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.
Liu
Ying Poi
YP
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
von Eije
Karin Jasmijn
KJ
Schopman
Nick C T
NC
Westerink
Jan-Tinus
JT
ter Brake
Olivier
O
Haasnoot
Joost
J
Berkhout
Ben
B
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
D034741:RNA, Small Interfering; D012367:RNA, Viral