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Virus-encoded microRNAs: novel regulators of gene expression
Review
TRENDS in Microbiology
Vol.14 No.4 April 2006
Virus-encoded microRNAs: novel regulators of gene expression Venugopal Nair1 and Mihaela Zavolan2 1 2
Viral Oncogenesis Group, Division of Microbiology, Institute for Animal Health, Compton, Berkshire, UK, RG20 7NN Division of Bioinformatics, Biozentrum, University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland
MicroRNAs (miRNAs) are a class of small RNAs that have recently been recognized as major regulators of gene expression. They influence diverse cellular processes ranging from cellular differentiation, proliferation, apoptosis and metabolism to cancer. Bioinformatic approaches and direct cloning methods have identified O3500 miRNAs, including orthologues from various species. Experiments to identify the targets and potential functions of miRNAs in various species are continuing but the recent discovery of virus-encoded miRNAs indicates that viruses also use this fundamental mode of gene regulation. Virus-encoded miRNAs seem to evolve rapidly and regulate both the viral life cycle and the interaction between viruses and their hosts. Discovery of microRNAs as regulators of gene expression The discovery that microRNAs (miRNAs) are key players in the micromanagement of gene expression in diverse cellular processes in many species is a remarkable breakthrough for the field of molecular biology. The first miRNA, lin-4, was discovered over 12 years ago in Caenorhabditis elegans [1], yet it was only when the first evolutionarily conserved miRNA, let-7, was shown to have a crucial role in C. elegans development that the versatility and the far-reaching influence of miRNAs on various cellular processes started to come to light. The latest release of the miRNA repository MiRBase (release 8.0; http://microrna.sanger.ac.uk) contains 3518 annotated miRNAs, including 326 human miRNA genes [2–4]. Within cells, the number of miRNA molecules seems to vary from only a few to as many as 50 000 [5]. Many miRNAs are ubiquitously expressed, whereas others are expressed in a cell-type-specific manner [6–11]. Because a single miRNA can target transcripts from multiple genes and, conversely, several miRNAs can control a single target [12], the miRNAs and their targets function as a complex regulatory network [13]. With recent computational predictions suggesting that miRNAs could target up to a third of all human genes [12,14], the potential of miRNAs to modulate the transcriptome is overwhelming. The profound influence of miRNAs on diverse regulatory pathways such as embryonic development, haematopoietic cell differentiation, apoptosis, cell proliferation and cancer has already been demonstrated Corresponding author: Nair, V. ([email protected]). Available online 13 March 2006
[15–20]. Here, we review the features of virus-encoded miRNAs and their potential involvement in the regulation of their own genes and in the subversion of cellular defence mechanisms. Biogenesis and functions of miRNAs The miRNA biogenesis pathway has been examined in detail (Figure 1). The sequential action of two doublestranded RNA-specific RNase III type ribonucleases (Drosha and Dicer) produces a 21-nucleotide RNA duplex from long primary transcripts (pri-miRNAs) [21]. Depending on the relative stability of their 5 0 ends, only one of the two strands of the duplex is incorporated into the RNAinduced silencing complex (RISC), which contains proteins such as members of the Argonaute (Ago) family, the RNA-binding proteins VIG and Fragile X-related protein and the nuclease Tudor-SN [22]. The RISC complex then becomes functional and inhibits the expression of specific cellular or viral proteins. There are two major mechanisms by which miRNAs regulate target gene expression. A high degree of complementarity between miRNAs and their cognate mRNAs in a RISC complex [which contains the appropriate Ago protein (Ago2 in humans) [23]] results in the cleavage of the target mRNA at a precise location, corresponding to the bond between the tenth and eleventh nucleotides of the miRNA [24]. This mode of silencing by direct cleavage of the target mRNA is most common in plants [25]. When there is insufficient complementarity with the target, the miRNA–RISC represses translation of the mRNA transcripts. Recent data demonstrate that this occurs by sequestration of target mRNA into distinct sites such as the P bodies in the cytoplasm, away from the translational machinery [26]. Because both effects can be observed with a given miRNA [27–29] and because different miRNAs can bind to multiple target sites on the same transcript, miRNAs might exert a dosage-dependent effect on the expression of individual genes. This extra tier of regulation has been compared with that of a dimmer switch or rheostat to increase or dampen the gene expression [30]. Perhaps best explored is the function of miRNAs in the regulation of developmental processes. Since the discovery of the first conserved miRNA, let-7 (which controls developmental timing in C. elegans [31]), evidence has accumulated regarding the differential expression of individual miRNAs in different cell lineages, in which they might determine cell fate. For example, miR-273 and lsy-6 are
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Figure 1. Schematic overview of miRNA biogenesis in eukaryotic cells. Processing of both host miRNAs (pink) and virus-encoded miRNAs (blue) is thought to take place through the same pathway. Initially, within the nucleus, the long primary transcripts of miRNA (pri-miRNAs) containing the hairpin structure are processed by the nuclear RNase III Drosha to release the precursor of miRNA (pre-miRNA). Following nuclear processing, pre-miRNAs are exported to the cytoplasm by one of the nuclear transport receptors, Exportin-5 (Exp-5), before being processed by the cytoplasmic RNase III Dicer into 21-nucleotide mature miRNAs. These miRNAs then associate with RISC to target the specific host- or virus-encoded transcripts to regulate gene expression.
a pair of miRNAs that function sequentially to control the laterality of the nematode chemosensory system [32]. In mammals, miR-223 and miR-342 are specifically expressed in myeloid and lymphoid lineages, respectively, and miR-181a is important in B-lymphoid lineage differentiation [8]. Furthermore, recent data suggest that miR-150 and miR-146 are differentially regulated during developmental stages of B and T cell maturation and during the transition from naı¨ve T cells to effector Th1–Th2 cells [33]. Thus, the miRNA milieu, which is unique to each cell type, could be instrumental in the establishment and maintenance of cell identity. Virus-encoded miRNAs Viruses are obligatory intracellular pathogens that are associated with many diseases in both animals and plants. The successful survival of viruses crucially depends on their ability to exploit the biosynthetic machinery of host cells and to inactivate the innate defence mechanisms of the host, such as the interferon and apoptosis responses. Although many large viruses achieve this by encoding proteins that specifically inactivate host-cell defences, the comparatively limited coding capacity of viruses makes the tiny miRNAs a particularly efficient and accessible tool to turn off the expression of specific genes. Indeed, recent studies show that viruses do exploit this pathway by generating their own miRNAs [34]. A computational and experimental survey of 29 virus genomes identified 33 novel viral miRNAs [35], which provide the opportunity to study the function and evolutionary conservation of viral miRNAs. MicroRNAs encoded by herpesviruses Among the various families of viruses, the herpesvirus family stands out in establishing long-standing latent infection as a major part of the viral life cycle. This large group of DNA viruses is classified into three subfamilies www.sciencedirect.com
(a, b or g) on the basis of their genome structure and biology, and includes pathogens such as herpes simplex virus, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), Kaposi’s sarcoma herpesvirus (KSHV) and murine herpesvirus 68 (MHV 68). Although the symptomatology associated with these viruses ranges from cold sores to lymphoproliferation to encephalopathy and cancer (particularly in immunocompromised hosts), they all share the ability to establish latency in the host after an early replication phase. During the latent and ‘active’ states, the intracellular virus must adapt to the multitude of events that occur in the cell. Much of this adaptation is achieved through specialized gene products that are encoded by the viruses. The herpesvirus gene repertoire includes homologues of genes such as Bcl-2, cyclin and chemokines that are thought to be ‘hijacked’ from the host itself to combat the hostile environment of the cell. MiRNAs are the most recent weapons found in the repertoire of herpesviruses. The first virus-encoded miRNAs that were discovered were those of EBV, from which five miRNAs were cloned [34]. These miRNAs are not homologous to any known host-cell miRNAs and an initial search in other herpesviruses did not reveal any orthologous miRNAs. These results indicated that viral miRNAs could not be identified with the existing miRNA prediction software, which relies heavily on cross-species sequence conservation. Using various combinations of novel computational prediction methods and a small-RNA cloning approach, several novel miRNAs have been identified in the genomes of many herpesviruses [35–38]. EBV-encoded miRNAs EBV (human herpesvirus years ago and is present population. EBV infection lymphoid and epithelial Burkitt’s lymphoma and
4) was discovered nearly 40 in O90% of the total world is thought to predispose for cell malignancies such as nasopharyngeal carcinoma.
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every stage of EBV infection, miR-BART-1 and 2 were detected during the lytic and the three latent stages. However, the expression of miR-BHRF1-1, 2 and 3 was related to the stage of latency. Their expression pattern also showed variations between cell lines, which provided the possibility of their classification based on the miRBHRF expression pattern. The functions of EBV miRNAs have not been fully established but, based on their location and on current knowledge regarding the function of animal miRNAs, the following types of functions can be distinguished. First, the mere processing of miRNAs could be a way to downregulate the expression of protein-coding genes within which the miRNAs reside. The three BHRF1 miRNAs, for example, require endonucleolytic processing of the BHRF1 transcript, thus, their expression should be anti-correlated with the expression of BHRF1. Second, miRNAs generated from the complementary strand of protein-coding genes could function as small interfering RNAs (siRNAs) against the transcripts of the proteincoding gene. (SiRNAs are a distinct class of 20–25nucleotide RNA molecules that interfere with gene expression by destroying the transcripts after binding to complementary target sequences.) This seems to be
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One of the unique features of EBV is its ability to transform resting B cells into proliferating cell lines. This system has been widely used as a model for the lymphomagenic potential of the virus [39]. EBV-induced B-cell proliferation is driven by a limited set of latent viral proteins that comprise six EBV nuclear antigens (EBNA 1, 2, 3A, 3B, 3C and LP) and three latent membrane proteins (LMP 1, 2A and 2B). Involvement of non-coding RNAs in EBV biology is not new: EBV-transformed cells show abundant expression of the small, non-polyadenylated RNAs named EBER1 and EBER2. The successful cloning of five novel EBV-encoded miRNAs from a B-cell line latently infected with EBV [34] demonstrates that EBV also has the potential to exploit RNA silencing as a convenient mechanism for the regulation of host and viral gene expression. EBV miRNAs are clustered in two distinct regions of the genome (Figure 2). The first miRNA cluster, which consists of miR-BHRF1-1, 2 and 3, is located within the 5 0 and 3 0 UTR of the transcript of BHRF1, a homologue of the antiapoptotic protein Bcl-2. The other EBV miRNAs (miRBART-1 and 2) are located within the BART gene (Figure 2). Consistent with the expression of BART in
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Figure 2. Schematic diagram showing the structure of EBV, KSHV, MHV 68 and HCMV genomes. Approximate genomic locations of miRNAs encoded by each of the viruses are indicated by the nucleotide positions shown in brackets. Positions of selected viral genes of each of the viruses are also shown. Names of individual miRNAs (red) are taken from Refs [34,35]. Asterisks mark the two additional miRNAs that are present in HCMV [45]. www.sciencedirect.com
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the case for the BART-2 miRNA, which is located on the complementary strand of the viral DNA polymerase BALF5. The description of a transcript that corresponds precisely to the predicted siRNA-dependent processing product of the viral DNA polymerase [40] indicates that BART-2 directs the cleavage of BALF5. Finally, it is thought that the virus-encoded miRNAs function in translational repression of viral and host genes, similar to host-encoded miRNAs. In this respect, computational predictions suggested that the targets of viral miRNAs might include regulators of cell proliferation, apoptosis, chemokines and cytokines – all molecules that have profound potential to influence the pathogenicity of the virus [34]. KSHV-encoded miRNAs KSHV (human herpesvirus 8), which was initially discovered in AIDS-related Kaposi’s sarcoma, is also associated with B-cell primary effusion lymphomas and some forms of multicentric Castleman’s disease. The most striking feature of KSHV is the extensive use of molecular piracy and the capture of several host genes including complement receptor 2, CC family chemokines, a CXC (GPCR) receptor homologue, interleukin-6 (IL-6) and Bcl-2 homologues, interferon regulatory factors, D-type cyclin, a FLICE-like caspase inhibitor and a transmembrane-spanning adhesion molecule. This complex array of host proteins enables KSHV to modulate the cellular regulatory network for KSHV replication and pathogenesis. The recent discovery of novel miRNAs encoded by KSHV further adds to the complexity of the gene regulation pathways in this virus. A cluster of 11 miRNAs confined to a 5-kb region, which also encoded the transforming protein kaposin, was identified in the KSHV-infected BCBL1 cell line [35]. This cluster included miR-K12-10 located within the K12 open reading frame (ORF). Other miRNAs are located within the intron of the larger kaposin primary transcript (Figure 2). These miRNAs were also identified in an independent study using another KSHV-infected cell line, BC-1, in which all of the miRNAs were expressed at levels detectable by northern blotting [36]. Because all KSHV-encoded miRNAs are located in a small region of the genome and are transcribed in the same direction, it is thought that they are all processed from a single promoter [41]. The recent isolation of these miRNAs from the KSHV-infected endothelial cell line TIVE-LTC demonstrates that the expression of KSHV miRNAs is not restricted to a particular cell type [38]. KSHV-encoded miR-K12-10 is located within the K12 ORF that encodes kaposin A, B and C, which have important functions in cell growth and cytokine production [37,42]. These proteins have to be tightly regulated during latency, and it remains to be determined whether the processing of miR-K12-10 miRNA has a role in downregulating kaposin expression. Computationally predicted targets for KSHV miRNAs include viral genes such as ORF 23, 27, 31, 52, 49, 61, 68, K7, K13 and K14, and several B-cell-specific genes involved in apoptosis and signalling [36]. Although these predictions remain to be www.sciencedirect.com
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validated, it is tempting to speculate that KSHV-encoded miRNAs have a role in KSHV replication and pathogenicity by regulating the expression of both viral and host genes. MHV 68-encoded miRNAs MHV 68 is a natural pathogen of small rodents. Based on its evolutionary relationship to the human g-herpesviruses, this virus is used in small animal models to study the biology of g-herpesviruses. As in the case of the human viruses, the MHV 68 genome encodes functional homologues of several host genes such as Bcl-2 (M11), IL8R (ORF74) and cyclin D (ORF72), all of which are used in the modulation of host gene expression. The recent discovery of nine miRNAs in the MHV 68 genome indicates that non-coding RNAs could be another means by which the virus modulates gene expression [35]. MHV 68 miRNAs, cloned from mouse B lymphoma S11 cells latently infected with MHV 68, are located within a 6-kb region near the M1 terminus of the linear MHV 68 genome (Figure 2), immediately downstream of predicted viral tRNA genes. This led to the speculation that MHV 68 premiRNAs are transcribed by pol III from tRNA promoters. Northern blot analysis on the three most abundant MHV 68 miRNAs (using probes that match the mature miRNA or the tRNA) further supports the hypothesis that miRNAs can be transcribed not only from pol II but also from pol III promoters that are active in many cell types. The unusual short-hairpin structures of the MHV 68 premiRNAs also suggest that their processing mechanism has distinct features compared with other miRNAs. The viral or cellular targets of these miRNAs remain to be identified. However, a mutant virus with a deletion that includes the miRNA cluster exhibited tissue-specific and route of infection-dependent alterations in latency and reactivation [43]. HCMV-encoded miRNAs HCMV (human herpesvirus 5) is a b-herpesvirus and an important pathogen that causes widespread primary and recurrent infections ranging from mild asymptomatic to severe congenital disease. HCMV is ubiquitous and, like other herpesviruses, can establish lifelong latency in infected individuals. HCMV-encoded proteins are thought to influence various cellular processes including senescence, cell cycle progression, apoptosis and transformation [44]. An initial bioinformatics and cloning approach identified nine miRNAs in the HCMV genome [35]. In contrast to MHV 68 and KSHV miRNAs, HCMV miRNAs are spread across the viral genome (Figure 2). Three of them are transcribed from the complementary strand of known ORFs, five are located in intergenic regions and one is located within an intron [35]. As with the other viral miRNAs, the targets remain to be identified. Nonetheless, the miRNAs that are transcribed from the complementary strand of protein-coding regions have the potential to function as siRNAs and regulate the level of viral proteins. For example, the miR-UL112-1 might target UL114, a homologue of the mammalian uracyl-DNA glycosylase. Because UL114 is required for efficient viral DNA replication, miR-UL112-1 has the potential to control
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viral replication. The expression of HCMV miRNAs was examined in two other recent studies. Grey et al. [45] found that the miRNAs expressed by the AD169 strain of HCMV (three of which were described previously [35] and the other two of which were novel) were expressed mostly with early kinetics in infected human fibroblasts [45]. A further study also demonstrated the expression of HCMV miRNAs during the productive lytic infection of four distinct human cell types, namely fibroblasts, endothelial cells, epithelial cells and astrocytes [46]. MiRNAs have also been computationally predicted in the genomes of a-herpesviruses, including seven miRNAs in HHV1, six in HHV2 [35] and nine in Marek’s disease virus (V. Nair and M. Zavolan, unpublished) but not in other human herpesviruses (HHV) such as HHV3, HHV6 and HHV7, which also have a similar life cycle. If these viruses also encode miRNAs, it would be interesting to find out how they differ from the EBV, KSHV and HCMV miRNAs such that they were not predicted computationally. MicroRNAs encoded by other viruses Simian virus 40 (SV40) and other polyomaviruses Polyomaviruses are a family of small double-stranded DNA viruses that infect various hosts including rodents, birds and human and non-human primates. Whereas some members of this family such as budgerigar fledgling disease virus cause lethal infections, others such as murine or hamster polyomaviruses induce tumours. In other cases, such as human polyomaviruses BK virus (BKV) and JC virus (JCV), infection results in a lifelong infection with no symptoms. Among polyomaviruses, SV40 is the best characterized and is an excellent model for oncogenesis. Most of the effects of SV40 result from the viral T antigen, which exhibits multiple functions that include triggering DNA replication and cell cycle progression [47]. As the major viral protein expressed in infected cells, T antigen is also the target of the host immune lymphocytes, which can destroy infected cells during immunosurveillance to prevent the virus spreading. Initial prediction of the existence of a miRNA in both BKV and SV40 indicated that polyomaviruses, which are known for long-term persistence and oncogenic potential similar to herpesviruses, might also express miRNAs [35]. A further experimental study characterized the SV40 miRNA located in the viral genome just 5 0 to the SV40associated small transcript [48]. The pre-miRNA and its mature form are expressed in late infection, which is consistent with their processing from a late viral transcript. The SV40 genome is circular and the region that encodes the miRNAs on the late strand overlaps with the early mRNAs produced from the opposite strand. This makes the early mRNAs targets for miRNA-dependent cleavage. Because the major early mRNAs encode for the viral T antigen, SV40 miRNA suppresses the T antigen expression in the later stages of infection when it is not actually required. In addition, downregulation of the T antigen, which is the main target of the cytotoxic T lymphocyte response, conveniently helps the virus to escape the host immune response. Thus, the SV40 miRNA www.sciencedirect.com
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is the first virus-encoded miRNA that has a definite major function in virus biology and interaction with the host. The identification of this miRNA in all SV40 isolates and in other primate polyomaviruses including BKV, JCV and SA12 further suggests that miRNAs represent an important mechanism in the biology of these virus infections. A recent study demonstrated the expression of the premiRNA precursor and the mature miRNA in SA12 polyomavirus-infected BSC40 monkey kidney epithelial cells, which makes SA12 the second polyomavirus confirmed to encode miRNAs [49]. Furthermore, a similar regulation of expression of the middle and large T antigens by a miRNA that emanates from a different region of the mouse polyomavirus has also been observed [50]. It is remarkable that two distinct miRNAs encoded by two unrelated viruses are expressed with the same kinetics with the principal function of downregulating T antigen. Human immunodeficiency virus-encoded miRNAs Retroviruses have the ability to establish persistent infection following integration into the host genome. Viral genes are expressed from proviral DNA copies using the cellular transcription machinery. The identification of miRNAs encoded by herpesviruses and polyomaviruses prompted investigation of whether the structures resembling miRNAs are also encoded by retroviruses. HIV-encoded miRNAs were not cloned from HIV-infected cells in one of the initial studies [35]. A subsequent search of regions of the HIV genome for premiRNA-like stem-loop structures uncovered a few potential candidates [51]. Recent evidence also suggests that HIV-1 might encode a viral siRNA precursor that elicits antiviral restriction in human cells, and the HIV-1 Tat protein has evolved to function as a suppressor of RNA silencing to combat this cellular defence [52]. In another study that used HIV-1-infected MT-4 T cells, a novel miRNA was isolated and designated miR-N367, corresponding to nucleotides 420–443 in the conserved region of Nef [53], a major HIV-encoded protein that assists in the long-term survival of infected cells. MiR-N367 downregulates HIV-1 transcription through the long terminal repeats (LTR) U3 region negative-response element, which demonstrates that HIV-1 might regulate its own transcription and replication by using miRNAs [54]. Well-defined stem-loop structures such as TAR (transactivation responsive RNA) and RRE (Rev-responsive element) have already been demonstrated in the HIV genome. TAR, which is required for the transcriptional activation of HIV, has a secondary stem-loop structure that resembles miRNA precursors, which raises the possibility that TAR is a pre-miRNA [55]. Recent studies on TAR have also led to fundamental insights into the potential mechanisms of discrimination between microRNA-mediated and siRNA-mediated silencing pathways [56]. The finding that TRBP (HIV-1 TAR RNA binding protein) is a regulator of cellular protein kinase R (PKR) and a partner of human dicer also raises important questions about the potential interplay and evolutionary relationship between RNAi and interferon-PKR pathways that have a major role in HIV-1 replication [57,58]. The discrepancy between the requirement of TRBP in RNAi
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and in HIV replication leads to the hypothesis that RNAi might be beneficial for HIV-1 replication or that HIV-1 could evade RNAi restriction by diverting TRBP from dicer and using it for its own benefit [59]. MiRNA-dependent regulatory interactions between viruses and hosts Although the current experimental evidence of virusencoded miRNAs is restricted to just three groups of viruses, computational approaches have suggested the existence of miRNAs in other viruses such as poxviruses and adenoviruses but not in most of the RNA viruses. However, there are several examples of modulation of viruses by host-encoded miRNAs. The best recent example is the remarkable positive regulation of replication of hepatitis C virus by the liver-specific miR-122 [60]. Impaired translation of target sequences from primate foamy virus 1 (PFV) by miR-32 has also been reported [61]. PFV counteracts this inhibitory effect by encoding a protein known as Tas, which is a representative of a growing family of proteins that are involved in blocking RNAi pathways [61]. The interaction of HIV with host T cells might also be modulated by miRNAs, as indicated by the fact that at least five of the miRNAs expressed in human T cells have highly conserved predicted target sites within the nef and vpr (virus protein R) transcripts of HIV [62]. Finally, latency type III EBV infections are associated with induction of miR-155 in B cells (M. Zavolan, unpublished; [63]). This miRNA is also highly expressed in Hodgkin’s, primary mediastinal and diffuse large cell lymphomas, which raises the question of whether miR-155 induction might, in fact, be responsible for some of the malignant disorders associated with EBV. Evolution of viral miRNAs One fascinating aspect of miRNA gene evolution that has been underscored by the discovery of viral miRNAs is that these genes can evolve rapidly. The miRNAs of the human herpesviruses do not share homologies with each other or with the host miRNAs, and their location in the viral genome also varies widely. Homologues have been found, however, for some of the HCMV miRNAs in a chimpanzee herpesvirus [35]. Similarly, BART-1, BHRF1-1 and BHRF1-2 miRNAs of EBV seem to be conserved in a Cercopithecine herpesvirus 1 (M. Zavolan, unpublished). These observations suggest that miRNA genes continue to emerge in genomes at high rates, consistent with the recent discovery of various eukaryotic miRNAs that have restricted phylogenetic distribution [64–66]. In this context, it is interesting to point out that miR-K12-11, which is encoded by KSHV [35], shares the first eight nucleotides with hsa-miR-155 but otherwise these miRNAs do not seem to be related. As this region of the mature miRNA is essential for the translational repression function of the miRNAs, the question arises of whether KSHV-encoded miR-K12-11 and hsa-miR-155 have undergone convergent evolution. The interaction between viruses and their hosts seems to involve host- and virus-encoded miRNAs, which continue to expand our knowledge about fundamental aspects of gene regulation. It will be fascinating to follow www.sciencedirect.com
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how the understanding of virus–host interactions is shaped by the discovery of the targets of these small RNAs. Acknowledgements Part of this work is funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC).
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28 Lim, L.P. et al. (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 29 Zeng, Y. et al. (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc. Natl. Acad. Sci. U. S. A. 100, 9779–9784 30 Bartel, D.P. and Chen, C.Z. (2004) Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat. Rev. Genet. 5, 396–400 31 Reinhart, B.J. et al. (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906 32 Chang, S. et al. (2004) MicroRNAs act sequentially and asymmetrically to control chemosensory laterality in the nematode. Nature 430, 785–789 33 Monticelli, S. et al. (2005) MicroRNA profiling of the murine hematopoietic system. Genome Biol. 6, R71 34 Pfeffer, S. et al. (2004) Identification of virus-encoded microRNAs. Science 304, 734–736 35 Pfeffer, S. et al. (2005) Identification of microRNAs of the herpesvirus family. Nat Methods 2, 269–276 36 Cai, X. et al. (2005) Kaposi’s sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells. Proc. Natl. Acad. Sci. U. S. A. 102, 5570–5575 37 McCormick, C. and Ganem, D. (2005) The kaposin B protein of KSHV activates the p38/MK2 pathway and stabilizes cytokine mRNAs. Science 307, 739–741 38 Samols, M.A. et al. (2005) Cloning and identification of a microRNA cluster within the latency-associated region of Kaposi’s sarcomaassociated herpesvirus. J. Virol. 79, 9301–9305 39 Young, L.S. and Rickinson, A.B. (2004) Epstein-Barr virus: 40 years on. Nat. Rev. Cancer 4, 757–768 40 Furnari, F.B. et al. (1993) Unconventional processing of the 3 0 termini of the Epstein-Barr virus DNA polymerase mRNA. Proc. Natl. Acad. Sci. U. S. A. 90, 378–382 41 Pearce, M. et al. (2005) Transcripts encoding K12, v-FLIP, v-cyclin, and the microRNA cluster of Kaposi’s sarcoma-associated herpesvirus originate from a common promoter. J. Virol. 79, 14457–14464 42 Kliche, S. et al. (2001) Signaling by human herpesvirus 8 kaposin A through direct membrane recruitment of cytohesin-1. Mol. Cell 7, 833–843 43 Clambey, E.T. et al. (2002) Characterization of a spontaneous 9.5kilobase-deletion mutant of murine gammaherpesvirus 68 reveals tissue-specific genetic requirements for latency. J. Virol. 76, 6532–6544 44 Castillo, J.P. and Kowalik, T.F. (2004) HCMV infection: modulating the cell cycle and cell death. Int. Rev. Immunol. 23, 113–139 45 Grey, F. et al. (2005) Identification and characterization of human cytomegalovirus-encoded microRNAs. J. Virol. 79, 12095–12099
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46 Dunn, W. et al. (2005) Human cytomegalovirus expresses novel microRNAs during productive viral infection. Cell. Microbiol. 7, 1684–1695 47 Saenz-Robles, M.T. et al. (2001) Transforming functions of Simian Virus 40. Oncogene 20, 7899–7907 48 Sullivan, C.S. et al. (2005) SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature 435, 682–686 49 Cantalupo, P. et al. (2005) Complete nucleotide sequence of polyomavirus SA12. J. Virol. 79, 13094–13104 50 Sullivan, C.S. and Ganem, D. (2005) MicroRNAs and Viral Infection. Mol. Cell 20, 3–7 51 Bennasser, Y. et al. (2004) HIV-1 encoded candidate micro-RNAs and their cellular targets. Retrovirology 1, 43 52 Bennasser, Y. et al. (2005) Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing. Immunity 22, 607–619 53 Omoto, S. et al. (2004) HIV-1 nef suppression by virally encoded microRNA. Retrovirology 1, 44 54 Omoto, S. and Fujii, Y.R. (2005) Regulation of human immunodeficiency virus 1 transcription by nef microRNA. J. Gen. Virol. 86, 751–755 55 Chendrimada, T.P. et al. (2005) TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740–744 56 Rossi, J.J. (2005) Mammalian Dicer finds a partner. EMBO Rep. 6, 927–929 57 Haase, A.D. et al. (2005) TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep. 6, 961–967 58 Ong, C.L. et al. (2005) Low TRBP levels support an innate human immunodeficiency virus type 1 resistance in astrocytes by enhancing the PKR antiviral response. J. Virol. 79, 12763–12772 59 Gatignol, A. et al. (2005) Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity? Retrovirology 2, 65 60 Jopling, C.L. et al. (2005) Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA. Science 309, 1577–1581 61 Lecellier, C.H. et al. (2005) A cellular microRNA mediates antiviral defense in human cells. Science 308, 557–560 62 Hariharan, M. et al. (2005) Targets for human encoded microRNAs in HIV genes. Biochem. Biophys. Res. Commun. 337, 1214–1218 63 Kluiver, J. et al. (2005) BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. J. Pathol. 207, 243–249 64 Bentwich, I. et al. (2005) Identification of hundreds of conserved and nonconserved human microRNAs. Nat. Genet. 37, 766–770 65 Chen, P.Y. et al. (2005) The developmental miRNA profiles of zebrafish as determined by small RNA cloning. Genes Dev. 19, 1288–1293 66 Giraldez, A.J. et al. (2005) MicroRNAs regulate brain morphogenesis in zebrafish. Science 308, 833–838
Articles of interest in other Trends and Current Opinion journals Kim, V.N. and Nam, J-W. (2006) Genomics of microRNA. Trends Genet. doi:10.1016/j.tig.2006.01.003 Meyers, B.C. et al. (2006) Sweating the small stuff: microRNA discovery in plants. Curr. Opin. Biotechnol. doi:10.1016/j.copbio.2006.01.008 Soto, C. et al. (2006) Amyloids, prions and the inherent infectious nature of misfolded protein aggregates. Trends Biochem. Sci. doi:10.1016/j.tibs.2006.01.002 McCarter, L.L. (2006) Regulation of flagella. Curr. Opin. Microbiol. doi:10.1016/j.mib.2006.02.001 www.sciencedirect.com
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Virus-encoded microRNAs: novel regulators of gene expression Venugopal Nair1 and Mihaela Zavolan2 1 2
Viral Oncogenesis Group, Division of Microbiology, Institute for Animal Health, Compton, Berkshire, UK, RG20 7NN Division of Bioinformatics, Biozentrum, University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland
MicroRNAs (miRNAs) are a class of small RNAs that have recently been recognized as major regulators of gene expression. They influence diverse cellular processes ranging from cellular differentiation, proliferation, apoptosis and metabolism to cancer. Bioinformatic approaches and direct cloning methods have identified O3500 miRNAs, including orthologues from various species. Experiments to identify the targets and potential functions of miRNAs in various species are continuing but the recent discovery of virus-encoded miRNAs indicates that viruses also use this fundamental mode of gene regulation. Virus-encoded miRNAs seem to evolve rapidly and regulate both the viral life cycle and the interaction between viruses and their hosts. Discovery of microRNAs as regulators of gene expression The discovery that microRNAs (miRNAs) are key players in the micromanagement of gene expression in diverse cellular processes in many species is a remarkable breakthrough for the field of molecular biology. The first miRNA, lin-4, was discovered over 12 years ago in Caenorhabditis elegans [1], yet it was only when the first evolutionarily conserved miRNA, let-7, was shown to have a crucial role in C. elegans development that the versatility and the far-reaching influence of miRNAs on various cellular processes started to come to light. The latest release of the miRNA repository MiRBase (release 8.0; http://microrna.sanger.ac.uk) contains 3518 annotated miRNAs, including 326 human miRNA genes [2–4]. Within cells, the number of miRNA molecules seems to vary from only a few to as many as 50 000 [5]. Many miRNAs are ubiquitously expressed, whereas others are expressed in a cell-type-specific manner [6–11]. Because a single miRNA can target transcripts from multiple genes and, conversely, several miRNAs can control a single target [12], the miRNAs and their targets function as a complex regulatory network [13]. With recent computational predictions suggesting that miRNAs could target up to a third of all human genes [12,14], the potential of miRNAs to modulate the transcriptome is overwhelming. The profound influence of miRNAs on diverse regulatory pathways such as embryonic development, haematopoietic cell differentiation, apoptosis, cell proliferation and cancer has already been demonstrated Corresponding author: Nair, V. ([email protected]). Available online 13 March 2006
[15–20]. Here, we review the features of virus-encoded miRNAs and their potential involvement in the regulation of their own genes and in the subversion of cellular defence mechanisms. Biogenesis and functions of miRNAs The miRNA biogenesis pathway has been examined in detail (Figure 1). The sequential action of two doublestranded RNA-specific RNase III type ribonucleases (Drosha and Dicer) produces a 21-nucleotide RNA duplex from long primary transcripts (pri-miRNAs) [21]. Depending on the relative stability of their 5 0 ends, only one of the two strands of the duplex is incorporated into the RNAinduced silencing complex (RISC), which contains proteins such as members of the Argonaute (Ago) family, the RNA-binding proteins VIG and Fragile X-related protein and the nuclease Tudor-SN [22]. The RISC complex then becomes functional and inhibits the expression of specific cellular or viral proteins. There are two major mechanisms by which miRNAs regulate target gene expression. A high degree of complementarity between miRNAs and their cognate mRNAs in a RISC complex [which contains the appropriate Ago protein (Ago2 in humans) [23]] results in the cleavage of the target mRNA at a precise location, corresponding to the bond between the tenth and eleventh nucleotides of the miRNA [24]. This mode of silencing by direct cleavage of the target mRNA is most common in plants [25]. When there is insufficient complementarity with the target, the miRNA–RISC represses translation of the mRNA transcripts. Recent data demonstrate that this occurs by sequestration of target mRNA into distinct sites such as the P bodies in the cytoplasm, away from the translational machinery [26]. Because both effects can be observed with a given miRNA [27–29] and because different miRNAs can bind to multiple target sites on the same transcript, miRNAs might exert a dosage-dependent effect on the expression of individual genes. This extra tier of regulation has been compared with that of a dimmer switch or rheostat to increase or dampen the gene expression [30]. Perhaps best explored is the function of miRNAs in the regulation of developmental processes. Since the discovery of the first conserved miRNA, let-7 (which controls developmental timing in C. elegans [31]), evidence has accumulated regarding the differential expression of individual miRNAs in different cell lineages, in which they might determine cell fate. For example, miR-273 and lsy-6 are
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Figure 1. Schematic overview of miRNA biogenesis in eukaryotic cells. Processing of both host miRNAs (pink) and virus-encoded miRNAs (blue) is thought to take place through the same pathway. Initially, within the nucleus, the long primary transcripts of miRNA (pri-miRNAs) containing the hairpin structure are processed by the nuclear RNase III Drosha to release the precursor of miRNA (pre-miRNA). Following nuclear processing, pre-miRNAs are exported to the cytoplasm by one of the nuclear transport receptors, Exportin-5 (Exp-5), before being processed by the cytoplasmic RNase III Dicer into 21-nucleotide mature miRNAs. These miRNAs then associate with RISC to target the specific host- or virus-encoded transcripts to regulate gene expression.
a pair of miRNAs that function sequentially to control the laterality of the nematode chemosensory system [32]. In mammals, miR-223 and miR-342 are specifically expressed in myeloid and lymphoid lineages, respectively, and miR-181a is important in B-lymphoid lineage differentiation [8]. Furthermore, recent data suggest that miR-150 and miR-146 are differentially regulated during developmental stages of B and T cell maturation and during the transition from naı¨ve T cells to effector Th1–Th2 cells [33]. Thus, the miRNA milieu, which is unique to each cell type, could be instrumental in the establishment and maintenance of cell identity. Virus-encoded miRNAs Viruses are obligatory intracellular pathogens that are associated with many diseases in both animals and plants. The successful survival of viruses crucially depends on their ability to exploit the biosynthetic machinery of host cells and to inactivate the innate defence mechanisms of the host, such as the interferon and apoptosis responses. Although many large viruses achieve this by encoding proteins that specifically inactivate host-cell defences, the comparatively limited coding capacity of viruses makes the tiny miRNAs a particularly efficient and accessible tool to turn off the expression of specific genes. Indeed, recent studies show that viruses do exploit this pathway by generating their own miRNAs [34]. A computational and experimental survey of 29 virus genomes identified 33 novel viral miRNAs [35], which provide the opportunity to study the function and evolutionary conservation of viral miRNAs. MicroRNAs encoded by herpesviruses Among the various families of viruses, the herpesvirus family stands out in establishing long-standing latent infection as a major part of the viral life cycle. This large group of DNA viruses is classified into three subfamilies www.sciencedirect.com
(a, b or g) on the basis of their genome structure and biology, and includes pathogens such as herpes simplex virus, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), Kaposi’s sarcoma herpesvirus (KSHV) and murine herpesvirus 68 (MHV 68). Although the symptomatology associated with these viruses ranges from cold sores to lymphoproliferation to encephalopathy and cancer (particularly in immunocompromised hosts), they all share the ability to establish latency in the host after an early replication phase. During the latent and ‘active’ states, the intracellular virus must adapt to the multitude of events that occur in the cell. Much of this adaptation is achieved through specialized gene products that are encoded by the viruses. The herpesvirus gene repertoire includes homologues of genes such as Bcl-2, cyclin and chemokines that are thought to be ‘hijacked’ from the host itself to combat the hostile environment of the cell. MiRNAs are the most recent weapons found in the repertoire of herpesviruses. The first virus-encoded miRNAs that were discovered were those of EBV, from which five miRNAs were cloned [34]. These miRNAs are not homologous to any known host-cell miRNAs and an initial search in other herpesviruses did not reveal any orthologous miRNAs. These results indicated that viral miRNAs could not be identified with the existing miRNA prediction software, which relies heavily on cross-species sequence conservation. Using various combinations of novel computational prediction methods and a small-RNA cloning approach, several novel miRNAs have been identified in the genomes of many herpesviruses [35–38]. EBV-encoded miRNAs EBV (human herpesvirus years ago and is present population. EBV infection lymphoid and epithelial Burkitt’s lymphoma and
4) was discovered nearly 40 in O90% of the total world is thought to predispose for cell malignancies such as nasopharyngeal carcinoma.
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every stage of EBV infection, miR-BART-1 and 2 were detected during the lytic and the three latent stages. However, the expression of miR-BHRF1-1, 2 and 3 was related to the stage of latency. Their expression pattern also showed variations between cell lines, which provided the possibility of their classification based on the miRBHRF expression pattern. The functions of EBV miRNAs have not been fully established but, based on their location and on current knowledge regarding the function of animal miRNAs, the following types of functions can be distinguished. First, the mere processing of miRNAs could be a way to downregulate the expression of protein-coding genes within which the miRNAs reside. The three BHRF1 miRNAs, for example, require endonucleolytic processing of the BHRF1 transcript, thus, their expression should be anti-correlated with the expression of BHRF1. Second, miRNAs generated from the complementary strand of protein-coding genes could function as small interfering RNAs (siRNAs) against the transcripts of the proteincoding gene. (SiRNAs are a distinct class of 20–25nucleotide RNA molecules that interfere with gene expression by destroying the transcripts after binding to complementary target sequences.) This seems to be
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One of the unique features of EBV is its ability to transform resting B cells into proliferating cell lines. This system has been widely used as a model for the lymphomagenic potential of the virus [39]. EBV-induced B-cell proliferation is driven by a limited set of latent viral proteins that comprise six EBV nuclear antigens (EBNA 1, 2, 3A, 3B, 3C and LP) and three latent membrane proteins (LMP 1, 2A and 2B). Involvement of non-coding RNAs in EBV biology is not new: EBV-transformed cells show abundant expression of the small, non-polyadenylated RNAs named EBER1 and EBER2. The successful cloning of five novel EBV-encoded miRNAs from a B-cell line latently infected with EBV [34] demonstrates that EBV also has the potential to exploit RNA silencing as a convenient mechanism for the regulation of host and viral gene expression. EBV miRNAs are clustered in two distinct regions of the genome (Figure 2). The first miRNA cluster, which consists of miR-BHRF1-1, 2 and 3, is located within the 5 0 and 3 0 UTR of the transcript of BHRF1, a homologue of the antiapoptotic protein Bcl-2. The other EBV miRNAs (miRBART-1 and 2) are located within the BART gene (Figure 2). Consistent with the expression of BART in
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Figure 2. Schematic diagram showing the structure of EBV, KSHV, MHV 68 and HCMV genomes. Approximate genomic locations of miRNAs encoded by each of the viruses are indicated by the nucleotide positions shown in brackets. Positions of selected viral genes of each of the viruses are also shown. Names of individual miRNAs (red) are taken from Refs [34,35]. Asterisks mark the two additional miRNAs that are present in HCMV [45]. www.sciencedirect.com
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the case for the BART-2 miRNA, which is located on the complementary strand of the viral DNA polymerase BALF5. The description of a transcript that corresponds precisely to the predicted siRNA-dependent processing product of the viral DNA polymerase [40] indicates that BART-2 directs the cleavage of BALF5. Finally, it is thought that the virus-encoded miRNAs function in translational repression of viral and host genes, similar to host-encoded miRNAs. In this respect, computational predictions suggested that the targets of viral miRNAs might include regulators of cell proliferation, apoptosis, chemokines and cytokines – all molecules that have profound potential to influence the pathogenicity of the virus [34]. KSHV-encoded miRNAs KSHV (human herpesvirus 8), which was initially discovered in AIDS-related Kaposi’s sarcoma, is also associated with B-cell primary effusion lymphomas and some forms of multicentric Castleman’s disease. The most striking feature of KSHV is the extensive use of molecular piracy and the capture of several host genes including complement receptor 2, CC family chemokines, a CXC (GPCR) receptor homologue, interleukin-6 (IL-6) and Bcl-2 homologues, interferon regulatory factors, D-type cyclin, a FLICE-like caspase inhibitor and a transmembrane-spanning adhesion molecule. This complex array of host proteins enables KSHV to modulate the cellular regulatory network for KSHV replication and pathogenesis. The recent discovery of novel miRNAs encoded by KSHV further adds to the complexity of the gene regulation pathways in this virus. A cluster of 11 miRNAs confined to a 5-kb region, which also encoded the transforming protein kaposin, was identified in the KSHV-infected BCBL1 cell line [35]. This cluster included miR-K12-10 located within the K12 open reading frame (ORF). Other miRNAs are located within the intron of the larger kaposin primary transcript (Figure 2). These miRNAs were also identified in an independent study using another KSHV-infected cell line, BC-1, in which all of the miRNAs were expressed at levels detectable by northern blotting [36]. Because all KSHV-encoded miRNAs are located in a small region of the genome and are transcribed in the same direction, it is thought that they are all processed from a single promoter [41]. The recent isolation of these miRNAs from the KSHV-infected endothelial cell line TIVE-LTC demonstrates that the expression of KSHV miRNAs is not restricted to a particular cell type [38]. KSHV-encoded miR-K12-10 is located within the K12 ORF that encodes kaposin A, B and C, which have important functions in cell growth and cytokine production [37,42]. These proteins have to be tightly regulated during latency, and it remains to be determined whether the processing of miR-K12-10 miRNA has a role in downregulating kaposin expression. Computationally predicted targets for KSHV miRNAs include viral genes such as ORF 23, 27, 31, 52, 49, 61, 68, K7, K13 and K14, and several B-cell-specific genes involved in apoptosis and signalling [36]. Although these predictions remain to be www.sciencedirect.com
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validated, it is tempting to speculate that KSHV-encoded miRNAs have a role in KSHV replication and pathogenicity by regulating the expression of both viral and host genes. MHV 68-encoded miRNAs MHV 68 is a natural pathogen of small rodents. Based on its evolutionary relationship to the human g-herpesviruses, this virus is used in small animal models to study the biology of g-herpesviruses. As in the case of the human viruses, the MHV 68 genome encodes functional homologues of several host genes such as Bcl-2 (M11), IL8R (ORF74) and cyclin D (ORF72), all of which are used in the modulation of host gene expression. The recent discovery of nine miRNAs in the MHV 68 genome indicates that non-coding RNAs could be another means by which the virus modulates gene expression [35]. MHV 68 miRNAs, cloned from mouse B lymphoma S11 cells latently infected with MHV 68, are located within a 6-kb region near the M1 terminus of the linear MHV 68 genome (Figure 2), immediately downstream of predicted viral tRNA genes. This led to the speculation that MHV 68 premiRNAs are transcribed by pol III from tRNA promoters. Northern blot analysis on the three most abundant MHV 68 miRNAs (using probes that match the mature miRNA or the tRNA) further supports the hypothesis that miRNAs can be transcribed not only from pol II but also from pol III promoters that are active in many cell types. The unusual short-hairpin structures of the MHV 68 premiRNAs also suggest that their processing mechanism has distinct features compared with other miRNAs. The viral or cellular targets of these miRNAs remain to be identified. However, a mutant virus with a deletion that includes the miRNA cluster exhibited tissue-specific and route of infection-dependent alterations in latency and reactivation [43]. HCMV-encoded miRNAs HCMV (human herpesvirus 5) is a b-herpesvirus and an important pathogen that causes widespread primary and recurrent infections ranging from mild asymptomatic to severe congenital disease. HCMV is ubiquitous and, like other herpesviruses, can establish lifelong latency in infected individuals. HCMV-encoded proteins are thought to influence various cellular processes including senescence, cell cycle progression, apoptosis and transformation [44]. An initial bioinformatics and cloning approach identified nine miRNAs in the HCMV genome [35]. In contrast to MHV 68 and KSHV miRNAs, HCMV miRNAs are spread across the viral genome (Figure 2). Three of them are transcribed from the complementary strand of known ORFs, five are located in intergenic regions and one is located within an intron [35]. As with the other viral miRNAs, the targets remain to be identified. Nonetheless, the miRNAs that are transcribed from the complementary strand of protein-coding regions have the potential to function as siRNAs and regulate the level of viral proteins. For example, the miR-UL112-1 might target UL114, a homologue of the mammalian uracyl-DNA glycosylase. Because UL114 is required for efficient viral DNA replication, miR-UL112-1 has the potential to control
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viral replication. The expression of HCMV miRNAs was examined in two other recent studies. Grey et al. [45] found that the miRNAs expressed by the AD169 strain of HCMV (three of which were described previously [35] and the other two of which were novel) were expressed mostly with early kinetics in infected human fibroblasts [45]. A further study also demonstrated the expression of HCMV miRNAs during the productive lytic infection of four distinct human cell types, namely fibroblasts, endothelial cells, epithelial cells and astrocytes [46]. MiRNAs have also been computationally predicted in the genomes of a-herpesviruses, including seven miRNAs in HHV1, six in HHV2 [35] and nine in Marek’s disease virus (V. Nair and M. Zavolan, unpublished) but not in other human herpesviruses (HHV) such as HHV3, HHV6 and HHV7, which also have a similar life cycle. If these viruses also encode miRNAs, it would be interesting to find out how they differ from the EBV, KSHV and HCMV miRNAs such that they were not predicted computationally. MicroRNAs encoded by other viruses Simian virus 40 (SV40) and other polyomaviruses Polyomaviruses are a family of small double-stranded DNA viruses that infect various hosts including rodents, birds and human and non-human primates. Whereas some members of this family such as budgerigar fledgling disease virus cause lethal infections, others such as murine or hamster polyomaviruses induce tumours. In other cases, such as human polyomaviruses BK virus (BKV) and JC virus (JCV), infection results in a lifelong infection with no symptoms. Among polyomaviruses, SV40 is the best characterized and is an excellent model for oncogenesis. Most of the effects of SV40 result from the viral T antigen, which exhibits multiple functions that include triggering DNA replication and cell cycle progression [47]. As the major viral protein expressed in infected cells, T antigen is also the target of the host immune lymphocytes, which can destroy infected cells during immunosurveillance to prevent the virus spreading. Initial prediction of the existence of a miRNA in both BKV and SV40 indicated that polyomaviruses, which are known for long-term persistence and oncogenic potential similar to herpesviruses, might also express miRNAs [35]. A further experimental study characterized the SV40 miRNA located in the viral genome just 5 0 to the SV40associated small transcript [48]. The pre-miRNA and its mature form are expressed in late infection, which is consistent with their processing from a late viral transcript. The SV40 genome is circular and the region that encodes the miRNAs on the late strand overlaps with the early mRNAs produced from the opposite strand. This makes the early mRNAs targets for miRNA-dependent cleavage. Because the major early mRNAs encode for the viral T antigen, SV40 miRNA suppresses the T antigen expression in the later stages of infection when it is not actually required. In addition, downregulation of the T antigen, which is the main target of the cytotoxic T lymphocyte response, conveniently helps the virus to escape the host immune response. Thus, the SV40 miRNA www.sciencedirect.com
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is the first virus-encoded miRNA that has a definite major function in virus biology and interaction with the host. The identification of this miRNA in all SV40 isolates and in other primate polyomaviruses including BKV, JCV and SA12 further suggests that miRNAs represent an important mechanism in the biology of these virus infections. A recent study demonstrated the expression of the premiRNA precursor and the mature miRNA in SA12 polyomavirus-infected BSC40 monkey kidney epithelial cells, which makes SA12 the second polyomavirus confirmed to encode miRNAs [49]. Furthermore, a similar regulation of expression of the middle and large T antigens by a miRNA that emanates from a different region of the mouse polyomavirus has also been observed [50]. It is remarkable that two distinct miRNAs encoded by two unrelated viruses are expressed with the same kinetics with the principal function of downregulating T antigen. Human immunodeficiency virus-encoded miRNAs Retroviruses have the ability to establish persistent infection following integration into the host genome. Viral genes are expressed from proviral DNA copies using the cellular transcription machinery. The identification of miRNAs encoded by herpesviruses and polyomaviruses prompted investigation of whether the structures resembling miRNAs are also encoded by retroviruses. HIV-encoded miRNAs were not cloned from HIV-infected cells in one of the initial studies [35]. A subsequent search of regions of the HIV genome for premiRNA-like stem-loop structures uncovered a few potential candidates [51]. Recent evidence also suggests that HIV-1 might encode a viral siRNA precursor that elicits antiviral restriction in human cells, and the HIV-1 Tat protein has evolved to function as a suppressor of RNA silencing to combat this cellular defence [52]. In another study that used HIV-1-infected MT-4 T cells, a novel miRNA was isolated and designated miR-N367, corresponding to nucleotides 420–443 in the conserved region of Nef [53], a major HIV-encoded protein that assists in the long-term survival of infected cells. MiR-N367 downregulates HIV-1 transcription through the long terminal repeats (LTR) U3 region negative-response element, which demonstrates that HIV-1 might regulate its own transcription and replication by using miRNAs [54]. Well-defined stem-loop structures such as TAR (transactivation responsive RNA) and RRE (Rev-responsive element) have already been demonstrated in the HIV genome. TAR, which is required for the transcriptional activation of HIV, has a secondary stem-loop structure that resembles miRNA precursors, which raises the possibility that TAR is a pre-miRNA [55]. Recent studies on TAR have also led to fundamental insights into the potential mechanisms of discrimination between microRNA-mediated and siRNA-mediated silencing pathways [56]. The finding that TRBP (HIV-1 TAR RNA binding protein) is a regulator of cellular protein kinase R (PKR) and a partner of human dicer also raises important questions about the potential interplay and evolutionary relationship between RNAi and interferon-PKR pathways that have a major role in HIV-1 replication [57,58]. The discrepancy between the requirement of TRBP in RNAi
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and in HIV replication leads to the hypothesis that RNAi might be beneficial for HIV-1 replication or that HIV-1 could evade RNAi restriction by diverting TRBP from dicer and using it for its own benefit [59]. MiRNA-dependent regulatory interactions between viruses and hosts Although the current experimental evidence of virusencoded miRNAs is restricted to just three groups of viruses, computational approaches have suggested the existence of miRNAs in other viruses such as poxviruses and adenoviruses but not in most of the RNA viruses. However, there are several examples of modulation of viruses by host-encoded miRNAs. The best recent example is the remarkable positive regulation of replication of hepatitis C virus by the liver-specific miR-122 [60]. Impaired translation of target sequences from primate foamy virus 1 (PFV) by miR-32 has also been reported [61]. PFV counteracts this inhibitory effect by encoding a protein known as Tas, which is a representative of a growing family of proteins that are involved in blocking RNAi pathways [61]. The interaction of HIV with host T cells might also be modulated by miRNAs, as indicated by the fact that at least five of the miRNAs expressed in human T cells have highly conserved predicted target sites within the nef and vpr (virus protein R) transcripts of HIV [62]. Finally, latency type III EBV infections are associated with induction of miR-155 in B cells (M. Zavolan, unpublished; [63]). This miRNA is also highly expressed in Hodgkin’s, primary mediastinal and diffuse large cell lymphomas, which raises the question of whether miR-155 induction might, in fact, be responsible for some of the malignant disorders associated with EBV. Evolution of viral miRNAs One fascinating aspect of miRNA gene evolution that has been underscored by the discovery of viral miRNAs is that these genes can evolve rapidly. The miRNAs of the human herpesviruses do not share homologies with each other or with the host miRNAs, and their location in the viral genome also varies widely. Homologues have been found, however, for some of the HCMV miRNAs in a chimpanzee herpesvirus [35]. Similarly, BART-1, BHRF1-1 and BHRF1-2 miRNAs of EBV seem to be conserved in a Cercopithecine herpesvirus 1 (M. Zavolan, unpublished). These observations suggest that miRNA genes continue to emerge in genomes at high rates, consistent with the recent discovery of various eukaryotic miRNAs that have restricted phylogenetic distribution [64–66]. In this context, it is interesting to point out that miR-K12-11, which is encoded by KSHV [35], shares the first eight nucleotides with hsa-miR-155 but otherwise these miRNAs do not seem to be related. As this region of the mature miRNA is essential for the translational repression function of the miRNAs, the question arises of whether KSHV-encoded miR-K12-11 and hsa-miR-155 have undergone convergent evolution. The interaction between viruses and their hosts seems to involve host- and virus-encoded miRNAs, which continue to expand our knowledge about fundamental aspects of gene regulation. It will be fascinating to follow www.sciencedirect.com
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how the understanding of virus–host interactions is shaped by the discovery of the targets of these small RNAs. Acknowledgements Part of this work is funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC).
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Articles of interest in other Trends and Current Opinion journals Kim, V.N. and Nam, J-W. (2006) Genomics of microRNA. Trends Genet. doi:10.1016/j.tig.2006.01.003 Meyers, B.C. et al. (2006) Sweating the small stuff: microRNA discovery in plants. Curr. Opin. Biotechnol. doi:10.1016/j.copbio.2006.01.008 Soto, C. et al. (2006) Amyloids, prions and the inherent infectious nature of misfolded protein aggregates. Trends Biochem. Sci. doi:10.1016/j.tibs.2006.01.002 McCarter, L.L. (2006) Regulation of flagella. Curr. Opin. Microbiol. doi:10.1016/j.mib.2006.02.001 www.sciencedirect.com
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