**9. miRNA biogenesis**

directly binds to dsRNA to inhibit DCR2 activity, to inert siRNA pathway [52]. In another study, researchers have found that plasmid-expressed La Crosse virus (LACV) NSs protein successfully inhibits interferon (IFN) and RNAi pathway in mammalian cells and mice, while fails to show any RNAi-suppressive effect in transfected U4.4 cells and C6/36 cells infected with LACV [53]. A recent publication has revealed that DENV NS4b functions as an RNAi suppressor in human Huh-7 cells via inhibition of dsRNA processing by Dicer [8]. However, whether NS4b behaves similarly in mosquitoes has not been investigated, and to date, no RNAi suppressor activity has been described for an arbovirus protein during mosquito infection. Given that DENV successfully develops persistent infection in mosquitoes despite their RNAi response, the precise mechanism of RNAi evasion is definitely a significant point of interest.

RNA activation (RNAa) is one of the nascent phenomena around the scientific field. RNAa is considered to be a potent emerging therapeutic strategy for the disease that can be cured by particular gene activation [54]. However, most of the studies relating to RNAa research have been carried out in human cell line. Till date, proper mechanism of RNAa is still not clear. However, it has been demonstrated that it is mostly a nuclear event that leads to chromatin restructuring [17]. RNA activation is still in its infancy, so its exact mechanism is still elusive. Recently, Portnoy et al. has proposed a model for the promoter-directed-small-activating-RNA (saRNAs)-mediated RNA activation. In this model, authors proposed that an exogenously introduced saRNA or endogenously produced small noncoding RNA, (e.g., miRNA) is loaded into an Ago protein in the cytoplasm. The Ago protein processes the saRNA by discarding its passenger strand to form an active Ago-RNA complex. Ago-RNA complex enters the nucleus through active transport/passive diffusion cell division. Guide strand directs Ago-RNA complex to complementary genomic DNA sequences usually in promoter region or to noncoding RNA (ncRNA) sequences, which tethers to the DNA. After that, the Ago-RNA complex initiates a process that differs from RNAi, which alter chromatin structure and epigenetic states of target gene via two different potential mechanistic models. In model A, the saRNA guide strand leads the Ago protein to its DNA target by constituting an RNA–DNA duplex or triplex structure, while Ago protein then serves as a docking platform to attract histone-modifying activities, like opening of chromatin structure and active transcription. On the other hand, in model B, the RNA guide strand binds to cognate promoter transcripts making saRNA-RNA complex and then Ago protein recruits histone modifiers to introduce active

chromatin marks on local chromosome, resulting in activation of transcription [17].

As the molecular biology, bioinformatics approaches and protocols of small RNA sequencing continue to grow and sophisticate, so is the number of miRNAs discovered in different species. Virus-encoded miRNAs have been known to be crucial for the viral replication. miRNAs encoded by DNA viruses have been well studied in comparison with those by RNA viruses because RNA

**7. RNA activation (RNAa)**

98 Current Topics in Tropical Emerging Diseases and Travel Medicine

**8. MicroRNAs (miRNAs)**

### **9.1. miRNAs genomic arrangement and transcription**

miRNAs are usually encoded by different regions of genome ranging from coding as well as noncoding [64]. Seventy percent of mammalian miRNAs are the intron products of protein coding genes, while rest comes from the noncoding transcription units. miRNAs have been found to be originated from both senses and antisense strands of DNA [65]. Studies have shown that about 30% of *C. elegans* miRNA genes are on the antisense strand overlapping protein coding region [66]. In Drosophila too, many miRNAs are originated from antisense strand overlapping protein coding region [67]. Multiple miRNAs can be transcribed as one long transcript named clusters [18].

Majority of miRNAs are transcribed by RNA polymerase II; however, some miRNAs usually located near Alu repeats are transcribed by RNA polymerase III, from independent genes having their own promoter or represent introns of protein-coding genes [18]. Nearly, all the independent miRNA promoters have particular features of Pol II promoters, including initiator elements and TATA boxes [18].

#### *9.1.1. Canonical pathway of miRNA biogenesis*

So far, a vast majority of reported miRNAs are produced through an RNase III enzyme-controlled canonical pathway. Transcription of primary transcript (i.e., pri-miRNA) harboring one or several stem loop structures by RNA polymerase II or otherwise RNA polymerase III kick start the biogenesis process in nucleus [68, 69]. pri-miRNAs are then further processed at stem loop sites by two RNase III proteins Drosha and DGCR8/Pasha (DiGeorge critical region 8) to produce about 70 nucleotide pre-miRNA [70]. This pre-miRNA is then exported to cytoplasm with the help of exportin-5 and RanGTP proteins [71]. Once in cytoplasm, pre-miRNA is chopped down into 22 nucleotide miRNA duplexes. Apart from above-mentioned proteins, many other proteins also work as cofactors to influence the outcome of miRNA biogenesis [65, 72].

fate [82]. The stability of the base pairs at the ends of the strands of miRNA duplex usually determines which strand will be degraded. Usually, less stable strand avoids degradation [82]. There are different modes of action of miRNAs that influence the expression of target gene ranging from mRNA degradation, translational repression to activation of transcription [16, 83, 84]. Whatever the outcome is, the general way of miRNA-mRNA interaction relies on the sufficient sequence complementarity between mRNA and 5′ seed region (usually 2–8 nucleotides) of miRNA [85]. Perfect complementarity between mRNA and miRNA is likely to downregulate the expression of target gene through its mRNA degradation by endonuclease activity of miRISC complex. This type of miRNA-mediated gene regulation is mostly evident in plants [86]. However, in animals, there is mostly imperfect complementarity between mRNA and miRNA, which usually leads to translational repression of target gene [86]. Interestingly, there are solid experimental evidences that miRNAs are also involved in upregulation of target genes as well. One of its well-characterized examples is host miR-122-mediated upregulation

RNA Association, RNA Interference, and microRNA Pathways in Dengue Fever Virus-Host…

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of HCV RNA replication by stabilizing HCV RNA and preventing its decay [87].

As most viruses need host genes to facilitate their replication, one cannot rule out the possibility of viruses encoding miRNAs in order to manipulate host gene expression or keep viral copy number under check to ensure persistent infection. Possible benefits of virus-encoded miRNAs are that they usually do not elicit immune response, require less coding ability, and they have ability to constantly evolve to target new transcripts. Till date, more than 200 viral miRNAs have been identified. Most of the well-characterized viral-encoded miRNA comes from DNA viruses as compared to RNA viruses, with herpes viruses as a major contributor having average copy number of more than 10/genome [58]. Different recent studies have identified novel microRNAs from different RNA viruses. Bovine leukemia virus (BLV), a retrovirus, also encodes microRNAs [57]. Hussain et al. has reported microRNA like small RNAs in both West Nile virus and Dengue virus [88]. These viral-encoded miRNAs are produced through canonical as well as noncanonical biogenesis pathways. Based on their function, viral

Important functions of viral miRNAs, which target host genes, include promoting cell survival through downregulation of apoptotic factors, thus promoting infected cells survival and proliferation, as well as modulating the immune response of the host cell [89]. It is becoming increasingly evident that one mechanism the viruses have evolved to facilitate regulatory control over their hosts is by generating transcripts, which outmatch cellular miR-NAs. Interestingly, Human Kaposi's sarcoma-associated herpes virus (KSHV) and chicken oncogenic Marek's disease virus (MDV) transcribe miR-K12-11 and miR-M4, respectively; these miRNAs appear to be an ortholog of miR-155 to identical seed region [90, 91]. miR-155 has been reported to be involved in many malignancies [92], so exploitation of its targets by

**11. Virus-encoded miRNAs**

encoded miRNAs can be classified into two broad classes:

**ii.** Virus-encoded miRNAs regulating virus copy number

**i.** Virus-encoded miRNA regulating host genes

#### *9.1.2. Noncanonical pathway of miRNA biogenesis*

Although, it was thought earlier that there is only one universal mechanism of all mature miRNAs biogenesis; however, multiple recent discoveries have led us to the conclusion that there may be several other miRNA biogenesis pathways as well. Biogenesis of quite a few miRNAs has been demonstrated to be not only Drosha independent, but also Dicer dependent [73]. It has been reported that in Drosophila and *C. elegans* along with some other vertebrates, mature miRNAs called "mirtrons" can be produced after splicing from intron hairpins independently of Drosha processing [74–76]. Recent studies have revealed that mature functional miRNAs can arise from highly conserved small nucleolar RNA (snoRNAs) in human and *Giardia lamblia* [77, 78] as well as DicerII-processed endogenous siRNAs mostly derived from transposable elements and load into AgoI in Drosophila [76]. Furthermore, researchers have identified miRNAs derived from transfer RNA (tRNA) in mouse embryonic stem cells [79].

Recent studies have led to the discovery of other types of miRNA noncanonical biogenesis pathway that is Dicer independent. One prime example of this is miR-451 biogenesis, which is Dicer independent as pre miR-451 has ~18 nucleotide stem duplex that is too short for Dicer activity. However, pre miR-451 requires AgoII for its maturation [80]. A few examples of other types of noncanonical biogenesis come from virus-encoded miRNAs. For example, several miRNAs are produced by a retrovirus, Bovine leukemia virus (BLV), based on DNA polymerase III transcription *in vivo* as well as *in vitro* [57]. The products of these transcripts were too small to be recognized by Drosha; therefore, these were directly processed by Dicer-1 to mature miRNAs.
