Student Perspectives on Contemporary Virology - Volume 1, 2007
microRNA and Viruses
(unreviewed)
Aleksandar Masic
Vaccine and Infectious Disease Organization and
Dept. of Veterinary Microbiology
University of Saskatchewan
for: Advanced Virology (VTMC 833)
alm858@mail.usask.ca


Table of Contents
Abstract

RNA interference (RNAi) is a natural response to the presence of double stranded RNA (dsRNA) which results in the sequence-specific silencing of gene expression. RNAi is a nucleic-acid based immune defense against viruses, transgenes and transposons. In eukaryotic cells, RNAi is triggered either by short interfering RNAs (siRNA) or by micro RNAs (miRNA) molecules. Recent findings reveal that certain viruses encode their own miRNAs that are processed by cellular RNAi machinery. However, it was unclear what the roles of these virus-encoded miRNAs play and whether some cellular miRNAs play a role in viral replication and phatogenicity. Here I have reviewed the current findings on virus-encoded miRNAs, and their roles in viral replication. I have also examined the role of cellular miRNA in the virus replicative cycle, mechanisms of virus countradefense as well as roles of viral miRNAs in cancer development and latest achievements in antiviral therapy using miRNA analogs.  


The mi/siRNA induced RNA interference pathway

RNAi was first discovered in the nematode Caeonrhabditis elegans in 1998 [1], but previous studies in plants and fungi had exposed homology-dependent gene silencing mechanism which can be triggered by viral replication or transgene expression [2]. The RNAi is a conserved sequence-specific gene silencing mechanism present in most eukaryotes that is induced by dsRNA. This natural response was initially shown to be posttranscriptional gene silencing (PTGS) resulting in sequence-specific degradation of targeting mRNA [1]. Beside PTGS, further research revealed that RNAi pathway could cause transcriptional gene silencing (TGS) and translational inhibition [3]. Presently, it has been shown that RNAi can be triggered by two similar mechanisms:

The first one is the regulation of cellular gene expression by miRNAs (Figure 1a.). Mammalian miRNA genes are found as single or clustered transcription units [4, 5]  which are expressed either from intron regions of protein-coding or exon non protein-coding genes [6]. miRNAs originate from a wide variety of monocistronic, dicistronic or polycistronic  primary transcripts (pri-miRNAs) [7, 8] that are generated by RNA polymerase II (pol II) in all eukaryotes or by RNA polymerase III (pol III) in some viruses [9]. Within the pri-miRNA, 21~23 nt mature miRNA forms part of one arm of ~70 nt imperfect stem-loop sequence [10]. Processing of pri-miRNAs requires the recognition and nuclear cleavage of the RNA stem-loop structures by nuclear RNase III enzyme Drosha. Drosha activity is supported by Di George-syndrome critical protein 8 (DGCR 8), which directly interacts with pri-miRNAs and stabilizes the junction between the helical RNA and single-stranded RNA in the pri-miRNA hairpin [11]. Cleavage of pri-miRNAs liberates a RNA hairpin intermediate (~70nt) containing a characteristic 2 nt 3’ overhang, named a premature miRNA (pre-miRNA) [12]. The next step in miRNA biogenesis requires nuclear export of the pre-miRNA. Exportin5 (Exp5) together with the GTP-bound form of cofactor Ran, recognize and bind the 2 nt 3’overhang and carry out the export of pre-miRNA from nucleus to cytoplasm [13] [14]. Once in the cytoplasm, GTP hydrolyses and provides release of the pre-miRNAs, which become targets for a second cellular RNase III enzyme, Dicer. Dicer rapidly cleaves pre-miRNAs and yield to the generation of mature miRNAs duplexes (~21-23nt). Dicer activity is aided by transactivating (TAR) RNA binding protein (TRBP) and PACT (interferon-inducible dsRNA dependent protein kinase (PKR) activator) which are both co-factors for strand selection [15, 16]. Dicer binds to the 2nt 3’overhang at the base of the pre-miRNA hairpin and cuts the terminal loop, leaving a second 2nt 3’overhang structure in the miRNA duplex intermediate. Further, Dicer facilitates assembly of the miRNA strand of the duplex into the RNA-induced silencing complex (RISC). The strand with the lower thermodynamic stability at the 5’end of miRNA is named “guide strand” and it is incorporated in RISC while the “passenger” strand is released and degraded [17]. The composition of RISC is not fully defined, but it is known that key component presents Argonaute protein (Ago). Besides Ago enzyme, eight different enzymes (FMRP, FXR1, FXR2, Dicer, Gemin-3, Gemin-4, MOV10, TNRC6B and PRMT5) have been identified as components of RISC involved in the recognition of target mRNA by the miRNA [18, 19]. The specific steps in how these proteins interact with each other and associate in RISC complex as well as their roles in the recognition and targeting mRNA is not fully understood. The incorporated miRNA strand act as a guide to direct RISC to the complementary mRNA. The most common scenario of target recognition by a miRNA in mammalian cells involves imperfect complementarity, which leads either to TGS or degradation of the target mRNA.
The second mechanism that triggers RNAi is related to the presence of viral genomes or transponsable elements and generation of siRNAs molecules (~19-21nt) (Figure 1b.). The processing of secondary RNA structures in the viral RNA genome or dsRNA replication intermediates by RNase III enzyme Dicer generates these siRNAs. After Dicer processing, siRNAs molecules follow the same pathway as miRNAs described previously. The only difference is that upon loading into the RISC complex, siRNAs trigger cleavage of the target mRNA, since they are perfectly complementary to the target mRNA.
  
Identification and verification of virus encoded miRNAs
 
Since miRNAs have been discovered and their role in gene regulation established, it has been theorized that viruses could generate miRNAs as well and that these viral encoded miRNAs could regulate cellular mechanisms and viral replication. There are several lines of evidence to support this theory:
      
The first being that miRNAs unlike viral proteins are not antigenic. Their small size is enough to avoid INF/PKR induced pathway triggered by long dsRNAs. Secondly, miRNAs are able to down-regulate the expression of host gene products, which might interfere with stages in viral life cycle. Finally, miRNAs are able to occupy ~200 nt or ~200bp of viral genome which is significant advantage given the tight constraints on viral genome size [20]. A comparison of the crucial stages in the miRNA biogenesis and viral replication has shown that there is no unique theory for the synthesis of viral miRNA. Most of the RNA viruses replicate in the cytoplasm while the replication site for the majority of DNA viruses is nucleus [21]. Initial processing of cellular miRNA occurs in the nucleus suggesting that only viruses that replicate in nucleus are able to generate miRNA. This theory is supported by recent reports [22-24]. However, there are some indications that Pox viruses, DNA viruses that replicate in the cytoplasm are also able to synthesis miRNA, what is inconsistent with the previous theory.
   
More than 60 viral miRNAs have been identified within four different virus families. Most of the reported virus encoded miRNAs are reported from Herpesvirues and small number within Adenovirus (1), Retrovirus (1) and Polyomavirus (1) families (Table1.)
Methods for identification and purification of viral miRNA     
  
  In all reports where the presence of viral encoded miRNA is confirmed, two methods appear to be the most reliable and efficient [25].
(i)    Small RNA cloning method is based on the isolation of size-fractionated small RNAs and their cloning from virus-infected cells. Briefly, 18 to 25 nt long RNAs are first purified by excision from acrylamide gel and individual linkers are ligated to the 3’ and 5’ ends of purified RNAs. Using the linkers, the small RNAs are reverse transcribed and amplified by PCR. Presence of the restriction sites within linkers allows the cloning of the small RNAs into plasmids. Sequencing of the cloned plasmids identifies small RNAs including miRNAs in the cells [26]. 
(ii)    Computer-driven predictions are the second way to identify virally encoded miRNAs. Currently, all miRNA prediction programs scan viral genomes for the presence of stem-loop structures present in the pri- and pre- miRNAs. The advantage of this approach is that the computer program can scan a large number of sequences and identify potential candidate stretches able to form stable secondary stem-loop structures. Further, predicted miRNAs-candidates are selected and validated by Northern blotting.
The internet and several RNA companies offer a variety of different miRNA prediction software (Promega, Ambion, ViTA, siVirus) which are usually connected with virus sequence databases (PubMed). These programs are quite reliable and efficient and most importantly, the majority are free. Although initially some viral miRNA were found using these methods, a number of problems can also be encountered. Small RNA cloning is highly dependent on the abundance of miRNA in virus-infected cells and the timing and expression pattern of viral miRNAs are unpredictable. Small RNA cloning depends on stage, robustness of infection and cell type as well. In addition, computer-driven predictions are not always accurate and at times misleading.
     
Viral encoded miRNA with known functions

       The role of most reported viral miRNAs (vi-miRNA) in the viral life cycle and cellular processing are still unknown. At present only four vi-miRNAs has been reported with, more or less established roles and functions (Table 2.).

Simian polyomavirus 40 (SV40) encodes a set of miRNAs whose in vivo targets have been defined [22]. Computer-based predictions identified one vi- pre-miRNA encoded by sequences located within 3’ of the late poly (A) cleavage site in the viral mRNA. Subsequent Northern blot analyses verified the presence of ~ 60nt pre-miRNAs processed from late SV40 transcripts [22]. The SV40 miRNAs were excised from both arms of pre-miRNAs and both found to be perfectly complementary to early SV40 transcripts, which encode the large “T” and small “t” SV40 tumor antigens. This finding was unusual because it is predicted to be energetically favorable for only one pre-miRNA strand to enter the RISC [17]. Further experiments confirmed that target mRNA (large “T” and small “t” early transcripts) were cleaved in cells infected with the wild type SV40 (wtSV40). Cells infected with mutant SV40, which contained mutations to disrupt the predicted pre-miRNA hairpin structure did not produce the cleaved early mRNA fragments and contained increased amounts of early viral mRNA transcripts. These observations insinuate that SV40 miRNAs have an autoregulatory function that regulates the amount of early viral transcripts. Interestingly, downregulation of the large “T” and small “t” antigens did not affect the final viral yield in infected cells. Cells infected with wtSV40 showed a lower susceptibility to lysis by T-antigen specific cytotoxic T lymphocytes and released less interferon-γ than cells infected with the mutant SV40 [22]. This indicates that vi-miRNAs induced downregulation of SV40 tumor antigens resulting in the reduced susceptibility to the activation of responsive cytotoxic T lymphocites [22].

Epstein-Barr virus (EBV) – Eighteen potential miRNAs were detected in small RNA cloned from a B cell line latently infected with EBV.  [25, 27]. The function of the EBV miRNAs has not been established, but the use of algorithms to predict possible targets of EBV miRNAs revealed cellular genes that are involved in cell proliferation and apoptosis as well as genes that encode B-cell specific chemokines and cytokines. In additon, one of the EBV miRNAs apparently has a viral target. miR-BART2 is antisense to a region in a lytic mRNA, BALF5 (BamHI L fragment 5) which encodes the EBV polymerase. Since it is fully complementary to that mRNA, it would be predicted to cleave the transcript. This hypothesis is supported by previous studies that detected a shortened version of BALF5 mRNA that showed a relationship in size with the predicted miR-BART2 mediated cleavage product [28]. At this point, what is unclear is the role of this cleavage. A reasonable explanation could be use of cleaved BART2 mRNA  to downregulate the expression of the EBV DNA polymerase, suggesting the possibility that viruses not only use miRNAs to downregulate host genes but also as autoregulatory tool.

Human Immunodeficiency virus (HIV-1)  - Although early attempts to clone virally encoded miRNA from cells infected with HIV-1 failed after the screening of more than 1500 clones [29], Omoto and colleagues, reported evidence for a miRNA within nef region of HIV-1 genome and proposed a role for it in the downregulation of viral transcription [23]. In addition, it was recently reported that HIV-1 encodes a siRNA precursor derived from hairpin structures composed of a 19 bp perfectly complementary stem and small loop [30]. Dicer processes this structure into functional siRNAs that target the HIV-1 genome. HIV nef derived miRNA (miR-N367) downregulates nef expression and suppress transcription by reducing HIV-promoter activity, which helps in maintaining low viremia in long-term non progressors. However, further studies will be needed to resolve the following questions (i) why these HIV siRNAs have not been cloned in previous studies and (ii) whether or not these hairpins occur endogenously during the natural infection. At present, it is unclear whether experimental designs influenced the outcomes of previous studies [23, 29, 30]

Herpes simplex virus-1 (HSV-1 ) - The most recently discovered miRNA with a known function is encoded by HSV-1 latency associated transcript (LAT) gene. miR-LAT targets genes of apoptotic pathway including those involved in TGF- pathway, thus protecting the cells from apoptosis [24] (see vi-miRNA and apoptosis).
Cellular miRNA with a viral function

       It is well established that cellular miRNAs play important roles in the regulation of cellular genes. Recent evidence indicates that cellular miRNAs can also target the genetic material of invading viruses. A perfect example comes from analysis of the replication of the retrovirus primate foamy virus type 1 (PFV-1) and hepatits C virus (HCV) [31, 32].
 
       mir-32 and PFV-1
       A comparison of PFV-1 genome and human miRNAs revealed several host cell miRNAs that could potentially shut down gene expression and viral replication of this virus [33]. Only cellular miR-32 showed complementarity to the 3’ UTR shared by five mRNA of PFV-1 indicating ability to down-regulate virus replication (Figure 2.). Mammalian miR-32 binds to ORF 2 of PFV-1 and restricts the viral RNA abundance in cultured human embryonic kidney cells (293T). When this miR-32 is knocked out, the virus doubles its replication rate [31]. Since the miR-32 is highly conserved in all vertebrates, its antiviral activity against PFV-1 is not due to evolutionary selection of miR-32 which confers an antiviral phenotype [33]. The target sequence for miR-32 is not highly conserved within other primate foamy viruses and it does not exist in non-primate foamy viruses. This is the reason for unrestricted replication of other primate/non-primate foamy viruses in different hosts.

      miR-122 and hepatitis C virus (HCV)
       A different mode of viral genome regulation by cellular miRNA occurs in HCV. Cellular miRNAs usually bind to the 3’UTR of mRNA, thereby repressing mRNA translation. Examination of the HCV RNA sequence led to the identification of two potential binding sites for liver specific miR-122; one in the 3’UTR and one in the 5’UTR. A study by [32] showed that miR-122 upregulates the expression of the HCV in cultured liver cells Huh7, by binding to 5’UTR of HCV genome. The evidence for this comes from the observation that sequestration of miR-122 by methylated oligonucleotides result in reduction of HCV RNA [32] (Figure 3.). In addition, mutation of the 3’UTR did not have an effect on virus replication whereas point mutation in the 5’UTR abolished viral RNA accumulation. The level of HCV replication was re-established by ectopic expression of miR-122 molecules that contained mutation to restore predicted base complementarity. Experiments with replication-deficient genomes of HCV indicated that miR-122-HCV interactions affected viral replication but not the mRNA translation [32].

Overall, these findings provide an illustration of how cellular miRNAs can be used in virus life cycle, either to downregulate or upregulate viral gene expression. Even though these results have enormous scientific weight, some questions remain. For instance, what pathway or RNAi mechanism does PFV-1 employ, to evade the cellular defense mechanism (see Viral vs Cellular Defense). In addition, what are the natural targets for cellular miR-122 in the liver and does the high concentration of virus in hepatic cells suppress miR-122 targeting?

Viral vs cellular defense  

From the aspect of cell virus interaction it is worth while to refresh our knowledge about the important mechanisms involved in cellular defense against viruses as well as the virus countradefense. 
        
Reflecting the intimate relationship between viruses and their host, infected cells have several signaling mechanisms to sense and respond to virus infection. These mechanisms involve cross-talk between different cellular pathways to modulate the expression and antiviral function of interferons (IFNs) and specific gene products. IFNs – an cytokines that are important regulators of innate and adaptive immune responses. Besides their antiviral role, which is well established, they are potent regulators of cell growth and have immunomodulatory activity. INFs mediate their effects through interactions with type specific receptors that are different and not redundant for the INF type I and INF type II (Figure 4.). Virus infection triggers the activation of INFs, probably through production of viral dsRNA and other virus specific signals. The most intensely studied pathway for INF production is the dsRNA-activated serine/threonine protein kinase (PKR). Activation of PKR occurs by the presence of cytoplasmic dsRNA, and leads to the rapid phosphorylation of eukaryotic initiation factor eIF2 and subsequent inhibition of both host and viral mRNA [34].
        
The discovery that the expression of small viral mi/siRNAs does not induce the mammalian interferon machinery, raises a number of questions (i) Can mammalian viruses be a target for cellular miRNAs in RNAi machinery and (ii) What mechanisms mammalian viruses employ to avoid and suppress such a sophisticated defense mechanism. A partial answer to the first question has been given through the example of miR-32 and PFV-1 virus, as well recent computational analysis conducted by Brahmachari and colleagues [35] who discovered two human miRNAs (miR-507 and miR-136) with potential binding sites in  polymerase B2 (PB2) and hemagglutinin (HA) genes of influenza H5N1 virus. These two influenza genes encode proteins crucial for viral pathogenicity and replication. Interestingly, miR-507 and mi-136 were not found in the chicken genome implicating the difference in infectivity and lethality of the H5N1 virus in chicken and humans. In an attempt to answer the second question, one should look into different virus families and their mechanisms to suppress RNAi. Currently, it has been reported that five mammalian viruses have mechanisms that suppress RNAi and cellular defenses (INF/PKR) in mammalian cells.
       
Adenoviruses use two countradefense mechanisms to shut down cellular innate immunity. VA1 is adenovirus encoded ~160nt non-coding RNA that is expressed at high levels during the late stage of lytic replication [36]. VA1 is transcribed by RNA polymerase III (polIII) in the nucleus and transported to cytoplasm by Exp5 and Ran. In the cytoplasm, VA1 binds to PKR preventing its activation and phosphorylation of eIF2, which results in no INF synthesis (Figure4. B). Another scenario is: VA1 like miRNAs is transported by Exp5 from nucleus and it shares the same sequence recognized by Exp5. This sequence is located in the 3’ overhang at the short RNA stem and it is recognized by Dicer-TRBP complex [37, 38] suggesting that VA1 act as a repressor of RNA silencing and cellular miRNA processing [39]. Dicer processes only 1% of VA1 into viral miRNAs but this percentage could give rise to up 10 VA1 encoded miRNAs with a possible role in the viral life cycle in virus-infected cells [40]  (Figure 5/1).
         
Recent in vitro studies on HIV-1 have shown that HIV-1 uses two pathways to repress RNA silencing. One pathway involve transactivation responsive RNA (TAR), a 59 nt secondary stem-loop structure located at the 5’end of all HIV-1 transcripts, which is essential for efficient viral replication. Because TAR is a high-affinity ligand for TRBP, it competes with Dicer for TRBP preventing TRBP incorporation in RISC. Elimination of TRBP results in destabilization of Dicer and loss in miRNA biogenesis [15] (Figure5/2a). Another pathway utilizes the HIV-1 protein Tat. Tat is a regulatory protein that binds viral RNA to facilitate initiation and elongation of viral transcripts. In the study conducted by Bennasser and colleagues it is shown that, Tat protein partially repress the processing activity of Dicer [30, 41] (Figure5/2b). Since TAR posses stem-loop structure which can be processed by Dicer into miRNA most likely HIV-1 use both mechanisms (Tat along with TAR) to increase suppression activity and provide safe virus assembling.
         
PFV-1 also employs the viral encoded protein as a suppressor of RNAi as mentioned earlier in chapter 4. miR-32 down-regulates virus gene expression and in order to counteract this RNAi pathway, PFV-1 uses Tas protein synthesized early in infection. Lecellier and their group reported that ectopic expression of Tas suppress miR-32 activity [31] (Figure 5/3).
        
Influenza NS 1 and vaccinia virus E3L proteins are known PKR inhibitors, but recent findings revealed that they are also potent inhibitors of  the host RNAi pathway [42] (Figure4.B). 
         
 
Virus encoded miRNA & apoptosis /latency

The latency-associated transcript (LAT) of the HSV-1 is involved in maintenance of the latent infection in neurons [43]. However, no protein product has been identified within the LAT gene and therefore the mechanism by which the LAT confers resistance to apoptosis was unknown until recently. Gupta et al. 2006 [24] reported that LAT codes for miRNA, named mir-LAT that is responsible for the observed antiapoptotic activity of LAT in infected cells. Deletion of miR-LAT resulted in more extensive apoptosis in infected cells upon stress. miR-LAT itself was able to suppress apoptosis significantly in stressed cells. In order to gain an insight into the mechanism how miR-LAT is able to suppress apoptosis, the authors searched the human genome for possible target genes by computational analysis. Using miRANDA computer prediction program, they identified TGF-ß and SMAD3 as a potential targets. Moreover, it is known that TGF-ß is a potent inhibitor of cell growth and inducer of apoptosis [44]. In further investigation into the correlation of miR-LAT, SMAD3 and TGF-ß, Gupta and colleagues observed that mRNA levels as well as the protein levels of these two genes were lower in LAT expressing cells. Furthermore, the activity of luciferase reporter gene of either TGF-ß or SMAD3 was significantly reduced upon cotransfection with LAT or a double stranded RNA oligonucleotide encoding miR-LAT itself. These findings lead to the conclusion that miR-LAT miRNA helps the virus to establish latency by allowing avoidance of TGF-B induced apoptosis following infection [24].

Another interesting study was published by Bagsara and colleagues [45] suggesting the involment of cellular miRNA in lentiviral latency. They hypothesized that in the case of lentivirus infection, cellular miRNAs are involved in the inhibition of these retroviruses by the formation of the intramolecular triplex formation between the polypurine track sequences in the viral genome and miRNAs. In addition, these triplex formations should block the viral replication at the preintegration stage, placing the viruses into a suspended latency. Using several latently and chronically infected cell lines and human PBMCs from HIV-1 infected individuals, they confirmed the presence of triplexes in lentivirus-infected cells. In addition, the number of triplexes decreased in cells with productive replication of lentivirus [45]. This correlation was further confirmed by stimulation of PBMCs and lentivirus-infected cell line with the appropriate mitogenes [45]. However, additional research has to be done to answer what cellular miRNAs are involved in triplex formation; could triplexes be formed in vivo in CD4+ T cells, and finally, what molecular interactions initiate/trigger their formation?
Viral miRNA and cancer

Cancer development has long been an enigma for researchers. Today we know that viruses represent one of the main factors that cause uncontrolled cell proliferation leading to the malignant transformation. The discovery that viruses are able to produce high level of viral miRNAs, currently with no determined specific roles in viral life cycle, indicated that vi-miRNAs might be involved in cancer development. Reports on precise roles of number identified vi-miRNAs have not been published as of yet. However, according to the current observations, we can hypothesize what roles vir-miRNAs might have in cancer development.
     (i) One possibility could be direct oncogene function. Vi-miRNAs could directly stimulate oncogenesis by inhibition of the major cellular tumor suppressor p53. Computational analysis, suggests that p53 could be a possible target for EBV encoded miR-BHRF1 [46]. Another EBV encoded miRNAs, miR-BART5-12, found in EBV associated gastric carcinoma cells but not in the latently infected B cell lymphomas, suggested that BART miRNAs may play important roles in EBV induced epithelia cell transformation but not in B-cell transformation [47]
     (ii) A second scenario might involve viral miRNAs in evasion of innate and adaptive immune responses during viral infection, which can result in chronic or latent infection, an environment conducive for cancer development. This scenario appears to be the most likely; there are examples of SV40 and MHV68, which use vi-miRNAs to evade cellular immune respond [22, 48].
    (iii) The third theory could suggest that viral miRNAs are involved in oncogenesis through the facilitation of tumor progression in tissues that are already prone to develop cancer in the absence of an infection. This hypothesis is based on research which has observed that the expression of the mir17-92 significantly accelerated lymphoma development in the mouse B-cell lymphoma model [49, 50]. There are some indications that some EBV-derived miRNAs could act in a similar manner to miR17-92 based on studies conducted by Taub et al.1982.
     (iv) Finally, it is possible that viral miRNAs can be expressed abundantly in infected cells resulting in alteration of miRNA processing, export or RISC function leading to oncogenesis. (example of adenovirus VA1). In addition, cellular miRNAs found in cancer tissue could be involved in maintenance of chronic viral infection and play role in viral affinity for specific organs (HCV and miR-122).
  
miRNA in therapy 
      
Since the discovery that miRNAs can silence gene expression in mammalian cells, their potential role as a therapeutic tool has been considered. Currently, application of miRNAs based therapy is focused on genetic inflammatory disorders, certain cancers as well as viral diseases. The advantage of this approach is that miRNAs have the potential to be more selective, more effective and less toxic than current drugs. The concept of miRNA therapy is based on computational prediction and design of artificial short hairpin RNAs molecules with a sequence complementarity to the target mRNA. These synthetic miRNAs are subsequently processed into active si/miRNAs following RNAi pathway in targeted cells. Administration of artificially miRNAs can be via different routs, such as direct injection, intravenous, intraperitoneal or by expression from polII/polIII or viral vectors. Recent reports showed that artificially designed miRNAs expressed from U6 or lentivirus vectors are able to induce RNAi and shutdown viral replication in vitro by targeting highly conserved regions or vital genes required for viral replication. (Table 3.)
      
However, despite some encouraging results, the miRNA-based therapy has some pitfalls. One of the drawbacks of synthetic miRNAs is transient nature of the inhibition in mammalian cells. Their activity is mainly dependent on the rate of cell growth and the turnover of targeted proteins (3 to 6 days). Perhaps a more serious problem is the unanticipated off-target effects that occur by synthetic miRNAs recognition of other mRNAs bearing partial homology. As well, considering the size of the human/animal genome and the number of known genes, it is difficult to imagine that artificially design miRNAs will be able of targeting a single gene of interest in vivo model. Hopefully ongoing and future preclinical studies in animal models will facilitate the development of highly efficient RNAi based therapies with minimal side effects.
References

1.    Montgomery MK, F.A., Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression. Trends Genet, 1998. 14(7): p. 255-8.
2.    Baulcombe, D., RNA silencing in plants. Nature, 2004. 431(7006): p. 356-63.
3.    Meister G, T.T., Mechanisms of gene silencing by double-stranded RNA. Nature., 2004 431(7006): p. 343-9.
4.    Lau, N.C., et al., An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science, 2001. 294(5543): p. 858-62.
5.    Lagos-Quintana M, R.R., Lendeckel W, Tuschl T. , Identification of novel genes coding for small expressed RNAs. Science 2001. 294(5543): p. 797-9.
6.    Rodriguez, A., et al., Identification of mammalian microRNA host genes and transcription units. Genome Res, 2004. 14(10A): p. 1902-10.
7.    Cai, X., C.H. Hagedorn, and B.R. Cullen, Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. Rna, 2004. 10(12): p. 1957-66.
8.    Lee Y, K.M., Han J, Yeom KH, Lee S, Baek SH, Kim VN. , MicroRNA genes are transcribed by RNA polymerase II.
. EMBO J. .
, 2004. 23(20): p. 4051-60.
9.    Aparicio, O., et al., Adenovirus virus-associated RNA is processed to functional interfering RNAs involved in virus production. J Virol, 2006. 80(3): p. 1376-84.
10.    Li, H.W. and S.W. Ding, Antiviral silencing in animals. FEBS Lett, 2005. 579(26): p. 5965-73.
11.    Han, J., et al., Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell, 2006. 125(5): p. 887-901.
12.    Lee, Y., et al., The nuclear RNase III Drosha initiates microRNA processing. Nature, 2003. 425(6956): p. 415-9.
13.    Yi, R., et al., Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev, 2003. 17(24): p. 3011-6.
14.    Lund, E., et al., Nuclear export of microRNA precursors. Science, 2004. 303(5654): p. 95-8.
15.    Chendrimada, T.P., et al., TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature, 2005. 436(7051): p. 740-4.
16.    Lee, Y., et al., The role of PACT in the RNA silencing pathway. Embo J, 2006. 25(3): p. 522-32.
17.    Maniataki, E. and Z. Mourelatos, A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev, 2005. 19(24): p. 2979-90.
18.    Mourelatos, Z., et al., miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev, 2002. 16(6): p. 720-8.
19.    Sasaki, T., et al., Identification of eight members of the Argonaute family in the human genome small star, filled. Genomics, 2003. 82(3): p. 323-30.
20.    Cullen, B.R., Transcription and processing of human microRNA precursors. Mol Cell, 2004. 16(6): p. 861-5.
21.    Harper, R.D., Virus structure and replication, in Molecular Virology
R.D. Harper, Editor. 1999Molecular Virology
BIOS Scientific Group: London. p. 25-29.
22.    Sullivan, C.S., et al., SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature, 2005. 435(7042): p. 682-6.
23.    Omoto, S., et al., HIV-1 nef suppression by virally encoded microRNA. Retrovirology, 2004. 1(1): p. 44.
24.    Gupta, A., et al., Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript. Nature, 2006. 442(7098): p. 82-5.
25.    Pfeffer, S., et al., Identification of virus-encoded microRNAs. Science, 2004. 304(5671): p. 734-6.
26.    Carmichael, G., RNA silencing : methods and protocols 2005: Totowa, N.J. : Humana Press, c2005.
27.    Cai, X., et al., Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS Pathog, 2006. 2(3): p. e23.
28.    Furnari, F.B., M.D. Adams, and J.S. Pagano, Unconventional processing of the 3' termini of the Epstein-Barr virus DNA polymerase mRNA. Proc Natl Acad Sci U S A, 1993. 90(2): p. 378-82.
29.    Pfeffer, S., et al., Identification of microRNAs of the herpesvirus family. Nat Methods, 2005. 2(4): p. 269-76.
30.    Bennasser, Y., et al., Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing. Immunity, 2005. 22(5): p. 607-19.
31.    Lecellier, C.H., et al., A cellular microRNA mediates antiviral defense in human cells. Science, 2005. 308(5721): p. 557-60.
32.    Jopling, C.L., et al., Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science, 2005. 309(5740): p. 1577-81.
33.    Linial, M.L., Foamy viruses are unconventional retroviruses. J Virol, 1999. 73(3): p. 1747-55.
34.    Kumar, K.U., S.P. Srivastava, and R.J. Kaufman, Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18. Mol Cell Biol, 1999. 19(2): p. 1116-25.
35.    Scaria, V., et al., Host-virus interaction: a new role for microRNAs. Retrovirology, 2006. 3: p. 68.
36.    Mathews, M.B. and T. Shenk, Adenovirus virus-associated RNA and translation control. J Virol, 1991. 65(11): p. 5657-62.
37.    Gwizdek, C., et al., Terminal minihelix, a novel RNA motif that directs polymerase III transcripts to the cell cytoplasm. Terminal minihelix and RNA export. J Biol Chem, 2001. 276(28): p. 25910-8.
38.    Zeng, Y. and B.R. Cullen, Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. Nucleic Acids Res, 2004. 32(16): p. 4776-85.
39.    Andersson, M.G., et al., Suppression of RNA interference by adenovirus virus-associated RNA. J Virol, 2005. 79(15): p. 9556-65.
40.    Sano, M., Y. Kato, and K. Taira, Sequence-specific interference by small RNAs derived from adenovirus VAI RNA. FEBS Lett, 2006. 580(6): p. 1553-64.
41.    Yeung, M.L., et al., Changes in microRNA expression profiles in HIV-1-transfected human cells. Retrovirology, 2005. 2: p. 81.
42.    Geiss, G.K., et al., Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. Proc Natl Acad Sci U S A, 2002. 99(16): p. 10736-41.
43.    Perng, G.C., et al., Virus-induced neuronal apoptosis blocked by the herpes simplex virus latency-associated transcript. Science, 2000. 287(5457): p. 1500-3.
44.    Schuster, N. and K. Krieglstein, Mechanisms of TGF-beta-mediated apoptosis. Cell Tissue Res, 2002. 307(1): p. 1-14.
45.    Bagasra, O., et al., Role of micro-RNAs in regulation of lentiviral latency and persistence. Appl Immunohistochem Mol Morphol, 2006. 14(3): p. 276-90.
46.    Soussi, T. and G. Lozano, p53 mutation heterogeneity in cancer. Biochem Biophys Res Commun, 2005. 331(3): p. 834-42.
47.    Kim do, N., et al., Expression of viral microRNAs in Epstein-Barr virus-associated gastric carcinoma. J Virol, 2007. 81(2): p. 1033-6.
48.    Bowden, R.J., et al., Murine gammaherpesvirus 68 encodes tRNA-like sequences which are expressed during latency. J Gen Virol, 1997. 78 ( Pt 7): p. 1675-87.
49.    He, H., et al., The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci U S A, 2005. 102(52): p. 19075-80.
50.    He, L., et al., A microRNA polycistron as a potential human oncogene. Nature, 2005. 435(7043): p. 828-33.
51.    Yeung, M.L., et al., siRNA, miRNA and HIV: promises and challenges. Cell Res, 2005. 15(11-12): p. 935-46.
52.    Sarnow, P., et al., MicroRNAs: expression, avoidance and subversion by vertebrate viruses. Nat Rev Microbiol, 2006. 4(9): p. 651-9.
53.    Grundhoff, A., C.S. Sullivan, and D. Ganem, A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses. Rna, 2006. 12(5): p. 733-50.
54.    Cai, X., et al., Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells. Proc Natl Acad Sci U S A, 2005. 102(15): p. 5570-5.
55.    Dunn, W., et al., Human cytomegalovirus expresses novel microRNAs during productive viral infection. Cell Microbiol, 2005. 7(11): p. 1684-95.
56.    Grey, F., et al., Identification and characterization of human cytomegalovirus-encoded microRNAs. J Virol, 2005. 79(18): p. 12095-9.
57.    Cui, C., et al., Prediction and identification of herpes simplex virus 1-encoded microRNAs. J Virol, 2006. 80(11): p. 5499-508.
58.    McCaffrey, A.P., et al., Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol, 2003. 21(6): p. 639-44.
59.    Cheng, T.L., et al., Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference. Biochem Biophys Res Commun, 2005. 336(3): p. 820-30.
60.    McCaffrey, A.P., et al., RNA interference in adult mice. Nature, 2002. 418(6893): p. 38-9.
61.    Korf, M., et al., Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors. J Hepatol, 2005. 43(2): p. 225-34.
62.    Song, E., et al., Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat Biotechnol, 2005. 23(6): p. 709-17.
63.    Park, W.S., et al., Specific HIV-1 env gene silencing by small interfering RNAs in human peripheral blood mononuclear cells. Gene Ther, 2003. 10(24): p. 2046-50.
64.    Novina, C.D., et al., siRNA-directed inhibition of HIV-1 infection. Nat Med, 2002. 8(7): p. 681-6.
65.    Palliser, D., et al., An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection. Nature, 2006. 439(7072): p. 89-94.
66.    Zhang, W., et al., Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nat Med, 2005. 11(1): p. 56-62.
67.    Yoshinouchi, M., et al., In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by E6 siRNA. Mol Ther, 2003. 8(5): p. 762-8.
68.    Jiang, M. and J. Milner, Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene, 2002. 21(39): p. 6041-8.
Figures


Figure 1. mi/siRNA induced RNA interference


A comparison of the miRNA and siRNA pathways. 1a) miRNAs are deriverd from highly-structured precursors pri-miRNAs transcribed by RNA Pol II/Pol III in the nucleus. Drosha and DGCR 8 processed pri-miRNAs into ~ 70nt hairpin structure with 2nt at the 3’ overhang (pre-miRNAs). pre-miRNAs are exported by Exp5 into the cytoplasm where are further processed by RNase III Dicer. Dicer trimmed loop structrures of pre-miRNAs leaving mature ~21nt long miRNAs . One strand of miRNAs is incorporated into RISC complex and acts as a guide for RNA silencing. The remaining passenger strand is degradated.  1b) siRNAs are derived from viral or artificial dsRNAs and share the common processing pathway which also involves Dicer/TRBP and RISC complex. mRNAs with perfect complementary to the si RNAs are targeted to the degradation while transcripts captured by miRNAs with incomplete complementary undergo translation repression. Figure created by using references [51, 52]


Figure 2. miR-32 and PFV-1

Binding  to the ORF2 of PFV-1, miR-32 restricts viral RNA synthesis and accumulation
         

Figure 3. miR-122 and HCV

Upregulation of HCV replication induced by binding of miR-122 to 5’UTR of HCV genome


Figure 4.  INF/PKR pathway and inhibition

A. viral dsRNA activate PKR and phosphorilation of eIF2α which results in translation inhibition and INF synthesis. B. Influenza NS1, Vaccinia E3L, and Adenovirus VA1 bind to PKR and block phosphorilation of eIF2α, which results in blocking INF synthesis and translation inhibition.

Figure 5. Viral mechanisms to suppress RNAi

5/1- Adenovirus VA1 is exported from nucleus by Exp5 to the cytoplasm where it binds to Dicer/TRBP and suppresses RNA silencing and cellular processing. 5/2a- HIV-1 Tar sequence is abundantly expressed; it competes with Dicer for TRBP and suppress RNAi. 5/2b HIV-TAT protein binds to Dicer and blocks its incorporation into RISC complex. 5/3-PFV-1 TAT protein binds to RISC complex with incorporated miR-32 and suppresses miR-32 activity

Figure 6. Overview