Inhibition of HIV-1 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

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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 lines and primary CD4؉ T cells. Inhibition of HIV replication was measured by p24 Gag content in supernatant, Northern blot analysis, and DNA PCR for products of reverse . 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 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 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 of viral . 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 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 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 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 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 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. 1A). These studies demonstrated that mRNA entering a cell Cells were cotransfected with TEV (the leader sequence from the tobacco through the plasma membrane could be specifically targeted by etch virus that promotes translation (9))-luciferase or TEV-gag encoding siRNA-mediated RNAi. The specificity of siRNA-mediated inhi- mRNA (0.1 ␮gin50␮l) and siRNA (0.1 ␮gin50␮l) complexed to Downloaded from lipofectin, as described previously (10). Luciferase-transfected cells were bition is demonstrated by the lack of effect observed with nonho- lysed with luciferase buffer (Promega, Madison, WI) and analyzed mologous siRNA. with luciferase substrate (Promega) in a Dynatech MLX luminometer The potential clinical use of siRNA-mediated inhibition of (Chantilly, VA). Supernatants were analyzed for p24 Gag protein content HIV-1 has many limitations. One is the ability to deliver siRNA in by ELISA (Beckman-Coulter, Miami, FL). vivo. siRNA were made using a T7 transcription reaction with HIV-1 infection

U87-CD4ϩ-CXCR4ϩ cells were infected with the HIV-1 IIIB virus. U87- http://www.jimmunol.org/ CD4ϩ-CCR5ϩ cells were infected with the HIV-1 Ba-L virus. T cell blasts were infected with both . HIV-1 viral stocks were acquired from the Center for AIDS Research at the University of Pennsylvania. Aliquots of 0.3 ng (9.8 infectious units (iu) of IIIB and 15 iu of Ba-L), 3 ng (98 iu of IIIB and 152 iu of Ba-L), or 10 ng (293 iu of IIIB and 456 iu of Ba-L) of virus in a final volume of 100 ␮l were added to cells. After2hat37°C, cells were washed extensively with PBS and cultured in complete medium, with added IL-2 (20 U/ml) for T cell blasts. Cells were transfected with siRNA (0.2 ␮gin50␮l) complexed with lipofectin, or siRNA was added

directly to serum-containing medium (4 ␮g/ml) either 24 h before or 3 days by guest on September 29, 2021 after infection. Infection was monitored by measuring supernatant-associ- ated p24 Gag protein content. Northern blot analysis RNA isolation and Northern blot analysis were performed as described (11). The blots were probed with gag (NcoI/BamHI fragment from pDAB72 (12)), HIV (BamHI fragment (13)), and GAPDH (pHcGAP from American Type Culture Collection, Manassas, VA). Bands were quanti- tated by PhosphorImager (Storm; Molecular Dynamics, Amersham Bio- sciences, Sunnyvale, CA). Quantitative PCR analysis Real time quantitative PCR analysis was performed 20–24 h after infec- tion. Virus used in PCR experiments was treated with DNase I (20 U/ml with5mMMg2ϩ) (Roche Diagnostics, Indianapolis, IN) for 30 min to remove contaminating DNA. Cells were lysed in 25 ␮l of lysis buffer (100 mM KCl, 20 mM Tris, pH 8.4, 0.1% Nonidet P-40, 500 ␮g/ml proteinase K (Sigma-Aldrich)). Real time PCR was performed in a PerkinElmer 7700 Sequence Detector (Boston, MA) with gag, strong stop, and GAPDH-spe- cific primers and probes, as described previously (14). Binding and inter- FIGURE 1. siRNA specifically suppresses protein production from lu- nalization of HIV were determined, as described previously (15). Briefly, ciferase or HIV-1 gag encoding mRNA. A, 293T cells were cotransfected cells were incubated with 10 ng HIV-1 Ba-L in 50 ␮lfor2hat37°C, with gag encoding mRNA, luciferase encoding mRNA, and gag- or lucif- washed three times in PBS, lysed, and assayed for p24 Gag protein content erase-specific chemically synthesized siRNA. Twenty-four hours later, su- by ELISA. pernatant was analyzed for p24 Gag protein content by ELISA, and cell lysates were analyzed for luciferase activity. Error bars represent SEM for Results duplicate wells and duplicate measurements. B, 293T cells transfected with siRNA suppresses protein production from transfected mRNA luciferasase encoding mRNA were treated with the indicated siRNA in the The delivery of dsRNA longer than 30 bp to mammalian cells presence (dark gray) or absence (light gray) of lipofectin complexing, suf- fix code 1-chemically synthesized, 2-T7 DNA oligos transcribed with stan- results in the activation of nonspecific mRNA degradation and ␣ dard nucleotides, and 3-T7 DNA oligos transcribed with fluorine-derivat- inhibition of protein synthesis through the IFN- system (4, 7). ized nucleotides. Three hours later, cells were lysed and luciferase activity The 21- to 23-bp siRNA are capable of inducing specific mRNA was quantitated. Data are expressed as the percentage of inhibition of li- degradation without activating this nonspecific pathway (4). PTGS pofectin or medium-treated luciferase mRNA-transfected 293T cells. Error in plants acts on cytoplasmic viral RNA (16). HIV-1 RNA is bars indicated the SEM. Data are representative of at least three experi- present in the cytoplasm after fusion and before reverse transcrip- ments for each condition. 5198 INHIBITION OF HIV REPLICATION BY RNAi

CTP and UTP replaced by 2Ј-F-dCTP and 2Ј-F-dUTP. This results in RNA that is resistant to RNase A (Epicentre, Madison, WI), but has the ability to be recognized by reverse transcriptases (Epicen- tre) and incorporated into RNA by RNA polymerases, and has the expected weight and structure by mass spectroscopy (19, 20). Lu- ciferase- and gag-specific siRNA made by chemical synthesis (luc1, gag1), T7 polymerase transcription of DNA oligomers (luc2, gag2), and T7 polymerase transcription with fluorine-deri- vatized nucleotides (luc3, gag3) were either complexed with lipo- fectin or added without complexing (naked) to 293T cells trans- fected with luciferase encoding mRNA. Three hours later, cells were lysed and luciferase activity was measured. Data are from at least three experiments for each condition and are expressed as percentage of inhibition of control (lipofectin- or saline-treated, luciferase mRNA-transfected 293T cells) luciferase levels (Fig. 1B). This demonstrated that similar inhibition was observed for each method of siRNA production, that fluorine-derivatized siRNA was effective and specific in inhibiting luciferase expres- sion, and that fluorine-derivatized siRNA could be delivered with- Downloaded from out lipofectin complexing and in the presence of serum. HIV-1-specific siRNA inhibits ongoing viral replication To test whether RNAi was capable of inhibiting ongoing viral replication, U87-CD4ϩ-CXCR4ϩ and U87-CD4ϩ-CCR5ϩ cell

lines capable of supporting T- and M-tropic HIV replication, re- http://www.jimmunol.org/ spectively, were infected with HIV-1 IIIB, T-tropic lab strain, and Ba-L, M-tropic virus. siRNA sequences targeting gag and the 3Ј- LTR were chosen to be completely homologous to both IIIB and Ba-L strains of HIV-1. The 3Ј-LTR sequence was chosen, as it is in the noncoding sequence before the poly(A) tail of all HIV-1 except nef encoding RNA, where it is within the coding sequence. Three days after infection, cells were transfected with enzymatically synthesized gag-specific or control siRNA or treated with lipofectin alone, and supernatants were collected to by guest on September 29, 2021 follow infection. p24 Gag protein analysis revealed that U87 cells transfected with siRNA homologous to gag showed a decrease in viral replication that lasted for 3 days, while control siRNA dis- played no depression of viral replication compared with lipofectin- treated cultures (Fig. 2, A and B). Loss of suppression of viral replication was observed by day 4 after siRNA treatment. The amount of specific suppression by gag-specific siRNA (0.2 ␮g/50 ␮l) ranged from 75 to 96% in U87 cells, which is similar to that observed for other mRNA targets by other investigators in mam- malian cell lines (1, 4, 21). The delivery of lower concentrations of siRNA resulted in a loss of viral suppression (Fig. 2C). FIGURE 2. HIV-1-specific siRNA suppresses ongoing HIV-1 replica- tion in U87 cells expressing CD4 and CCR5 or CXCR4. A, U87-CD4ϩ- To demonstrate that dsRNA was mediating the suppression of ϩ HIV-1 replication, U87-CCR5ϩ-CD4ϩ cells infected with HIV-1 CCR5 cells were infected with HIV-1 Ba-L strain (10 ng, 456 iu, in 100 ␮l) for 2 h and washed. Three days later, cells were transfected withT7 Ba-L were treated with the single-stranded sense or antisense RNA polymerase-transcribed gag-specific or control siRNA complexed chemically synthesized RNAs used to make the siRNAs. Suppres- with lipofectin (arrow). B, U87-CD4ϩ-CXCR4ϩ cells were infected with sion of HIV-1 replication was only observed when cells were HIV-1 IIIB strain (10 ng, 293 iu, in 100 ␮l) and treated, as described above. treated with gag-specific dsRNA (Fig. 3). Similar studies using T7 Supernatants were collected at the indicated time points and assayed for RNA polymerase-transcribed RNA also demonstrated specific in- p24 Gag protein content. C, U87-CD4ϩ-CCR5ϩ cells were infected with hibition only with gag-specific dsRNA (data not shown). HIV-1 Ba-L and treated with the indicated concentration of siRNA 3 days after infection. Three days later, p24 Gag protein content was measured. Inhibition of HIV-1 replication by siRNA occurs in primary Error bars represent SEM for duplicate wells and duplicate measurements. ϩ CD4 T cells Data are representative of at least three experiments. The inability to transfect primary cells with is a major obstacle in the field of gene therapy. Our previous results studying the delivery of mRNA by lipofectin transfection demonstrated ef- siRNA homologous to gag or the 3Ј-LTR of HIV-1 3 days after ficient delivery to primary dendritic cells. Over 95% of transfected HIV-1 Ba-L infection demonstrated suppression of viral replica- cells expressed the encoded protein (22). Given the success in tion, while control siRNA showed no effect on viral replication delivering RNA to primary cells, the ability of siRNA to suppress compared with lipofectin-treated cells (Fig. 4A). siRNA could be viral replication in PHA plus IL-2-activated primary CD4ϩ T cells added between 2 and 5 days postinfection and result in inhibition (T cell blasts) was then assessed. T cell blasts transfected with of viral replication. Once high levels of viral replication were The Journal of Immunology 5199

FIGURE 3. Suppression of HIV-1 replication is only observed when dsRNA homologous to gag is used. U87-CD4ϩ-CCR5ϩ cells were infected with 10 ng, 456 iu, of HIV-1 Ba-L in 100 ␮l. Three days later, cells were transfected with the indicated chemically synthesized siRNA, and two days later, p24 Gag protein content in the supernatant was measured. Error bars represent SEM for duplicate wells and duplicate measurements. Data are representative of five experiments. Downloaded from

present in the culture, day 6–9, inefficient inhibition was observed (data not shown). Sense and antisense ssRNA used to make the

HIV-specific siRNA did not inhibit HIV infection in primary T cell http://www.jimmunol.org/ blasts (data not shown). The 3Ј-LTR siRNA targets sequences at the 3Ј-terminus of the nef coding sequence before the poly(A) tail. This sequence is present in all spliced and unspliced RNA pro- duced from the provirus, while the sequences targeted by the gag- specific siRNA are only present in the unspliced viral RNA. siRNA specific for the 3Ј-LTR of HIV-1 gave similar suppression of viral replication compared with gag-specific siRNA. The com- Ј

bination of gag and 3 -LTR siRNAs at one-half the concentration by guest on September 29, 2021 of each gave greater inhibition of viral replication, suggesting a synergistic effect of targeting multiple sites. Unlike siRNA inhi- bition of HIV-1 replication in U87 cells, continued suppression of HIV-1 replication could sometimes be observed in T cell blasts. The amount of rebound in viral replication varied between 22 and 74% of the peak value of viral replication in the luciferase-specific FIGURE 4. HIV-1-specific siRNA inhibit HIV-1 replication in PHA- siRNA-treated cultures. No obvious cause(s) for the varied re- ϩ activated primary CD4 T cells. A, PBMC from an HIV-1-uninfected sub- bound in viral replication was evident. ject was stimulated for 3 days with PHA, washed, cultured in IL-2, and Gag and control ssRNA and siRNA were made with fluorine- infected for 2 h with HIV-1-Ba-L (10 ng, 456 iu, in 100 ␮l). Three days derivatized RNA and used to inhibit HIV-1 replication in T cell later, cells were transfected with T7 RNA polymerase-transcribed lucif- blasts. RNA without complexing to lipofectin was directly added erase or HIV-1-specific siRNA complexed with lipofectin (arrow). B,T to 3-day HIV-1-infected T cell blasts in serum-containing medium. cell blasts infected as in A were treated with fluorine-derivatized T7 RNA Three days later, culture supernatant was analyzed for p24 Gag polymerase-transcribed gag-specific ssRNA or siRNA or other siRNA in protein content. The gag-specific fluorine-derivatized siRNA ef- the absence of lipofectin complexing in the presence of serum. Superna- tants were collected at the indicated time points (A) or 3 days post-siRNA fectively inhibited HIV-1 replication (Fig. 4B). These data dem- treatment (B) and assayed for p24 Gag protein content. Error bars represent onstrate that HIV-specific siRNA made resistant to RNase A could be SEM for duplicate wells and duplicate measurements. C, Day 3 HIV-in- added to cells in the presence of serum without complexing to lipo- fected T cell blasts were treated with fluorine-derivatized control and HIV- fectin and mediate sequence-specific silencing of HIV replication. specific siRNA. Four days later, total RNA was obtained and analyzed for RNAi acts to specifically cleave RNA with sequence homology gag- and GAPDH-specific RNA by Northern blot. Data are representative to the 21-bp siRNA. Total cellular RNA was obtained from HIV- of at least six (A and B) or three (C) experiments. 1-specific or control siRNA-treated HIV-1-infected T cell blasts 1–4 days after siRNA treatment and analyzed by Northern blot for gag- and GAPDH-specific RNA. A significant, average 73%, re- duction in gag RNA was observed by gag and 3Ј-LTR-specific siRNA inhibits HIV-1 infection before reverse transcription siRNA, when normalized for GAPDH RNA (Fig. 4C). Inhibition We observed that siRNA-mediated RNAi could inhibit protein was observed for both Ba-L and IIIB strains of HIV-1 and on days production from mRNA that entered the cell through the plasma 1–4 post-siRNA treatment. Similar inhibition of HIV-1 RNA ac- membrane, and we sought to determine whether viral infection cumulation was obtained when a probe including Tat, Nef, and could be inhibited after viral entry and before reverse transcription. Env was used to probe RNA from control and HIV-1-specific U87-CD4ϩ-CXCR4ϩ and U87-CD4ϩ-CCR5ϩ cells were trans- siRNA-treated primary T cell blasts (data not shown). fected with control and HIV-1-specific siRNA. Twenty-four hours 5200 INHIBITION OF HIV REPLICATION BY RNAi

servation that certain plant viruses encode inhibitors of the PTGS silencing pathway (reviewed in Ref. 23). The role of RNAi in controlling viral infection in vertebrates is unknown. The predom- inant response to viruses with a dsRNA intermediate is mediated by the induction of IFN-␣ (reviewed in Ref. 24) and activation of enzymes that nonspecifically cleave mRNA and inhibit protein synthesis. Whether RNAi in mammals is functional in the control of viral replication is an important question. The enzymes used in RNAi are used by other systems, including nonsense-mediated RNA decay, which is an important system controlling mRNA fi- delity (25) and processing of small temporal RNA that are highly conserved and important in gene regulation (26), suggesting that their presence does not equate with functional use in RNAi. We demonstrate that siRNA-mediated inhibition of HIV-1 in- fection occurred at two points in the viral life cycle. After binding of virions to CD4 and chemokine receptor expressing target cells and fusion, the HIV-1 core containing viral genomic RNA, reverse transcriptase, and proteins necessary for nuclear import and chro-

FIGURE 5. HIV-1-specific siRNA suppresses infection before reverse Downloaded from ϩ ϩ mosomal integration is released into the cytoplasm. We demon- transcription. A, U87-CD4 -CXCR4 cells were transfected with lucif- strate that HIV-1-specific siRNA inhibit infection before the com- Ј erase- or HIV-1-specific chemically synthesized siRNA except for 3 -LTR, pletion of reverse transcription, most likely by cleaving genomic which was produced from T7 RNA polymerase-transcribed DNA oli- RNA in the cytoplasm. A second site of inhibition of HIV-1 rep- gomers, siRNA, followed by infection with DNase-treated HIV-1 IIIB vi- rus (10 ng, 293 iu, in 100 ␮l). B, PHA blasts were transfected with lucif- lication is also identified. After integration, the provirus becomes erase- or HIV-1-specific chemically synthesized siRNA and then infected a eukaryotic transcriptional element producing full-length viral with DNase-treated HIV-1 Ba-L (10 ng, 456 iu, in 100 ␮l). Twenty (A)or RNA that is cleaved to produce RNA encoding all of the viral http://www.jimmunol.org/ twenty-four (B) hours later, cells were lysed and analyzed for HIV-1 gag proteins necessary for replication. HIV-1-specific siRNA targets and GAPDH DNA content by real time quantitative PCR. Copies of HIV-1 this viral RNA for destruction. Although the studies presented can gag were normalized for copies of GAPDH and expressed as copies of definitively demonstrate inhibition at a preintegration stage by 5 HIV-1 gag DNA/10 cells. Data were obtained from at least four experi- RNAi, they cannot exclude that the suppression of viral replication ments for each condition. observed when cells are treated 3 days after infection occurs through a targeting of viral RNA made from the integrated provi- rus. A recently reported study using an infectious molecular clone later, cells were infected with HIV-1 strains IIIB and Ba-L, re- demonstrates that viral RNA made from the provirus (infectious spectively. Twenty hours later, cells were lysed and analyzed by molecular clone) can be targeted by RNAi (27), and our prelimi- by guest on September 29, 2021 real time quantitative PCR for gag DNA (14). Cell numbers for nary data are in agreement using siRNA (K. Kariko´, J. Capodici, each experimental condition were determined using GAPDH prim- and D. Weissman, unpublished observations). ers and probe, and copies of HIV-1 DNA per sample were cor- The ability of HIV-1 infection and replication to be inhibited by rected for cell number. Cells transfected with siRNA targeting gag RNAi mediated by siRNA presents a new approach to both study or the 3Ј-LTR had less gag DNA per cell (Fig. 5A and data not and treat HIV-1 and potentially other infectious and neoplastic shown), indicating inhibition before the completion of reverse diseases. Although not specifically addressed in this study, the transcription. Primary T cell blasts were treated with HIV-1- or ability to knock down specific viral proteins during different stages luciferase-specific siRNA 2 (data not shown) or 24 h before in- of infection is potentially achievable. This would allow the dis- fection with HIV-1 Ba-L virus. Twenty-four hours later, cells were section of the role of specific proteins during various stages of lysed and analyzed for gag and GAPDH DNA. HIV-1-specific infection, including latency and reactivation without concern for siRNA reduced the number of copies of HIV-1 gag DNA per cell, the effect of that protein during earlier stages of infection. indicating inhibition of viral infection before reverse transcription Similar to and gene therapy for HIV, many difficul- ϩ in primary CD4 T cells (Fig. 5B). Analysis with primers for ties in the delivery of siRNA in vivo can be envisioned. Although 5Ј-negative strand strong stop DNA gave similar results, suggest- we cannot measure the transfection efficiency of siRNA in primary ing that RNAi inhibited early and late stages of reverse transcrip- CD4ϩ T cells, we believe it is quite high, given that we can inhibit tion. To control for equal viral uptake by siRNA-treated cells, HIV infection and replication. Studies are ongoing to measure the ϩ ϩ U87-CD4 -CCR5 cells treated with HIV-specific and control efficiency of the generation of active RISC in primary cells after siRNA were incubated with HIV-1 Ba-L for 2 h, washed, lysed, siRNA transfection. The in vivo delivery of siRNA complexed to and analyzed for bound and taken up HIV by p24 Gag protein lipofectin presents an additional challenge. Studies using a tran- content analysis. Untreated, HIV-specific, and control siRNA- scription reaction with CTP and UTP replaced by 2Ј-F-dCTP and treated cells had similar levels of p24 Gag protein. 2Ј-F-dUTP for generation of siRNA, previously demonstrated to result in RNA resistant to RNase A, demonstrated that siRNA Discussion could be delivered to cells without lipofectin complexing and in RNAi is an antiviral response found in many organisms. The en- the presence of serum. Thus, in addition to demonstrating that zymes that mediate specific cleavage of mRNA through RNAi siRNA can inhibit viral infection in mammalian cells, we also have been observed in organisms from fungi to mammals (1). The show that primary CD4ϩ T cells can be efficiently targeted and that role of RNAi-like activities in controlling viral infection and trans- functional siRNA can be delivered in the absence of complexing posable elements has been established in plants and other inver- with a carrier, such as cationic lipids, and in the presence of serum. tebrate organisms. This includes analyses of RNAi/PTGS gene These observations, in addition to demonstrating inhibition of HIV knockouts that demonstrate increased viral replication and the ob- replication in primary CD4ϩ T cells, begin to address the multiple The Journal of Immunology 5201 complications likely to be encountered in the use of siRNA in 13. Kinter, A. L., P. Biswas, M. Alfano, J. S. Justement, B. Mantelli, C. Rizzi, animal models. A. R. Gatti, E. Vicenzi, P. Bressler, and G. Poli. 2001. Interleukin-6 and glu- cocorticoids synergistically induce human immunodeficiency virus type-1 ex- pression in chronically infected U1 cells by a long terminal repeat independent Acknowledgments post-transcriptional mechanism. Mol. Med. 7:668. 14. Scales, D., H. Ni, F. Shaheen, J. Capodici, G. Cannon, and D. Weissman. 2001. We thank Dr. Stuart Isaacs for critical review of the manuscript. The fol- Nonproliferating bystander CD4ϩ T cells lacking activation markers support HIV lowing reagents were obtained through the AIDS Research and Reference replication during immune activation. J. Immunol. 166:6437. Reagent Program, Division of AIDS, National Institute of Allergy and 15. Pohlmann, S., G. J. Leslie, T. G. Edwards, T. Macfarlan, J. D. Reeves, Infectious Diseases, National Institutes of Health: U87-CD4ϩ-CCR5ϩ and K. Hiebenthal-Millow, F. Kirchhoff, F. Baribaud, and R. W. Doms. 2001. 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