ESF-EMBO Symposium: Antiviral Applications of RNA Interference

ABSTRACT: The first ESF-EMBO symposium on applications of antiviral RNA interference (RNAi) was held in the spring of 2008 in Sant Feliu de Guixols at the Costa Brava in Spain. Some 60 participants from the field of RNAi and virology came together to present their latest findings on RNAi-virus interactions, as well as the progress in the development of RNAi-based antiviral therapeutics. One of the big topics concerned the role of RNAi in natural antiviral defence mechanisms in mammals. In addition, new solutions to improve the efficacy and safety of RNAi-based antiviral drugs were presented. The combined expertise of researchers studying RNAi in plants, insects and mammalian systems greatly stimulated the overall discussion. The meeting was funded by the European Science Foundation (ESF) in partnership with the European Molecular Biology Organisation (EMBO

Efficient inhibition of HIV-1 expression by LNA modified antisense oligonucleotides and DNAzymes targeted to functionally selected binding sites

BACKGROUND: A primary concern when targeting HIV-1 RNA by means of antisense related technologies is the accessibility of the targets. Using a library selection approach to define the most accessible sites for 20-mer oligonucleotides annealing within the highly structured 5'-UTR of the HIV-1 genome we have shown that there are at least four optimal targets available. RESULTS: The biological effect of antisense DNA and LNA oligonucleotides, DNA- and LNAzymes targeted to the four most accessible sites was tested for their abilities to block reverse transcription and dimerization of the HIV-1 RNA template in vitro, and to suppress HIV-1 production in cell culture. The neutralization of HIV-1 expression declined in the following order: antisense LNA > LNAzymes > DNAzymes and antisense DNA. The LNA modifications strongly enhanced the in vivo inhibitory activity of all the antisense constructs and some of the DNAzymes. Notably, two of the LNA modified antisense oligonucleotides inhibited HIV-1 production in cell culture very efficiently at concentration as low as 4 nM. CONCLUSION: LNAs targeted to experimentally selected binding sites can function as very potent inhibitors of HIV-1 expression in cell culture and may potentially be developed as antiviral drug in patients

Trans-inhibition of HIV-1 by a long hairpin RNA expressed within the viral genome

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) can be inhibited by means of RNA silencing or interference (RNAi) using synthetic short interfering RNAs (siRNAs) or gene constructs encoding short hairpin RNAs (shRNAs) or long hairpin RNAs (lhRNAs). The use of siRNA and shRNA as antiviral therapeutic is limited because of the emergence of viral escape mutants. This problem is theoretically prevented by intracellular expression of lhRNAs generating multiple siRNAs that target the virus simultaneously, thus reducing the chance of viral escape. However, gene constructs encoding lhRNA molecules face problems with delivery to the right cells in an infected individual. In order to solve this problem, we constructed an HIV-1 variant with a 300 bp long hairpin structure in the 3' part of the genome corresponding to the Nef gene (HIV-lhNef). RESULTS: Intriguingly, HIV-lhNef potently inhibited wild-type HIV-1 production in trans. However, HIV-lhNef demonstrated a severe production and replication defect, which we were able to solve by selecting spontaneous virus variants with truncated hairpin structures. Although these escape variants lost the ability to trans-inhibit HIV-1, they effectively outgrew the wild-type virus in competition experiments in SupT1 cells. CONCLUSION: Expression of the lhNef hairpin within the HIV-1 genome results in potent trans-inhibition of wild-type HIV-1. Although the mechanism of trans-inhibition is currently unknown, it remains of interest to study the molecular details because the observed effect is extremely potent. This may have implications for the development of virus strains to be used as live-attenuated virus vaccines

The Ebola Virus VP35 Protein Is a Suppressor of RNA Silencing

RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences

Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence. Recently, artificial miRNAs have been shown to be potent RNAi inducers due to their efficient processing by the RNAi machinery. Furthermore, miRNAs may be more proficient in suppressing imperfect targets than shRNAs. In this study, we tested the knockdown efficiency of miRNAs and shRNAs against wild-type and RNAi-escape HIV-1 variants with one or two mutations in the target sequence. ShRNAs and miRNAs can significantly inhibit the production of HIV-1 variants with mutated target sequences in the open reading frame. More pronounced mutation-tolerance was measured for targets in the 3′ untranslated region (3′ UTR). Partially complementary sequences within the 3′ UTR of the HIV-1 RNA genome efficiently act as target sites for miRNAs and shRNAs. These data suggest that targeting imperfect target sites by antiviral miRNAs or shRNAs provides an alternative RNAi approach for inhibition of pathogenic viruses

Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron

RNA interference (RNAi) is a powerful approach to inhibit human immunodeficiency virus type 1 (HIV-1) replication. However, HIV-1 can escape from RNAi-mediated antiviral therapy by selection of mutations in the targeted sequence. To prevent viral escape, multiple small interfering RNAs (siRNAs) against conserved viral sequences should be combined. Ideally, these RNA inhibitors should be expressed simultaneously from a single transgene transcript. In this study, we tested a multiplex microRNA (miRNA) expression strategy by inserting multiple effective anti-HIV siRNA sequences in the miRNA polycistron mir-17-92. Individual anti-HIV miRNAs that resemble the natural miRNA structures were optimized by varying the siRNA position in the hairpin stem to obtain maximal effectiveness against luciferase reporters and HIV-1. We show that an antiviral miRNA construct can have a greater intrinsic inhibitory activity than a conventional short hairpin (shRNA) construct. When combined in a polycistron setting, the silencing activity of an individual miRNA is strongly boosted. We demonstrate that HIV-1 replication can be efficiently inhibited by simultaneous expression of four antiviral siRNAs from the polycistronic miRNA transcript. These combined results indicate that a multiplex miRNA strategy may be a promising therapeutic approach to attack escape-prone viral pathogens

Combinatorial RNAi Against HIV-1 Using Extended Short Hairpin RNAs

RNA interference (RNAi) is a widely used gene suppression tool that holds great promise as a novel antiviral approach. However, for error-prone viruses including human immunodeficiency virus type 1(HIV-1), a combinatorial approach against multiple conserved sequences is required to prevent the emergence of RNAi-resistant escape viruses. Previously, we constructed extended short hairpin RNAs (e-shRNAs) that encode two potent small interfering RNAs (siRNAs) (e2-shRNAs). We showed that a minimal hairpin stem length of 43 base pairs (bp) is needed to obtain two functional siRNAs. In this study, we elaborated on the e2-shRNA design to make e-shRNAs encoding three or four antiviral siRNAs. We demonstrate that siRNA production and the antiviral effect is optimal for e3-shRNA of 66 bp. Further extension of the hairpin stem results in a loss of RNAi activity. The same was observed for long hairpin RNAs (lhRNAs) that target consecutive HIV-1 sequences. Importantly, we show that HIV-1 replication is durably inhibited in T cells stably transduced with a lentiviral vector containing the e3-shRNA expression cassette. These results show that e-shRNAs can be used as a combinatorial RNAi approach to target error-prone viruses

Design of extended short hairpin RNAs for HIV-1 inhibition

RNA interference (RNAi) targeted towards viral mRNAs is widely used to block virus replication in mammalian cells. The specific antiviral RNAi response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular expression of short hairpin RNAs (shRNAs). For HIV-1, both approaches resulted in profound inhibition of virus replication. However, the therapeutic use of a single siRNA/shRNA appears limited due to the rapid emergence of RNAi-resistant escape viruses. These variants contain deletions or point mutations within the target sequence that abolish the antiviral effect. To avoid escape from RNAi, the virus should be simultaneously targeted with multiple shRNAs. Alternatively, long hairpin RNAs can be used from which multiple effective siRNAs may be produced. In this study, we constructed extended shRNAs (e-shRNAs) that encode two effective siRNAs against conserved HIV-1 sequences. Activity assays and RNA processing analyses indicate that the positioning of the two siRNAs within the hairpin stem is critical for the generation of two functional siRNAs. E-shRNAs that are efficiently processed into two effective siRNAs showed better inhibition of virus production than the poorly processed e-shRNAs, without inducing the interferon response. These results provide building principles for the design of multi-siRNA hairpin construct

RNA interference against viruses: strike and counterstrike

RNA interference (RNAi) is a conserved sequence-specific, gene-silencing mechanism that is induced by double-stranded RNA. RNAi holds great promise as a novel nucleic acid-based therapeutic against a wide variety of diseases, including cancer, infectious diseases and genetic disorders. Antiviral RNAi strategies have received much attention and several compounds are currently being tested in clinical trials. Although induced RNAi is able to trigger profound and specific inhibition of virus replication, it is becoming clear that RNAi therapeutics are not as straightforward as we had initially hoped. Difficulties concerning toxicity and delivery to the right cells that earlier hampered the development of antisense-based therapeutics may also apply to RNAi. In addition, there are indications that viruses have evolved ways to escape from RNAi. Proper consideration of all of these issues will be necessary in the design of RNAi-based therapeutics for successful clinical intervention of human pathogenic viruse

doi:10.1093/nar/gkm596 Design of extended short hairpin RNAs for HIV-1 inhibition

RNA interference (RNAi) targeted towards viral mRNAs is widely used to block virus replication in mammalian cells. The specific antiviral RNAi response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular expression of short hairpin RNAs (shRNAs). For HIV-1, both approaches resulted in profound inhibition of virus replication. However, the therapeutic use of a single siRNA/shRNA appears limited due to the rapid emergence of RNAi-resistant escape viruses. These variants contain deletions or point mutations within the target sequence that abolish the antiviral effect. To avoid escape from RNAi, the virus should be simultaneously targeted with multiple shRNAs. Alternatively, long hairpin RNAs can be used from which multiple effective siRNAs may be produced. In this study, we constructed extended shRNAs (e-shRNAs) that encode two effective siRNAs against conserved HIV-1 sequences. Activity assays and RNA processing analyses indicate that the positioning of the two siRNAs within the hairpin stem is critical for the generation of two functional siRNAs. E-shRNAs that are efficiently processed into two effective siRNAs showed better inhibition of virus production than the poorly processed e-shRNAs, without inducing the interferon response. These results provide building principles for the design of multi-siRNA hairpin constructs