilippis, D. Malouli, K. Fruh, Cytomegalovirus immune evasion, Curr. Top. Microbiol. Immunol. 325 (2008) 333–359. [65] G.W. Wilkinson, P. Tomasec, R.J. Stanton, M. Armstrong, V. Prod'homme, R. Aicheler, B.P. McSharry, C.R. Rickards, D. Cochrane, S. Llewellyn-Lacey, E.C. Wang, C.A. Griffin, A.J. Davison, Modulation of natural killer cells by human cytomegalovirus, J. Clin. Virol. 41 (2008) 206–212. [66] N. Elefant, A. Berger, H. Shein, M. Hofree, H. Margalit, Y. Altuvia, RepTar: a database of predicted cellular targets of host and viral miRNAs, Nucleic Acids Res. 39 (2011) D188–D194. [67] N. Stern-Ginossar, N. Elefant, A. Zimmermann, D.G. Wolf, N. Saleh, M. Biton, E. Horwitz, Z. Prokocimer, M. Prichard, G. Hahn, D. Goldman-Wohl, C. Greenfield, S. Yagel, H. Hengel, Y. Altuvia, H. Margalit, O. Mandelboim, Host immune system gene targeting by a viral miRNA, Science 317 (2007) 376–381. [68] S.A. Welte, C. Sinzger, S.Z. Lutz, H. Singh-Jasuja, K.L. Sampaio, U. Eknigk, H.G. Rammensee, A. Steinle, Selective intracellular retention of virally induced

622

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77] [78] [79]

[80]

[81] [82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

L. Tuddenham, S. Pfeffer / Biochimica et Biophysica Acta 1809 (2011) 613–622 NKG2D ligands by the human cytomegalovirus UL16 glycoprotein, Eur. J. Immunol. 33 (2003) 194–203. J. Wu, N.J. Chalupny, T.J. Manley, S.R. Riddell, D. Cosman, T. Spies, Intracellular retention of the MHC class I-related chain B ligand of NKG2D by the human cytomegalovirus UL16 glycoprotein, J. Immunol. 170 (2003) 4196–4200. N. Stern-Ginossar, C. Gur, M. Biton, E. Horwitz, M. Elboim, N. Stanietsky, M. Mandelboim, O. Mandelboim, Human microRNAs regulate stress-induced immune responses mediated by the receptor NKG2D, Nat. Immunol. 9 (2008) 1065–1073. D. Nachmani, N. Stern-Ginossar, R. Sarid, O. Mandelboim, Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells, Cell Host Microbe 5 (2009) 376–385. X. Cai, S. Lu, Z. Zhang, C.M. Gonzalez, B. Damania, B.R. Cullen, Kaposi's sarcomaassociated herpesvirus expresses an array of viral microRNAs in latently infected cells, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 5570–5575. M.A. Samols, J. Hu, R.L. Skalsky, R. Renne, Cloning and identification of a microRNA cluster within the latency-associated region of Kaposi's sarcomaassociated herpesvirus, J. Virol. 79 (2005) 9301–9305. D. Nachmani, D. Lankry, D.G. Wolf, O. Mandelboim, The human cytomegalovirus microRNA miR-UL112 acts synergistically with a cellular microRNA to escape immune elimination, Nat. Immunol. 11 (2010) 806–813. J.D. Keene, J.M. Komisarow, M.B. Friedersdorf, RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts, Nat. Protoc. 1 (2006) 302–307. L.P. Tan, E. Seinen, G. Duns, D. de Jong, O.C. Sibon, S. Poppema, B.J. Kroesen, K. Kok, A. van den Berg, A high throughput experimental approach to identify miRNA targets in human cells, Nucleic Acids Res. 37 (2009) e137. G. Malterer, L. Dolken, J. Haas, The miRNA-targetome of KSHV and EBV in human B-cells, RNA Biol. 8 (2011). S.W. Chi, J.B. Zang, A. Mele, R.B. Darnell, Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps, Nature 460 (2009) 479–486. D.D. Licatalosi, A. Mele, J.J. Fak, J. Ule, M. Kayikci, S.W. Chi, T.A. Clark, A.C. Schweitzer, J.E. Blume, X. Wang, J.C. Darnell, R.B. Darnell, HITS-CLIP yields genome-wide insights into brain alternative RNA processing, Nature 456 (2008) 464–469. M. Hafner, M. Landthaler, L. Burger, M. Khorshid, J. Hausser, P. Berninger, A. Rothballer, M. Ascano Jr., A.C. Jungkamp, M. Munschauer, A. Ulrich, G.S. Wardle, S. Dewell, M. Zavolan, T. Tuschl, Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP, Cell 141 (2010) 129–141. Y. Huang, Y. Qi, Q. Ruan, Y. Ma, R. He, Y. Ji, Z. Sun, A rapid method to screen putative mRNA targets of any known microRNA, Virol. J. 8 (2011) 8. F. Grey, R. Tirabassi, H. Meyers, G. Wu, S. McWeeney, L. Hook, J.A. Nelson, A viral microRNA down-regulates multiple cell cycle genes through mRNA 5′UTRs, PLoS Pathog. 6 (2010) e1000967. F. Moretti, R. Thermann, M.W. Hentze, Mechanism of translational regulation by miR-2 from sites in the 5′ untranslated region or the open reading frame, RNA 16 (2010) 2493–2502. M. Schnall-Levin, Y. Zhao, N. Perrimon, B. Berger, Conserved microRNA targeting in Drosophila is as widespread in coding regions as in 3′UTRs, Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 15751–15756. E.M. Borst, G. Hahn, U.H. Koszinowski, M. Messerle, Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants, J. Virol. 73 (1999) 8320–8329. M. Messerle, I. Crnkovic, W. Hammerschmidt, H. Ziegler, U.H. Koszinowski, Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 14759–14763. L. Dolken, A. Krmpotic, S. Kothe, L. Tuddenham, M. Tanguy, L. Marcinowski, Z. Ruzsics, N. Elefant, Y. Altuvia, H. Margalit, U.H. Koszinowski, S. Jonjic, S. Pfeffer, Cytomegalovirus microRNAs facilitate persistent virus infection in salivary glands, PLoS Pathog. 6 (2010) e1001150. H.R. Smith, J.W. Heusel, I.K. Mehta, S. Kim, B.G. Dorner, O.V. Naidenko, K. Iizuka, H. Furukawa, D.L. Beckman, J.T. Pingel, A.A. Scalzo, D.H. Fremont, W.M. Yokoyama, Recognition of a virus-encoded ligand by a natural killer cell activation receptor, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 8826–8831. H. Arase, E.S. Mocarski, A.E. Campbell, A.B. Hill, L.L. Lanier, Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors, Science 296 (2002) 1323–1326.

[90] C.H. Lecellier, P. Dunoyer, K. Arar, J. Lehmann-Che, S. Eyquem, C. Himber, A. Saib, O. Voinnet, A cellular microRNA mediates antiviral defense in human cells, Science 308 (2005) 557–560. [91] J. Huang, F. Wang, E. Argyris, K. Chen, Z. Liang, H. Tian, W. Huang, K. Squires, G. Verlinghieri, H. Zhang, Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes, Nat. Med. 13 (2007) 1241–1247. [92] M. Otsuka, Q. Jing, P. Georgel, L. New, J. Chen, J. Mols, Y.J. Kang, Z. Jiang, X. Du, R. Cook, S.C. Das, A.K. Pattnaik, B. Beutler, J. Han, Hypersusceptibility to vesicular stomatitis virus infection in Dicer1-deficient mice is due to impaired miR24 and miR93 expression, Immunity 27 (2007) 123–134. [93] C.L. Jopling, M. Yi, A.M. Lancaster, S.M. Lemon, P. Sarnow, Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA, Science 309 (2005) 1577–1581. [94] R.E. Lanford, E.S. Hildebrandt-Eriksen, A. Petri, R. Persson, M. Lindow, M.E. Munk, S. Kauppinen, H. Orum, Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection, Science 327 (2010) 198–201. [95] E.S. Machlin, P. Sarnow, S.M. Sagan, Masking the 5′ terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex, Proc. Natl. Acad. Sci. U.S.A. (2011). [96] J.E. Cameron, Q. Yin, C. Fewell, M. Lacey, J. McBride, X. Wang, Z. Lin, B.C. Schaefer, E.K. Flemington, Epstein–Barr virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a modulator of lymphocyte signaling pathways, J. Virol. 82 (2008) 1946–1958. [97] Q. Yin, J. McBride, C. Fewell, M. Lacey, X. Wang, Z. Lin, J. Cameron, E.K. Flemington, MicroRNA-155 is an Epstein-Barr virus-induced gene that modulates Epstein-Barr virus-regulated gene expression pathways, J Virol 82 (2008) 5295–5306. [98] G. Gatto, A. Rossi, D. Rossi, S. Kroening, S. Bonatti, M. Mallardo, Epstein–Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-kappaB pathway, Nucleic Acids Res. 36 (2008) 6608–6619. [99] E. Anastasiadou, F. Boccellato, S. Vincenti, P. Rosato, I. Bozzoni, L. Frati, A. Faggioni, C. Presutti, P. Trivedi, Epstein–Barr virus encoded LMP1 downregulates TCL1 oncogene through miR-29b, Oncogene 29 (2010) 1316–1328. [100] A.L. Ellis-Connell, T. Iempridee, I. Xu, J.E. Mertz, Cellular microRNAs 200b and 429 regulate the Epstein–Barr virus switch between latency and lytic replication, J. Virol. 84 (2010) 10329–10343. [101] G. Li, Z. Wu, Y. Peng, X. Liu, J. Lu, L. Wang, Q. Pan, M.L. He, X.P. Li, MicroRNA-10b induced by Epstein–Barr virus-encoded latent membrane protein-1 promotes the metastasis of human nasopharyngeal carcinoma cells, Cancer Lett. 299 (2010) 29–36. [102] E. Gottwein, N. Mukherjee, C. Sachse, C. Frenzel, W.H. Majoros, J.T. Chi, R. Braich, M. Manoharan, J. Soutschek, U. Ohler, B.R. Cullen, A viral microRNA functions as an orthologue of cellular miR-155, Nature 450 (2007) 1096–1099. [103] R.L. Skalsky, M.A. Samols, K.B. Plaisance, I.W. Boss, A. Riva, M.C. Lopez, H.V. Baker, R. Renne, Kaposi's sarcoma-associated herpesvirus encodes an ortholog of miR155, J. Virol. 81 (2007) 12836–12845. [104] D. Lagos, G. Pollara, S. Henderson, F. Gratrix, M. Fabani, R.S. Milne, F. Gotch, C. Boshoff, miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator, Nat. Cell Biol. 12 (2010) 513–519. [105] F.Z. Wang, F. Weber, C. Croce, C.G. Liu, X. Liao, P.E. Pellett, Human cytomegalovirus infection alters the expression of cellular microRNA species that affect its replication, J. Virol. 82 (2008) 9065–9074. [106] D. Santhakumar, T. Forster, N.N. Laqtom, R. Fragkoudis, P. Dickinson, C. AbreuGoodger, S.A. Manakov, N.R. Choudhury, S.J. Griffiths, A. Vermeulen, A.J. Enright, B. Dutia, A. Kohl, P. Ghazal, A.H. Buck, Combined agonist-antagonist genomewide functional screening identifies broadly active antiviral microRNAs, Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 13830–13835. [107] A.H. Buck, J. Perot, M.A. Chisholm, D.S. Kumar, L. Tuddenham, V. Cognat, L. Marcinowski, L. Dolken, S. Pfeffer, Post-transcriptional regulation of miR-27 in murine cytomegalovirus infection, RNA 16 (2010) 307–315. [108] D. Cazalla, T. Yario, J.A. Steitz, Down-regulation of a host microRNA by a Herpesvirus saimiri noncoding RNA, Science 328 (2010) 1563–1566. [109] I.K. Guttilla, B.A. White, Coordinate regulation of FOXO1 by miR-27a, miR-96, and miR-182 in breast cancer cells, J. Biol. Chem. 284 (2009) 23204–23216. [110] S.L. Ameres, M.D. Horwich, J.H. Hung, J. Xu, M. Ghildiyal, Z. Weng, P.D. Zamore, Target RNA-directed trimming and tailing of small silencing RNAs, Science 328 (2010) 1534–1539. [111] S. Chatterjee, H. Grosshans, Active turnover modulates mature microRNA activity in Caenorhabditis elegans, Nature 461 (2009) 546–549.