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Interference Interfering RNA-Mediated RNA Inhibition Of Inhibition of HIV-1 Infection by Small Interfering RNA-Mediated RNA Interference John Capodici, Katalin Karikó and Drew Weissman This information is current as J Immunol 2002; 169:5196-5201; ; of September 29, 2021. doi: 10.4049/jimmunol.169.9.5196 http://www.jimmunol.org/content/169/9/5196 Downloaded from References This article cites 27 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/169/9/5196.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Inhibition of HIV-1 Infection by Small Interfering RNA-Mediated RNA Interference1 John Capodici,* Katalin Kariko´,† and Drew Weissman2* RNA interference (RNAi) is an ancient antiviral response that processes dsRNA and associates it into a nuclease complex that identifies RNA with sequence homology and specifically cleaves it. We demonstrate that RNAi mediated by 21-bp dsRNA spe- cifically inhibits HIV-1 infection of permanent cell lines and primary CD4؉ T cells. Inhibition of HIV replication was measured by p24 Gag protein content in supernatant, Northern blot analysis, and DNA PCR for products of reverse transcription. The inhibition occurred at two points in the viral life cycle, after fusion and before reverse transcription and during transcription of viral RNA from integrated provirus. Treatment of HIV-infected activated CD4؉ T cells with a fluorine-derivatized siRNA that is resistant to RNase A yielded similar inhibition of HIV infection. In addition, the derivatized siRNA could be delivered without lipofectin complexing and in the presence of serum. The identification of RNAi activity against HIV-1 presents a new approach Downloaded from to study viral infections and a proof of concept of RNAi antiviral activity in mammalian cells. The Journal of Immunology, 2002, 169: 5196–5201. ibonucleic acid interference (RNAi)3 is an ancient and some. The integrated provirus is organized as a eukaryotic tran- conserved activity that controls viral infection and the scriptional unit with the 5Ј-long terminal repeat (LTR) containing expression of transposable elements and repetitive se- a strong enhancer/promoter region and the 3Ј-LTR encoding a R http://www.jimmunol.org/ quences (1). Originally described as an antiviral mechanism in polyadenylation site. A primary transcript of the HIV provirus plants, known as posttranscriptional gene silencing (PTGS), RNAi serves as the genomic RNA for future generations of HIV and is activity has been demonstrated in many organisms including hu- processed to ensure efficient translation of viral proteins. During mans (2–5). The most important characteristic of RNAi is that it is the viral life cycle, viral RNA is present in the cytoplasm of cells specific. dsRNA, when acting as part of RNAi, reduces expression after fusion and before reverse transcription, which presents a tar- of genes with sequence homology, but has no effect on the expres- get that when acted on can inhibit infection before proviral inte- sion of genes with unrelated sequence (1, 5, 6). RNAi functions as gration. A second recently described necessity for transcription of follows: 1) by identifying dsRNA and cleaving it into 21- to 23-bp the viral DNA in the cytoplasm after reverse transcription and fragments called small interfering (si) RNA by an enzyme called before nuclear import and integration offers an additional target by guest on September 29, 2021 Dicer with RNase III activity; 2) the siRNA fragments associate (8). Cells harboring proviral HIV, such as reservoirs or acutely with a multicomponent nuclease forming a complex known as the infected cells that have progressed past proviral integration, can RNA-induced silencing complex (RISC); 3) the RISC hybridizes also be targeted by RNAi-mediated inhibition of viral replication to target mRNA with a complementary region; 4) the target mRNA by targeting viral RNA transcripts produced from the provirus. is cleaved in the middle of the 21- to 23-bp complementary region (1, 5, 6). Present studies of RNAi in mammalian cells have dem- Materials and Methods onstrated that exogenous genes delivered by DNA transfection as Cell culture well as endogenous gene expression can be suppressed by the de- The 293T and U87-CD4ϩ-CCR5ϩ and CXCR4ϩ cells (AIDS Reference livery of siRNA (4, 7). In this study, we demonstrate suppression and Reagent Program) were propagated in DMEM supplemented with 10% of HIV-1 infection and replication in permanent cell lines and FBS, 15 mM HEPES buffer, and 2 mM L-glutamine (Life Technologies, ϩ primary activated CD4 T cells by siRNA specific for different Gaithersburg, MD) (DMEM complete) at 37°C in a 5% CO2 incubator. regions of HIV-1. PBMC were obtained under an Institutional Review Board-approved pro- tocol. PBMCs were stimulated with PHA (Sigma-Aldrich, St. Louis, MO) HIV is a ssRNA virus in the lentivirus family that reverse tran- at a concentration of 4 ␮g/ml for 3 days in RPMI 1640 medium supple- scribes and integrates its genetic material into the host chromo- mented with 10% FBS, 15 mM HEPES, and 2 mM L-glutamine (RPMI complete). Activated T cell blasts were washed three times in PBS and plated in RPMI complete medium supplemented with 20 U/ml IL-2 (AIDS † Departments of *Medicine and Neurosurgery, University of Pennsylvania, Philadel- Reference and Reagent Program). S2 cells were grown in DES expression phia, PA 19104 medium (Life Technologies) at 25°C. Received for publication June 6, 2002. Accepted for publication August 19, 2002. siRNA generation The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance siRNAs were constructed using two approaches. The first method used with 18 U.S.C. Section 1734 solely to indicate this fact. chemical synthesis of ssRNA (Dharmacon, Lafayette, CO). The RNA se- 1 This work was supported in part by a grant from the Pediatric AIDS Foundation, quences corresponded to conserved regions of the HIV-1 core protein Gag 27-PG-51257. and the coding sequence of firefly luciferase. The sequences were: luc1, Ј Ј Ј 2 Address correspondence and reprint requests to Dr. Drew Weissman, University of 5 -CAUUCUAUCCUCUAGAGGAUGdTdT-3 , and gag1,5-GAGAAC Pennsylvania, 522 B Johnson Pavilion, Philadelphia, PA 19104. E-mail address: CAAGGGGAAGUGACAdTdT-3Ј (HXB2 position 1475). The second ap- [email protected] proach used DNA oligomers containing a T7 dsRNA polymerase promoter, 3 Ј followed by 21 bp of ssDNA homologous to the sense or antisense regions Abbreviations used in this paper: RNAi, RNA interference; CTP, cytidine 5 - Ј triphosphate; iu, infectious unit; LTR, long terminal repeat; PTGS, posttranscriptional to be targeted and terminated with2Ctoform a 3 -overhang as a template gene silencing; RISC, RNA-induced silencing complex; siRNA, small interfering for transcription with T7 RNA polymerase (Megashortscript; Ambion, RNA. Austin, TX). Regions of sequence chosen for the production of siRNA by Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 5197 enzymatic transcription contained a C and a G separated by 19 nt. This tion, and transcription of the provirus before nuclear import and allowed a G to be present as the first nucleotide downstream from the T7 integration may be required for efficient infection (8). Thus, these promoter in both sense and antisense DNA oligomers, ensuring efficient intermediates present targets for RNAi-mediated suppression. As a transcription. siRNA made by T7 transcription with standard nucleotides were designated gag2, luc2, and 3Ј-LTR2. The third approach used fluo- model of these targets, we sought to determine whether siRNA- rine-derivatized cytidine 5Ј-triphosphate (CTP) and UTP to generate mediated RNAi could specifically suppress translation from RNA ssRNA with T7 polymerase (DuraScribe T7 transcription kit; Epicentre, delivered to the cytoplasm through the plasma membrane. We pre- Madison, WI) using the same DNA oligomers described above. These pared siRNA by two methods, one chemical synthesis as described siRNA were called gag3, luc3, 3Ј-LTR3. The sequences were: luc2 and 3, 5Ј-CAUUCUAUCCUCUAGAGGAUG-3Ј; gag2 and 3, 5Ј-GGGCAAG (17), and the second through T7 RNA polymerase-mediated tran- CAGGGAGCUAGAAC-3Ј (HXB2 position 896); and 3Ј-LTR2 and 3, 5Ј- scription from DNA oligomers (18). GAUGGUGCUUCAAGCUAGUAC-3Ј (HXB2 position 9215). RNA oli- Two mRNAs, one encoding the HIV-1 core protein Gag and the gomers in water were annealed at a concentration of 50 mM each with an second encoding firefly luciferase, were cotransfected into 293T initial hold at 90°C for 1 min, followed by a time-controlled cooling to cells along with luciferase- or gag-specific siRNA. Gag expression room temperature over 60 min. Annealing was confirmed on 15% Tris- borate-EDTA polyacrylamide gels (Bio-Rad, Hercules, CA), followed by was reduced by siRNA homologous to gag with no effect on lu- ethidium bromide staining and UV illumination. ciferase production, while siRNA homologous to luciferase did not affect Gag protein production while inhibiting luciferase activity RNA transfection and analysis (Fig.
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