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sdarticle-2 Molecular Cell, Vol. 20, 3–7, October 7, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.09.012 MicroRNAs and Viral Infectio with them will come new insights into miRNA biogene- sis and function. Here we review what has been learned to date ...

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Molecular Cell, Vol. 20, 3–7, October 7, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.09.012 MicroRNAs and Viral Infectio with them will come new insights into miRNA biogene- sis and function. Here we review what has been learned to date from the study of such miRNAs. MicroRNA Biogenesis and Function miRNAs are derived from larger RNA precursors by the sequential action of two ribonucleases of the RNase III *Correspondence: ganem@cgl.ucsf.edu lation remain unknown. Clearly, miRNA-based regula- tion has the potential to be staggeringly complex. Virally Encoded miRNAs Given the important role that miRNAs play in gene regu- lation, it was a safe bet that viruses would employ them to modulate both their own gene expression and that of their host cells. There are several ways in which virus- infected cells have been screened for miRNAs. Most studies begin with bioinformatic analyses aimed at Christopher S. Sullivan and Don Ganem* Howard Hughes Medical Institute Departments of Microbiology and Medicine G.W. Hooper Foundation University of California, San Francisco San Francisco, California 94143 MicroRNAs (miRNAs) play a pivotal role in the regula- tion of genes involved in diverse processes such as development, differentiation, and cellular growth con- trol. Recently, many viral-encoded miRNAs have been discovered, for the most part in viruses transcribed from double-stranded DNA genomes. As with their cel- lular counterparts, the functions of most viral-derived miRNAs are unknown; however, functions have been documented or proposed for viral miRNAs from three different viral families—herpesviruses, polyomaviruses, and retroviruses. Several virus-encoded miRNAs have unique aspects to their biogenesis, such as the poly- merase that transcribes them or their location within the precursor transcript. Additionally, viral interac- tions with cellular miRNAs have also been identified, and these have substantially expanded our apprecia- tion of miRNA functions. MicroRNAs (miRNAs) are small (approximately 22 nt) RNAs that regulate gene expression by a variety of mechanisms. Initially discovered in C. elegans, they are now known to be widespread in nature. Thus far, over 1000 miRNAs have been identified, with greater than 320 found in humans (MiRBase) (Griffiths-Jones, 2004). Recently, based on the technique of phylogenetic shadowing, it has been suggested that the actual num- ber of human miRNAs could be as high as 1000 (Berezi- kov et al., 2005). miRNAs are predicted to play a pivotal role in the regulation of many genes, especially those at nodes of signaling pathways involved in such pro- cesses as development and growth control. It therefore comes as no surprise that viruses, which typically em- ploy many components of the host gene expression machinery, also encode miRNAs. Potential functions have been suggested for several viral miRNAs; in addition, host-encoded miRNAs have recently been described that can modulate viral replication via in- teraction with target sites in viral transcripts. It seems certain that many new virus-encoded miRNAs will be identified in the coming years, and equally certain that Short Reviewn family, known as Drosha and Dicer (Kim, 2005). The lat- ter enzyme was discovered as part of the RNA interfer- ence (RNAi) machinery, which generates small interfer- ing RNAs (siRNAs) from cytoplasmic RNA duplexes. In fact, the late steps in miRNA generation share many commonalties with siRNA biogenesis, but the nature and location of the early steps differ substantially. miRNAs are generated by excision from a hairpin struc- ture which is, in turn, typically derived from a longer Pol II transcript (typically hundreds to several thousands of nucleotides) called a pri-miRNA (see Figure 1). In the nucleus, the pri-miRNA is cleaved by Drosha into an approximately 60–80 nt imperfect hairpin called a pre- miRNA, which in turn is bound and exported from the nucleus by the complex of the karyopherin exportin 5 and Ran (Bohnsack et al., 2004; Lund et al., 2004; Yi et al., 2003). In the cytoplasm, the pre-miRNA is then recognized and cleaved by Dicer into a (presumably transient) partially dsRNA structure that is then un- wound, leaving one strand energetically favored to en- ter the multiprotein RNA-induced silencing complex (RISC). RISC then directs either cleavage or transla- tional inhibition of its target mRNA, depending on the degree of complementarity between the RISC bound miRNA and target mRNA. Perfect complementarity generally results in cleavage; imperfect complementar- ity usually leads to impaired translation, most often when the target sequences are located in the 3#UTR of the mRNA. Prediction of miRNA targets is still an imperfect sci- ence. It is known that pairing of nucleotides 2–8 from the 5# end of the miRNA (the so-called “seed” region) with the target is especially important for target rec- ognition; mutational disruption of this region can ablate miRNA activity. But there is more to target recognition than just seed complementarity, since any given 7-mer would have thousands of complements in the mamma- lian genome. Progress has been made by using infor- matic approaches that impose additional requirements for evolutionary conservation and localization of the target in the 3#UTR, but these still indicate that thou- sands of human genes are potential miRNA targets. Re- lated to this, it was recently shown that, with overex- pression of individual miRNAs, the levels of 100–200 transcripts promptly declined, as judged by array-based expression profiling (Lim et al., 2005). Since many of the downregulated mRNAs had seed complementarity at their 3#UTRs, it is unlikely that this result was due solely to secondary effects of inhibition of a small num- ber of directly targeted transcription factors. However, details of the actual mechanisms underlying this regu- linruxian 高亮 linruxian 高亮 Molecular Cell 4 oped for cellular miRNAs, with high-throughput se- quencing of large numbers of the resulting clones. Northern blotting of total cellular RNA with the appro- priate sequence from the clones provides additional confirmation. Extensive cDNA cloning studies by the Tuschl group across many families of RNA viruses have failed to identify miRNAs from viruses with RNA genomes (Pfef- fer et al., 2005). This negative finding is subject to all the usual interpretive caveats of negative results but is consistent with the prominent role of the DNA-depen- dent RNA polymerase II in the biogenesis of pri-miRNAs. Of course, it remains formally possible to envision Drosha acting on RNA templates produced by viral RNA- dependent RNA polymerases, especially for virus fami- lies (e.g., myxoviruses) in which genomic replication or transcription occurs in the nucleus (where Drosha re- sides). For viruses whose RNA-based replication cycle is cytoplasmic (e.g., cornonaviruses, picornaviruses), however, segregation away from the Drosha machinery provides an additional theoretical barrier to miRNA bio- genesis. To date, miRNAs have been identified in the following classes of viruses: Herpesviruses. Herpesviruses are large, enveloped viruses with dsDNA genomes that typically encode 100–200 genes. There are eight human herpesviruses, all of them linked to important disease syndromes in replication is characterized by the temporally regulated expression of most of the viral genes, with extensive viral DNA replication, cell death, and release of infec- tious progeny. Lytic replication can be evoked from lat- ency, a phenomenon that accounts for the relapsing nature of many herpesviral disorders. Herpesviruses can be classified into three subfamilies (α, β, or γ) based on sequence relatedness and virus biology. To date, three members of the γ (lymphotropic) subfamily—EBV and KSHV of humans and MHV68 of mice—and one β-herpesvirus (HCMV) have been shown to encode miRNAs; additionally, two α viruses, HSV-1 and -2, have been predicted but not experimentally proven to en- code miRNAs (Cai et al., 2005; Pfeffer et al., 2004, 2005; Samols et al., 2005). None of these miRNAs appear to be conserved among the different viruses (Pfeffer et al., 2005). EBV, causative agent of infectious mononucleosis and etiologically linked to several malignant lymphomas, was the first virus demonstrated to encode miRNAs (Pfef- fer et al., 2004). Cloning from a B cell line latently in- fected with EBV identified five miRNAs. The function of the EBV miRNAs has not been established, but compu- tational predictions suggest that some of these miRNAs may target chemokines, cytokines, and apoptotic and cell growth control genes such as p53. Additionally, one of the miRNAs (mir-BART2) apparently has a viral target (Table 1): it is antisense to a region in a lytic mRNA Figure 1. Viral Utilization of miRNAs Diagram of miRNA biogenesis and activity. The outer rectangular boxes h systems. identifying stem-loop structures compatible with pre- miRNAs. Experimental screening for miRNAs usually proceeds via cDNA cloning strategies originally devel- e g e man. A defining feature of herpesviral biology is the presence of two alternative genetic lifestyles—a cryptic or latent infection and a patent, or lytic, replicative cy- cle. During latency, only a handful of viral genes are ighlight some miRNA properties that have been identified in viral xpressed, and no viral progeny are produced; the viral enome is persistently maintained over many cell gen- rations as a low copy number nuclear plasmid. Lytic (BALF5) encoding the viral DNA polymerase. Because it is fully complementary to that mRNA, it would be pre- dicted to cleave the transcript. Since the experiment that identified the miRNA was conducted in a latently Short Review 5 (kaposins A, B, and C) with important roles in latency: kaposin A affects cell growth control, and kaposin B enhances cytokine production by infected cells (Kliche et al., 2001; McCormick and Ganem, 2005). Both pro- teins are tightly regulated in latency, as overexpression of either is deleterious to cells. Is it possible that Drosha- mediated cis cleavage of the transcript during pri- miRNA formation serves to downregulate the steady- state level of kaposin mRNA during latency? If so, this regulation would have to be nullified during lytic infec- tion, since levels of kaposin mRNA and protein become Polyomaviruses. The polyomavirus family is com- posed of nonenveloped viruses with small dsDNA ge- nomes. The best-known polyomavirus is simian virus 40 (SV40), which replicates in simian cells and produces a subclinical but persistent infection of monkeys. SV40 has recently been found to encode a set of miRNAs whose in vivo target has been clearly defined (Sullivan et al., 2005). The miRNAs originate from a single pre- miRNA that maps to the late strand of the viral genome (the strand transcribed after the onset of viral DNA rep- lication) and is found downstream of the late polyade- Table 1. miRNAs with Reported Viral Functions Virus miRNA Origin Function SV40 SVmiRNAs Viral Cleave and downregulate earl EBV miR-BART2 Viral Modify BALF5 transcript HIV miR-N367 Viral Downregulate LTR-driven tran PFV1 miR-32 Host Downregulate translation of v HCV miR-122 Host Upregulate viral RNA levels aConserved in other viruses. infected cell, such a cleavage product would not be expected to be found in that context. But, strikingly, earlier cDNA cloning of lytic-cycle transcripts for the EBV DNA polymerase detected an “aberrant” mRNA whose sequence had undergone a rearrangement pre- cisely at the site of complementarity to the miRNA (Furnari et al., 1993)! It seems highly likely that cleavage of the transcript did indeed occur in vivo—with subse- quent repair/rescue of the cleaved transcript by an as- yet-uncertain mechanism. Less clear is what the bio- logical role of this cleavage might be. The cleaved and modified RNA remains fairly abundant, is polyadeny- lated, and retains its full coding potential. Moreover, in- fected cells also produce a second mRNA for polymer- ase that is unaffected by this miRNA. So early notions that the miRNA might extinguish polymerase expres- sion and thereby block lytic replication seem unlikely, and its biological role remains something of an enigma. KSHV is the causative agent of Kaposi’s sarcoma (an inflammatory and proliferative lesion of the endothe- lium) and is also linked to several rare lymphoprolif- erative syndromes. Several groups, using traditional RACE-like cloning (Cai et al., 2005; Pfeffer et al., 2005; Samols et al., 2005) have identified miRNAs in KSHV. There is broad agreement among these studies that a majority of the KSHV miRNAs are encoded in a single 4.5 kb region of the genome. Most derive from a non- coding region that is represented in an intron of a latent mRNA (Li et al., 2002). Computational predictions sug- gest that some of these miRNAs may have cellular tar- gets involved in such activities as apoptosis, signaling, and B cell regulation (Cai et al., 2005), which, if proven, would have important implications for KSHV-associ- ated disease. Two of the KSHV miRNAs map within the body of a latent transcript that is strongly upregulated during lytic replication. This mRNA encodes a family of proteins very high as lytic replication proceeds. MHV68 is a murine γ herpes virus that is much sim- pler to grow and to genetically manipulate than either EBV or KSHV; however, it is less pathogenic in its native Conserveda Reference y gene expression Y (Sullivan et al., 2005) N (Pfeffer et al., 2004) scription? N (Omoto et al., 2004) iral transcripts NA (Lecellier et al., 2005) NA (Jopling et al., 2005) host and does not recapitulate many of the pathologic features of infection by its human counterparts. MHV68 encodes at least nine miRNAs as demonstrated by mo- lecular cloning (Pfeffer et al., 2005). All cluster within a 6 kb region near the M1 terminus of the linear genome. A mutant that deletes a region that includes miR-M1-1, miR-M1-2, miR-M1-3, and miR-M1-4 was completely viable and displayed no demonstrable phenotype in vivo (Simas et al., 1998). Thus, any function supplied by these miRNAs must be nonessential or redundant. Perhaps the most noteworthy aspect of the MHV68 miRNAs concerns their biogenesis: at least some of them are thought to be derived from RNA Pol III tran- scripts (Pfeffer et al., 2005). This suggests that not all mammalian pre-miRNAs need to be processed from Pol II transcripts, and it serves as a reminder that the pathways to miRNAs may be more varied than pres- ently assumed. Human cytomegalovirus (HCMV) infection is often subclinical in normal hosts but is more serious in immu- nocompromised patients, in whom it can precipitate retinitis, pneumonitis, hepatitis, and colitis; additionally, intrauterine infection can result in birth defects. HCMV encodes for at least nine microRNAs (Pfeffer et al., 2005), three of which are complementary to regions encoding ORFs, suggesting the possibility of miRNA- induced viral gene autoregulation. However, no direct functional studies of these miRNAs have yet been re- ported. Importantly, all of the HCMV miRNAs were cloned from lytically infected cells, indicating that her- pesviral utilization of miRNAs is not solely associated with the latency program. Thus, many more miRNAs may await discovery, since, for the other herpesviral family members where miRNA cloning was attempted, only cells that were predominantly latently infected (and therefore transcribing only restricted portions of the genome) were used as a source for RNA. nylation site. Since no other promoter is evident in this region, the pri-miRNA is thought to emanate from the viral late pre-mRNA (the primary transcript produced prior to cleavage and polyadenylation). Processing of Molecular Cell 6 the pri-miRNA generates a 57 nt pre-miRNA that dis- plays two unusual features. First, multiple miRNAs are generated from it, deriving from both arms of the pre- miRNA hairpin. Second, the processing of this miRNA appears to be remarkably inefficient, resulting in large amounts of pre-miRNA accumulating in the cell relative to the processed miRNAs. The SV40 miRNAs are the first viral miRNAs whose target is known with certainty (Table 1). The SV40 ge- nome is circular, and the region encoding the miRNAs on the late strand overlaps the body of the viral early mRNAs produced from the opposite strand. The prod- uct miRNAs are, therefore, completely complementary to early mRNA and thus would be predicted to cleave that transcript. In fact, the 3# (polyadenylated) product fragment predicted from such cleavage is readily de- monstrable in cells, and mutational disruption of the pre-miRNA results in the disappearance of these frag- ments, confirming that they result from the action of the miRNAs. The net result is that the expression of the miRNAs reduces the expression of early mRNAs (and their major translation product, the viral large T antigen) at late times in the replicative cycle. T antigen is a dom- inant target of host cytotoxic T lymphocyte (CTL) re- sponses, and miRNA-mediated downregulation of T an- tigen synthesis diminishes the susceptibility of infected cells to CTL-mediated lysis in vitro. This suggests that one function of the SV40 miRNAs may be to help evade immune detection during its long persistence in the host; additional functions are certainly conceivable. Whatever the in vivo function of this pre-miRNA, it is presumably important, as it is conserved with three other polyomaviruses: the baboon virus SA12 and the human viruses, BK and JC (Sullivan et al., 2005). The finding that the miRNAs target a viral transcript does not, in principle, exclude the possibility that it might also target host RNAs as well. However, studies of the related mouse polyoma virus (Py) bear impor- tantly on this issue. No homology exists between Py and SV40 in the region of the SV40 pre-miRNA, and Py is predicted to encode no miRNAs from this region. However, we have recently identified a Py miRNA that emanates from a different region of the late pre-mRNA. Like the SV40 miRNA, it, too, is complementary to early viral mRNA, resulting in cleavage of the mRNAs for both middle and large T antigens (C.S.S., A. Grundhoff, R. Treisman, C.K. Sung, T. Benjamin, and D.G., unpublished data). Thus, these two completely unrelated miRNAs are expressed with the same kinetics and target the same viral mRNA but would be expected to share no cellular targets; this strongly suggests that the principal func- tion of these miRNAs is indeed to downregulate T anti- gen mRNA. Retroviruses. Retroviruses are small, enveloped RNA viruses that replicate by reverse transcription, deposit- ing a dsDNA copy of their genome into the host chro- mosome. This integrated provirus serves as the tem- plate for viral gene expression via host Pol II-mediated transcription. This makes retroviruses the likeliest of all RNA viruses to encode miRNAs, since all retroviral tran- m o l w i i l 2 c r c H t t i e ( o g n b s W I r a v o D a c ( s u i t o m f s t e i V S o t s p S C p i r t c a q m a scription emanates from host machinery similar to that directing expression of cellular miRNAs. Nonetheless, Tuschl and colleagues did not identify any virally encoded miRNAs from HIV-infected cells by olecular cloning approaches, even after screening ver 1500 amplicons (Pfeffer et al., 2005). Fuji and col- eagues, however, have reported evidence for a miRNA ithin the nef region of the genome, as judged by clon- ng and Northern blot analysis, and proposed a role for t in downregulation of viral transcription based on pre- iminary studies with reporter genes (Omoto and Fujii, 005; Omoto et al., 2004). But the mechanism that ould account for such a phenotype is unclear, as is the eason for the discrepancy between the two different loning studies. Finally, it was recently reported that IV encodes small RNAs d
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