The RNA interference (RNAi) phenomenon is a recently discovered process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of mRNA containing the same sequence. We designed mammalian expression vectors that direct the synthesis of small interfering RNA (siRNA)-like transcripts and examined them for their siRNA-mediated gene interference targeting the env gene (NL4–3:7490–7508, E7490). We constructed siRNA expression vectors for two different strands (sense and antisense; tandem promoter) and for siRNA expressed from the short hairpin RNA (shRNA). The inhibition efficacy on HIV–1 replication differed between these two vectors. Notably, the shRNA vector pU6-env-shRNA inhibited p24 production more effectively than the tandem promoter expression vector pU6-env-siRNA. Furthermore, we examined the ability of lentiviral vectors expressing shRNA to suppress HIV-1 expression in HIV-1-infected SupT1 cells. The env-shRNA (E 7490) almost completely suppressed HIV-1 expression in infected cells for up to 15 days.
BrummelkampTRBernardsR & AgamiR (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science296: 550–553.
2.
ElbashirSMLendeckelW & TuschlT (2001a) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes & Development15: 188–200.
3.
ElbashirSMMartinezJPatkaniowskaALendeckelW & TuschlT (2001b) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO Journal20: 6877–6888.
4.
ElbashirSMHarborthJLendeckelWYalcinAWeberK & TuschlT (2001c) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature411: 494–498.
5.
FollenziAAillesLEBakovicSGeunaM & NaldiniL (2000) Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nature Genetics2: 217–222.
6.
JacqueJMTriquesK & StevensonM (2002) Modulation of HIV-1 replication by RNA interference. Nature418: 435–438.
7.
LeeNSDohjimaTBauerGLiHLiMJEhsaniASalvaterraP & RossiJJ (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnology20: 500–505.
8.
LiMJBauerGMichienziAYeeJKLeeNSKimJLiSCastanottoDZaiaJ & RossiJJ (2003) Inhibition of HIV-1 infection by lentiviral vectors expressing pol III-promoted anti-HIV RNAs. Molecular Therapy8: 196–206.
9.
ManganiniMSerafiniMBambacioniFCasatiCErbEFollenziANaldiniLBernasconiSGaipaGRambaldiABiondiAGolayJ & IntronaM (2002) A human immunodeficiency virus type 1 pol gene-derived sequence (cPPT/CTS) increases the efficiency of transduction of human nondividing monocytes and T lymphocytes by lentiviral vectors. Human Gene Therapy13: 1793–1807.
10.
MautinoMR & MorganRA (2002) Enhanced inhibition of human immunodeficiency virus type 1 replication by novel lentiviral vectors expressing human immunodeficiency virus type 1 envelope antisense RNA. Human Gene Therapy13: 1027–1037.
11.
McManusMTPetersenCPHainesBBChenJ & SharpPA (2002) Gene silencing using micro-RNA designed hairpins. RNA8: 842–850.
12.
MichienziACastanottoDLeeNLiSZaiaJA & RossiJJ (2003) RNA-mediated inhibition of HIV in a gene therapy setting. Annals of the New York Academy of Sciences1002: 63–71.
13.
MontanerJS & MellorsJW (1999) Effective salvage therapy for HIV-1 infection — An unmet challenge. Antiviral Therapy4: 59–60.
14.
MukhtarMDukeHBouHamdanM & PomerantzRJ (2000) Anti-human immunodeficiency virus type 1 gene therapy in human central nervous system-based cells: An initial approach against a potential virus reservoir. Human Gene Therapy11: 347–359.
15.
MyslinskiEAmeJCKrolA & CarbonP (2001) An unusually compact external promoter for RNA polymerase III transcription of the human H1 RNA gene. Nucleic Acids Research29: 2502–2509.
16.
NaldiniLBlomerUGallayPOryDMulliganRGageFHVermaIM & TronoD (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science272: 263–267.
PaddisonPJCaudyAABernsteinEHannonGJ & ConklinDS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Development16: 948–958.
19.
PandyaSBoris-LawrieKLeungNKAkkinaR & PlanellesV (2001) Development of an Rev-independent, minimal simian immunodeficiency virus-derived vector system. Human Gene Therapy12: 847–857.
20.
ParkWSMiyano-KurosakiNHayafuneMNakajimaEMatsuzakiTShimadaF & TakakuH (2002) Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference. Nucleic Acids Research30: 4830–4835.
21.
ParkWSHayafuneMMiyano-KurosakiN & TakakuH (2003) Specific HIV-1 env gene silencing by small interfering RNAs in human peripheral blood mononuclear cells. Gene Therapy10: 2046–2050.
22.
PaulCPGoodPDWinerI & EngelkeDR (2002) Effective expression of small interfering RNA in human cells. Nature Biotechnology20: 505–508.
23.
QinXFAnDSChenIS & BaltimoreD (2003) Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proceedings of the National Academy of Sciences USA100: 183–188.
24.
RubinsonDDillonCPKwiatkowskiAVSieversCYangLKopinjaJRooneyDLIhrigMMMcManusMTGertlerFBScottML & Van ParijsL (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nature Genetics33: 401–406.
25.
SakaiAHirabayashiYAizawaSTanakaMIdaS & OkaS (1999) Investigation of a new p24 antigen detection system by the chemiluminescence-enzyme-immuno-assay. Journal of Japanese Association for Infectious Diseases73: 205–212.
26.
SchroersRDavisCMWagnerHJ & ChenSY (2002) Lentiviral transduction of human T-lymphocytes with a RANTES intrakine inhibits human immunodeficiency virus type 1 infection. Gene Therapy9: 889–897.
27.
SongELeeSKDykxhoornDMNovinaCZhangDCrawfordKCernyJSharpPALiebermanJManjunathN & ShankarP (2003) Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. Journal of Virology77: 7174–7181.
28.
StewartSDykxhoornDMPalliserDMizunoHYuEYAnDSSabatiniDMChenISHahnWCSharpPAWeinbergRA & NovinaCD (2003) Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA9: 493–501.
29.
SuiGSoohooCAffarelBGayFShiYForresterWC & ShiY (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proceedings of the National Academy of Sciences USA99: 5515–5520.
30.
TavernarakisNWangSLDorovkovMRyazanovA & DriscollM (2000) Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genetics24: 180–183.
31.
YuJYDeRuiterSL & TurnerDL (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proceedings of the National Academy of Sciences USA99: 6047–6052.
