Small noncoding RNA: novel targets for antiviral therapy

Abstract

Future MicrobiologyVol. 3, No. 6 EditorialFree AccessSmall noncoding RNA: novel targets for antiviral therapyLars Dölken & Jürgen HaasLars DölkenMax von Pettenkofer-Institute, Ludwig-Maximilians Universitaet Muenchen, Pettenkoferstrasse 9a, 80336 Munich, Germany. Search for more papers by this authorEmail the corresponding author at doelken@mvp.uni-muenchen.de & Jürgen Haas† Author for correspondenceMax von Pettenkofer-Institute, Ludwig-Maximilians Universitaet Muenchen, Pettenkoferstrasse 9a, 80336 Munich, Germany and, Division of Pathway Medicine, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, Scotland, UK. Search for more papers by this authorEmail the corresponding author at juergen.haas@ed.ac.ukPublished Online:11 Dec 2008https://doi.org/10.2217/17460913.3.6.585AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit miRNAs are abundantly expressed, small noncoding RNAs generated from both RNA polymerase II and III transcripts by two sequential cleavage events mediated by the nuclear RNAse III enzyme Drosha and cytoplasmic Dicer. Mature miRNAs are integrated into RNA-induced silencing complexes, mainly targeting 3´-untranslated regions of a large number of target mRNAs. When there is perfect or near-perfect complementarity, the target is cleaved in a position corresponding to the center of the miRNA-target duplex upon which the cleaved RNA is degraded by exonucleases. While this mode of miRNA action is common for plant miRNAs, current data suggest that the preferred mode of action of animal miRNAs is translation repression upon imperfect base pairing [1,2].While miRNAs have only recently been identified as key regulators in nearly all biological processes, they provide an ancient but continuously adapting mechanism for regulation of gene expression. Viruses have co-evolved with their hosts over millions of years. With the identification of viral miRNAs in 2004 [3], another layer of complexity has been added in our understanding of the interaction between viruses and their hosts. Whereas RNA silencing is used by plants and invertebrates to protect themselves from viral infection [4], miRNAs are also ideal tools for viruses to regulate their own and host gene expression, as no potentially immunogenic protein expression is required for their function. In addition, they are very small and offer exceptional flexibility, as single point mutations may already dramatically alter target specificity. It is therefore not very surprising that many of the large DNA viruses have been found to express miRNAs.Most, but not all herpesviruses express miRNAs. Currently, they are unique in expressing multiple miRNAs. Herpesviruses are large (∼110–240 kb) DNA viruses which, upon primary infection, establish lifelong, latent infection, leaving the infected individual at risk for subsequent reactivation and disease. They can be divided into three subfamilies based on sequence homologies and unique biological features (α-, β- and γ-herpesviruses). Upon primary infection, herpes simplex virus (HSV)-1, a member of the α-herpesvirus family, establishes lifelong latent infection in sensory ganglia. The mechanisms regulating establishment of latency and reactivation are poorly understood. The only abundant viral gene product in latently-infected cells is the noncoding latency-associated transcript (LAT). LAT expression has been shown to increase the number of neurons in which latency is successfully established by promoting survival of infected cells [5]. LAT has now been found to give rise to at least four different miRNAs that are also expressed during lytic infection, two of which target the main viral transactivators ICP0 and ICP4 [6]. Thus, HSV-1 may use the cellular miRNA machinery to facilitate the establishment and maintenance of latency in infected neurons by post-transcriptionally regulating the key regulators of viral gene expression.The human cytomegalovirus (HCMV) belongs to the subfamily of β-herpesviruses and is the major cause of morbidity in immunocompromised patients and allogenic bone marrow or organ transplant recipients. During lytic HCMV infection, at least 11 miRNAs are expressed, with miRNA levels strongly accumulating throughout infection. HCMV miRNA loci are scattered throughout the entire genome. With the identification of 18 miRNAs of the murine cytomegalovirus, the role of miRNA in cytomegalovirus infection and latency can now be studied in the natural host. In murine cytomegalovirus-infected cells, viral miRNAs contribute more than two-thirds of the cellular miRNA pool late in infection. This implies an important function of cytomegalovirus miRNAs during productive infection. However, the effect of this overwhelming miRNA biogenesis on the miRNA machinery and function still remains to be elucidated. Thus far, none of the viral miRNAs have been found to be essential for virus replication, with most of the viral miRNAs even being completely dispensable for virus replication in fibroblasts in vitro. However, this is also true for a large number of other cytomegalovirus genes. Some of these carry out important functions in modulating innate and adaptive immunity, with knockout mutants showing severe loss of fitness. Therefore, animal models are required to study viral miRNA function. Studies are ongoing to look for in vivo phenotypes of the respective miRNA knockout mutants and elucidate their role in productive infection, latency and reactivation.Viral miRNA may function by regulation of cellular and viral genes. HCMV miR-UL112 regulates both the expression of major viral transactivators [7], implying a similar function in establishment or maintenance of latency as for HSV-1, as well as MICB [8], a stress-induced ligand of the natural killer (NK) cell-activating receptor, NKG2D, which is critical for NK cell killing of virus-infected cells and tumor cells. Interestingly, MICB is also directly targeted by another viral protein (HCMV UL16), emphasizing the need of the virus to modulate NK cell-mediated killing of infected cells by multiple and at least partially redundant mechanisms.In theory, perfect sequence complementarity would ensure the most efficient downregulation of any miRNA target. Intriguingly, none of the viral miRNAs identified so far show perfect sequence complimentarity to any cellular or viral gene, unless a viral gene is expressed from the same gene locus in antisense orientation to the miRNA [6]. This implies that not highly efficient targeting of a single gene but concomitant targeting of a spectrum of relevant targets is the rule and not the exception for viral miRNA function. Unless the target sequence is part of a cellular mechanism utilized for the regulation of a certain cellular pathway, the existence of suitable miRNA target sequences is hard to imagine. Interestingly, the target site of HCMV miR-UL112 in the 3´-untranslated region of MICB is conserved in MHC class I polypeptide-related sequence A (MICA), another NK ligand. It has been found that this site is also targeted by other cellular miRNAs, indicating that the virus has usurped a cellular mechanism to regulate expression of these ligands for its own needs [9]. Vice versa, analyzing the function of a viral miRNA has led to the identification of a novel evolutionarily conserved strategy utilized by the host to regulate stress-induced ligands and probably a number of other proteins involve in NK cell-mediated killing. A similar phenomenon was elucidated for Kaposi’s sarcoma herpesvirus miR-K12-11, which appears to be an ortholog of cellular miR-155, a miRNA involved in cell growth and tumorigenesis [10]. It will be interesting to see whether this represents a more generalized mechanism used by viral miRNAs.As noted above, recent findings indicate that herpesviruses use the cellular miRNA machinery to promote establishment or maintenance of latency by downregulating the expression of their major transactivating genes. This phenomenon is also seen in polyomaviruses. In simian virus (SV)40 and both JC and BK virus infection, a miRNA expressed in antisense orientation to the large T antigen leads to cleavage of its mRNA and downregulation of large T antigen late in infection [11]. It was shown that this miRNA-mediated autoregulation of early gene expression can reduce cytotoxic T cell-mediated recognition and lysis of SV40-infected cells. These data suggest a model where polyomaviral miRNAs may function to evade the cellular immune response. Antisense drugs targeting these miRNAs may therefore represent a novel strategy for antiviral therapy by enhancing the visibility of infected cells to the immune system. This would not only promote T cell-mediated killing of the infected cells in which the miRNAs are efficiently targeted but also result in a general enhancement and sharpening of the immune response against the virus.During co-evolution with their hosts, viruses have become entwined in a dense interaction network of host and viral genes. This is best exemplified by the interaction of HCV with the cellular miRNA machinery. HCV is a positive-strand RNA virus of approximately 9.4 kb, belonging to the Flaviviridae. Upon primary infection, persistent infection is established in more than 70% of cases, resulting in approximately 3% of the world population being chronically infected. As a result, HCV infection is one of the major causes of chronic liver disease, including cirrhosis and liver cancer.HCV demonstrates a surprising phenomenon. Its replication is strongly dependent on cellular miR-122 and a functional RNA machinery [12]. miR-122 is the most abundant miRNA in hepatocytes, constituting more than 70% of the total cellular miRNA pool in this cell type. As miR-122 expression is strictly confined to hepatocytes it may, at least in part, be responsible for the restriction of HCV replication to this cell type. miR-122 binds to two adjacent sites in the HCV 5´-noncoding region separated by 14 nucleotides, which are conserved among all six HCV genotypes. Mutational analysis of both miR-122 and its binding sites revealed that binding to both sites is required to promote HCV replication. The exact mechanism, however, still needs to be elucidated. In mice, miR-122 can be efficiently knocked down in hepatocytes by systemic administration of chemically modified antisense oligomers, so-called antagomirs [13]. Knockdown of miR-122 in mice is well tolerated with positive side effects, including lower serum cholesterol levels. It should be noted that targeting a cellular instead of a viral factor is much more likely to prevent development of drug resistance by the virus. Antagomirs against miR-122, therefore, are the most promising candidates for new miRNA-targeting antiviral drugs.Interferons (IFNs) provide the backbone of all current HCV treatment regimens. IFNs counteract viral infections by an array of different mechanisms involving innate and adaptive immunity. The finding by itself – that IFN also regulates the expression of a number of miRNAs – is not very surprising. It seems plausible that such an ancient and highly specialized system uses all available mechanisms to provide an optimal antiviral state, for example, by reducing the threshold for apoptosis upon infection. Surprisingly, Pedersen and colleagues not only observed that IFN-β downregulates miR-122 expression but also induces two cellular miRNAs that inhibit HCV replication by directly targeting the HCV genome, thereby inhibiting HCV protein expression [14]. The development of such a mechanism appears counter-intuitive, considering the relatively low prevalence of HCV in the human population worldwide. Further studies on the conservation of these miRNA target sites in the HCV genome of different isolates, genotypes and other flaviviruses, as well as potential effects on IFN resistance, are required.In summary, it has become apparent that viruses are entwined in a dense functional network of cellular and viral miRNAs at all stages of infection. During co-evolution with their host they have learned to usurp and modulate the cellular miRNA machinery for their needs. We have only begun to glimpse at the complex network they are involved in. Individual miRNAs can be specifically and effectively targeted by chemically modified antisense oligonucleotides (so-called antagomirs) in vivo. Identification of viral miRNA targets including the actual miRNA-binding sites and the assessment of their contribution to different stages of the virus life cycle, as well as optimizing the chemical structure and delivery of antagomirs, are milestones on the way to new antiviral drugs targeting either viral or cellular miRNAs. In addition, this will help to unravel the mechanisms involved in the control of many biological processes and substantially reshape our understanding of the dense interaction between viruses and their hosts.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. 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