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Jpn. J. Infect. Dis., 64, 357-366, 2011 Invited Review Vertebrate Virus-Encoded MicroRNAs and Their Sequence Conservation Kahori Takane1,2 and Akio Kanai1,2* 1Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017; and Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa 252-8520, Japan 2Systems (Received June 27, 2011) CONTENTS: 1. Introduction 2. Vertebrate virus-encoded miRNAs 3. Nucleotide sequence conservation among viral miRNAs 4. Viral RNAs targeted by viral miRNAs 5. Host mRNAs targeted by viral miRNAs 6. Conclusion SUMMARY: An increasing number of studies have reported that approximately 400 microRNAs (miRNAs), encoded by vertebrate viruses, regulate the expression of both host and viral genes. Many studies have used computational and/or experimental analyses to identify the target genes of miRNAs, thereby enabling us to understand miRNA functions. Here, we suggest that important aspects become apparent when we focus on conserved viral miRNAs, although these miRNA sequences generally show little similarity among viral species. Reliable viral miRNA–target gene pairs can be efficiently identified using evolutionary information. In this review, we summarize information on (i) the nucleotide sequence conservation among viral miRNAs and (ii) the RNAs targeted by viral miRNAs. Recent advances in these topics are discussed. 1. Introduction The discovery of huge amounts of noncoding RNAs (ncRNAs) has indicated the importance of RNA molecules in many steps of gene regulation (1). Among the ncRNAs, microRNAs (miRNAs) are very small molecules of approximately 20 ribonucleotides, which function posttranscriptionally by hybridizing to their target mRNAs. Typically, miRNAs repress the translation or trigger the degradation of their target mRNAs (2). An increasing number of studies have shown that not only animals and plants but also viruses encode miRNAs, which target both viral and host mRNAs to control their expression (3–6). Because an understanding of viral miRNAs is necessary for both basic science and the development of therapeutic agents, we focus on viral miRNAs in this review. In Fig. 1, we summarize existing knowledge regarding the regulatory relationships between viruses and host cells mediated by miRNAs. Basically, this figure shows the flow of genetic information from DNA to protein, which is the ``central dogma'' of molecular biology. It is noteworthy that both viral and host miRNAs can control the flow of genetic informa- Fig. 1. Schematic representation of the regulatory relationships mediated by miRNA in the genetic information flow between virus and host cell. The flow of genetic information from DNA to protein, which is the central dogma of molecular biology, is shown for both the virus and host cell. A recent study demonstrated that miRNAs are not only encoded in the genomes of host cells, but also by certain viral genomes, and play important roles in their gene regulation. The arrows indicate the regulatory interactions that affect the genetic information flow. Four examples of miRNA-mediated regulatory interactions, represented by bold arrow (A–D), are described in the text. *Corresponding author: Mailing address: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan. Tel: +81-235-29-0524, Fax: +81-23529-0525, E-mail: akio@sfc.keio.ac.jp tion according to the central dogma. As examples, virus-encoded miRNAs target viral RNAs and host cell RNAs (regulatory relationships A and B, respectively, in Fig. 1) (4–6), whereas host cell-encoded miRNAs target viral RNAs (regulatory relationship C in Fig. 1) (4). It is also noteworthy that a viral protein can interact with a host protein (regulatory relationship D in Fig. 1). This article is an Invited Review based on a lecture presented at the 20th Symposium of the National Institute of Infectious Diseases, Tokyo, May 21, 2010. 357 A viral protein has been reported to bind the Argonaute (Ago) protein, one of the most important factors involved in the functioning of the RNA-induced silencing complex (RISC) (7). Based on these findings, the miRNA regulatory systems of viruses and their host cells seem to mediate the conflicts between them. Understanding this complex regulatory relationship is important for the development of antiviral measures. In this paper, we review the recent progress in virusencoded miRNAs, focusing particularly on vertebrate viruses, and list the known virus-encoded miRNAs. The nucleotide sequence features of these miRNAs are then discussed based on sequence conservation analysis. To understand the functions of these miRNAs, it is necessary to identify their target mRNAs. Therefore, we provide information on the cellular and viral targets of these viral miRNAs. 2. Vertebrate virus-encoded miRNAs Five miRNAs encoded by a viral genome were first reported in the Epstein-Barr virus (EBV) in 2004 (6), and 400 viral miRNAs encoded by 22 viruses are currently registered in miRBase (release 17.0, April 2011). The miRBase is one of the most useful databases, providing an integrated interface for comprehensive miRNA sequence data, annotations, and predicted gene targets (8). In most cases, just as viral miRNAs are synthesized in host cells, they are thought to be processed by the host miRNA-processing machinery (9,10). Before discussing recently reported virus-encoded miRNAs in detail, we will briefly describe the general miRNA biogenesis pathway in host cells (Fig. 2). Generally, several RNA-processing steps are required to produce mature miRNAs. Initially, the primary miRNAs (pri-miRNAs) are transcribed from their genes in the nucleus (11). PrimiRNAs are processed into approximately 70-nucleotide hairpin RNAs by Drosha, a nuclease of the RNase III family, and DGCR8 (12,13). The processed RNAs are called ``precursor miRNAs'' (pre-miRNAs), and are exported to the cytoplasm by Exportin-5, where the miRNA duplexes produced are cleaved by another RNase III nuclease, Dicer (14,15). The mature miRNAs (``guide strands'') are then incorporated into the miRNA-induced silencing complex (miRISC) to play a role in regulating gene expression, whereas the other strands (``passenger strands'') are immediately degraded, although it has also been reported that both the guide and passenger strands are potentially functional in some cases (16). Two approaches are generally used to identify viral miRNAs. The first involves experimental validation, such as molecular cloning and nucleotide sequencing of the small RNA fraction (6), whereas in the second, a bioinformatics methodology is used. This approach is based on computational prediction, such as predicting the secondary structures of pre-miRNAs from viral genomes, or distinguishing positive from false miRNAs to identify their nucleotide sequences and structural characteristics using a machine learning technique (17,18). It has been suggested that high-throughput analyses combined with computational analyses constitute the most effective approach (19,20). In Table 1, we summarize the currently known miRNAs encoded by vertebrate Fig. 2. General miRNA processing pathway. Initially, miRNA genes are transcribed to produce primary miRNAs (primiRNAs) in nucleus. RNase III enzyme Drosha and its binding partner DiGeorge-syndrome critical-region protein 8 (DGCR8), also known as Microprocessor, processes primiRNAs into precursor miRNAs (pre-miRNAs). After being exported to cytoplasm by a complex of Exportin-5 and RanGTP, pre-miRNAs are cleaved into miRNA duplexes by another RNase III enzyme Dicer with its cofactor transactivating region RNA-binding protein (TRBP). One strand of miRNA duplexes is incorporated into miRNA-induced silencing complex (miRISC) containing Argonaute 2 (AGO2) for mRNA degradation or translational repression. Triangles in both pri-miRNA and pre-miRNA indicate the cutting sites of RNase III enzymes. viruses. The number of viral miRNAs ranges from 2 to 68 (approximately 16 per viral genome). Among these viruses, the Herpesviridae encode large numbers of miRNAs, and rhesus lymphocryptovirus (RLCV) encodes 68 miRNAs. In contrast, only 1–4 miRNAs are found in the families Polyomaviridae, Papillomaviridae, Adenoviridae, and Retroviridae. We note that most viruses encoding miRNAs belong to doublestranded DNA virus families, such as the Herpesviridae, Polyomaviridae, Papillomaviridae, and Adenoviridae. In contrast, there have been no reports of RNA viruses encoding miRNAs, except for Retroviridae human immunodeficiency virus 1 (HIV-1). This is probably because the replication systems of the double-stranded DNA viruses and RNA viruses differ. In general, double-stranded DNA viruses replicate their genomes in the host nuclei, whereas RNA viruses replicate their genomes in the host cytoplasm. As described above, host nuclear factors such as Drosha and DGCR8 are required for the pre-miRNA processing step. Therefore, many 358 Table 1. List of vertebrate virus-encoded miRNAs Family Herpesviridae Subfamily Name a-Herpesvirinae Herpes simplex virus-1 (HSV-1) Herpes simplex virus-2 (HSV-2) Marek's disease virus-1 (MDV-1) Marek's disease virus-2 (MDV-2) Turkey herpesvirus (HVT) Infectious laryngotracheitis virus (ILTV) Bovine herpesvirus (BHV1) Herpes B virus (HBV) Human cytomegalovirus (HCMV) Mouse cytomegalovirus (MCMV) Epstein-Barr virus (EBV) Rhesus lymphocryptovirus (RLCV) Kaposi's sarcoma-associated herpesvirus (KSHV) Rhesus monkey rhadinovirus (RRV) Mouse gamma herpesvirus-68 (MHV68) b-Herpesvirinae g-Herpesvirinae Polyomaviridae — Retroviridae 1): — Lentivirinae Host Reference 25 24 26 36 28 10 12 3 17 29 44 68 25 11 28 (16) (18) (14) (18) (17) (7) (10) (3) (11) (18) (25) (36) (12) (7) (15) Human Human Avian Avian Avian Avian Cattle Simian Human Murine Human Simian Human Simian Murine 50–52 50, 53, 54 55, 56 23, 24 24, 57 24, 58 59 60 17, 61 62, 63 6, 64–66 64, 67, 68 17, 65, 69, 70 71 17 2 2 2 2 2 2 (1) (1) (1) (1) (1) (1) Simian Simian Human Human Human Murine 18 72 73 74 74 75 1 (1) Bandicoot 76 1 (1) Bandicoot 76 Human adenovirus (AV) 3 (2) Human 77 Human immunodeficiency virus 1 (HIV-1) 4 (3) Human 21, 22 Simian virus 40 (SV40) Simian agent 12 (SA12) Merkel cell polyomavirus (MCV) BK polyomavirus (BKV) JC polyomavirus (JCV) Murine polyomavirus (PYV) Bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1) Bandicoot papillomatosis carcinomatosis virus type 2 (BPCV2) Papillomaviridae Adenoviridae No. miRNAs1) Number of precursor miRNAs is shown in parentheses. Table 2. Nucleotide sequence conservation of viral miRNAs in miRBase viral miRNAs may be found in double-stranded DNA viruses. However, miRNAs have been reported in HIV-1 of the family Retroviridae (21,22). This retrovirus can integrate into the host nuclear genome as double-stranded DNA using the virally encoded enzymes RNA reverse transcriptase and integrase. Therefore, miRNAs are produced from the genome of this retrovirus as they are from double-stranded DNA viruses. 3. Nucleotide sequence conservation among viral miRNAs As discussed above, the number of studies on viral miRNAs is increasing. Recent studies have also reported that viral miRNA sequences are less similar to one another than are nonviral miRNAs, although the genomic locations of the viral miRNAs are often conserved (23,24). However, some questions remain regarding how many viral miRNA sequences are conserved in the same viruses or individual viruses, and whether any sequence characteristics exist in these miRNAs. To address these questions, we comprehensively analyzed the nucleotide sequence conservation among viral miRNAs. For this purpose, we first aligned the 400 viral miRNA sequences registered in miRBase in all combinations (79,800 pairs in total). The levels of conservation among these pairs are listed in Table 2. The sequence similarities of the 79,800 miRNA pairs were calculated. No. of combinations (without considering seed sequence match) No. of combinations (with complete match of seed sequence) Total 79,800 79,800 Æ30z Æ40z Æ50z Æ60z Æ70z Æ80z Æ90z 100z 56,083 19,078 3,390 277 67 34 17 4 90 89 72 50 34 22 14 4 Numbers of combinations of conserved viral miRNAs in various viruses are shown with their similarities. In the left column, only the sequence similarity is considered, whereas in the right column, both the sequence identity and the seed sequence matches are considered (nucleotides 1–7, 2–8, or 3–9 from the 5? end of the miRNA). The ``seed'' sequences include nucleotides 2–8 from the 5? end of the miRNAs, and are important for target recognition (25). Therefore, we considered two sets of data: the degree of similarity between sequence pairs when the seed sequence matches were not considered and the degree of similarity in sequence pairs when their seed sequences showed complete matches. As is evident 359 spectively (Table 2). Notably, among the same or related viruses, the viral miRNA sequences are 100z identical for four pairs (bkv-miR-B1-3p and jcv-miR-J1-3p, ebv-miR-BART1-3p and rlcv-miR-rL1-6, hvt-miR-H93p and hvt-miR-H12-3p, and bpcv1-miR-B1 and bpcv2miR-B1), although the pre-miRNA sequences are not completely conserved (Fig. 3). This strongly suggests that both these miRNAs and their possible targets have been highly conserved, and have influenced viral function throughout viral evolution. For instance, the identical miRNAs of the polyomaviruses (bkv-miR-B1-3p and jcv-miR-J1-3p) have functions that allow the viruses to evade immune cell attack (26). Specifically, these two miRNAs target the 3? untranslated region (3?-UTR) of ULBP3 mRNA, which encodes a stress-induced ligand recognized by the killer receptor NKG2D of natural killer (NK) cells. Focusing on the conserved miRNA– target gene pairs is also very important for an understanding of the evolution of miRNA-mediated regulation. Recently, we reported that miRNA–target gene pairs can be effectively identified based on the evolutionary analysis of bilaterian animals (27). Let us examine the characteristics of miRNA pairs with more than 80z sequence similarity. Twenty-two pairs with this degree of this similarity have completely matched seed sequences, whereas 34 pairs have this degree of this similarity if we do not consider the seed matches (Table 2). Therefore, 12 pairs have Æ80z sequence similarity but incompletely matched seed sequences. Three representative examples of these 12 pairs are illustrated in Fig. 4. We have identified a rule for the pattern of nucleotide sequences in the seed region. As shown in Fig. 4, these miRNA pairs contain nucleotide mismatches in the seed region. When G–U wobble pairs are permitted, it is conceivable that these pairs recognize identical or orthologous target mRNAs. Indeed, it has been reported that miRNA sequences that form G–U pairs recognize target mRNAs and reduce their expres- from this table, the numbers of conserved miRNA pairs (80z–100z sequence similarity) are relatively small. We also note that the numbers of conserved miRNA pairs differ depending on whether we ignore the seed sequence matches or consider the completely matched seed sequences. The number of conserved miRNA pairs (Æ70z sequence similarity) is considerably larger when we do not consider the seed sequence matches than when we consider the completely matched seed sequences. Assuming that the seed sequence is important for the recognition of target mRNAs, the large numbers of pairs with only 30z–60z sequence similarity are considered to be false-positive pairs. Therefore, we focus on the evolutionarily conserved miRNAs in this review and describe the importance of analyzing pairs with high sequence similarity. For example, when considering the seed sequence matches, the number of miRNA pairs with more than 70z, 80z, and 90z sequence similarities are 34, 22, and 14, re- Fig. 3. Secondary structures of virus-encoded pre-miRNAs sharing the same mature miRNA sequences. (A–D) All pairs of premiRNAs with 100z identical mature miRNA sequences are shown. The mature miRNA sequences are shown in bold uppercase letters. Notably, both the secondary structure and nucleotide sequence of the precursor miRNA differ slightly, despite the complete match of the mature miRNA sequences in these four pairs. bkv, polyomavirus BK; jcv, polyomavirus JC; ebv, Epstein-Barr virus; rlcv, rhesus lymphocryptovirus; hvt, turkey herpesvirus; bpcv1, bandicoot papillomatosis carcinomatosis virus type 1; bpcv2, bandicoot papillomatosis carcinomatosis virus type 2. Fig. 4. Three examples of highly conserved miRNAs encoded by viruses. (A–C) Nucleotide sequences of mature viral miRNAs with Æ80z conservation are aligned. Conserved nucleotide residues are indicated with an asterisk. Gaps are inserted for maximum homology. The miRNA seed region is bracketed. Note that the miRNA sequences differ in their seed regions but possibly bind the same target mRNAs when G–U wobble pairs are permitted. See the text for details. ebv, Epstein-Barr virus; mdv1, Marek's disease virus 1; hsv1, herpes simplex virus 1. 360 miRNAs play important roles in the maintenance of viral latency. miRNAs involved in the avoidance of the host immune system are classified in group II. An example of this is polyomavirus simian virus 40 (SV40) miRNA (sv40-miR-S1) downregulation of the expression of the viral T-antigen, which is a target of the cytotoxic T-lymphocyte response. sv40-miR-S1 accumulates during the late stages of infection and targets early viral mRNAs for cleavage, resulting in the reduced expression of the viral T antigen (18). One possible function of sv40-miRS1 is to allow the virus to escape from the host immune system, increasing the probability of successful infection. The group III miRNAs are related to the regulation of cellular apoptosis, limiting host cell death during viral proliferation. It has been reported that overexpression of the EBV LMP1 gene promotes host cell apoptosis (35). These authors showed that three EBV miRNAs (miR-BART1-5p, miR-BART16, and miR-BART17-5p) target the 3?-UTR of LMP1 mRNA and downregulate the expression of LMP1 protein, reducing the proapoptotic effect. Therefore, EBV miRNAs have an impact on the host cell-death pathway, enhancing viral survival. Viral miRNAs associated with the control of viral replication are classified to group IV. EBV expresses different replication systems during latent and lytic infection (36). The authors reported that cellular replication factors are recruited during latent infection, whereas viral replication factors, including viral polymerase BALF5, are required during lytic infection. It has been shown that miR-BART2 regulates the transition from latent to lytic viral replication (6,81). During latent infection, EBV miR-BART2 cleaves the 3?-UTR of the BALF5 mRNA, whereas induction of the lytic replication cycle causes a reduction in miR-BART2 levels. Another example is the HIV-1 protein Nef. The viral miRNA miR-N367 suppresses Nef expression through the regulatory U3 region in the 5? long terminal repeat (5?-LTR) (22). Because Nef is considered a regulatory factor for HIV-1 replication, miR-N367 may control viral replication by limiting Nef expression. Among other HIV-1 miRNAs, TAR miRNA is thought to be processed from the HIV-1 TAR element by the Dicer enzyme in host cells (21,37). Although the function of TAR miRNA is unclear, it is believed to repress viral gene expression through the viral LTR (group V). sion (28). It is noteworthy that this pattern of nucleotide sequences associated with G–U pairs was observed in 11 of the 12 pairs. Among these 11 pairs, four pairs are encoded by the genomes of related viruses (two by herpes simplex virus 1 [HSV-1] and herpes simplex virus 2 [HSV-2], and two by EBV and RLCV). Moreover, seven pairs are encoded by the genomes of identical viruses (a pair each in EBV, HSV-1, HSV-2, and Marek's disease virus 1 [MDV-1], and three pairs in turkey herpesvirus [HVT]). When these conserved miRNA pairs are encoded in the genomes of the same viruses, they may play a role in a backup system, or may be useful in avoiding host cell attack. If these pairs recognize the same target sites, the part of nucleotide sequences on the target sites are restricted to ``G'' or ``U'' (Fig. 4). Therefore, it is presumed that reliable target sites can be extracted using this conserved miRNA sequence information. It has also been reported that one mRNA can have several different miRNA-binding sites. For instance, C/EBPb p20 (LIP) mRNA, which encodes a negative transcriptional regulator of specific cytokines, including interleukin 6 (IL6) and IL10, has target sites for two Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs (miR-k12-3 and miR-k12-7) (29). These miRNAs control the expression of LIP mRNA, resulting in the induction of cytokine (IL6 and IL10) secretion by macrophages. In this situation, IL6 and IL10 play important roles in KSHV-associated cancer (30,31). 4. Viral RNAs targeted by viral miRNAs Recent reports have suggested that viral miRNA targets can be categorized into two classes: viral RNA targets (regulatory relationship A in Fig. 1) and cellular mRNA targets (regulatory relationship B in Fig. 1), which are described in this section and in Section 5 of this review, respectively. Viral RNAs regulated by viral miRNAs are listed in Table 3. Currently, 21 viral mRNA targets have been experimentally identified, and this number is increasing. Viral miRNA functions are categorized into five groups: (I) latent and lytic viral infection, (II) immune evasion, (III) prevention of apoptosis, (IV) viral replication, and (V) others. More than half of these viral miRNAs are associated with latent and lytic viral infections (group I). For instance, infectious laryngotracheitis virus (ILTV) miR-I5 targets the transcriptional activator ICP4 mRNA, which is essential for viral growth and is repressed during latent infection. Therefore, miR-I5 is involved in modulating the balance between the lytic and latent status of the virus (32). miR-I5 is located antisense to the ICP4 mRNA in the ILTV genome. With the complete hybridization of miRNA–mRNA pairs, miR-I5 regulates ICP4 mRNA in an siRNA-like manner, cleaving ICP4 mRNA rather than inhibiting its translation. In another example, KSHV-encoded miR-k12-9* suppresses the expression of the viral replication and transcription activator (RTA), which is the major lytic switch protein, by hybridizing directly with the 3?-UTR of its mRNA to regulate lytic reactivation (33). A recent report mentioned that miR-k12-7-5p also targets the 3?-UTR of RTA mRNA to prevent the production of progeny virus (34). These findings suggest that KSHV 5. Host mRNAs targeted by viral miRNAs In this section, we focus on recent research that has described the regulation of cellular mRNAs by viral miRNAs. Thirty-two cellular mRNA targets of viral miRNAs are listed in Table 4. The cellular mRNAs regulated by viral miRNAs can be categorized into six groups: (I) latent and lytic viral infection, (II) immune evasion, (III) prevention of apoptosis, (IV) viral replication, (V) cell cycle, and (VI) others. This classification is essentially the same as that in Section 4, except for group V. One of the roles of viral miRNAs is the maintainance viral latency via repression of the cellular factors involved in viral lytic reactivation (group I). It has been 361 Table 3. Summary of viral mRNA targets of viral miRNAs Virus Family Herpesviridae Viral mRNA target Subfamily Name a-Herpesvirinae HSV-1 miR-H2-3p ICP0 HSV-1 miR-H6 ICP4 HSV-2 miR-I, II ICP34.5 HSV-2 miR-III ICP0 MDV1 miR-M4 UL28 MDV1 miR-M4 UL32 ILTV miR-I5 ICP4 b-Herpesvirinae g-Herpesvirinae Papillomaviridae Retroviridae — — Lentivirinae Name Possible function Group1) Host Reference Transcriptional activator: thought to have a role in reactivation Transcriptional activator: required for expression of most HSV1 genes during productive infection Neurovirulence factor: required to control viral replication in neuronal cells Transcriptional activator: important for HSV reactivation DNA packing protein: involved in cleavage/packing of herpesvirus DNA DNA packing protein: involved in cleavage/packing of herpesvirus DNA Transcriptional activator: essential for viral growth and repressed during latency Transcriptional activator: critical for gene expression and required for viral replication Uracil DNA glycosylase: important for viral replication DNA polymerase: required for lytic viral replication Latent membrane protein: potent immunogenic viral antigen recognized by cytotoxic T cells Latent membrane protein: induces cell growth I Human 51 I Human 51 I, IV Human 53, 78 I Human 53 I Avian 49 I Avian 49 I Avian 32 I, IV Human 79 I, IV Human 80 I, IV Human 6, 81 II Human 82 III Human 35 HCMV miR-UL112-1 IE1/IE72 HCMV miR-UL112-1 UL114 EBV miR-BART2 BALF5 EBV miR-BART22 LMP2a EBV miR-BART1-5p, miR-BART16, miR-BART17-5p miR-k12-9*, miR-k12-7-5p LMP1 RTA Lytic switch protein: controls viral reactivation from latency I Human 33, 34 SV40 miR-S1 LT-Ag II Simian 18 JCV miR-J1 LT-Ag II Human 74 BKV miR-B1 LT-Ag II Human 74 PYV miR-P1 LT-Ag Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response II Murine 75 BPCV1 miR-B1 LT-Ag II Bandicoot 76 BPCV2 miR-B1 LT-Ag Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response II Bandicoot 76 HIV-1 miR-N367 NEF IV Human 22 HIV-1 miR-TAR LTR Accessory protein: important, but not essential for viral replication Important for viral replication V Human 37 KSHV Polyomaviridae Viral miRNA For abbreviations, see Table 1. Roman numerals indicate the following possible functions: (I) latent and lytic viral infection, (II) immune evasion, (III) prevention of apoptosis, (IV) viral replication, and (V) others. 1): 362 Table 4. Summary of cellular mRNA targets of viral miRNAs Virus Family Herpesviridae Cellular mRNA target Subfamily Name a-Herpesvirinae MDV1 MDV1 MDV1 MDV1 MDV1 MDV1 MDV1 b-Herpesvirinae MCMV HCMV HCMV HCMV HCMV g-Herpesvirinae EBV EBV EBV EBV KSHV KSHV KSHV KSHV KSHV KSHV KSHV KSHV KSHV KSHV KSHV KSHV Polyomaviridae — BKV JCV Retroviridae Lentivirinae HIV-1 HIV-1 Viral miRNA mdv1-miR-M3 Name SMAD2 Possible function A critical factor in the transforming growth factor b signal pathway mdv1-miR-M4 PU.1 — mdv1-miR-M4 GPM6B — mdv1-miR-M4 RREB1 — mdv1-miR-M4 c-Myb — mdv1-miR-M4 MAP3K7IP2 — mdv1-miR-M4 C/EBP — miR-M23-2 CXCL16 Chemokine expressed in both soluble and transmembrane forms miR-US25-1 CCNE2 G1/S cyclin E2 miR-US25-1 H3F3B H3 histone, family 3B miR-US25-1 TRIM28 Transcriptional silencer miR-UL112-1 MICB Stress-induced ligand of the natural killer cell activating receptor NKG2D miR-BHRF1-3 CXCL11 CXC chemokine ligand for CXCR3 miR-BART2-5p MICB Stress-induced ligand of the natural killer cell activating receptor NKG2D miR-BART5 PUMA Induces apoptosis in response to a wide variety of stimuli miR-BART6 Dicer RNase III enzyme, which is a component of the miRNA processing pathway miR-k12-1 P21 A key inducer of cell-cycle arrest An inhibitor of the NF-kB miR-k12-1 IkBa complex miR-k12-3 NFIB Activates the promoter of the viral RTA gene miR-k12-7 MICB Stress-induced ligand of the natural killer cell activating receptor NKG2D miR-k12-4-5p Rbl2 A known repressor of DNA methyltransferase 3a and 3b mRNA miR-k12-10a TWEAKR Tumor-necrosis-factor-like weak inducer of apoptosis receptor miR-k12-11 Fos — miR-k12-11 BACH1 Transcriptional repressor of heme oxygenase 1, which promotes cell survival miR-k12-3, C/EBPb p20 An isoform of C/EBPb known miR-k12-7 (LIP) to function as a negative transcriptional regulator miR-k12-1, MAF Cellular transcription factor, miR-k12-6-5p, which has a role in tissue miR-k12-11 specification and the terminal differentiation of a wide variety of cell types miR-k12-5, BCLAF-1 Bcl2-associated transcription miR-k12-9, factor miR-k12-10 miR-k12-1, THBS1 Potent inhibitor of blood vessel miR-k12-3-5p, growth miR-k12-6-3p, miR-k12-11 miR-B1-3p ULBP3 Stress-induced ligand recognized by the killer receptor NKG2D miR-J1-3p ULBP3 Stress-induced ligand recognized by the killer receptor NKG2D TAR miRNA ERCCI Involved in serum-starvationinduced apoptosis TAR miRNA IER3 Involved in serum-starvationinduced apoptosis Group1) Host Reference III Avian 83 VI VI VI VI VI VI II Avian Avian Avian Avian Avian Avian Murine 49 49 49 49 49 49 42 V VI VI II Human Human Human Human 47 47 47 39 II II Human Human 41 40 III Human 43 I Human 84 V IV Human Human 46 45 I Human 38 II Human 40 VI Human 48 III Human 85 VI VI Human Human 86 87 VI Human 29 VI Human 88 VI Human 89 VI Human 90 II Human 26 II Human 26 III Human 44 III Human 44 For abbreviations, see Table 1. Roman numerals indicate the following functions: (I) latent and lytic viral infection, (II) immune evasion, (III) prevention of apoptosis, (IV) viral replication, (V) cell cycle, and (VI) others. 1): 363 Finally, the ``others'' of group VI include an interesting example. Viral miRNA regulates DNA methylation at several sites in both the viral and host genomes. Viral genomic methylation by viral miRNA has been described. The KSHV miRNA miR-k12-4-5p reduces the expression of retinoblastoma (Rb)-like protein 2 (Rbl2) mRNA. Rbl2 is a repressor of DNA methyltransferases 3a and 3b (DNMT); consequently, this miRNA increases the number of methylated DNA sites in the viral genome (48). The promoter region of the viral RTA gene is methylated, which helps to maintain the latent status of the virus. In Sections 4 and 5, we discussed viral and cellular mRNA regulation by individual viral miRNAs. However, it should be noted that the same viral miRNA sometimes targets both viral and cellular mRNAs. For instances, the MDV1 miRNA miR-M4, which is known to be an orthologue of the host miR-155, targets cellular mRNAs (of PU.1, GPM6B, RREB1, c-Myb, MAP3K7IP2, and C/EBP) and viral RNAs (of UL28 and UL32) (49). Furthermore, HCMV miR-UL112-1 regulates both viral RNAs (IE1/IE72 and UL114) and a cellular mRNA (MICB), as described above (39,40). Because the number of virus-encoded miRNAs is generally small, it is conceivable that the same viral miRNAs have evolved to regulate both host and viral genes for efficient viral infection. However, this is observed in a minority of cases, with only a dozen miRNA target genes having been identified to date. With advances in molecular virology, it is probable that more examples of miRNAs that target both viral and cellular mRNAs will be identified. reported that the KSHV miRNA miR-k12-3 targets the 3?-UTR of nuclear factor I/B (NFIB) mRNA and downregulates its expression (38). NFIB enhances the promoter activity of the viral RTA gene, which is involved in KSHV reactivation. Therefore, miR-k12-3 stabilizes viral latency through the regulation of NFIB. In Section 4, we described how KSHV miRNAs (miRk12-9* and miR-12-7-5p) directly target and suppress the expression of viral RTA. These data together suggest that KSHV miRNAs suppress both viral and cellular RTA-mediated factors to maintain viral latency. As mentioned in Section 4, some viral miRNAs can potentially allow the virus to evade the host immune system by targeting viral mRNAs. It is also true that cellular mRNAs are targeted by viral miRNAs for the same purpose (group II). In fact, translation of the mRNA of MICB, which is the stress-induced ligand recognized by NKG2D on NK cells, is repressed by viral miRNAs, allowing the virus to evade the host immune response (39,40). It is noteworthy that the downregulation of MICB expression mediated by viral miRNAs has been confirmed in three types of herpesviruses (HCMV miRUL112-1, EBV miR-BART2-5p, and KSHV miR-k127), although these three miRNAs have no nucleotide sequence conservation. In another example, polyomavirus miRNAs are able to evade the host immune system by targeting the ULBP3 ligand, as described Section 3 (26). The chemokines CXCL11 and CXCL16 are also suppressed by EBV miR-BHRF1-3 and mouse cytomegalovirus (MCMV) miR-M23-2 miRNAs, respectively (41,42). These studies indicate that the targeting of ligands by viral miRNAs is a major viral strategy for avoiding the host immune system, at least in the polyomaviruses and herpesviruses. Viral miRNAs involved in the resistance to cellular apoptosis are classified in group III. A recent study demonstrated that miR-BART5 targets and represses the expression of proapoptotic PUMA (p53 upregulated modulator of apoptosis) mRNAs, protecting the virus from cellular apoptosis (43). HIV-1 TAR miRNA is also reported to attenuate host apoptosis (44). Briefly, TAR miRNA downregulates the expression of both ERCC1 (excision repair cross complementing group 1) and IER3 (intermediate early response 3) mRNAs, the products of which are known to induce apoptosis in response to serum starvation. Viral replication is regulated by viral miRNAs of group IV via the cellular NF-kB pathway. It has been shown that the deletion of the KSHV miRNA cluster reduces NF-kB activity, resulting in increased lytic replication. Detailed research has shown that miR-k1, in particular, represses the expression of IkBa mRNA, which encodes an inhibitor of the NF-kB complex (45). The host cell cycle is regulated by the viral miRNAs of group V. KSHV miRNA attenuates p21-mediated cellcycle arrest and miR-k1 represses p21 mRNA, a key inducer of cell-cycle arrest (46). Another example of host cell-cycle regulation is HCMV miR-US25-1 targeting and downregulation of the expression of cyclin E2 (CCNE2) mRNA (47). Cyclin E protein is expressed in the G1 phase, and binds to and activates CDK2 protein, resulting in progression to the S phase. Therefore, the inhibition of CCNE2 mRNA by miR-US25-1 may block cell-cycle progression. 6. Conclusion To determine the functions of viral miRNAs, it is important to consider the following two characteristics: (i) the nucleotide sequence conservation among viral miRNAs, and (ii) the target mRNAs of the viral miRNAs. By focusing on the miRNA sequences that are highly conserved among these viruses, reliable miRNA– target genes can be identified. It is noteworthy that different miRNAs can bind to the same mRNAs. Therefore, it is necessary to consider not only the highly conserved miRNAs but also other miRNAs that may bind to the same mRNAs. Viral miRNAs tend to regulate functions important for viral survival, such as the evasion of the host immune system and the regulation of viral replication, by targeting both host and viral mRNAs. Furthermore, one viral miRNA can regulate both host and viral mRNAs because there are only a limited number of viral miRNAs. Finally, we proposed that these two characteristics could contribute to the development of therapeutic applications. Developing drugs from these conserved miRNAs could allow us to (a) target several viruses by antagonizing these conserved miRNAs, and (b) effectively repress viral growth by targeting genes essential for viral survival. Acknowledgments The authors would like to thank the members of the RNA Group at the Institute for Advanced Biosciences, Keio University, Japan, for their helpful discussions. K. 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