Functions of DENV genome structural and nonstructural proteins.
Dengue fever is a fatal vector transmitted disease and is one of the most significant health problems which have magnified its impact globally by afflicting 390 million people across 110 countries. The causative agent of this life-threatening disease is a positive single-stranded RNA arbovirus known as dengue virus (DENV), which uses Aedes aegypti mosquito as an intermediate host. It has been well demonstrated that virus evades mosquito’s RNA interference (RNAi)-mediated antiviral defense and manipulates host microRNA (miRNA) profile to its own benefit. However, the exact mechanisms are still not exclusively elucidated. The molecular mechanisms which characterize the role of novel DENV-encoded small RNAs and other viral proteins in host miRNA modulation and evasion of RNA interference are still elusive. Furthermore, the possibility of small activating RNAs-(RNAa)-mediated activation in mosquitoes in conjunction with dengue virus genes is not fully explored. This book chapter pragmatically overviews intricate interplay between virus-host interactions, how virus invades host antivirus defense mechanisms, and possibly the potential emerging therapeutic role of RNA activation (RNAa) and RNAi for the infections, which can be cured by specific gene activation and gene silencing, respectively.
- dengue fever
- dengue fever virus
- Aedes aegypti
- RNA interference
- RNA association
- host-virus interaction
- DENV therapy
Dengue virus (DENV) is a major arbovirus that uses
RNA interference (RNAi) is evolutionarily conserved phenomenon, constituting a major component of mosquito innate immune response to virus infections . It has been demonstrated that dengue virus elicits RNAi response in
Apart from small interference RNAs (siRNA) reputation to inhibit gene expression through a phenomenon called RNAi, the target gene can also be induced by a mechanism called RNA activation (RNAa). The term RNAa was first coined by Li, who named them after he surprisingly found up regulation of human E-cadherin, p21, and VEGF genes by synthetic dsRNAs designed to target promoters of these genes . He further named those promoters targeting small RNAs as short activating RNAs (saRNAs). Recent studies in which authors evaluated RNAa of above-said genes in monkey, rat, and mouse found that RNAa is highly conserved among all mammals . However, the term RNA activation is not necessarily limited to transcriptional activation by saRNA, targeting promoter region only, rather it should be used to describe all small RNA-mediated gene and epigenome activation mechanisms, including transcriptional activation by targeting 3′ terminal regions of genes with saRNAs , piwi-interacting RNA (piRNAs)-mediated epigenetic activation , and microRNA (miRNA)-mediated translation activation . One of the key differences between RNAi and RNAa is their kinetics. RNAi effect is known to occur within a couple of hours and disappears 5–7 days following siRNA transfection, while RNAa effect does not appear until about 48 h posttransfection and lasts much longer (at least 2 weeks) . This delay may be due to the more complex mechanism of RNAa involving nuclear access and chromatin restructuring as compared to RNAi .
microRNAs (miRNAs) represent one of the shortest functional classes of noncoding RNAs, depicting hottest spot of gene regulation research. They were first reported in 1993 in small round worms
This pragmatic book chapter overviews the possibility of RNAi suppressor activity of DENV in the context of different viral genes. By characterizing the role of different DENV genes in RNAi suppression, we will review to gather valuable lead into the mechanism that governs DENV RNAi evasion in mosquitoes and the viral genes that are responsible for it. We will also explicit the possibility of RNA activation (RNAa) in insect, especially mosquitos. It is interesting to characterize whether RNAa has any role in host pathogen interaction because, despite the difference in their mode of action, RNAa use same RNAi machinery to induce gene activation. Although very few virus-encoded miRNAs have been fully characterized, it is understandable that they can target both viral and host genes. The information will produce a better understanding of microRNAs role in DENV replication and further deeper insight into characterization of DENV-mediated differential expression of
2. Dengue virus genome organization
The dengue virus (DENV) genome comprises a single open reading frame (ORF) of approximately 10.7 kilobases flanked by 5′ and 3′ nontranslated regions (NTRs) . The nontranslated regions contain highly conserved secondary structures and coding regions in ORF, which play integral roles in viral life cycle. The composition of secondary structures is based on the sequencing of DENV-2 (a southeast Asian strain) strain, which is similar to other different viral strains and serotypes. Although most of the DENV proteins are with known functions but some remains to be explored (Table 1) .
3. Viral entry
DENV is mostly introduced in human by infected
4. Viral replication
DENV like other flaviviruses, after adsorption to receptors, is endocytosed by cells in clathrin-dependent manner and then is further transported to endosomes . However, according to one study, DENV can adopt clathrin-independent pathways as well to gain entry into mammalian cells in particular . DENV nucleocapsid is released into cytosol after acidic pH triggered rearrangements of envelope protein in late endosomes leading to fusion of viral envelope and cellular endosome membrane [44, 45]. The nucleocapsid disassembles, and viral genome is translated by endoplasmic reticulum (ER)-located ribosomes generating numerous copies of viral proteins. NS5 and other viral nonstructural proteins along with various host proteins establish replication complexes . At replication complex, the viral polymerase (NS5) transcribes negative viral RNA, which serves as a template for synthesis of subsequent positive viral RNA copies. Viral replication is regulated by 5′-3’ UTR sequences (upstream of AUG region) present in 5′-3’ UTRs, which are actively involved in the circularization of DENV RNA . These newly synthesized positive DENV RNAs interact with core proteins to assemble into nucleocapsids . The nucleocapsid buds into ER lumen, thereby getting enveloped in a membrane bilayer carrying the viral prM and E proteins . From ER lumen, immature viral particles are transported through cellular secretary pathways, furin protease cleaves prM, and results in the formation of mature viral particles capable of infecting naïve cells .
5. Virus-host interactions
In nature, almost every organism engages in ecological or molecular interactions whether antagonistic or mutualistic with other organisms of different species to thrive and excel. These interactions are major drivers of diversification and adaptive evolution. Among these interactions, most fascinating examples are those involved in invertebrate susceptibility to pathogens . According to a recent study, the susceptibility of
6. RNA interference (RNAi)
RNA interference (RNAi) is a major component of innate defense of invertebrates against pathogens. Most arboviruses cause persistent infections within their arthropod vectors. However, how viruses maintain persistent infection in the face of a robust RNAi response is still not fully understood. Many plants and animal viruses have evolved molecular mechanisms for subverting the host RNAi response. For example, Flock house virus (FHV) encodes B2 protein, which directly binds to dsRNA to inhibit DCR2 activity, to inert siRNA pathway . 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 . 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 . 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.
7. RNA activation (RNAa)
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 . 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 . 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 .
8. MicroRNAs (miRNAs)
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 viruses lack nuclear access . However, recent discovery of noncanonical miRNA biogenesis pathways that utilize Argonaute (Ago) 2 for processing of pre-miRNA to mature miRNA has highlighted the fact that RNA viruses may opt similar pathway to generate functional miRNAs. Similar noncanonical pathways might be used to generate functional miRNAs derived from RNA viruses that replicate in the cytoplasm. Several examples up to now have been accumulated like cellular miRNAs, miR-124, is expressed from a cytoplasmic RNA virus, Sindbis virus (SINV) . Several miRNAs were found to be produced by a retrovirus, Bovine leukemia virus (BLV), based on DNA polymerase III transcription
Flaviviruses are well reported to manipulate host miRNA machinery to facilitate their replication. It has been well reported that DENV induce differential expression of miRNAs in human peripheral blood mononuclear cells (PBMCs) . It has been previously reported that miR-124a, -128a, -218, and -let-7c may be important for neurological symptoms caused by a chimeric tickborne encephalitis/dengue virus . Additional studies have shown that miR-122 and miR-142 of the host cells are involved in restricting dengue virus [61, 62]. However, most of the above work has been done in human. Very little is known about the differential miRNA expression of
9. miRNA biogenesis
9.1. miRNAs genomic arrangement and transcription
miRNAs are usually encoded by different regions of genome ranging from coding as well as noncoding . 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 . Studies have shown that about 30% of
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 . Nearly, all the independent miRNA promoters have particular features of Pol II promoters, including initiator elements and TATA boxes .
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 . This pre-miRNA is then exported to cytoplasm with the help of exportin-5 and RanGTP proteins . 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].
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 . It has been reported that in Drosophila and
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 . 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
10. Mode of action of miRNAs
After the production of mature miRNA by either canonical or noncanonical pathway, the resulting mature miRNA duplex is loaded into miRNA inducing silencing complex (miRISC) having Ago as core component . After degradation of one strand, other strands of duplex, representing mature miRNA, then guide miRISC complex to target mRNA to determine its fate . 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 . 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 . 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 . However, in animals, there is mostly imperfect complementarity between mRNA and miRNA, which usually leads to translational repression of target gene . 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 of HCV RNA replication by stabilizing HCV RNA and preventing its decay .
11. Virus-encoded miRNAs
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 . Different recent studies have identified novel microRNAs from different RNA viruses. Bovine leukemia virus (BLV), a retrovirus, also encodes microRNAs . Hussain et al. has reported microRNA like small RNAs in both West Nile virus and Dengue virus . These viral-encoded miRNAs are produced through canonical as well as noncanonical biogenesis pathways. Based on their function, viral encoded miRNAs can be classified into two broad classes:
Virus-encoded miRNA regulating host genes
Virus-encoded miRNAs regulating virus copy number
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 . 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 miRNAs. 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 , so exploitation of its targets by viruses may contribute to viral oncogenesis. Recently, it has been reported that a flavivirus named West Nile virus encode an miRNA KUN-miR-1 that specifically induces the expression of GATA4 in
Interestingly, virus-encoded miRNAs can regulate their own replication as well their own benefit. For example, simian virus 40 (SV40)-encoded miRNA miR-S1, expressed during late infection, has been shown to downregulate viral T-antigen, which is crucial to evade cytotoxic T cell response . Another example is HvAV-miR-1 encoded by
12. miRNA role in host-viral interactions
Regulative control of miRNAs over gene expression in every organism is the core reason why pathogens try to hijack them. This takeover may include disruption of miRNA biogenesis pathway, inhibiting a specific host miRNA that hinders pathogen propagation or differential miRNA expression. In case of human, it has been well documented that miR-146 and miR-155 are involved in upregulating immune function in response to various bacterial infections including
12.1. miRNA role in
Aedes aegyptiwith an insect -specific flavivirus
Previous studies demonstrated that mosquito miRNAs could be affected by different viral infections. However, those studies were restricted to pathogenic viruses, mainly restricted to the role of differently expressed miRNAs with limited characterization. Limited knowledge is available about the modulation of host miRNAs with respect to insect-specific flaviviruses (ISFs, e.g., Palm Creek virus (PCV)). ISFs share the similar genome organization with other flaviviruses; however, their rate of vertical transmission between insect hosts is higher than other vertebrate-infecting flaviviruses. Although it is still unclear that why ISFs fail to replicate in mammalian cells, it is assumed that ISFs potentially lack to antagonize the host interferon system. Similarly, ISFs express a high adaptation level in insect hosts, which may further limit their replication in vertebrate hosts. Recently, Lee et al. explicated the potential role of miRNAs in Palm Creek virus (PCV) infection using the
13. RNA interference in
Aedes aegyptiwith an insect -specific flavivirus
RNA interference is considered a vital antiviral defense response in mosquitoes as lot of studies reported the production of viral-specific small RNAs for different viruses. However, several reports also confirm the establishment of persistent viral infection only in mosquitoes either infected by pathogenic mosquito-borne viruses or insect-specific viruses that produce these small RNAs. While doing so, viral-derived DNA (vDNA) is produced by reverse transcription during persistent infection and may survive in extrachromosomal or integrated forms. Such vDNAs increase RNAi-mediated antiviral response in mosquitoes to boost mosquito tolerance to arbovirus infection while establishing persistent infection. In one study, Lee et al. detected PCV-specific 21 nucleotide small-RNAs in mosquitoes at different time points of infection (2, 6, and 12 days p.i.) as a potential indicator of active viral replication in the mosquitoes . These findings complied with other studies where the researchers demonstrated the similar phenomenon for other pathogenic mosquito-borne flaviviruses (e.g., DENV) and insect-specific flaviviruses (e.g., cell fusing agent viruses). Their presence as hot spots suggests that either those are the potential targets of Dicer-2, or are more stable or can be reverse-transcribed into vDNA. All these might be the potential cause to enhance RNAi antiviral response. However, the authors are also afraid of bias, which could be due to technical issues such as library preparation. In addition to RNAi, piRNAs (piwi RNAs) have been reported in mosquitoes and knockdown of piRNAs-related specific proteins indicates their endogenous antiviral activity instead of well-established exogenous RNAi activities in mosquitoes. Similarly, virus-specific piRNAs with typical A10 bias in sense RNA and U1 bias in antisense RNA have been reported to be produce by Bunya viruses and alphaviruses. However, the piRNAs expressed by dengue virus and cell fusing agent virus have only A10 bias in sense RNA. Interestingly, the PCV-specific small RNAs do not show either A10 or U1 bias features. In this scenario, it is uncertain that either they are piRNAs or just viral degradation products. This situation is very similar seen in other flavivirus-specific piRNAs, which map to very small number of sequences in the genome where the presence of one copy of genome indicates more specific targeting than random RNA degradation. The production of virus small RNAs and piRNAs through vDNA synthesis is also documented in mosquitoes or in their derived cell lines. It is worth mentioning to note the contribution of vDNA in the production of small RNAs of virus and piRNAs in mosquitoes, although insect-specific viruses like PCV establish persistent infection in their hosts .
14. Long intergenic noncoding RNAs (lincRNAs) and their association with DENV-host interaction
Considering as another important class of regulatory RNAs, lincRNAs (sometimes known as dark matter) have various biological functions including genomic imprinting and cell differentiation specifically in host-pathogen interaction . A growing number of evidences also reflect their possible role in gene regulation either epigenetics or nonepigenetics. However, their role in immune cell differentiation and activation is poorly understood, but the recent discoveries show their potential role in defense system as well as rapid responses to various stimuli and stress factors. Some studies also predict their active role to enhance viral replication or decrease antiviral immunity. It was also depicted that some lincRNAs also interact with other noncoding RNAs like miRNAs. The
Etebari et al. identified lincRNAs in dengue fever vector
The authors also described lincRNAs potential involvement in mosquito-pathogen interaction by determining its association with host-endogenous small RNAs and its direct interaction with DENV-2 infection. lincRNA_1317 was not found to be located in any of the known piRNA clusters; however, no differences were found in the mapping pattern and mapped read length distribution when reads from DENV-infected and noninfected small RNA libraries were mapped to lincRNA_1317. Gene silencing like role of piRNAs on lincRNA_1317 transcriptome was also speculated. However, a little information is available about piRNA-mediated lincRNA although some recent studies predict piRNA-mediated degradation of lincRNAs in mouse’s late spermatocytes .
The researchers also tested the hypothesis that
15. IsomiRs and their impact on miRNAs in dengue fever vector
microRNAs may exist in various lengths and sequence variations, which are known as isomiRs . Earlier, those were considered as sequencing errors; however, some studies predicted to be physiologically relevant and posttranscriptionally modified miRNA variants. IsomiRs may express affinities for different targets than their canonical miRNA counterparts. Furthermore, nucleotide heterogeneity could be found at both ends of miRNA sequence in the form of nucleotide substitutions despite the variations are more frequent at 3′ end. The molecular biology, genome structure, and epigenetics of isomiRs are still poorly understood, and the mechanisms of their biogenesis are also considered very complex and even cell-type specific. Some variations in miRNA sequences are supposed to be the product of template variations, which might be brought by the exonuclease activity of Drosha and Dicer .
It is a well-documented fact that miRNAs may play a vital role to modulate the capability of vectors to propagate the infection for widely damaging arboviruses (e.g., dengue virus) . It has been demonstrated that miRNA could be modified in mosquitoes upon the induction of DENV infection. As genetic variations in isomiR prevalence have significant impact on gene regulation, a clear understanding of the role of isomiR profile of mosquitoes in DENV and other arboviruses infection propagation is urgently needed. Etebari et al. in one study found this altered posttranscriptional modification role of miRNAs in
An over-growing number of reports and continuous publication of journals and books illustrate an intricate interplay between virus-host interactions in dengue fever virus infection. Similarly, an increase in morbidity and mortality rate with DENV infection and still unavailability of standard care to treat the infection have diverted the DENV-research to explore new paradigms in treatment and to map/identify cellular/molecular pathways to better understanding of disease progression in infected vectors, either mosquitoes or humans. Small interference RNA and microRNA as anti-mRNA-based treatment strategies with strong evidences are evolutionary, but unfortunately still not a successful and reliable treatment line in viral infections with certain issues of drug delivery and long-term therapeutic effects. The involvement of lincRNAs and variations in miRNA epigenetic profile in disease-specific arboviruses (e.g., dengue virus) during infection also indicate complex genome interactions between virus and hosts. RNA association as a novel therapeutic approach to treat infection by gene activation, to find out some specific modulators of gene regulation in viral replication as well as in disease progression is still in an explanatory phase specifically for disease-specific or insect-specific flaviviruses. In conjunction, the molecular approaches to find out disease progression pathways in disease-specific arboviruses are still investigative and antiviral treatment approaches in the form of siRNAs, microRNAs as well as RNA association still need to be explored fully before their practical implementations in diagnostics and as some antiviral therapeutics.
Conflict of interest
The author declare that there is no conflict of interest.