title: Combinatorial RNAi against HIV-1 using extended short hairpin RNAs.
abstract: 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.
authors: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors: Ying Poi
authors: YP
authors: Liu
authors: Karin Jasmijn
authors: KJ
authors: von Eije
authors: Nick C T
authors: NC
authors: Schopman
authors: Jan-Tinus
authors: JT
authors: Westerink
authors: Olivier
authors: O
authors: ter Brake
authors: Joost
authors: J
authors: Haasnoot
authors: Ben
authors: B
authors: Berkhout
mesh_terms: D015152:Blotting, Northern; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000821:virology
chemical_list: D034741:RNA, Small Interfering; D012367:RNA, Viral