Viruses and micrornas

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1 Viruses and micrornas Bryan R Cullen The discovery of RNA interference and cellular micrornas (mirnas) has not only affected how biological research is conducted but also revealed an entirely new level of posttranscriptional gene regulation. Here, I discuss the potential functions of the virally encoded mirnas recently identified in several pathogenic human viruses and propose that cellular mirnas may have had a substantial effect on viral evolution and may continue to influence the in vivo tissue tropism of viruses. Our increasing knowledge of the role and importance of virally encoded mirnas will probably offer new insights into how viruses that establish latent infections, such as herpesviruses, avoid elimination by the host innate or adaptive immune system. Research into viral mirna function might also suggest new approaches for treating some virally induced diseases. mirnas probably have an important role in the post-transcriptional regulation of gene expression in every somatic cell of every metazoan eukaryote (plant and animal) 1. More than 300 distinct human mirnas have been identified so far, and similar numbers are probably expressed in all other vertebrate species. Many mirnas have unique tissue-specific or developmental expression patterns such that each human tissue is characterized by a specific set of mirnas that may form a defining characteristic of that tissue 1. mirnas have had a considerable effect on cellular mrna evolution by promoting both selection for relevant mirna target sites on mrnas whose downregulation is advantageous in a particular tissue, and selection against potential mirna target sites on mrnas whose expression is required in that same tissue 2,3. The existence of mirna-mediated post-transcriptional gene regulation provides both opportunities and potential problems for infecting viruses and therefore has probably exerted a substantial influence on viral evolution. In this perspective, I review our current understanding of the expression and function of virally encoded mirnas and propose that cellular mirnas have a significant role in regulating the tissue tropism of pathogenic human viruses. Biogenesis and function of animal mirnas Any understanding of the potential role of mirnas in the viral life cycle requires an appreciation of how mirnas are transcribed and processed Bryan R. Cullen is at the Center for Virology and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA. culle002@mc.duke.edu Published online 30 May 2006; doi: /ng1793 and how they normally function in animal cells 1,4. All cellular mirnas characterized so far are transcribed by RNA polymerase II (pol II) as part of a long RNA precursor called a primary mirna (pri-mirna; Fig. 1). A single pri-mirna may contain a cluster of distinct mirnas or only a single mirna and may be from 200 nt to several thousand nucleotides in length 5,6. Within the pri-mirna, the 22-nt mature mirna forms part of one arm of an 80-nt imperfect stem-loop sequence 4. The first step in mirna processing involves the recognition and nuclear cleavage of this RNA stem-loop structure by a heterodimer consisting of the cellular RNase III enzyme Drosha and a cofactor called DGCR8 in vertebrates (Fig. 1) 7 9. This cleavage liberates a 60-nt RNA hairpin intermediate, bearing a characteristic 2-nt 3 overhang, called a pre-mirna 7. Drosha cleavage results in the separation of the pri-mirna into at least three segments: the pre-mirna and flanking 5 and 3 sequences 5,7. Because these flanking sequences are generally degraded in the nucleus 5, the presence of a pri-mirna stem-loop in cis is predicted to inhibit the ability of a pri-mirna precursor to also function as an mrna. Presumably as a result, cellular pri-mirna stem-loops are generally found either in unspliced noncoding RNAs or within the introns of coding or noncoding pol II transcripts 4. The next step in mirna biogenesis is nuclear export of the premirna hairpin by a heterodimer consisting of Exportin 5 (Exp5) and the GTP-bound form of its cofactor Ran, which together recognize and bind the 2-nt 3 overhang and adjacent stem that are characteristic of pre-mirnas (Fig. 1) Upon reaching the cytoplasm, GTP hydrolysis results in release of the pre-mirna, which is then bound by a second cellular RNase III enzyme called Dicer, acting in concert with its cofactor TRBP 1,4,13,14. Dicer binds the 2-nt 3 overhang at the base of the premirna hairpin and removes the terminal loop, leaving a second 2-nt 3 overhang and generating the mirna duplex intermediate. Dicer then facilitates assembly of the mirna strand of the duplex into the RNAinduced silencing complex (RISC) 13,15,16, and the passenger strand is released and degraded. The composition of RISC remains incompletely defined, but a key component is an Argonaute protein, four of which are found in human cells 1, The mirna acts as a guide RNA to direct RISC to complementary mrna species 19. If RISC encounters an mrna bearing extensive homology to the mirna, and if the RISC contains Argonaute-2, then the mrna will be cleaved and degraded Degradation of an mrna by RISC, a process indistinguishable from RNA interference, requires only a single highly complementary target site (Fig. 1). More commonly, RISC will bind to an mrna bearing a partially complementary sequence; this can induce translational repression of that mrna But translation inhibition seems to require several RISC complexes NATURE GENETICS SUPPLEMENT VOLUME 38 JUNE 2006 S25

2 Figure 1 Biogenesis and function of human mirnas. For simplicity, not all cellular factors involved in mirna processing are shown. Ago, Argonaute. bound to identical or distinct partially complementary target sites 25 (Fig. 1). The strong cooperativity, and hence combinatorial potential, of translational inhibition by mirnas may be a key attribute of this latter mechanism 1. Which viruses might encode mirnas? From a viral perspective, mirnas offer several potential advantages as tools for reshaping the cellular environment to maximize viral replication. First, mirnas provide a highly specific way of downregulating the expression of host cell gene products that otherwise might interfere with some aspect of the viral replication cycle. Second, each mature mirna, together with all the necessary cis-acting RNA processing signals, could occupy <200 nt, or <200 bp, of a viral genome 4, a potentially large advantage given the tight constraints on viral genome size. Third, mirnas, unlike viral proteins, are not antigenic. Despite these potential advantages, inspection of the mirna biogenesis pathway also exposes some serious potential difficulties. The initial step in mirna processing, cleavage of the pri-mirna by Drosha, occurs in the nucleus 4,7,26 (Fig. 1). But most RNA viruses, as well as one group of DNA viruses, replicate and express their genome in the cytoplasm. Moreover, as noted above, Drosha cleavage of the pri-mirna genome cuts the pri-mirna into three pieces, only one of which, the pre-mirna, Katie Ris is then exported to the cytoplasm (Fig. 1) 5. Therefore, if an RNA virus encodes an mirna, then this would be predicted to result in the cleavage and destruction of the viral RNA genome (or the viral genome minus strand) if that RNA were to enter the nucleus. On the basis of these considerations, one can predict that RNA viruses, as well as poxviruses, a cytoplasmic DNA virus family, probably do not encode viral mirnas. Because it is possible to conceive of arcane ways to circumvent these problems, however, this is not an absolute prediction. mirnas act at the mrna level, not the protein level 1. The development of the relevant null phenotype can therefore be delayed by hours, or even days, because the existing pool of the encoded protein must undergo normal decay. Viral mirnas could target mrnas that are induced only after infection, but this consideration suggests that viruses that undergo rapid lytic replication cycles are less likely to encode mirnas. We are then left with the prediction that the viruses most likely to encode mirnas are nuclear DNA viruses that establish long-term latent or persistent infections. This is almost a definition of herpesviruses, which are characterized by their ability to establish long-term latent infections in vivo. Virally encoded mirnas Every herpesvirus that has been analyzed encodes several viral mirnas, and the other viruses that have so far been shown to encode a single mirna (i.e., simian virus 40 (SV40) and adenovirus) are also nuclear DNA viruses In contrast, the RNA viruses yellow fever virus, human immunodeficiency virus and hepatitis C virus (HCV) do not seem to encode any mirnas 27. A listing of currently known viral mirnas (Table 1) shows that several herpesviruses express a large number of distinct mirnas, with Epstein-Barr virus (EBV) holding the current record of 23. But these different viral mirnas show no sequence similarity to each other, with the exception of eight mirnas that are conserved between EBV and the related rhesus lymphocryptovirus 27,34,35. The lack of conservation of mirnas between different human herpesviruses could be viewed as evidence that these mirnas do not have an important role in virus replication. But mirnas are only 22 nt in length, and inhibition of target mrna expression may require only limited homology 1. Therefore, mirnas could rapidly evolve new mrna target specificities after mutation of only a few nucleotides. As EBV, human cytomegalovirus (hcmv) and Kaposi s sarcoma associated herpesvirus (KSHV) are separated by tens of millions of years of evolution, this lack of conservation seems predictable. A more surprising result is that two herpesviruses separated by >13 million years of evolution, EBV and rhesus lymphocryptovirus, have retained several closely similar mirnas 34. This observation suggests that the integrity of these viral mirnas has been subject to ongoing positive selection and, therefore, that their expression confers a selective advantage. Though not yet confirmed by expression data, several of the mirnas encoded by hcmv seem to be conserved chimpanzee CMV; these viruses again are separated by a substantial evolutionary distance 27,30. In addition to the herpesviruses listed in Table 1, single mirnas have also been reported for two lytic DNA viruses: SV40 and human adenovirus In the case of SV40, the existence of the viral mirna mir-s1 is well documented, and a role for this mirna in the SV40 life cycle has been described. In contrast, it remains uncertain whether the adenovirus mirna has a role in the adenovirus replication cycle. Adenovirus encodes a 160-nt noncoding RNA called VA1 that is expressed at very high levels (up to 10 8 copies per cell) during the late stage of its lytic replication cycle 36. VA1 RNA is transcribed in the nucleus by RNA polymerase III (pol III) and then transported by Exp5 and Ran to the cytoplasm, where it inhibits the antiviral enzyme PKR Because VA1, like pre-mirnas, uses Exp5 to exit the nucleus, it shares the RNA S26 VOLUME 38 JUNE 2006 NATURE GENETICS SUPPLEMENT

3 Table 1 Summary of known virus-encoded mirnas Virus family Species Number of validated mirnas mirnas References Herpesviridae hcmv 11 mir-ul22-1, mir-us5-1, mir-us5-2, mir-ul36-1, mir-ul112-1, mir-us25-1, mir-us25-2, mir-ul148d-1, mir-us33-1, mir-ul70-1, mir-us4-1 structural motif recognized by Exp5; that is a 3 overhang at the base of a short RNA stem 12,38. This is the same structure recognized by the Dicer-TRBP heterodimer 4,39 ; therefore, it is not unexpected that VA1 is subject to processing by Dicer, although this processing is (perhaps by design) very inefficient, with only ~1% of VA1 being processed into adenovirus-derived mirnas 33. In fact, evidence suggests that VA1 is a potent inhibitor of Dicer function and, hence, cellular mirna processing 32,40. Although the adenoviral mirna is active on artificial mrna targets 32,33, it remains somewhat uncertain whether this mirna has a role in the viral life cycle or instead is simply a byproduct of inadvertent processing of VA1 by Dicer. Nevertheless, a processing efficiency of only 1% would be predicted to give rise to up to 10 6 VA1-derived mirnas in an adenovirus-infected cell; this number is probably higher than that of almost all endogenous mirnas. Therefore, it remains possible that VA1-derived mirnas have a role in the adenovirus life cycle 41. The list of viral mirnas provided in Table 1 is certainly far from complete, and it is of interest to speculate briefly as to which other pathogenic human viruses might encode mirnas. One obvious omission is any of the herpesviruses, such as herpes simplex virus type 1 (HSV-1), that establish long-term latent infections in the peripheral nervous system of humans 42. Within infected sensory or autonomic ganglia, the HSV- 1 DNA genome is entirely quiescent except for expression of a set of overlapping viral RNAs called latency-associated transcripts (LATs) 42. Although LAT RNAs are unstable and do not encode any proteins, LAT expression affects the ability of HSV-1 to initiate lytic infection and renders expressing cells resistant to apoptotic stimuli 42,43. If LAT is an HSV-1 pri-mirna, then the instability of LAT could be explained by its cleavage by nuclear Drosha, and the phenotypes associated with LAT could be due to the expression of the resultant viral mirnas. Of note, computer analysis of LAT predicts the presence of several primirna-like hairpins 27 ; therefore, it seems probable that LAT is a viral pri-mirna. A final viral family worthy of mention is the human papilloma viruses, which are nuclear DNA viruses that establish long-term persistent infections in the subcutaneous layers of the skin and in the lining of the uterus. Although human papilloma viruses would thus seem to be good candidates to encode viral mirnas, computer analysis does not predict any appropriate RNA stem-loop structures, at least in human papilloma virus serotype 18 (ref. 27). Because these computer predictions are not 27, 30 KSHV 12 mir-k1 to mir-k12 27, 28, 35, 45 EBV 23 mir-bhrf1-1 to mir-bhrf1-3 and mir-bart1 to mir-bart20 29, 34, 35 MHV68 9 mir-m1-1 to mir-m rlcv 16 mir-rl1-1 to mir-rl Adenoviridae hav 1 Unnamed 32, 33, 41 Polyomaviridae SV40 1 mir-s1 31 rlcv, rhesus lymphocryptovirus; hav, human adenovirus. entirely accurate at present, however, this conclusion awaits experimental confirmation. Viral mirna expression Cellular mirnas are transcribed by pol II and are generally located in noncoding pri-mirna transcripts or in introns 4. This is also true for most of the viral mirnas described so far 27,29,34,44. One exception is the adenovirus mirna, which forms part of the VA1 noncoding RNA transcribed by pol III 32,33. All nine mirnas encoded by mouse herpesvirus 68 (MHV68) are also transcribed by pol III 27. The MHV68 mirnas are all transcribed as unusual precursor RNAs consisting of a normal looking pri-mirna hairpin attached to the 3 end of a trna-like molecule 27. It is currently uncertain whether the processing of MHV68 mirnas proceeds as described for cellular mirnas (Fig. 1). Another exception to the above generalization that mirnas are derived from noncoding RNAs or introns is the KSHV mirna mir-k10, which is located in the middle of the viral K12 open reading frame 27,28,45. K12 translation and mir-k10 processing must be mutually exclusive. One reasonable explanation is that mir-k10 processing is simply inefficient, so that a substantial percentage of K12 mrna is able to exit the nucleus before Drosha cleavage. Alternatively, the efficiency of mir- K10 processing and, hence, the level of K12 protein expression may be regulated somehow during the viral life cycle. Viral mirnas have also been identified in viral open reading frames, or predicted mrnas, in EBV and hcmv 27,29,30, so it is possible that regulation of viral mrna expression by modulation of Drosha cleavage efficiency has a role in several different virus replication cycles. I propose above that viral mirnas might be particularly useful during long-term latent or persistent infections; in fact, all the herpesvirus mirnas that have been analyzed so far are expressed in latently infected cells 27 29,34. Nevertheless, at least one of the KSHV mirnas, and almost all of the EBV mirnas, show enhanced expression after induction of lytic replication 27,34. Whether this increased expression is functionally relevant, or instead simply reflects the higher level of viral RNA transcription and increased genome copy number seen during lytic replication, is currently unknown. Although the 12 mirnas encoded by KSHV are all expressed coordinately, at least in latently infected cells 44, the 23 mirnas encoded by EBV are found in two distinct clusters, one of 3 mirnas and one of NATURE GENETICS SUPPLEMENT VOLUME 38 JUNE 2006 S27

4 Figure 2 Potential mechanisms by which mirnas can affect virus replication. (a) Viral mirnas may inhibit the expression of viral mrnas. As shown, these are perfectly complementary mrnas that are transcribed from the strand of a viral DNA genome that lies opposite the mirna gene, but inhibition of mrnas transcribed from other regions of the viral genome is possible. dsdna, double-stranded DNA. (b) Viral mirnas may also inhibit the expression of cellular mrnas. (c) Finally, cellular mirnas could inhibit the expression of viral mrnas, as reported for PFV and mir-32 (ref. 51). It has also been reported that the human liver-specific mirna mir-122 facilitates HCV replication 53. The mechanism underlying this phenomenon, and whether it involves RISC, remains unknown. 20 mirnas 29,34,35, whereas the mirnas encoded by hcmv are scattered throughout the viral genome 27,30. In the case of EBV, these two mirna clusters are differentially expressed, with the 3-member BHRF- 1 mirna cluster expressed only in EBV-infected B cells undergoing stage III latency, and the 20-member BART mirna cluster predominantly expressed in transformed cells undergoing EBV stage II latency 34. The importance of this differential expression is not yet known, but this observation suggests that these mirnas act at different steps of the EBV replication cycle. Function of viral mirnas Transcripts targeted by viral mirnas could, in principle, derive from one of two sources: the host cell or the virus itself (Fig. 2). Because of the small size of viral genomes, it is easier to identify target mrnas of viral origin, and two such viral mrna targets are now well supported by experimental data. In both cases, these mrna targets are transcribed from the strand of the viral double-stranded DNA genome that lies directly opposite to the mirna 29,31 (Fig. 2a). As they are therefore fully complementary to the viral mirna, RISC binding results in mrna cleavage (Fig. 1). The first viral mrna target to be described encodes the EBV DNA polymerase, which is transcribed from the DNA strand opposite to EBV mir-bart2 (ref. 29). EBV DNA polymerase mrnas cleaved at the predicted position, precisely 10 nt from the 5 end of the region of homology to mir-bart2, have been identified in infected cells 46. The purpose of this mrna cleavage is not fully understood; it could inhibit activation of lytic EBV replication. A second mrna target has been defined for the SV40 mir-s1 mirna. This mirna is encoded immediately 3 to the polyadenylation site used by SV40 late transcripts and, in the circular SV40 genome, overlaps with, and is fully complementary to, viral early mrnas encoding the viral T antigens 31. Analysis has confirmed that these viral early mrnas are degraded by mir-s1, which is expressed only late in the viral replication cycle. This late reduction in T antigen expression has no effect on virus replication but does protect SV40-infected cells against killing by cytotoxic T-lymphocytes specific for SV40 T antigen 31. Therefore, mir-s1 does not enhance SV40 replication directly but instead protects infected cells from elimination by the host immune system. Although viral mrnas are regulated by EBV mir-bart2 and SV40 mir-s1, and hcmv also contains three open reading frames that lie directly opposite to hcmv mirnas 27, it seems obvious that most viral mirna probably regulate cellular mrnas (Fig. 2b). Unfortunately, no host cell mrna targets for any viral mirna have been identified thus far. On the basis of the activities associated with viral nonstructural proteins, however, one can predict that viral mirnas probably inhibit aspects of the host adaptive immune response, including antigen presentation, or the innate immune response, including induction of apoptosis or the interferon system 47,48. Viral mirnas may also modulate aspects of signal transduction or regulate cellular proliferation. I would argue that it is unlikely that viral mrnas are essential for virus replication per se, as has been demonstrated in SV40 (ref. 31), and that they are more likely to have a role in inhibiting protective antiviral responses in the infected host. Although no viral mutants selectively lacking one or more mirnas have as yet been analyzed in vivo, there is one published report examining the replication in mice of an MHV68 deletion mutant, called MHV76, that lacks all nine viral mirnas as well as four adjacent protein-coding genes 49. The MHV76 mutant grew as well as MHV68 in tissue culture, Katie Ris S28 VOLUME 38 JUNE 2006 NATURE GENETICS SUPPLEMENT

5 but its growth was substantially attenuated in vivo, with lower levels of virus replication and pathogenesis, a greater inflammatory response and more rapid clearing by the immune system. Moreover, MHV76 was much less able to establish long-term latent infections in mice 49. This phenotype could be due, in whole or in part, to the loss of the proteincoding genes, but it does suggest that the MHV68 mirnas may have a role in modulating host immune responses. It is not certain whether this single result with a murine herpesvirus is predictive of what may be seen with human or primate herpesviruses. Viruses and cellular mirnas All metazoan eukaryotes encode mirnas, which may be expressed at >1,000 copies per cell 1. Moreover, each differentiated human cell expresses multiple mirnas, and the pattern of mirna expression can vary markedly across different human tissues 1. Some evidence suggests that cellular mrnas are under selection to avoid the presence of target sequences complementary to mirnas present in cells where translation of these mrnas is required 2,3. So, are viruses also subject to evolutionary pressure to avoid regions of complementarity to mirnas present in their normal target tissues? At present, this question remains unanswered. But all vertebrate viruses that have been analyzed thus far are susceptible to inhibition by RNA interference 50, and it therefore seems likely that viral mrnas would also be susceptible to translational inhibition by host cell mirnas. If this is the case, then one can predict that viruses that replicate in a specific tissue, for example, human immunodeficiency virus in CD4 + lymphoid cells, would have been selected to contain few sites of complementarity to mirnas expressed in that tissue. In contrast, regions of complementarity to other human mirnas, expressed in other irrelevant tissues, might remain present. If this is the case, then viruses may be tailored by evolution to avoid inhibition by the mirnas present in their normal target tissue yet would remain susceptible to mirna-mediated inhibition when they infect, or are induced to infect, a nonphysiological target cell. If this hypothesis is correct, then mirnas could have an important role in determining the tissue tropism of animal viruses. Evidence in favor of the above hypothesis comes from analysis of the replication of the retrovirus primate foamy virus (PFV) in the human embryonic kidney cell line 293T, which showed inhibition of PFV replication by human mir-32 (ref. 51; Fig. 2c). This inhibition is not due to the evolutionary selection of mir-32 to confer an antiviral phenotype against PFV, as mir-32 is highly conserved in all vertebrates, including chickens, and PFV normally infects only chimpanzees 52. Moreover, the target sequence for mir-32 in PFV is not well conserved in other primate foamy viruses and is entirely lacking in nonprimate foamy viruses. I therefore hypothesize that the observed inhibition is fortuitous and may be due to the fact that PFV normally replicates primarily in salivary glands in vivo 52, where I speculate that mir-32 is likely not expressed, and not in 293T cells, where mir-32 is expressed. Although the effect of host cell mirnas on viral replication is therefore likely, in most instances, to be either nonexistent or inhibitory, one report has documented a strong positive effect of the liver-specific mirna mir-122 on the level of expression of HCV mrna 53. The mechanism underlying this effect is obscure, but mir-122 has been shown to act primarily by enhancing viral RNA replication rather than viral mrna translation or stability. A direct interaction between mir-122 and a region of complementarity in the 5 noncoding region of HCV is required for this enhancing effect to be observed 53. This unexpected result emphasizes the fact that our knowledge of mirna function is far from complete. But the observation that specific cellular mirnas can facilitate virus replication, together with the finding that several pathogenic human viruses encode mirnas that undoubtedly are important in the virus life cycle in vivo, suggests that technologies that allow the specific inhibition of mirna function, such as the recently described antagomir molecules 54, could have a future role in the treatment of virally induced diseases. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. ACKNOWLEDGMENTS Research from my laboratory described in this manuscript was supported by grant GM from the US National Institutes of Health. 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