Arch Virol DOI 10.1007/s00705-013-1883-4

BRIEF REVIEW

The role of microRNAs in hepatitis C virus RNA replication K. Dominik Conrad • Michael Niepmann

Received: 1 August 2013 / Accepted: 28 September 2013 Ó Springer-Verlag Wien 2013

Abstract Replication of hepatitis C virus (HCV) RNA is influenced by a variety of microRNAs, with the main player being the liver-specific microRNA-122 (miR-122). Binding of miR-122 to two binding sites near the 50 end of the 50 untranslated region (UTR) of the HCV genomic RNA results in at least two different effects. On the one hand, binding of miR-122 and the resulting recruitment of protein complexes containing Argonaute (Ago) proteins appears to mask the viral RNA0 s 50 end and stabilizes the viral RNA against nucleolytic degradation. On the other hand, this interaction of miR-122 with the 50 -UTR also stimulates HCV RNA translation directed by the internal ribosome entry site (IRES) located downstream of the miR122 binding sites. However, it is suspected that additional, yet undefined roles of miR-122 in HCV replication may also contribute to HCV propagation. Accordingly, miR-122 is considered to contribute to the liver tropism of the virus. Besides miR-122, let-7b, miR-196, miR-199a* and miR448 have also been reported to interact directly with the HCV RNA. However, the latter microRNAs inhibit HCV replication, and it has been speculated that miR-199a* contributes indirectly to HCV tissue tropism, since it is mostly expressed in cells other than hepatocytes. Other microRNAs influence HCV replication indirectly. Some of those are advantageous for HCV propagation, while others suppress HCV replication. Consequently, HCV up-regulates or down-regulates, respectively, the expression of most of these miRNAs.

K. D. Conrad  M. Niepmann (&) Institute of Biochemistry, School of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392 Giessen, Germany e-mail: [email protected]

Introduction Hepatitis C virus (HCV) belongs to a large family of single-stranded RNA viruses that replicate exclusively in the cytoplasm [1]. Their plus-strand RNA genome allows them to be translated directly in the cytosol to produce the viral gene products involved in the viral replication cycle. Replication of the HCV RNA genome then takes place at specialized membrane structures (called the membranous web) in the cytosol that are elicited by viral proteins [2–4]. The HCV RNA genome contains a single large open reading frame (ORF) coding for a polyprotein that is coand posttranslationally processed to the functional gene products. These include the core protein and the E1 and E2 envelope proteins, which comprise the structural proteins of the virus. Furthermore, seven nonstructural proteins are formed, called p7 and NS2 through NS5B, which is the viral replicase [3]. The polyprotein ORF is flanked by the 50 and 30 untranslated regions (UTR), which contain signals necessary for replication and translation of the viral RNA (Fig. 1) [1, 4]. The viral RNA has two functions. On the one hand, it serves as a template for replication to generate progeny virus genomes. Therefore, the very ends of the viral RNA provide cis signals involved in initiation of minus-strand and subsequently plus-strand RNA replication. In the 50 UTR, the stem-loops I and II as well as some sequences and secondary structures in the core coding region are involved in replication [5, 6], whereas in the 30 -UTR virtually all regions are necessary for replication in concert with two other cis-acting replicative elements (CREs) within the NS5B coding region (for an overview, see ref. [4]). On the other hand, the genomic RNA serves as mRNA for expression of the viral proteins. Thus, in or near the 50 -UTR, there must also be the signals for the initiation of

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Fig. 1 The HCV genome with the 50 - and 30 -UTRs. The polyprotein ORF is flanked by the 50 - and 30 -untranslated regions (UTRs), which contain several stem-loops (SLs). In the 30 -UTR, the variable region (VR), the poly(U/C)-tract (p(U/C)), and the highly conserved 30 -X

region are shown. Cis-acting replicative elements (CREs) and their interactions as well as the stem-loops in the core-coding region are indicated

translation. However, these signals used for translation initiation should not interfere with replication. HCV belongs to a large group of viruses that have solved this problem by utilizing a specialized RNA structure for translation initiation. These viruses do not use a cap nucleotide at the 50 end but instead employ a so-called internal ribosome entry site (IRES) in the 50 -UTR to recruit ribosomes to an internal start site of translation. By the use of such an IRES element, the virus avoids the need for any nuclear RNA processing events like capping. This IRES element directs the cap-independent initiation of translation of the viral genome (reviewed in refs. [7, 8]). In the HCV RNA, the IRES includes the stem-loops II to IV and thereby partially overlaps with the replication signals. The IRES extends a few nucleotides into the core coding region, since the AUG start codon is located in the apical loop of stem-loop IV. In addition to the core IRES, the sequences and possibly RNA secondary structures in the core coding region also contribute to efficient translation [6] (designated stem-loops V and VI in Fig. 1). In analogy to the function of the poly(A)-tail at the 30 end of capped cellular mRNAs, the HCV 30 -UTR stimulates HCV translation in cis [9–12], most likely to allow efficient translation only of undegraded viral RNAs that are suitable for the production of progeny virus. In addition to viral structural and non-structural proteins, HCV also employs cellular factors to improve translation and replication of its genome. On the one hand, a variety of cellular RNA binding proteins are recruited by the HCV IRES RNA to modulate its translation efficiency (reviewed in refs. [13, 14]). On the other hand, Jopling and coworkers reported in 2005 that the liver-specific microRNA-122 (miR-122) facilitates HCV RNA accumulation [15], a break-through finding that complemented the previous observation that viruses can encode their own microRNAs to modulate their host cell [16]. When acting on normal cellular mRNAs, microRNAs (miRNAs) regulate eukaryotic gene activity at the posttranscriptional level [17–19]. After processing of miRNA precursors to *22-bp miRNA duplexes with 30 -overhangs,

one of the strands of the duplex is incorporated into a microRNA/protein (miRNP) complex and used as a sequence-specific guide to identify its target mRNA(s). In humans, these complexes contain proteins of the Argonaute (Ago) family, which includes four members (Ago1-4), of which only Ago2 possesses ‘‘slicer’’ activity for endonucleolytic cleavage of RNA [17]. In complex with Ago protein, the very 50 nucleotide of the miRNA resides in the so-called MID (middle) domain of the protein, and the last two 30 nucleotides of the miRNA reside in the PAZ (PIWI/ Argonaute/Zwille) domain of the protein [20, 21]. Thus, these nucleotides may not be available for base pairing with the target, while the so-called seed region near the miRNA0 s 50 end (usually miRNA nucleotides 2 to 8) is made accessible for base pairing with the target sequence in the 30 -UTR of the mRNA [20, 21]. In addition, other regions of the miRNA may also be involved in base pairing [22]. The function of this Ago-containing complex then depends on the extent of base pairing between the small RNA and the mRNA target. When the small RNA matches perfectly to its target, an RNA-induced silencing complex (RISC) is formed, and Ago2, as part of the RISC, cleaves the target mRNA opposite to the guide strand, resulting in degradation of the target mRNA. In contrast, when base pairing between miRNA and target mRNA is imperfect, the interaction of the miRNA-protein complex with the target mRNA usually results in repression of translation or degradation [17–19]. In contrast to the canonical suppression of mRNA translation by miRNAs as described above, the finding that miR-122 stimulates HCV propagation [15] pointed to a completely different mode of microRNA action. Firstly, miR-122 was found to stimulate propagation of HCV RNA instead of repressing it. Secondly, this action of miR-122 was found to be conferred by two target sites near the 50 end of the HCV RNA, while the possible roles of two other potential miR-122 binding sites in the 30 -UTR and in the NS5B coding region are not yet clear. Moreover, miR-122 is expressed preferentially in the liver, where it constitutes about 60-70% of the microRNAs [23–28]. Thus, besides the function of surface receptors in HCV entry [29, 30],

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The role of microRNAs

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Fig. 2 Binding of miR-122 to the HCV 50 -UTR. (a) The secondary structure of the HCV 50 -UTR is shown with the two miR-122 molecules (green) and their binding sites 1 and 2. The interaction between stem-loops (SL) II and IV is indicated by a thin line. In the expanded view, HCV nucleotides (sequence of genotype 1b, Con1

isolate) pairing with miR-122 molecules are shown in red for target site 1 and in blue for target site 2. Optional base pairing is indicated by grey bars. (b) Alignment of HCV sequences of different isolates. Asterisks indicate conserved residues. The color code is as in (a) (color figure online)

miR-122 can be regarded as an intracellular tissue-specific factor that may contribute to the liver tropism of HCV by promoting its replication in hepatocytes but not in other cells. More recently, other microRNAs have been characterized that also bind directly to the HCV RNA but have inhibitory effects on HCV replication. Furthermore, HCV was found to influence the activity of various cellular miRNAs to modulate the response of the host cell. In this review, we discuss the molecular details of miR-122 binding and function on the HCV RNA, while possible treatment options using miR-122 antagonists have been discussed in detail elsewhere [31]. In addition, we briefly describe other miRNAs that have either direct or indirect

effects on HCV replication, focusing on miRNAs that are regulated by HCV and in turn actually also regulate HCV replication, emphasizing the mutual interactions between HCV and miRNAs and their advantages for HCV replication.

microRNA-122 The anatomy of the interaction of microRNA-122 with its two target sites in the HCV 50 -UTR Between the stem-loops I and II in the highly conserved 50 UTR of HCV, there are two sequence stretches of six

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(CACUCC) or seven nucleotides (ACACUCC), respectively, which are complementary to the seed sequence of miR-122 (see Fig. 2). In HuH-7 hepatoma cells that are most permissive for HCV replication, HCV genome amplification was shown to be decreased by mutations in the seed target sequence of the first target site in the 50 UTR. Compensatory mutations in the microRNA restored efficient HCV genome amplification, indicating a direct interaction between miR-122 and HCV RNA [15]. Exchanging the sequence of the first miR-122 target site for the target sequence of miR-21 abolished stimulation of HCV replication by miR-122, underlining the importance of this first miR-122 target site [32]. Relocation of the first 45 nucleotides of the HCV RNA, including both miR-122 target sites, to the HCV 3’-UTR led to translational repression of a HCV reporter construct, indicating that the biological function of the miR-122 target sites in the HCV 50 -UTR depends on their position in the RNA [32]. In that study, Jopling and coworkers also demonstrated the function of the second miR-122 binding site in the 50 -UTR [32]. Although the second miR-122 seed target sequence comprises only six nucleotides, it was shown to enhance HCV genome amplification when present in the HCV 50 -UTR, while it also confers translation repression when placed in the 30 -UTR of an artificial reporter mRNA [32]. These findings, which by now were also supported by data from others [33], led to a model in which both miR-122 target sites in the HCV 50 -UTR can functionally bind miR-122. In addition, subtle variations in the short sequence between the two miR-122 seed target sites were found to impair HCV replication [32], suggesting that binding of miR-122 to target site 2, not only with the seed sequence but also with other nucleotides, is important for the interaction. This idea was tested in detail by Machlin and coworkers [34], who showed that binding of miR-122 to HCV binding site 2 includes a second hybridization area involving miR-122 nucleotides 14-16, 30 of the seed sequence. These results were confirmed by two other studies [35, 36], while Shimakami and coworkers [36] extended these findings by showing that nucleotides 13 and 14 (tested only in combination) are also of some importance for binding of miR-122 to target site 2. The model proposed by Machlin and coworkers stated that binding of miR-122 also to HCV site 1 includes base pairing outside the seed sequence of the microRNA. They showed that for efficient miR-122 binding to target site 1 an additional hybridization of miR-122 nucleotides 14-17 to nucleotides 1-4 of the HCV RNA is also necessary. This interaction would reach across stem loop I (see Fig. 2). Moreover, binding of miR-122 to the 50 -UTR RNA has been analyzed by in vitro structure probing (in the absence of proteins) using a method called ‘‘SHAPE’’ (selective 2’-

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hydroxyl acylation analyzed by primer extension) [37]. In the absence of miR-122, the second miR-122 target site upstream of stem-loop II was found to be largely singlestranded [38, 39], whereas the structure of the seed target region of miR-122 target site 1 could not be analyzed by using SHAPE. Binding of miR-122 results in decreased accessibility of the 50 -UTR RNA at miR-122 binding site 2 and at the second hybridization site of miR-122 binding site 1 at the very 50 end of the HCV RNA. However, in extension of the model proposed by Machlin and coworkers [34], not only nucleotides 14-16 but also nucleotide 13 of miR122 bind to the second target site in vitro [38, 39]. Nucleotide 17 of miR-122 was shown to be not important for binding to target site 2, whereas it is important for binding to target site 1 [34, 36]. In addition, miR-122 nucleotides 9 and 11 were also proposed to base pair to the target when analyzed in vitro [38, 39], even though this requires the single unpaired U at position 8 of miR-122 to flip out of the duplex (see Fig. 2a). In the study of Machlin and coworkers [34], nucleotides 19-22 of miR-122 also appeared to be of functional importance. However, these findings were not confirmed in another study [36]. In summary, these interactions would lead to much stronger miR-122 binding to target site 2 in the HCV 50 UTR than to target site 1. The different KD values measured in vitro by isothermal titration calorimetry (ITC) for the interaction of miR-122 molecules with the two binding sites [39] reflect this situation. Although the classical seed target sequence at binding site 1 is seven nucleotides long instead of six nucleotides at binding site 2, binding of miR122 to target site 2 is much stronger, with a KD of about 90 nM compared to 845 nM for binding to site 1. We have aligned the sequences spanning from the 50 end up to the base of stem-loop II of HCV isolates from a variety of different genotypes (Fig. 2b) (compare also the sequence alignment in [36]). For miR-122 target site 1, this alignment shows that while the seven nucleotide seed target sequence ACACUCC is conserved, miR-122 nucleotides 14-17 can pair to the very 50 -terminal four nucleotides of the HCV RNA in some isolates, but not in all. In many isolates, a U or C residue at position 4 cannot base pair to the U residue at position 14 of miR-122, resulting in the situation that only three base pairs are involved in this hybridization site. This suggests that other constraints may be more important in these strains to maintain a U or C instead of an A (or G) at HCV position no. 4. In contrast, at target site 2, the six-nucleotide seed target sequence CACUCC can indeed be extended by one upstream U nucleotide (or a C in some isolates) when miR122 binds with nucleotide 9 (G) to this position, flipping out the single U at miR-122 position 8. Moreover, the second binding site appears to be well conserved, allowing the 30 region of miR-122 to pair to five nucleotides. The

The role of microRNAs

nucleotides in between allow one additional base pair with the C residue at miR-122 position 11, but only in isolates that have a G at this position. Most isolates, however, carry a non-pairing A instead of a G. In addition, functional assays revealed that the pairing of miR-122 position 11 to the target is not very important [34]. Rather than forming a perfectly matching binding site that allows strong binding of most of miR-122, the sequence between the two hybridization sites at miR-122 binding site 2 may have been evolved to not base pair completely, presumably to avoid cleavage of the HCV RNA by Ago2. In summary, miR-122 can bind to two separate binding sites in the HCV 50 -UTR, forming miRNA:RNA complexes that appear unusual when compared with canonical binding of miRNAs to the 30 -UTRs of mRNAs [22]. Several studies have shown that both miR-122 binding sites in the HCV 50 -UTR actually bind miR-122 [32–36, 40–42]. This has led to the general assumption that two molecules of miR-122 bind simultaneously to one molecule of HCV RNA. This hypothesis, however, was only tested in one study using in vitro gel shift experiments in the absence of any proteins. In that study, increasing amounts of miR-122 successively formed two discrete miRNA:RNA complexes with the 50 end of the HCV 50 -UTR [39]. Although it is generally assumed that this is also the case in vivo, this remains to be shown, since we do not yet definitely know if the proteins associated with these miR-122:RNA complexes (see below) actually allow simultaneous binding of the two miR-122 molecules. Functional consequences of miR-122 action on the HCV 50 -UTR The interaction of miR-122 with its two target sites in the 50 -UTR results in the formation of one or two miR-122/ RNA complexes at the very 50 end of the HCV 50 -UTR. This direct interaction of miR-122 with the HCV RNA has been shown to enhance HCV RNA accumulation in HuH-7 hepatoma cells that contain endogenous miR-122 [15, 32, 43, 44]. In addition, ectopic supplementation of miR-122 in hepatoma cell lines that do not express significant amounts of miR-122, like HepG2 cells [45], as well as in nonhepatic cell lines [46], enhanced replication of intracellular HCV RNA, showing that miR-122 is a key factor for HCV replication. Accordingly, the requirement for miR-122 for efficient production of infectious virus was demonstrated [45, 47–49]. In summary, the requirement for miR-122 for efficient overall replication of HCV was confirmed in several studies. From analyses aiming at the elucidation of the molecular mechanisms of miR-122 action on HCV, more and more evidence emerges that miR-122 has more than one function in the HCV replication cycle. In a first attempt to identify the

molecular mechanisms of miR-122 action, Jopling and coworkers tested a possible effect of miR-122 on HCV translation [15]. Using a dicistronic HCV replicon system they found that translation was not stimulated by miR-122. Based on this finding, the authors proposed that miR-122 most likely positively influences HCV RNA replication [15]. However, this idea was extended when Henke, Goergen and coworkers reinvestigated the possible stimulation of HCV translation by miR-122 using a monocistronic reporter system in order to avoid the collateral translation activation of the HCV IRES by additional IRES elements in cis ([10, 40, 50], discussed in ref. 51). The result of that study was that miR-122 has a positive effect on HCV translation, while other, additional modes of miR-122 action were not excluded [40]. Both miR-122 target sites in the HCV 50 -UTR were shown to be involved in translation stimulation, and stimulation of translation by miR-122 was shown to be independent of viral RNA synthesis using a replication-defective NS5B polymerase mutant genome [40]. This was one of the first reports of stimulation of translation by microRNAs [40, 52, 53], challenging the concept that microRNAs always repress translation by acting on the RNA0 s 30 -UTR. The positive effect of miR-122 on HCV translation was then confirmed by other studies [33, 35, 41, 44, 54, 55]. During the cell cycle, the stimulation of HCV translation by miR-122 is superimposed onto the constitutional translation efficiency directed by the HCV 50 -UTR, which is highest during the G0 and G1 phases of the cell cycle. Stimulation by miR-122 is high in G0, G1 and G2/M [54], making the overall translation efficiency highest in the normal (non-dividing) G0 state of hepatocytes. Interestingly, in vitro binding of miR-122 to binding site 2 results not only in decreased accessibility at this binding site but also in SL IV, where the polyprotein AUG start codon is located [38, 39]. Furthermore, in the base of stem-loop II at position 55 [38, 39] and at position 80-85 [38], near the apical loop region of stem-loop II, the accessibility in hydroxyl printing assays was shown to increase upon miR-122 binding. Stem-loop II is in close contact with the ribosomal surface [56]. Moreover, the apical loop of stem-loop II is also involved in tertiary interactions with SL IV, which induce conformational changes in the 40S subunit and help to place the AUG in the ribosome entry channel [57, 58]. Therefore, it can be speculated that binding of miR-122 may be indirectly involved in long-range changes of IRES tertiary structure that could modulate translation initiation in a yet unknown way, perhaps by inducing changes in 40S conformation. Consistently, binding of miR-122 to its two target sites in the 50 -UTR has higher KD values when analyzed in the context of the complete 50 -UTR than when the measurements are made using only a small RNA containing the miR-122 target sites [39]. This supports the idea that miR-122 induces long-range structural changes in the IRES that require an energy input

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that is higher than for the mere binding of miR-122 to the HCV RNA. Such long-range changes in IRES structure induced by miR-122 may then, in turn, induce conformational changes in the ribosome. In accordance with this idea, stimulation of translation of HCV IRES activity by miR-122 is disabled when the HCV sequence downstream of stemloop II is replaced by the corresponding sequences from the CSFV IRES, which is similar but not identical to the HCV IRES [41]. However, the fact that insertions between the miR-122 binding sites and stem-loop II do not influence HCV translation significantly [41, 55] argues against the idea that the IRES tertiary structure confers miR-122-induced long-range interactions that have an influence on the conformation of the small ribosomal subunit. Nonetheless, translation stimulation is not fully sufficient to explain miR-122 action in the HCV replication cycle. Mutations in the miR-122 target sites were found to have a much more pronounced effect on HCV virus yield than mutations in the IRES stem-loop IIId that have a quantitatively similar effect on translation alone [44]. This led to the conclusion that an additional mechanism of miR122 action must also contribute to the positive effect of miR-122 on the overall replication of HCV. The elongation phase of HCV RNA synthesis was tested but found not to contribute to stimulation by miR-122 [59]. The likely simultaneous binding of two molecules of miR-122 with their seed sequences and also internal miRNA nucleotides to the 50 -end of the HCV RNA then led to the hypothesis that binding of miR-122 to the HCV RNA may result in a largely double-stranded RNA complex at the 50 end of the HCV RNA [34] that serves to protect the HCV RNA against nucleolytic degradation. This idea was corroborated by Shimakami and coworkers [36, 42], who showed that HCV RNA is stabilized by miR-122. This stabilization was dependent on the presence of the HCV 50 -UTR, but not on downstream sequences, indicating that the two miR-122 binding sites in the HCV 50 -UTR confer this effect and that this interaction protects against degradation starting in the HCV 50 -UTR [42]. Accordingly, Li and coworkers [60] showed that replicating HCV RNA within infected cells is degraded primarily from the 50 end by the 50 -30 -exonuclease Xrn1, but this degradation is impaired in the presence of miR-122. Li and coworkers [60] argue that the positive effect of miR-122 on translation may be conferred indirectly by stabilizing the HCV RNA and maintaining its abundance as a translation template, since they observed no increase in viral protein expression following miR-122 supplementation in Xrn1-depleted cells, and no defects in ribosome loading by a mutant HCV RNA defective in miR122 binding. Moreover, in the latter study, Xrn1 knockdown did not rescue replication of a viral mutant defective in miR-122 binding, suggesting that miR-122 may have yet

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uncharacterized additional functions in the viral life cycle. The finding that HCV NS5B, miR-122 and Ago2 colocalize in replication complexes [61] would also be compatible both with the idea that the miR-122-protein complex at the HCV RNA0 s 50 end may contribute to increased stability of the HCV RNA and with other yet undefined roles of miR122 in HCV replication. Thus, further studies may explore other roles of miR-122 in HCV replication, e.g., in regulation of RNA minus- or plus-strand synthesis initiation or genome encapsidation. Involvement of protein components in miR-122 action on the HCV 50 -UTR When considering which proteins may be involved in conferring miR-122 action on the HCV RNA, for three reasons it was an obvious assumption that Ago proteins could be involved. Firstly, cellular microRNAs can be assumed to be routinely associated with complexes including Ago proteins. Secondly, the HCV sequence could be regarded as having evolved to largely avoid binding of the terminal nucleotides of miR-122, which are required to interact with the Ago protein [20, 21]. Specifically, the 50 terminal nucleotide of at least the miR-122 molecule binding to the second target site in the HCV 50 -UTR as well as the two 30 -terminal nucleotides ends of both miR-122 molecules appear not to be involved in the interaction with the HCV 50 -UTR. Thirdly, the HCV sequence at miR-122 target site 2 obviously evolved to avoid binding to the internal nucleotides of miR-122 to escape cleavage by Ago2 (see Fig. 2). Consistently, in a systematic RNAi knockdown approach, Randall and coworkers identified several proteins involved in HCV replication that are also part of the miRNA maturation process [47]. Knockdown of these components such as Dicer, DGCR8 and Ago1-4 resulted in reduced HCV RNA levels and virus production, suggesting that Ago proteins are indirectly or directly involved in the HCV life cycle. In subsequent studies, the involvement of Ago proteins in the stimulation of HCV replication and translation was confirmed. Knockdown of Ago2 resulted in drastic reduction of HCV replication and translation [33]. The effect of Ago2 knockdown on translation, however, was less pronounced than that on overall HCV replication, supporting the idea that miR-122 may have more than one function in the HCV life cycle. In addition, Roberts and coworkers [41] confirmed the involvement of Ago proteins, mainly Ago2, in HCV translation regulation by miR-122. Knockdown of Ago4 resulted in the most severe reduction of overall HCV replication in one study [47], whereas knockdown of Ago1-4 in another study did not have a very much more pronounced effect on HCV translation than the knockdown of Ago2 alone [41]. The exact contributions of

The role of microRNAs

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II Fig. 3 A model for the action of miR-122 at the HCV 50 -UTR. Agoprotein-containing complexes most likely load two molecules of miR122 to the HCV 50 -UTR. The duplex miR-122 is unwound during the loading process. The Ago-containing protein complexes remain associated with miR-122 after binding to the HCV 50 -UTR and confer protection of the HCV RNA against nucleolytic degradation (mainly by the 50 -30 -exonuclease Xrn1). Most likely, an interaction of these complexes also directly confers stimulation of translation directed by the HCV IRES. The interaction between stem-loops (SL) II and IV is indicated by a thin line (color figure online)

the various Ago proteins remain to be analyzed, in particular since the intracellular levels of Ago proteins in the cell are quite different [62]. The first evidence for miR-122-dependent direct binding of Ago2 protein to the HCV 50 -UTR was reported by Shimakami and coworkers [42]. These authors performed immunoprecipitations with an anti-Ago2 antibody. Using RT-PCR to detect co-precipitated HCV RNA, they showed that HCV RNA could be recovered only in the presence of wild-type but not mutant miR-122. This was confirmed in another study [55] employing direct detection of the HCV RNA after recovery without amplification steps. The above results suggest that Ago2 indeed binds a considerable fraction of the HCV RNA, indicating that Ago2 is involved in conferring miR-122 activity on HCV translation and replication. Moreover, the interaction of Ago2 with miR122 and HCV 50 -UTR RNA was found to be stable for more than 3 hours during immunoprecipitation [55], suggesting that Ago2 remains associated with miR-122 after loading it to the HCV RNA. The idea that Ago proteins directly

mediate the interaction of miR-122 with the HCV RNA was supported by the finding that, in cells, duplex, but not single-stranded, miR-122 precursors stimulate HCV translation, suggesting that the miRNA duplex must be unwound and loaded to Ago protein containing effector complexes [35]. Moreover, only miR-122 duplex precursors of 22 nt in length function effectively in stimulating HCV translation, whereas shorter or longer duplex precursors are less efficient [55]. This observation supports the idea that the Ago proteins fit microRNAs of appropriate length in their binding pocket to confer miRNA action [20, 21]. In addition to Ago protein, the RISC protein component TRBP also appears to be closely involved in miR-122mediated stimulation of HCV translation and RNA accumulation [63]. GW182/TNRC6 may also be involved in the miRNP complexes, since siRNA knockdown of GW182 reduced the ability of miR-122 to stimulate HCV translation [41]. Moreover, IGF2BP1 is associated with HCV RNAs containing the miR-122 target sites in the 50 -UTR in higher amounts than with HCV RNA not containing these target sites, suggesting that the miR-122/protein complexes also involve binding and possibly a function of IGF2BP1 [64]. In contrast, the RNA helicase DDX6 appears not to be involved in mediating miR-122 action [65]. Taken together, Ago protein may act as a component of a larger miR-122/protein complex, and loads miR-122 to the two binding sites in the HCV 50 -UTR (Fig. 3), and it is thereby possible that both target sites in the HCV 50 -UTR are occupied simultaneously by miR-122 miRNP complexes. These miRNP complexes then confer enhanced stability of the HCV RNA by protecting its 50 end against exonucleolytic degradation, stimulation of translation, and perhaps other functions in the HCV life cycle. Interaction of miR-122 with other target sites in the HCV RNA There are two other miR-122 target consensus sites in the HCV genome. One of these sites is located in the variable region of the 30 -UTR (VR in Fig. 4), the other lies in the NS5B coding region. Jopling and coworkers [15] found that mutation of the potential miR-122 target site in the 30 -UTR had no significant effect on HCV genome amplification. In a study focusing on translation, mutations in the target consensus sequence in the 30 -UTR were found to affect the overall efficiency of translation stimulation by the 30 -UTR but showed no influence on the ability of miR-122 to stimulate or repress translation [40]. Furthermore, Nasheri and colleagues [66] analyzed the accessibility and affinity of the miR-122 binding sites on the HCV RNA for miR-122 in vitro and found that the site in the variable region of the 30 -UTR showed a higher affinity for miR-122 binding compared to the second site in the 50 -UTR. In contrast, in another study,

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Fig. 4 Direct interaction of microRNAs with the HCV RNA genome. The positive role of miR-122 on HCV replication by binding to the

HCV UTR is indicated in green. Inhibiting roles of miR-122 and of other microRNAs are indicated in red (color figure online)

no significant binding of miR-122 to the 30 -UTR site was found [55]. Thus, currently, no clear role can be attributed to the potential miR-122 target site in the 30 -UTR. The second miR-122 binding site outside the 50 -UTR is located in the NS5B coding region (around position 8870 in the JFH-1 isolate of genotype 2a and around position 8805 in the Con1 isolate of genotype 1b; see Fig. 4) [66]. miR-122 was reported to bind to this site with high affinity and to exert a slightly inhibiting effect on HCV replication in the replicon system, leading to the suggestion that this binding site has a repressor function for HCV translation and replication [66].

Even though there is hope that anti-miR-122 treatment could support the reduction of HCV load in combination therapy, it must be suspected that anti-miR-122 treatment on its own does not result in virus elimination from the liver. Another obstacle for long-term treatment with miR-122 antagonists is the fact that miR-122 is required for the regulation of hepatocyte cholesterol and fatty acid synthesis [70, 71] which gives rise to the assumption that miR-122 may be indirectly involved in HCV assembly by regulating the expression of ApoE and other proteins involved in VLDL production [72–75]. However, it is not yet known if and how the corresponding genes are regulated (most likely indirectly) by miR-122 [76, 77]. Moreover, the long-term consequences of anti-miR-122 treatment on liver metabolism and a possible dedifferentiation of hepatocytes were not yet extensively investigated, but a recent study reported that miR-122 depletion can result in severe liver disease [78]. Nevertheless, liver injury by HCV infection as well as the success of treatment with interferon and ribavirin can be followed by analyzing the serum level of miR-122, which increases upon HCV-induced liver damage and decreases upon therapy of chronic HCV infection [79, 80]. Therefore, detection of miR-122 serum levels is an even more sensitive method than measuring changes in alanine aminotransferase (ALT) [79].

Effects of therapeutic sequestration of miR-122 in vivo Since miR-122 appears to be a prime determinant of intracellular HCV replication, sequestration of miR-122 in vivo is considered to be a therapeutic option. A few studies have been published up to now that investigated the effects of miR-122 sequestration in vivo. Even though in one study the intrahepatic and serum HCV-RNA levels in human patients were found not to correlate with intrahepatic miR-122 expression [67], the sequestration approach had been principally explored for miR-122 in chimpanzees, with the result that sequestration of miR-122 in the liver can reduce HCV replication [68]. However, serum HCV RNA increased again after relief of anti-miR-122 treatment, indicating that the virus was not eliminated from the liver of the chimpanzees. In humans, sequestration of miR-122 by antisense locked nucleic acids (MiravirsenÓ) in a phase 2a trial also showed partially promising results concerning viral clearance and emergence of viral resistance [69]. However, miR-122 sequestration did not result in sustained reduction of plasma HCV RNA levels in all patients. In some cases, the HCV RNA levels in plasma decreased after treatment, whereas in other cases HCV RNA levels increased again after treatment relief [69]. In both of the above studies, no escape mutants were found in the miR122 binding sites in the 50 -UTR, but the whole genomes were not analyzed for possible secondary-site mutations.

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Other microRNAs that interact directly with the HCV RNA Besides miR-122, which is almost exclusively expressed in the liver, other microRNAs, such as miR-199a*, let-7b, miR-196 and miR-448, have also been reported to interact directly with the HCV RNA. In all of these cases, a physical interaction of the microRNAs with the HCV RNA was suggested since mutations in either the HCV sequence or the miRNA sequence disabled the interactions. However, restoration of the original effects by compensatory mutations in both the HCV and the miRNA sequence has not yet been shown.

The role of microRNAs

Directly downstream of the second miR-122 binding site in the HCV 50 -UTR there is a binding site for miR-199a* located in stem-loop (SL) II (see Fig. 4). This miR-199a* binding site includes seed matches as well as some additional matches that extend down to the base of SL II. It can be expected that base pairing of miR-199a* to this region would at least partially alter the secondary structure of SL II, which is involved in both translation and replication [14]. Accordingly, Murakami and coworkers [81] discovered reduction of HCV replication efficiency in the presence of miR-199a*. A possible effect of miR-199a* on HCV translation, however, remains to be determined. It has been speculated that miR-199a* contributes to the liver tropism of HCV [51, 82]. miR-199a* appears to be expressed at moderate levels in several human tissues [23, 27, 83], but its expression in human liver tissue is rather low [81, 83]. Even though the tissue specificity of HCV may also be dependent on the metabolism of lipoprotein particles, the low-density lipoprotein receptor [84, 85] and the promotion of HCV propagation by miR-122 [15, 40], the suppression of HCV replication by miR-199a* in tissue other than liver may also contribute indirectly to HCV liver tropism. In addition to miR-199a*, another microRNA called let-7b was found to interact directly with the HCV RNA. Let-7b is expressed in various tissues including liver and spleen, as well as part of the brain [23], and it has been shown to interact directly with the HCV RNA genome at several positions [86]. One target site is located in SL IV of the 50 -UTR and overlaps with the initiator AUG (see Fig. 4). Two other target sites are located in the NS5B coding region, upstream of stem-loops 5BSL3.1 and 5BSL3.2. The latter stem-loop is also called CRE 9266 and is involved in kissing-loop interactions with the 30 -X region of the 30 -UTR (see Fig. 1). Hybridization of let-7b to these three sites slightly represses HCV replication, but not translation, whereas another potential target sequence in the very stable stem-loop SL 1 at the 30 -end of the 30 UTR does not appear to be functional at all in these assays [86]. Two other microRNAs, miR-196 and miR-448, have direct target sites in the HCV RNA as well: miR-196 at position 6880 in the NS5A sequence and miR-448 at position 801 in the core coding sequence [43]. Overexpression of miR-196 and miR-448 reduces HCV replication [43], and it has been shown that interferon enhances miR196 expression [87, 88], suggesting that it is involved in the interferon response. In concordance with this idea, miR196 was reported to repress HCV gene expression [87, 88]. Furthermore, HCV infection represses miR-196 expression, but it is not yet clear whether there is an additional direct or indirect involvement of miR-196 in HCV replication. Thus, the propagation of HCV appears to underlie a

more complex control than previously expected, exerted by at least five microRNAs that interact directly with the HCV RNA.

Several microRNAs are indirectly involved in HCV replication During infection, HCV remodels the expression pattern of the host cell to facilitate its replication or escape from the immune system; several studies indicate that HCV infection can alter the microRNA expression profile of the host cell. For example, Liu and coworkers identified 42 microRNAs that were differentially expressed upon HCV infection, of which 22 were up- and 20 down-regulated [89]. Some of these miRNAs have been suspected to influence HCV replication or to modulate the response of the immune system of the host without direct interaction with the HCV RNA. However, the molecular details of their mode of action have been identified for only a few of them so far. Some microRNAs are advantageous for HCV propagation: for example, miRNAs that are involved in downregulation of apoptosis, the interferon (IFN) response or other aspects of innate immunity. The expression of some of these miRNAs is up-regulated by HCV (Table 1). miRNAs that are differentially expressed upon HCV infection, such as miR-24, miR-149* and miR-638, have been shown to be involved in HCV entry, replication and propagation [89]. Of these, expression of miR-149 and miR-638 was found to be advantageous for HCV propagation since it results in up-regulation of HCV production (while miR-24 has the opposite effect, see below). Accordingly, HCV up-regulates expression of miR-149 and miR-638 [89]. miR-491, a miRNA that is up-regulated by HCV infection, was shown to enhance HCV replication, presumably by suppressing the PI3 kinase/Akt signaling pathway, which is known to regulate apoptosis [90, 91]. Similar results were obtained for miR-320c and miR-483-5p. These miRNAs were shown to be up-regulated by HCV infection and were shown to have putative targets in PI3 kinase/Akt signaling, the MAP kinase and NF-jB pathway [92], all playing major roles in cell proliferation, apoptosis or the inflammation response. However, in another study contradicting results were reported for miR-320c, which was found to be down-regulated in patients chronically infected with HCV [93]. HCV also induces expression of miR-21, which in turn contributes to evasion of HCV from the host immune response by targeting expression of NF-jB [94], MyD88 and IRAK1 [95], all of which are key components of interferon signaling in the innate immune response.

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K. D. Conrad, M. Niepmann Table 1 microRNAs that are indirectly involved in HCV replication. microRNA

Cellular target(s)

References

microRNAs that up-regulate HCV replication and are up-regulated by HCV miR-21

NFjB, MyD88, IRAK1

[94, 95]

miR-27b

Lipid production, affects a late stage of HCV replication

[98]

miR-130a

IFITM1 ?

[96]

miR-149*

[89]

miR-192

[90]

miR-215

[90]

miR-320c

PI3K/Akt, MAPK, NFjB

[92]

miR-483-3p

PI3K/Akt, MAPK, NFjB

[92]

miR-491

Suppressing PI3K/Akt action

[89–91]

miR-638

[89]

microRNAs that down-regulate HCV replication and are down-regulated by HCV miR-24 miR-29

[47, 89] COX-2 ?, IFN k ?

miR-30(a-d)

[89, 91, 93, 99] [89, 97]

miR-130a

[89, 97]

miR-192

[97]

miR-296

[43]

miR-301

[89, 97]

miR-324-5p

[97]

miR-351

[43]

miR-431

[43]

miR-565

[97]

Only those microRNAs are listed for which it is known that they regulate HCV expression and, in turn, HCV also regulates microRNA expression in order to support its replication. The effects of miR-27a are discussed in the text ? indicates presumed cellular targets

Conflicting results have been published for miR-130a. miR-130a was found to be up-regulated in HCV infected cells [96]. Experimental sequestration of miR-130a in that study resulted in a lower efficiency of HCV replication, most likely accomplished by repression of the interferoninduced transmembrane protein, IFITM1, a protein that is known for its antiviral function [96]. However, other studies found miR-130a to be down-regulated upon HCV infection [89, 97]. Thus, the actual role of miR-130a in HCV infection remains to be determined. Other microRNAs positively regulate the host interferon response or are involved in other aspects of cellular metabolism. Several of these miRNAs were shown to be down-regulated by HCV to counteract measures of the innate immune system. The miR-30(a-d) cluster as well as miR-192, miR-301 and miR-324-5p have been reported to be down-regulated by HCV infection. This regulation is advantageous for the virus, since these

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miRNAs are up-regulated in response to IFN-a [89, 97], and some of these miRNAs are assumed to have an impact on endocytosis as well as TGF-b signaling, a pathway involved in cell growth, and on apoptosis [97]. For miR-30c it has been shown that its down-regulation enhances HCV genome replication. Other examples of down-regulation of miRNAs that interfere with HCV replication are miR-24, miR-27, miR-29, miR-296, miR351 and miR-431. Of these, miR-296, miR-351 and miR431 are up-regulated by interferon and suppress HCV replication [43]. The mechanism by which miR-24 acts on HCV replication is not known, but it has been shown that miR-24 reduces HCV replication and thus is in turn repressed during HCV infection [47, 89]. miR-29 probably down-regulates HCV replication by acting on cycloxygenase-2 (COX-2) and interferon-lambda (IFN k), and in turn, HCV down-regulates miR-29 expression [89, 91, 93, 99]. In contrast to the above miRNAs, which are involved regulation of the innate immune system or related pathways, two members of the miR-27 family that are involved in the regulation of lipid metabolism have been reported to affect HCV replication. The human miR-27a and miR-27b sequences differ by two nucleotides. Contradictory results have been published for miR-27a. One study reported that miR-27a reduces the cellular triacylglyceride and cholesterol content, and it was speculated that it counteracts the production of lipids required for HCV production [100]. In contrast, another study showed that miR-27a expression increases the cellular lipid content [98]. In either case, miR-27a was found to exert a negative effect on HCV replication and infectivity [100]. Nevertheless, HCV induces the expression of miR27a [98, 100], a finding that was speculated to perhaps establish a negative feedback loop that involves downregulation of the cholesterol transporter ABCA1 [100]. The authors of the latter study speculate that down-regulation of miR-27a by HCV may contribute to escape from host immune surveillance and establishment of a persistent chronic HCV infection. In contrast, miR-27b overexpression inhibits genotype 1b HCV RNA replication, whereas miR-27b up-regulates production of extracellular infectious HCV [98]. Thus, a late stage of HCV replication or assembly may be affected by miR-27b, and the increased HCV production may relate to the increase of cellular lipid content by miR-27b [98]. Consequently, HCV was shown to up-regulate miR-27b expression [98], a result that is contradictory to the previous finding that HCV down-regulates miR-27b expression [101]. Taken together, the regulation of miR-27a and miR-27b by HCV and their roles in the various steps of HCV particle formation in relation to the regulation of cellular lipids awaits further investigation.

The role of microRNAs

Concluding remarks Overall, the alterations in the miRNA expression profile in the host cell induced by HCV infection seem to facilitate HCV reproduction and maintain its persistence. It remains to be elucidated if a combination of drugs targeting HCV proteins with anti-microRNA treatment will possibly be able to support virus elimination, but it can be expected that there will be many obstacles (including adverse effects of anti-miRNA treatment for the host cells) before a curative anti-microRNA treatment against HCV will become effective. For understanding the molecular details of miR-122 action on HCV replication, it remains to be shown which proteins are contained in these complexes, how they interact with each other and with the HCV RNA, and what their exact functions in enhancing HCV replication are. Acknowledgments We apologize to those investigators whose work has not been discussed due to space limitations. Work in MN0 s lab is supported by grants of the Deutsche Forschungsgemeinschaft, DFG (SFB 1021, IRTG 1384, Ni 604/2-2). Conflict of interest

The authors declare no conflict of interest.

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The role of microRNAs in hepatitis C virus RNA replication.

Replication of hepatitis C virus (HCV) RNA is influenced by a variety of microRNAs, with the main player being the liver-specific microRNA-122 (miR-12...
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