- Short report
- Open Access
Optimal design and validation of antiviral siRNA for targeting HIV-1
© Naito et al; licensee BioMed Central Ltd. 2007
- Received: 06 August 2007
- Accepted: 08 November 2007
- Published: 08 November 2007
We propose rational designing of antiviral short-interfering RNA (siRNA) targeting highly divergent HIV-1. In this study, conserved regions within HIV-1 genomes were identified through an exhaustive computational analysis, and the functionality of siRNAs targeting the highest possible conserved regions was validated. We present several promising antiviral siRNA candidates that effectively inhibited multiple subtypes of HIV-1 by targeting the best conserved regions in pandemic HIV-1 group M strains.
- Infectious Molecular Clone
- Polypurine Tract
- Nucleotide Sequence Level
- Effective siRNAs
- Viral Mutational Escape
RNA interference (RNAi) is now widely used to knockdown gene expression in a sequence-specific manner, making it a powerful tool not only for studying gene function, but also for therapeutic applications including antiviral treatments [1, 2]. The replication of a wide range of viruses can be successfully inhibited using RNAi with both short interfering RNA (siRNA) and siRNA expression vectors [3, 4]. However, for RNA viruses such as HIV-1, designing functional siRNAs that target viral sequences is problematic because of their extraordinarily high genetic diversity. We analyzed 495 entries of near full-length HIV-1 group M sequences available in the Los Alamos HIV Sequence Database, and selected the highest-possible conserved target sites for designing optimal antiviral siRNAs. It is known that RNAi-resistant viral mutants emerge rapidly when targeting viral sequences due to their high mutation rate [5–7]. Since highly conserved sequences are likely to contain structurally or functionally constrained elements, our approach is anticipated to resist viral mutational escape.
However, our analysis has identified several distinct regions that are highly conserved in the HIV-1 genome (Figure 1B). Such regions include the regulatory domains responsible for the viral gene expression, such as the TATA sequence and polyadenylation signal (AAUAAA). In addition, several regions essential for the regulation of viral replication were also highly conserved, including the primer activation signal (PAS), primer binding site (PBS), packaging signal (Ψ), central polypurine tract (cPPT), central termination sequence (CTS), and 3' polypurine tract (3' PPT). All of these highly conserved sequences are constrained at the nucleotide sequence level or by their RNA secondary structure in order to execute their functions. In contrast, regions constrained by amino acid sequences were not necessarily conserved at the nucleotide sequence level due to the wobbling of the third base in the codon (data not shown). siRNAs targeting the highly conserved regions are expected to overwhelm the high level of sequence diversity of the HIV-1 genome, and also to reduce the chances of viral mutational escapes.
Total of 216 highly conserved (>70%) siRNA targets identified in this study are listed in Additional file 3. In mammalian RNAi, the efficacy of each siRNA varies markedly depending on its sequence. According to our guidelines for the selection of effective siRNAs [11, 12], 31 out of 216 siRNAs were predicted to be functional. Similarly, 30 and 44 siRNAs are functional according to the algorithms reported by Reynolds et al. , and Amarzguioui et al. , respectively (Additional file 3). This suggests that only a limited fraction of 21-mers is best suited for use as functional antiviral siRNAs.
Next, siRNAs were evaluated for their antiviral efficacy against three evolutionary-distant groups of HIV-1: subtypes B and B' (Thailand variant of subtype B ); subtype C; and CRF01_AE. Each siRNA was cotransfected into HeLa cells at 5 nM with one of the four infectious molecular clones: pNL4-3 (subtype B); 95MM-yIDU106 (subtype B'); 93IN101 (subtype C); or 93JP-NH1 (CRF01_AE). Culture supernatants were collected 48 h after transfection and the viral reverse transcriptase activity was measured (Additional file 5 and ). The results show that 26 of the 41 siRNAs effectively inhibited viral replication of all four strains by >80% (Figure 2, marked with red or orange circles). Of the remaining 15 siRNAs, 13 of them (except si4794/4888) were shown to be functional in the target mRNA cleavage assay, and 12 of them (except si690/4794/4888) inhibited the replication of at least one viral strain by >80%, indicating that the designed siRNAs have the potential to induce RNAi. In several viral strains, nucleotide substitutions in their target sites essentially abolished the inhibition of viral replication (Figure 2, blue bars with arrowheads). However, mismatches near the ends of the target sites (see Additional file 6) did not necessarily abolish the siRNA efficacy (Figure 2, blue bars with asterisks). si689 and si690 did not inhibit viral replication even though these siRNAs perfectly matched to their target sites (confirmed by DNA sequencing of the infectious molecular clones). This is probably due to the stable secondary structure at the si689-690 target sites in both BMH (branched multiple hairpin) conformation and LDI (long distance interaction) conformation of the HIV-1 leader RNA  (see Additional file 4). It should be noted that the efficacy of si575 differed when targeting pNL4-3 and 93IN101. One possible explanation for this is the secondary structure differences among HIV-1 subtypes, which may alter the accessibility of the si575 target site.
The approach described here enabled us to select highly effective siRNAs against divergent HIV-1 strains at a high rate. The highly effective siRNAs (>90% inhibition) with maximal conservation (>70%) identified in our study include si521 (poly A site; 94% conservation), si764/770 (Ψ; 88%), si510 (TAR/poly A; 84%), si2075 (ribosomal slip site; 70%), si2329/2330/2333 (protease region; 77%), and si4750/4751/4753 (integrase region; 71–74%). These sites are found mostly in the 5' LTR, protease, and integrase regions (Figure 2). However, the extraordinarily high genetic diversity of HIV-1 obviously prevents us from designing a single siRNA that can nullify all HIV-1 strains currently circulating worldwide (Additional file 7). One possible approach is to combine multiple siRNAs targeting different conserved regions [19, 20]. The siRNAs selected and validated in this study have the potential to target >99% of HIV-1 strains by combining only two siRNAs (Additional file 7), and also considered to resist viral mutational escape. Our approach is expected to be highly applicable to therapeutic intervention for other pathogens of public health importance, including HCV, influenza virus, and SARS coronavirus, that are known to show high genetic diversity.
This study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to YN, KU-T, KS, and YT), the Ministry of Health, Labour and Welfare of Japan (to YT), and the Japan Health Sciences Foundation (to YT).
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998, 391: 806-811. 10.1038/35888.View ArticlePubMedGoogle Scholar
- Hannon GJ, Rossi JJ: Unlocking the potential of the human genome with RNA interference. Nature. 2004, 431: 371-378. 10.1038/nature02870.View ArticlePubMedGoogle Scholar
- Nielsen MH, Pedersen FS, Kjems J: Molecular strategies to inhibit HIV-1 replication. Retrovirology. 2005, 2: 10-10.1186/1742-4690-2-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Leonard JN, Schaffer DV: Antiviral RNAi therapy: emerging approaches for hitting a moving target. Gene Ther. 2006, 13: 532-540. 10.1038/sj.gt.3302645.View ArticlePubMedGoogle Scholar
- Boden D, Pusch O, Lee F, Tucker L, Ramratnam B: Human immunodeficiency virus type 1 escape from RNA interference. J Virol. 2003, 77: 11531-11535. 10.1128/JVI.77.21.11531-11535.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Das AT, Brummelkamp TR, Westerhout EM, Vink M, Madiredjo M, Bernards R, Berkhout B: Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol. 2004, 78: 2601-2605. 10.1128/JVI.78.5.2601-2605.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Westerhout EM, Ooms M, Vink M, Das AT, Berkhout B: HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res. 2005, 33: 796-804. 10.1093/nar/gki220.PubMed CentralView ArticlePubMedGoogle Scholar
- Naito Y, Ui-Tei K, Nishikawa T, Takebe Y, Saigo K: siVirus: web-based antiviral siRNA design software for highly divergent viral sequences. Nucleic Acids Res. 2006, 34: W448-W450. 10.1093/nar/gkl214.PubMed CentralView ArticlePubMedGoogle Scholar
- ter Brake O, Berkhout B: A novel approach for inhibition of HIV-1 by RNA interference: counteracting viral escape with a second generation of siRNAs. J RNAi Gene Silencing. 2005, 1: 56-65.PubMed CentralPubMedGoogle Scholar
- Beerens N, Groot F, Berkhout B: Initiation of HIV-1 reverse transcription is regulated by a primer activation signal. J Biol Chem. 2001, 276: 31247-31256. 10.1074/jbc.M102441200.View ArticlePubMedGoogle Scholar
- Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K: Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 2004, 32: 936-948. 10.1093/nar/gkh247.PubMed CentralView ArticlePubMedGoogle Scholar
- Naito Y, Yamada T, Ui-Tei K, Morishita S, Saigo K: siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Res. 2004, 32: W124-W129. 10.1093/nar/gkh442.PubMed CentralView ArticlePubMedGoogle Scholar
- Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A: Rational siRNA design for RNA interference. Nat Biotechnol. 2004, 22: 326-330. 10.1038/nbt936.View ArticlePubMedGoogle Scholar
- Amarzguioui M, Prydz H: An algorithm for selection of functional siRNA sequences. Biochem Biophys Res Commun. 2004, 316: 1050-1058. 10.1016/j.bbrc.2004.02.157.View ArticlePubMedGoogle Scholar
- Ui-Tei K, Naito Y, Saigo K: Guidelines for the selection of effective short-interfering RNA sequences for functional genomics. Methods Mol Biol. 2007, 361: 201-216.PubMedGoogle Scholar
- Kalish ML, Baldwin A, Raktham S, Wasi C, Luo CC, Schochetman G, Mastro TD, Young N, Vanichseni S, Rübsamen-Waigmann H, von Briesen H, Mullins JI, Delwart E, Herring B, Esparza J, Heyward WL, Osmanov S: The evolving molecular epidemiology of HIV-1 envelope subtypes in injecting drug users in Bangkok, Thailand: implications for HIV vaccine trials. AIDS. 1995, 9: 851-857. 10.1097/00002030-199508000-00004.View ArticlePubMedGoogle Scholar
- Willey RL, Smith DH, Lasky LA, Theodore TS, Earl PL, Moss B, Capon DJ, Martin MA: In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity. J Virol. 1988, 62: 139-147.PubMed CentralPubMedGoogle Scholar
- Huthoff H, Berkhout B: Two alternating structures of the HIV-1 leader RNA. RNA. 2001, 7: 143-157. 10.1017/S1355838201001881.PubMed CentralView ArticlePubMedGoogle Scholar
- Nishitsuji H, Kohara M, Kannagi M, Masuda T: Effective suppression of human immunodeficiency virus type 1 through a combination of short- or long-hairpin RNAs targeting essential sequences for retroviral integration. J Virol. 2006, 80: 7658-7666. 10.1128/JVI.00078-06.PubMed CentralView ArticlePubMedGoogle Scholar
- ter Brake O, Konstantinova P, Ceylan M, Berkhout B: Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol Ther. 2006, 14: 883-892. 10.1016/j.ymthe.2006.07.007.View ArticlePubMedGoogle Scholar
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