Design of extended short hairpin RNAs for HIV-1 inhibition.
RNA interference (RNAi) targeted towards viral mRNAs is widely used to block virus replication in mammalian cells. The specific antiviral RNAi response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular expression of short hairpin RNAs (shRNAs). For HIV-1, both approaches resulted in profound inhibition of virus replication. However, the therapeutic use of a single siRNA/shRNA appears limited due to the rapid emergence of RNAi-resistant escape viruses. These variants contain deletions or point mutations within the target sequence that abolish the antiviral effect. To avoid escape from RNAi, the virus should be simultaneously targeted with multiple shRNAs. Alternatively, long hairpin RNAs can be used from which multiple effective siRNAs may be produced. In this study, we constructed extended shRNAs (e-shRNAs) that encode two effective siRNAs against conserved HIV-1 sequences. Activity assays and RNA processing analyses indicate that the positioning of the two siRNAs within the hairpin stem is critical for the generation of two functional siRNAs. E-shRNAs that are efficiently processed into two effective siRNAs showed better inhibition of virus production than the poorly processed e-shRNAs, without inducing the interferon response. These results provide building principles for the design of multi-siRNA hairpin constructs.
Liu
Ying Poi
YP
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, Amsterdam, The Netherlands.
Haasnoot
Joost
J
Berkhout
Ben
B
D019380:Anti-HIV Agents / Q000737:chemistry* / Q000494:pharmacology; D002460:Cell Line; D017930:Genes, Reporter; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D016899:Interferon-beta / Q000096:biosynthesis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000494:pharmacology; D022661:RNA, Untranslated / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
D019380:Anti-HIV Agents; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; D016899:Interferon-beta
Gene Ther. 2006 Oct;13(19):1403-13
16708080
Nat Genet. 2003 Jul;34(3):263-4
12796781
Nat Cell Biol. 2003 Sep;5(9):834-9
12942087
Mol Biosyst. 2005 Dec;1(5-6):382-90
16881007
Retrovirology. 2006;3:2
16398936
J Biomol Tech. 2002 Dec;13(4):265-71
19498992
Retrovirology. 2006;3:57
16948865
Genes Dev. 2002 Apr 15;16(8):948-58
11959843
Nat Biotechnol. 2002 May;20(5):500-5
11981565
Nature. 1998 Feb 19;391(6669):806-11
9486653
J Virol. 2002 Sep;76(18):9225-31
12186906
J Virol. 2006 Aug;80(15):7658-66
16840344
Nature. 2000 Mar 16;404(6775):293-6
10749213
RNA. 2005 May;11(5):674-82
15811921
Nucleic Acids Res. 2005;33(2):796-804
15687388
Cell. 2000 Mar 31;101(1):25-33
10778853
Mol Cell Biol. 1992 Nov;12(11):5238-48
1357546
J Virol. 2003 Nov;77(21):11531-5
14557638
Nature. 2001 May 24;411(6836):494-8
11373684
Virology. 1991 Dec;185(2):661-72
1683726
Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):183-8
12518064
Virology. 2006 Feb 20;345(2):457-67
16297951
Biochem Soc Trans. 1997 May;25(2):509-13
9191145
Nature. 2002 Jul 25;418(6896):435-8
12087358
Nat Med. 2002 Jul;8(7):681-6
12042777
J Gene Med. 2004 Jul;6(7):715-23
15241778
Science. 2002 Apr 19;296(5567):550-3
11910072
Nat Biotechnol. 2005 Feb;23(2):227-31
15619616
J Virol. 2005 Oct;79(20):12893-904
16188991
J Invest Dermatol. 2003 Jun;120(6):990-7
12787126
Science. 1999 Oct 29;286(5441):950-2
10542148
Gene Ther. 2006 Jun;13(11):883-92
16496015
J Virol. 2004 Mar;78(5):2601-5
14963165
J Virol. 2000 Apr;74(8):3740-51
10729149
Mol Ther. 2007 Mar;15(3):534-41
17213835
Mol Ther. 2006 Dec;14(6):883-92
16959541
Nature. 2006 May 25;441(7092):537-41
16724069
Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
12824337