Combinatorial RNAi Against HIV-1 Using Extended Short Hairpin RNAs

• Published in: Molecular therapy Vol. 17; no. 10; pp. 1712 - 1723
• Main Authors: Liu, Ying Poi, von Eije, Karin Jasmijn, Schopman, Nick CT, Westerink, Jan-Tinus, Brake, Olivier ter, Haasnoot, Joost, Berkhout, Ben
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2009; Elsevier Limited; Nature Publishing Group
• Subjects: Blotting, Northern; Cell Line; Design; Genetic Vectors - genetics; Genomes; HIV-1 - genetics; Humans; Lentivirus - genetics; Mutation; Original; Plasmids; RNA Interference - physiology; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; RNA, Viral - genetics; RNA, Viral - metabolism; T-Lymphocytes - virology; Virology; Viruses; Netherlands
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1038/mt.2009.176

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.