A novel approach for inhibition of HIV-1 by RNA interference: counteracting viral escape with a second generation of siRNAs

RNA interference (RNAi) is an evolutionary conserved gene silencing mechanism in which small interfering RNA (siRNA) mediates the sequence specific degradation of mRNA. The recent discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the possibility to use this technology as a therapeutic tool against pathogenic viruses. Indeed, it has been shown that siRNAs can be used effectively to inhibit virus replication. The focus of this review is on RNA interference strategies against HIV-1 and how this new technology may be developed into a new successful therapy. One of the hallmarks of RNAi, its sequence specificity, also presents a way out for the virus, as single nucleotide substitutions in the target region can abolish the suppression. Strategies to prevent the emergence of resistant viruses have been suggested and involve the targeting of conserved sequences and the simultaneous use of multiple siRNAs, similar to current highly active antiretroviral therapy. We present an additional strategy aimed at preventing viral escape by using a second generation of siRNAs that recognize the mutated target sites

The virion-associated incoming HIV-1 RNA genome is not targeted by RNA interference

BACKGROUND: RNA interference (RNAi) has proven to be a powerful tool to suppress gene expression and can be used as a therapeutic strategy against human pathogenic viruses such as human immunodeficiency virus type 1 (HIV-1). Theoretically, RNAi-mediated inhibition can occur at two points in the replication cycle, upon viral entry before reverse transcription of the RNA genome, and on the newly transcribed viral RNA transcripts. There have been conflicting results on whether RNAi can target the RNA genome of infecting HIV-1 particles. We have addressed this issue with HIV-1-based lentiviral vectors. RESULTS: We determined the transduction efficiency of a lentiviral vector, as measured by GFP expressing cells, which reflects the number of successful integration events in a cell line stably expressing shNef. We did not observe a difference in the transduction efficiency comparing lentiviral vectors with or without the Nef target sequence in their genome. The results were similar with particles pseudotyped with either the VSV-G or HIV-1 envelope. Additionally, no reduced transduction efficiencies were observed with multiple other shRNAs targeting the vector genome or with synthetic siNef when transiently transfected prior to transduction. CONCLUSION: Our findings indicate that the incoming HIV-1 RNA genome is not targeted by RNAi, probably due to inaccessibility to the RNAi machinery. Thus, therapeutic RNAi strategies aimed at preventing proviral integration should be targeting cellular receptors or co-factors involved in pre-integration events

Anticipating and blocking HIV-1 escape by second generation antiviral shRNAs

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of target mRNAs. RNAi can be used to inhibit HIV-1 replication by targeting the viral RNA genome. However, the error-prone replication machinery of HIV-1 can generate RNAi-resistant variants with specific mutations in the target sequence. For durable inhibition of HIV-1 replication the emergence of such escape viruses must be controlled. Here we present a strategy that anticipates HIV-1 escape by designing 2^ndgeneration short hairpin RNAs (shRNAs) that form a complete match with the viral escape sequences.</p> <p>Results</p> <p>To block the two favorite viral escape routes observed when the HIV-1 integrase gene sequence is targeted, the original shRNA inhibitor was combined with two 2^ndgeneration shRNAs in a single lentiviral expression vector. We demonstrate in long-term viral challenge experiments that the two dominant viral escape routes were effectively blocked. Eventually, virus breakthrough did however occur, but HIV-1 evolution was skewed and forced to use new escape routes.</p> <p>Conclusion</p> <p>These results demonstrate the power of the 2^ndgeneration RNAi concept. Popular viral escape routes are blocked by the 2^ndgeneration RNAi strategy. As a consequence viral evolution was skewed leading to new escape routes. These results are of importance for a deeper understanding of HIV-1 evolution under RNAi pressure.</p

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

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

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

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

SHAPE-directed Discovery of Potent shRNA Inhibitors of HIV-1

The RNA interference (RNAi) pathway can be exploited using short hairpin RNAs (shRNAs) to durably inactivate pathogenic genes. Prediction of optimal target sites is notoriously inaccurate and current approaches applied to HIV-1 show weak correlations with virus inhibition. In contrast, using a high-content model for disrupting pre-existing intramolecular structure in the HIV-1 RNA, as achievable using high-resolution SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing information, we discovered strong correlations between inhibition of HIV-1 production in a quantitative cell-based assay and very simple thermodynamic features in the target RNA. Strongest inhibition occurs at RNA target sites that both have an accessible “seed region” and, unexpectedly, are structurally accessible in a newly identified downstream flanking sequence. We then used these simple rules to create a new set of shRNAs and achieved inhibition of HIV-1 production of 90% or greater for up to 82% of designed shRNAs. These shRNAs inhibit HIV-1 replication in therapy-relevant T cells and show no or low cytotoxicity. The remarkable success of this straightforward SHAPE-based approach emphasizes that RNAi is governed, in significant part, by very simple, predictable rules reflecting the underlying RNA structure and illustrates principles likely to prove broadly useful in understanding transcriptome-scale biological recognition and therapeutics involving RNA

Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions

BACKGROUND: HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. METHODS: Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. RESULTS: RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. CONCLUSIONS: Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector productio