key: cord-310141-2jofy8fo authors: Qureshi, Abid; Tantray, Vaqar Gani; Kirmani, Altaf Rehman; Ahangar, Abdul Ghani title: A review on current status of antiviral siRNA date: 2018-04-15 journal: Rev Med Virol DOI: 10.1002/rmv.1976 sha: doc_id: 310141 cord_uid: 2jofy8fo Viral diseases like influenza, AIDS, hepatitis, and Ebola cause severe epidemics worldwide. Along with their resistant strains, new pathogenic viruses continue to be discovered so creating an ongoing need for new antiviral treatments. RNA interference is a cellular gene‐silencing phenomenon in which sequence‐specific degradation of target mRNA is achieved by means of complementary short interfering RNA (siRNA) molecules. Short interfering RNA technology affords a potential tractable strategy to combat viral pathogenesis because siRNAs are specific, easy to design, and can be directed against multiple strains of a virus by targeting their conserved gene regions. In this review, we briefly summarize the current status of siRNA therapy for representative examples from different virus families. In addition, other aspects like their design, delivery, medical significance, bioinformatics resources, and limitations are also discussed. Because of their high mutation rates, viruses have the potential to elude host defense systems as well as antiviral drugs and vaccines. Thus, development of new and alternate antiviral therapies has become important. 1, 2 During the past decade, scientists have widely used the cellular RNA interference (RNAi) pathway approach to target a number of viral genes to restrain their expression. 3, 4 In this pathway, long double-stranded RNA (dsRNA) precursors are split into short interfering RNAs (siRNAs) following which the RNA-induced silencing complex includes one of the siRNA strands and slices the complementary target mRNA using ATP. 5,6 Short interfering RNA technology has been exploited to target disease-causing genes as well as for functional studies. 7, 8 Also, this strategy can target diverse types of viruses as even a tiny viral genome can provide several targetable regions. For example, siRNAs directed against different genes of deadly viruses like human immunodeficiency virus (HIV), 9, 10 influenza virus (INFV), 11, 12 hepatitis B virus (HBV), 13 SARS coronavirus (SARS-CoV), 14, 15 human papillomavirus (HPV), 16 and West Nile virus (WNV) 17 in infected cells displayed encouraging results in inhibiting viral replication. Researchers have also used multiple siRNAs simultaneously to augment viral inhibition in a coordinated approach. 18, 19 Short interfering RNAs for various human viruses like respiratory syncytial virus (RSV), hepatitis C virus (HCV), HBV, and HIV are also appearing in clinical trials, which further elucidate their importance in inhibiting viral infections. 20 Thus, siRNAs have emerged as practically modular and adaptable therapeutics for treating viral infections. In this review, we will discuss the use of siRNAs against different viral families, their therapeutic applications, and design and delivery considerations. Further limitations of siRNAs as antivirals and the remedial measures are also discussed. Viruses are tiny obligate intracellular parasites, having either an RNA or a DNA genome enclosed by a virus-coded protein coat. Viruses depend on host cells for proliferation. They are classified on account of shape, genome structure, or mode of replication, and many classes of these pathogens cause a large number of diseases in different organisms. 21 Different species of viruses have varying types of infection processes; however, there are many general steps 22 Figure 1 . Short interfering RNAs directed against specific viral/host genes can block the virus life cycle at any of the steps. RNAi is a cellular mechanism wherein small molecules of RNA hinder the expression of a particular gene(s) via counteracting the corresponding mRNA molecules that possess nucleotide sequences complimentary to the small RNA. 24 Historically, RNAi was identified by different terms such as quelling, cosuppression, and posttranscriptional gene silencing. In 1998, Mello and Fire illustrated a strong gene silencing caused by injecting dsRNA into Caenorhabditis elegans. 25 Further findings in the field of RNAi have been pictorially summarized in Figure 2 . The RNAi pathway processes dsRNA into 21 to 30 nucleotidelong RNA molecules that act as a module of a silencing machinery to distinctively suppress expression/function of an intended gene/genomic region ( Figure 3 ). In particular, the silencing pathway involves chopping of dsRNA into siRNA that are typically 21 to 25 base pairs long dsRNA having dinucleotide overhangs on the 3′ termini. One of the siRNA strands (guide strand) is then incorporated into an RNAinduced silencing complex that degrades the target mRNA. 5, 26 The siRNAs resulting from the original longer dsRNA are different from microRNAs as the latter in general have partial base pairing with a target mRNA and restrain the expression of several different genes having related sequences. Short interfering RNAs also differ from short hairpin RNAs as the latter have a hairpin turn and their expression in cells is usually achieved by means of bacterial/viral vectors. 27 Short interfering RNAs characteristically base-pair completely and cause mRNA cleavage in a precise target region. 28 MicroRNAs are generated from introns or their own genes. Gene silencing can take place through mRNA cleavage or thwarting mRNA translation. The role of microRNAs is to regulate gene expression. In the plant kingdom, RNAi is broadcasted by the transportation of siRNAs amid cells via plasmodesmata. 29 RNAi was described as the "breakthrough of the year" in 30 2002 . RNAi is also believed to be a component of a primitive immune system based on nucleic acid recognition. 31 show any harmful effect on control cells, which indicates meager off-target effects. 87 This illustrates that siRNA treatment has the potential to suppress the progression of cervical cancer. Polyomaviruses have a double-stranded circular DNA genome. 88 Repression of the T-Ag oncogene has been shown to hinder the transformation of the cells. Short interfering RNAs designed to target the BKV T-Ag were reported to restrain its expression in pRPc cell lines. Blocking of T-Ag results in diminished growth rate of BKV-transformed cells and thus suppresses tumorigenicity. 38 Poxviruses have a single, linear, double-stranded DNA and infect both vertebrates and invertebrates. Vaccinia virus (VACV) is the quintessential member of the Poxviridae. 89 The VACV produces a dsRNA FIGURE 2 Timeline depicting the sequential progression in the field of RNA interference (RNAi). shRNA, short hairpin RNA; siRNA, short interfering RNA In the RNA interference mechanism, double-stranded RNA (dsRNA) is chopped into short (21-25 nucleotides) interfering RNA (siRNA) molecules possessing dinucleotide overhangs on the 3′ termini. One siRNA strand is integrated into the RNA-induced silencing complex (RISC) that finally degrades the complementary target mRNA This group of viruses contains linear, single-stranded, positive-sense RNA. 105 One of its members, the Chikungunya virus, is the causative agent of chikungunya fever, which has emerged as an important arboviral infection of public health concern. Short interfering RNAs targeting the conserved segments of nsP3 and E1 mRNAs of the pathogen were able to drastically reduce the virus titer in Vero cells. 71 Severe acute respiratory syndrome, also known as SARS-CoV, is a member of the Coronaviridae family whose members have single- Zika virus had recently posed as a global health threat prompting intense research to control the pathogen. 112 Scientists have found that the conserved 3′ UTR of the virus genome plays a crucial function in its replication. Therefore, rationally designed siRNAs against the 3′ UTRs have been predicted to inhibit the pathogen. 113, 114 Studies have also shown that siRNA-directed silencing of host endoplasmic reticulum membrane complex protein components halted the replication of multiple Zika virus strains in HeLa cells. 115 Hepeviridae mainly includes hepatitis E virus, a key source of waterborne hepatitis in adults that has particularly high mortality in pregnant women. Short interfering RNAs developed against the helicase and replicase genes of hepatitis E virus were found to be effective in inhibiting virus replication in A549 as well as HepG2 cells. 78,79 Retroviridae is a family of enveloped single-stranded RNA viruses that replicate in a host cell through the process of reverse transcription. 116 It has been noted that siRNAs can inhibit HIV-1 growth by aiming the genes for the host CD4 cell receptor, or the viral Gag and Nef proteins Designing siRNAs with high antiviral activity is a challenging task. There are many features that influence siRNA efficacy including GC (guanine-cytosine) content, nucleotides at siRNA termini, thermodynamic properties, siRNA structure, and accessibility of the target site. 127, 128 Also, the siRNAs should be developed against conserved target sites to prevent viral escape due to mutations. 129 It has been suggested that a single target site might not be enough for durable viral silencing so using multiple siRNAs against several target sites can also prevent viral escape. 19 In addition, chemically modified nucleotides can also be used to enhance the stability or reduce off-target effects of siRNAs, 130 Short interfering RNAs are delivered in a number of ways, eg, by encapsulation in synthetic vehicles such as cationic liposomes/nanoparticles or siRNAs conjugated to cell penetrating peptides or specific antibodies against the infected cells. 134 Liposomes are frequently used as delivery mediums for a wide range of drugs including siRNAs. This method often involves cationic lipids to overcome the negative charge associated with siRNAs. 135 Lately, stable nucleic acid lipid particles have been used to efficiently stabilize and transport siRNAs. 136 Morrissey et al used stable nucleic acid lipid particles to deliver siRNAs against HBV, which efficiently inhibited the virus in mice. 137 Next, the polymer-based siRNA delivery vehicles (polyplexes) afford high structural and physicochemical flexibility while shielding the siRNAs against nucleases. 138 Likewise, inorganic nanoparticles generated from calcium phosphate, gold, carbon, and iron oxides are sometimes preferred to transport siRNAs due to their small size and increased permeability in comparison with liposomes and polyplexes. Occasionally, surface ligands are incorporated with the nanoparticles to enhance selective targeting of the diseased cells. 139 Plasmid and viral vectors are also used as expression cassettes for sustained silencing effect. However, use of plasmids limits the delivery efficiency as compared with the viral vectors. 140, 141 Alternatively, direct injection into the infected tissue may also help in targeting the specific cells. 134 Although a large number of databases, design, and prediction algo- 144 Reynolds et al, 145 Ui-Tei et al, 146 Amarzguioui et al, 147 and Jagla et al. 148 Rates of evolutionary change in viruses: patterns and determinants Viral mutation rates Antiviral RNAi therapy: emerging approaches for hitting a moving target Nucleic acids-based therapeutics in the battle against pathogenic viruses RNAi: the nuts and bolts of the RISC machine RNA interference: new mechanisms for targeted treatment? ASPsiRNA: a resource of ASP-siRNAs having therapeutic potential for human genetic disorders and algorithm for prediction of their inhibitory efficacy. G3 (Bethesda) vhfRNAi: a web-platform for analysis of host genes involved in viral infections discovered by genome wide RNAi screens RNA interference and its therapeutic potential against HIV infection RNAi as a treatment for HIV-1 infection RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription PA subunit of RNA polymerase as a promising target for anti-influenza virus agents Inhibition of HBV replication by siRNA in a stable HBV-producing cell line Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes Inhibition of cervical cancer cell growth by human papillomavirus virus-like particles packaged with human papillomavirus oncoprotein short hairpin RNAs A single siRNA suppresses fatal encephalitis induced by two different flaviviruses siRNA pool targeting different sites of human hepatitis B surface antigen efficiently inhibits HBV infection Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition Antiviral RNAi: translating science towards therapeutic success Cell-to-cell transmission of viruses Adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs Hepatitis C virus: virology and life cycle Molecular mechanisms of RNA interference Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Ribo-gnome: the big world of small RNAs A system for stable expression of short interfering RNAs in mammalian cells Repression of protein synthesis by miRNAs: how many mechanisms? MicroRNA-196 inhibits HOXB8 expression in myeloid differentiation of HL60 cells Breakthrough of the year. Small RNAs make big splash Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing Short interfering RNA confers intracellular antiviral immunity in human cells A cellular microRNA mediates antiviral defense in human cells The role of RNA interference in heterochromatic silencing Gene silencing with siRNA targeting E6/E7 as a therapeutic intervention in a mouse model of cervical cancer Identification of cellular targets for the human papillomavirus E6 and E7 oncogenes by RNA interference and transcriptome analyses Design of potential siRNA molecules for T antigen gene silencing of Merkel cell polyomavirus Anticancer activity of an adenoviral vector expressing short hairpin RNA against BK virus T-ag In vitro evaluation of antisense RNA efficacy against filovirus infection, by use of reverse genetics Inhibition of Marburg virus protein expression and viral release by RNA interference Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates Effective small interfering RNAs targeting matrix and nucleocapsid protein gene inhibit influenza A virus replication in cells and mice Baculovirus-mediated bispecific short-hairpin small-interfering RNAs have remarkable ability to cope with both influenza viruses A and B Inhibitory effect of small interfering RNA specific for a novel candidate target in PB1 gene of influenza A virus Inhibition of enterovirus 71 in virus-infected mice by RNA interference Selective inhibition of enterovirus 71 replication by short hairpin RNAs Complete cure of persistent virus infections by antiviral siRNAs RNA interference mediated inhibition of Chikungunya virus replication in mammalian cells Antiviral RNA interference responses induced by Semliki Forest virus infection of mosquito cells: characterization, origin, and frequency-dependent functions of virusderived small interfering RNAs Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference Inhibition of severe acute respiratory syndrome virus replication by small interfering RNAs in mammalian cells Targeted delivery of small interfering RNA to human dendritic cells to suppress dengue virus infection and associated proinflammatory cytokine production Silencing early viral replication in macrophages and dendritic cells effectively suppresses flavivirus encephalitis Use of RNA interference to prevent lethal murine West Nile virus infection Inhibition of hepatitis E virus replication using short hairpin RNA (shRNA) Effective inhibition of hepatitis E virus replication in A549 cells and piglets by RNA interference (RNAi) targeting RNA-dependent RNA polymerase RNA interference inhibits hepatitis E virus mRNA accumulation and protein synthesis in vitro Optimal design and validation of antiviral siRNA for targeting HIV-1 RNA interference approaches for treatment of HIV-1 infection Efficacy analysis of combinatorial siRNAs against HIV derived from one double hairpin RNA precursor Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1 Classification of papillomaviruses Hoppe-Seyler F. siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells Highly potent and specific siRNAs against E6 or E7 genes of HPV16-or HPV18-infected cervical cancers Taxonomical developments in the family Polyomaviridae Insights into the evolution of a complex virus from the crystal structure of vaccinia virus D13 Vaccinia virus double-stranded RNA-binding protein E3 does not interfere with siRNA-mediated gene silencing in mammalian cells Primate hepatitis B viruses-genetic diversity, geography and evolution Small interfering RNA inhibits hepatitis B virus replication in mice siRNAs against the Epstein Barr virus latency replication factor, EBNA1, inhibit its function and growth of EBVdependent tumor cells Short interfering RNA-mediated inhibition of herpes simplex virus type 1 gene expression and function during infection of human keratinocytes RNA composition of Junin virus Identification and molecular characterization of 18 paramyxoviruses isolated from snakes RNA interference inhibits respiratory syncytial virus replication and disease pathogenesis without inhibiting priming of the memory immune response Emerging viruses: the Bunyaviridae An siRNA against JC virus (JCV) agnoprotein inhibits JCV infection in JCV-producing cells inoculated in nude mice Filoviridae: a taxonomic home for Marburg and Ebola viruses? Use of siRNAs to prevent and treat influenza virus infection Inhibition of influenza virus production in virus-infected mice by RNA interference Molecular detection and typing of human picornaviruses A small interfering RNA targeting coxsackievirus B3 protects permissive HeLa cells from viral challenge Viruses of the Bunya-and Togaviridae families: potential as bioterrorism agents and means of control Inhibition of SARS-CoV replication by siRNA Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque New drug targets for hepatitis C and other Flaviviridae viruses Small interfering RNAmediated inhibition of hepatitis C virus replication in the human hepatoma cell line Huh-7 Effective siRNA targeting of the 3′ untranslated region of the West Nile virus genome Inhibition of dengue virus infections in cell cultures and in AG129 mice by a small interfering RNA targeting a highly conserved sequence An update on Zika virus infection A computational approach to design potential antiviral RNA for 3'UTR post transcriptional gene silencing of different strains of Zika virus ZikaVR: an integrated Zika virus resource for genomics, proteomics, phylogenetic and therapeutic analysis Identification of Zika virus and dengue virus dependency factors using functional genomics Emergence of vertebrate retroviruses and envelope capture siRNA-directed inhibition of HIV-1 infection A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus RNA interference and its potential applications to chronic HBV treatment: results of a phase I safety and tolerability study SPC3649) can inhibit the biogenesis of miR-122 Current prospects for RNA interferencebased therapies Emerging targets and novel approaches to Ebola virus prophylaxis and treatment Targeting Marek's disease virus by RNA interference delivered from a herpesvirus vaccine Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo RNA interference acts as a natural antiviral response to O'nyongnyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae The role of RNAi and microRNAs in animal virus replication and antiviral immunity siRNA efficiency: structure or sequence-that is the question Selection of hyperfunctional siRNAs with improved potency and specificity Universal and mutation-resistant antienteroviral activity: potency of small interfering RNA complementary to the conserved cis-acting replication element within the enterovirus coding region Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing siRNA-optimized modifications for enhanced in vivo activity siRNAmod: a database of experimentally validated chemically modified siRNAs Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication Delivery of siRNA therapeutics: barriers and carriers siRNA delivery strategies: a comprehensive review of recent developments. Nanomaterials (Basel) Tumor-targeted delivery of siRNA by non-viral vector: safe and effective cancer therapy Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs Lipoplexes versus nanoparticles: pDNA/siRNA delivery Delivery materials for siRNA therapeutics Knocking down barriers: advances in siRNA delivery Delivering small interfering RNA for novel therapeutics VIRsiRNAdb: a curated database of experimentally validated viral siRNA/shRNA HIVsirDB: a database of HIV inhibiting siRNAs Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Rational siRNA design for RNA interference Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference An algorithm for selection of functional siRNA sequences Sequence characteristics of functional siRNAs siVirus: web-based antiviral siRNA design software for highly divergent viral sequences Predicting the efficacy of short oligonucleotides in antisense and RNAi experiments with boosted genetic programming Design of a genome-wide siRNA library using an artificial neural network Improving model predictions for RNA interference activities that use support vector machine regression by combining and filtering features VIRsiRNApred: a web server for predicting inhibition efficacy of siRNAs targeting human viruses Potential clinical applications of siRNA technique: benefits and limitations Strategies for ocular siRNA delivery: potential and limitations of non-viral nanocarriers Applications of RNA interference: current state and prospects for siRNA-based strategies in vivo siRNA function in RNAi: a chemical modification analysis siRNA and isRNA: two edges of one sword Development and validation of vectors containing multiple siRNA expression cassettes for maximizing the efficiency of gene silencing RNAi as a bioinformatics consumer A review on current status of antiviral siRNA Authors are thankful to Biomedical Informatics Center, Sher-i-Kashmir The authors have no competing interest.ORCID Abid Qureshi http://orcid.org/0000-0002-1140-1649