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Gene Therapy (1998) 5, 166516761998 Stockton Press All rights reserved 0969-7128/98 $12.00

http://www.stockton-press.co.uk/gt

High level inhibition of HIV replication with combinationRNA decoys expressed from an HIV-Tat induciblevector

C Fraisier1, A Irvine2, C Wrighton2, R Craig3 and E Dzierzak11Erasmus University Rotterdam, Department of Cell Biology and Genetics, Rotterdam, The Netherlands; 2Cobra Therapeutics Ltd,and 3Therexsys Ltd, The Science Park, University of Keele, UK

Intracellular immunization, an antiviral gene therapy combination TAR+RRE decoy when compared with theapproach based on the introduction of DNA into cells to single decoys or the tat-ribozyme. We also show that thestably express molecules for the inhibition of viral gene Tat-inducible HIV promoter directs a higher level of steady-expression and replication, has been suggested for inhi- state transcription of decoys and inhibitors and that higher

bition of HIV infection. Since the Tat and Rev proteins play levels of expression directly relate to increased levels ofa critical role in HIV regulation, RNA decoys and ribozymes inhibition of HIV infection. Furthermore, a stabilization ofof these sequences have potential as therapeutic molecu- the 3 end of TAR+RRE inhibitor transcripts using a-glo-lar inhibitors. In the present study, we have generated sev- bin 3 UTR sequence leads to an additional 15-folderal anti-HIV molecules; a tat-ribozyme, RRE, RWZ6 and increase in steady-state RNA levels. This cassette whenTAR decoys and combinations of decoys, and tested them used to express the best combination decoy inhibitorfor inhibition of HIV-1 replication in vitro. We used T cell TAR+RRE, yields high level HIV inhibition for greater thanspecific CD2 gene elements and regulatory the HIV 3 weeks. Taken together, both optimization for high levelinducible promoter to direct high level expression and a 3 expression of molecular inhibitors and use of combinationsUTR sequence for mRNA stabilization. We show that HIV of inhibitors suggest better therapeutic application in limit-replication was most strongly inhibited with the ing the spread of HIV.

Keywords:HIV; RNA decoys; ribozyme; HIV-inducible vector; gene therapy

Introduction

Over the past 10 years, molecular approaches to combatHIV infection have been suggested as potential clinicaltherapies and are generally referred to as intracellularimmunization.1 This method relies on the introduction ofDNA into the cells which can be, or are already, HIV-infected. DNA constructs thought to be useful in intra-cellular immunization strategies are those which expressinhibitor or antiviral molecules such as: dominant nega-tive regulatory proteins or receptors; RNA inhibitorssuch as antisense molecules or ribozymes; RNA decoy;and anti-HIV-specific antibodies. With some exceptions,

most of the antiviral strategies are based on moleculesencoded by HIV itself, mainly the regulatory or structuralproteins. Tat and Rev are two of the HIV regulatory pro-teins that are critical to HIV replication and have beenmost extensively characterized, thus offering great poten-tial for molecular inhibition strategies.2

The Tat regulatory protein acts as a potent transactiv-ator of HIV long terminal repeat (LTR) directed transcrip-tion. Tat binds directly to the Tat activation region (TAR)which is located immediately 3of the site of initiation of

Correspondence: EA Dzierzak, Erasmus University Rotterdam, Depart-ment of Cell Biology and Genetics, PO Box 1738, 3000 DR Rotterdam,The Netherlands

Received 20 May 1998; accepted 20 July 1998

HIV-1 transcription3 and increases both the initiation andelongation of HIV RNAs. Tat-mediated transactivation ofHIV promoter-driven transcription has been shown toincrease reporter gene expression 10- to 1000-fold.49

While the HIV-1 Tat protein binds with high affinity toboth HIV-1 and HIV-2 TAR RNAs and consequently cantransactivate both HIV-1 and HIV-2 LTRs, the HIV-2 Tatprotein binds with high affinity to and transactivates onlyits own HIV-2 LTR.10,11 Rev is a small nuclear regulatoryprotein expressed from multiply spliced HIV RNA. Incontrast to Tat, Rev acts post-transcriptionally by bindingto a cis-acting RNA sequence, the Revresponse element(RRE). The Rev-RRE interaction strongly enhances HIV-

1 production by facilitating the extranuclear transport ofunspliced (9 kb) and single spliced (4 kb) mRNAs thatencode HIV-1 structural proteins.12

Since the Tat and Rev regulatory proteins play a criticalrole in the HIV infectious cycle, protein-based gene ther-apy strategies with transdominant mutants have beendeveloped by several laboratories. These studies demon-strate that Tat and Rev mutant proteins inhibit HIV repli-cation in lymphoid cell lines1316 as well as in CD4+ pri-mary hematopoietic cells15,17,18 and CD34+ hematopoieticprecursors.19,20 However, such mutant proteins ifexpressed inin vivo gene therapy applications may elicitan immune response against themselves. Thus, RNA-based inhibition strategies have been proposed2 so as to

eliminate the risk of rejection.


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Regulated expression of HIVmolecular inhibitors

C Fraisier et al

1666RNA decoy strategies use short RNA oligonucleotides

which mimic critical regulatory sequences in HIV.Decoys such as TAR21 and RRE22 could inhibit the actionof Tat and Rev regulatory proteins in HIV replication bysequestering these RNA binding proteins. TAR RNAfrom HIV-1 forms a stable stem-loop structure (from +1to +60), the maintenance of which is critical for Tat-

mediated transactivation.23,24

The HIV-2 TAR element ismore complex than HIV-1 TAR and the secondary struc-ture consists of an additional loop within the predictedTat recognition sequence. The requirement for recog-nition of two such loops by HIV-2 Tat may explain theincomplete reciprocal transactivation by HIV-1 and HIV-2 Tat.25,26 Located within the envgene is the HIV-1 RRE.RRE RNA is predicted to form a central stem-loop andfive stem-loop structures.27,28 Biochemical analyses haveidentified a high affinity Rev-binding site in stem-loop IIof HIV-1 RRE.29,30 To date, several studies have shownthe inhibition of HIV replication by the expression ofeither TAR or RRE RNA decoy sequences.21,22,31,32

Another category of RNA inhibitor consists of HIV

RNA specific ribozymes. Ribozymes are described ascatalytic RNA molecules capable of recognizing andcleaving a specific target RNA.33 Classically defined,hammerhead ribozymes are RNA molecules thathybridize to complementary RNA sequences in whichthe central part of the sequence forms a specific second-ary structure where reactive groups are located thatmediate specific cleavage of the target RNA at a consen-sus GUC target.34 Ribozymes are potent genetic therapiesagainst HIV, as they cleave both incoming HIV genomicRNA and newly transcribed viral mRNA.35 HIV-specificribozymes targeting the 5 U5 region,36,37 Tat,38,39 Rev,Env40 or Gag41 have been made and have been shown toinhibit HIV replication. To optimize the inhibiting effectsof the protein-based and RNA-based strategies, manystudies have used combinations of these diverse mol-ecules: Tat and Rev transdominant mutants,14,42 trans-dominant mutant and RNA decoy43 and RNA decoyand ribozyme.44,45

In exploring the potential of RNA-based strategies forinhibiting the spread of HIV for possible clinical appli-cations, we tested several molecular inhibitors; RNAdecoys corresponding to RRE, RWZ6 (a part of RRE stem-loop II which binds three molecules of Rev)46 and TARand a combination of these. We also tested the inhibitingpotential of a hammerhead ribozyme targeted to theentire Tat RNA. The aim of our study was to determinethe potency of these inhibitors using CD2-basedexpression vectors designed to give high level virus-

inducible and T lymphocyte-specific expression. Wepresent data showing that Tat-inducibility together withmRNA stabilization provide the highest level ofexpression of inhibitor molecules. Moreover, we demon-strate for the most effective inhibitor, the combinationTAR+RRE decoy, a clear correlation between increasedexpression level and increased inhibition of HIVreplication.

Results

The aims of our study were: (1) to construct efficientexpression cassettes, including a Tat-inducible expressioncassette, for the high level transcription of HIV RNA-

based molecular inhibitors; (2) to determine which mol-

ecular constructs alone or in combination wouldefficiently inhibit HIV production; (3) to attain the highestexpression of the best single or combination HIV molecu-lar inhibitor and determine quantitatively whether higherexpression levels correlate with greater inhibition of HIV.

Generation of expression cassettes, HIV inhibitors andstably transfected cell linesGenerally, promoters directing ubiquitous and constitut-ive transcription have been utilized for expression of HIVinhibitors.14,21,22,36,3840,42,43,45 However, control elementsknown as locus control regions (LCRs) have been shownto confer high level tissue specific, chromosomal positionindependent expression.47 In the case of the human-globin locus LCR, high level, erythroid-specificexpression is obtained.48 Likewise the human CD2(hCD2) gene LCR yields high level, sustained copy num-ber dependent T cell-specific gene expression which isindependent of the chromosomal integration site of thetransgene.49 Thus, the use of strong LCRs for high level

tissue specific transcription holds great promise for sus-tained expression of transgenes. Since CD4 T cells are thepredominant target of HIV infection we used the hCD2gene promoter, 3untranslated region (UTR) and LCR asour first generation expression vector: pVA-CD2described by Zhumabekovet al.50 In a strategy to obtaininducible higher level expression of HIV inhibitory mol-ecules, an HIV-Tat inducible vector was constructed bysubstitution of the hCD2 promoter by the HIV-2 LTR toyield pVA-HIV. To increase steady state levels of RNAfurther, a derivative of pVA-HIV was used which con-tains the human -globin second intron (betaIVS-II) andpolyadenylation sequences in place of the endogenousCD2 3 UTR to provide RNA stabilization.5153 Hence a

third vector was constructed, pVA-HIV-3, which con-tains the HIV2LTR,8 the human -globin 3UTR and thehCD2 LCR. The construction and characterization of thisconstruct is described elswhere (Wrighton et al, manu-script in preparation). These vectors pVA-CD2, pVA-HIVand pVA-HIV-3 (Figure 1a) were used to express HIVmolecular inhibitors in stably transfected CEM humanT cells.

Several RNA-based molecular inhibitor sequences ofHIV were constructed as described in the Materials andmethods section and in Figure 1b. The Tat regulatory pro-tein serves as a direct transcriptional transactivator ofHIV through its physical association with its cis-actingtarget, TAR. Overexpression of TAR RNA has been

shown previously by others to inhibit HIV replication.21

Similarly, regulatory protein Rev acts through a physicalassociation with its cis-target, RRE, to induce theexpression of unspliced HIV-1 mRNAs. Hence TAR andRRE decoys (and RWZ6, a shortened version of RRE)were constructed. Also, since a hammerhead ribozyme ofthe HIVtat gene was shown previously to cleave specifi-cally targeted HIV-RNA sequences, we constructed a Tat-specific ribozyme from the HIV-1 tatcoding region. Thesemolecular inhibitor constructs were inserted singly or incombination into the above-described expression cas-settes containing the puromycin selectable marker gene.Human CD4-positive CEM T cells were stablytransfected. Transfectant populations were selected with

puromycin and used for HIV inhibition studies.


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Figure 1 (a) Schematic representation of the pVA vector used for expression of the HIV inhibitory genes. HIV inhibitory genes encoding RNA decoysor Tat-ribozyme (1) were inserted in the multiple cloning site of the pVA vector containing the human CD2 (hCD2) promoter (pVA-CD2) or theHIV2LTR (pVA-HIV) promoter (2), a 3 untranslated region (UTR) from the hCD2 gene or from the human -globin gene so as to stabilize RNAs(3) and the hCD2 locus control region (LCR) (4) to direct copy number-dependent and position-independent specific expression in T cells. (b) Schematicrepresentation of the RNA decoys (RRE, RWZ6, TAR) and the tat-specific ribozyme stably expressed in CEM cells for infection experiments. Criticalnucleotides forming the high affinity binding site in RRE and RWZ6, and the consensus GUC sequence at the cleavage site for the ribozyme are high-lighted.


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1668Combination RNA decoys inhibit HIV infection of CEMcell transfectants most efficientlyTo determine if the inhibitor sequences generated in ourlaboratory were effective and to determine which inhibi-tors or combination inhibitors were best in preventing thespread of HIV, puromycin-selected stable populations oftransfected cells were infected with HIV at several MOIs

and tested for the production of either p24 or reversetranscriptase (RT) at various times after infection. Controland transfectant populations were analyzed for the pres-ence of CD4 surface antigen. All cell lines were 91% to99% CD4-positive with similar intensity of staining (datanot shown).

Transfected CEM populations carrying pVA-CD2-RWZ6, pVA-CD2-TAR or pVA-CD2-TAR+RWZ6 wereinfected with HIV-1IIIB/LAI at an MOI of 0.0004. Figure 2ashows that CEM transfectants carrying the RWZ6 decoyare almost as infectable as control CEM cells as determ-ined by RT activity at day 9 (2.0 0.29 c.p.m./105 mlcompared with 3.8 0.67 for the control cells). The TARdecoy on its own was able to reduce RT levels only up

to day 9 (0.68

0.07 c.p.m./10

5

ml) when compared withCEM control cells. In contrast, transfectants carrying aTAR decoy in addition to RWZ6 (TAR + RWZ6) keep RTlevels to less than 50% of maximum for at least 3 daysas compared with the control (8.3 0.48 c.p.m./105 ml atday 13 compared with 17.1 3.0 for the control cells).

To verify this finding, these transfectant populationswere infected with five-fold more HIV-1IIIB/LAI (MOI,0.002) (Figure 2b). At this MOI the RWZ6 and TAR trans-fected cells only slightly resist HIV infection. At day 9of culture CEM control cells are maximally infected (RTactivity 15.1 0.7 c.p.m./105 ml) while RWZ6 and TARtransfectant populations are 75% infected (11.61.9c.p.m./105 ml and 11.0 1.1 c.p.m./105 ml,respectively). Similar to the result at the lower MOI, theTAR+RWZ6 transfectants inhibit best. At day 9 only 40%RT level (5.8 2.6 c.p.m./105 ml) was found in super-natants of TAR+RWZ6 cells as compared with that ofcontrol cells. All three transfectant populations reachpeak level infection at day 13 of culture. Hence, theTAR+RWZ6 combination decoy appears to be the bestinhibitor of HIV-1 replication.

Molecular inhibitors were next tested in the context ofthe pVA-HIV expression cassette. Since the single RNAdecoys were unable to inhibit HIV replication efficientlyat the high MOIs, we compared these and other molecu-lar inhibitor constructs in cell transfectants infected withHIV-1IIIB/LAI at a very low MOI of 0.0001 (Figure 3). HIVinfection was determined by sensitive p24 immunoassay

on samples taken every 34 days up to 23 days afterinfection. CEM transfected populations carrying RRE,RWZ6 or tat ribozyme sequences all inhibit HIV infectionto a similar degree, giving 9095% inhibition at day 20as compared with control CEM infected cells. Infectionin these populations increases at day 23 giving similarlevels of p24 (2.7 to 4.4 g/ml). In contrast, the CEMpopulation carrying the RRE sequence in combinationwith a TAR decoy (TAR+RRE) shows complete inhi-bition, with no or barely measurable p24 up to day 23 (6ng/ml). This strong inhibition was confirmed when theTAR+RRE transfectant population was tested at higherMOIs (see next section). Thus, the TAR+RRE double RNAdecoy combination sequence is the best molecular inhibi-

tor of HIV infection. It is interesting to note that HIV

Figure 2Inhibition of HIV replication in CEM transfectants populationscontaining the combination TAR+RWZ6 RNA decoy. CEM cells stablytransfected with pVA-CD2-TAR (), RWZ6 (), TAR+RWZ6 ()were in vitro infected with HIV-1IIIB/LAI and at MOI of 0.0004 (a) andMOI of 0.002 (b). Cell-free supernatants were tested every 34 days forthe presence of reverse transcriptase activity (RT). HIV replication wascompared with that in control infected CEM cells (). Uninfected CEMcells () were used as the negative control. Triplicate samples were ana-lyzed for all time-points and the average of the mean is shown. At the

MOI of 0.0004 (a), values of RT activity (c.p.m./105

ml) at day 13 afterinfection are 17.1 3.0 for CEM cells, 17.7 3.1 for RWZ6, 14.6 1.8for TAR and 8.3 0.5 for TAR+RWZ6 cells. At the MOI of 0.002 (b),values of RT activity (c.p.m./105 ml) at day 9 after infection are 15.1 0.7for CEM cells, 11.61.9 for RWZ6, 11.0 1.1 for TAR and 5.8 2.6for TAR+RWZ6 cells.

infection is inhibited in pVA-HIV vector transfectants ascompared with the pVA-CD2 vector transfectants andnontransfected CEM cell controls. This inhibition is mostlikely due to the presence of the TAR sequence in pVA-HIV, acting as a molecular decoy as suggested byothers.54 Our results showing slight inhibition of HIVinfection with a single TAR decoy in the CD2 cassette

further support this suggestion (Figure 2a and b).


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Figure 3 Inhibition of HIV replication in CEM transfectant populationscontaining the combination TAR+RRE RNA decoy. CEM cells stablytransfected with pVA-HIV-Rbz tat (), RWZ6 (), RRE () andTAR+RRE () were challenged with HIV-1IIIB/LAIat an MOI of 0.0001.Cell-free supernatants were tested every 34 days for the presence of HIV-1 p24 core protein. HIV replication was compared with that in controlCEM cells () and with that in pVA-CD2 () and pVA-HIV ()-trans-fected cells. Uninfected CEM cells () were used as the negative control.

Quantitative expression of RNA decoys and the tat-ribozyme in stably transfected cellsWhile initial experiments demonstrated the effectivenessof the molecular inhibitors in delaying HIV infection, wesought to determine the quantitative levels of expressionof each of the inhibitors in the various cassettes. Wefocused on the inhibitors tat-ribozyme, RRE andTAR+RRE. To confirm the presence of the inhibitor con-structs in the puromycin-resistant transfectant cell popu-lations and to determine the copy number, Southern blothybridization was performed. Using a puromycin gene-specific probe to detect a 1.4 kb XhoI fragment containedin the vector sequence of pVA-CD2 and pVA-HIV con-structs, we found copy numbers in the transfectant popu-lations ranging from 1.1 to 2.0 (Figure 4a). The CEM cellpopulations were found to be similarly heterogeneous asdetermined by a smear of hybridizing bands in Southernblot analysis using a restriction enzyme which cut onlyonce within the transfected constructs (not shown).

RNA was isolated from the stably transfected cells,treated with RNase-free DNase to eradicate potentiallycontaminating DNA and reverse-transcribed usingoligo(dT) oligonucleotides. We performed semi-quanti-tative RT-PCR analysis by using serially diluted cDNAs.The cDNAs were amplified in the presence of RRE, Tatand human -actin specific primers. As shown inFigure 4b, the presence of a 184 bp PCR product detectedafter hybridization with a Tat specific probe indicates theexpression of the tat-ribozyme in pVA-CD2-Rbztat andpVA-HIV-RbztatCEM transfectants. The presence of a 245bp PCR product hybridizing with an RRE probe indicatesthe expression of the RRE RNA decoy in CEM transfec-tants containing pVA-HIV-RRE, pVA-HIV-TAR+RRE or

pVA-HIV-3-TAR+RRE. No corresponding fragments

were amplified from untransfected CEM cells, from CEMcells transfected with vector sequences or from samplesthat were not subjected to reverse-transcriptase treat-ment. As an internal control for RNA quantification, thesteady state RNA levels of-actin were determined simi-larly by RT-PCR. All samples showed a 590 bp PCR pro-duct characteristic of-actin expression.

After phosphorimager analysis, normalization with -actin and copy number consideration, RT-PCR analysisdemonstrates that the pVA-HIV-Rbztattransfectant popu-lation expresses the Rbz tat RNA at a 5.4-fold higher levelthan the pVA-CD2-Rbztat transfectants (Figure 4 andTable 1) suggesting that the HIV promoter is more activethan the CD2 promoter. Since the stability of mRNA mayalso influence steady state levels of mRNA, weexchanged the hCD2 3UTR in pVA-HIV with the human-globin gene 3 UTR to make the pVA-HIV-3 cassetteand directly compared the steady state levels of RREmRNA in pVA-HIV-TAR+RRE transfectants with pVA-HIV-3-TAR+RRE transfectants. As shown in figure 4 andTable 1, the level of steady state RRE mRNA in the trans-

fectants with the 3 -globin UTR, as determined afternormalization to the -actin control (Table 1) was 9.6-fold

higher than that found in the transfectants with the 3CD2 UTR. After further normalization to the transgenecopy number, a 15.8-fold increase of TAR+RREexpression was obtained with the pVA-HIV-3 vector ascompared with the pVA-HIV vector. Thus, in comparisonto the other vectors, the pVA-HIV-3 expression cassettepromotes the highest levels of decoy mRNA production.

A further advantage to the pVA-HIV-3expression cas-sette is that the HIV promoter is inducible. HIV promotermediated transcription has been shown to be increased10 to 1000 times in the presence of Tat.49 Thus, we testedfor an increase in transcription from pVA-HIV-TAR+RREand pVA-HIV-3-TAR+RRE in the presence of Tat. A Tatpeptide corresponding to the first exon of Tat protein (aa172) was added to cultures of CEM transfectants andRNA was prepared after 3 days. Semi-quantitative RT-PCR analysis was performed as described above, usingthe RRE specific oligo primers (Figure 5). In the presenceof Tat we find a four-fold increase in inhibitor geneexpression in pVA-HIV-TAR+RRE transfectants and atwo-fold increase in inhibitor gene expression in pVA-HIV-3-TAR+RRE transfectants. Therefore, the HIV-2 pro-moter in these expression constructs is Tat-inducible andleads to higher levels of RNA decoy expression.

Inhibition of HIV infection is related to levels of inhibitorexpression

After determining the relative steady state mRNA levelsof the transfectant populations, we sought to determinewhether quantitatively higher expression levels of themolecular inhibitors would correlate with greater inhi-bition of HIV. To ensure that each transfectant populationwas equally infectible with HIV, CD4 levels were determ-ined by FACScan analysis. Before infection, control andtransfectants CEM cells were 91% to 99% CD4-positivewith equivalent intensity of staining (data not shown).Inhibition of HIV infection due to Rbztatexpression fromthe pVA-CD2 and pVA-HIV cassettes was compared inthe transfectant populations (Figure 6). After challengewith an MOI of 0.002, a substantial inhibition of HIV rep-lication was observed in the pVA-HIV-Rbztattransfectant

population. HIV replication was reduced by a factor of 8


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F igure4(a) Determination of copy number of the constructs in transfected cells. Cellular DNA was digested with BamHI and XhoI, separated on a0.8% agarose gel, transferred onto a nylon membrane and hybridized with a puro-specific probe, giving a 1.4 kb signal. A 3.4 kb BamHI human -globin LCR fragment from pLCR was used as probe for internal single copy loading control. Increasing amounts of pVA-HIV plasmid DNA digestedwith BamHI and XhoI were also added to negative control genomic DNA from nontransfected CEM cells and analyzed simultaneously as copy numbercontrols. Quantification was performed by Phosphorimager analysis. *The DNA in this lane is from the pVA-HIV-3 -TAR+RRE transfectant population.(b) Expression of Tat ribozyme and RRE in stably transfected cells. RNA was extracted from control CEM cells, CEM cells transfected with pVA-CD2and pVA-HIV control plasmid DNAs, and from CEM cells stably transfected with pVA-CD2-Rbz tat, pVA-HIV-Rbz tat, pVA-HIV-RRE, pVA-HIV-TAR+RRE and pVA-HIV-3-TAR+RRE. Specific RNA expression was determined by RT-PCR using tat- and RRE-specific primers, and primers specificto human -actin mRNA as internal control, giving PCR products of 184, 245 and 590 bp, respectively. No corresponding Rbz tat or RRE fragmentwas amplified from negative cell lines or from samples not subjected to the RT (not shown). For semi-quantitative RT-PCR, serial two-fold (Rbz) andfour-fold (RRE and -actin) dilutions of cDNA samples were amplified. PCR products were electrophoresced on a 2% agarose gel and stained withethidium bromide. For Southern blotting, PCR products were transferred on to a nylon membrane and hybridized with 32P-labeled Tat-, RRE- and -actin-specific probes. Relative signal intensitites were quantitated with a Phosphorimager.

at day 6 and an 84% inhibition was observed at day 9(RT activity 2.5 0.25 c.p.m./105 ml compared with15.1 0.73 for control cells). Infection peaked in thispopulation at day 13, compared with day 9 for controlcells. In contrast, the pVA-CD2-tat ribozyme transfectantpopulation showed only a very slight inhibition at day 6(RT levels 1.52 0.31 c.p.m./105 ml compared with2.15 0.09 for control cells) and infection peaked at day9 (14.6 1.05 c.p.m./105 ml). Thus, inhibition of HIV rep-lication appears to be directly related to increasedexpression of the Rbztatfrom the pVA-HIV cassette; a 5.4-fold increase in basal level RNA expression from the HIVcassette as compared with the CD2 cassette and a furthertwo- to four-fold increase in Tat-inducible RNA

expression from the HIV promoter giving an estimated

total 10- to 20-fold increase in Rbztat steady-state RNAlevels.

To examine this correlation between expression levelsand inhibition further, we compared the inhibition ofHIV infection in transfectant populations carrying pVA-HIV-RRE, pVA-HIV-TAR+RRE and pVA-HIV-3-TAR+RRE (Figure 7a). When infected at an MOI of 0.002,all three transfectant populations show inhibition. Theexpression of RRE alone in the pVA-HIV cassette reducedRT activity by 60% at day 6 (0.8 0.26 c.p.m./105 mlcompared with 2.15 0.09 for control cells) and peakedat day 13 after infection (as compared with day 9 for con-trol CEM cells). The expression of RRE in combinationwith TAR in pVA-HIV-TAR+RRE transfectants yielded a

further reduction in RT activity, resulting in 95% inhi-


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Table 1 Quantification values for Rbz tat and RRE expressionin CEM transfectant populations

Transfectant -actina Rbz Normalization Copy Normalizationpopulations tatb to -actind number to copy

numbere

pVA-CD2-Rbz 1:14 192 13.7 1.7 8.1pVA-HIV-Rbz 1:2 177 88.5 2.0 44.2

-actina RREcNormalization Copy Normalizationto -actind number to copy

numbere

pVA-HIV-RRE 1:13.5 220 16.3 1.7 9.6pVA-HIV- 1:16.5 440 26.7 1.8 14.8TAR+RREpVA-HIV-3- 1:3.5 900 257.1 1.1 233.7TAR+RRE

After RT-PCR using serial dilutions of cDNA and Southern blot-ting with Rbz tat-, RRE- and -actin specific probes, quantifi-cation was performed by Phosphorimager analysis and nor-

malization to the -actin control. (a) Dilution of cDNA givingthe same value for -actin signal and falling within the linearrange. Quantitation value for Rbz tat (b) and RRE (c) signal ata 1:2 dilution of cDNA for each sample. All values fall withinthe linear range. These values were then normalized to the -actin control (d) and to the copy number of the construct in thetransfected cell population (e).

bition of infection at day 9 (0.71 0.08 c.p.m./105 mlcompared with 15.1 0.73 for control cells) and peakingonly at day 16. Most impressively, the TAR+RRE combi-nation in the pVA-HIV-3 cassette yielded the greatestinhibition. RT levels were reduced by 9095% up to day13 (0.97 0.21 c.p.m./105 ml) and never approachedeven the 50% level of RT seen in the other transfectantsor the control CEM cells. RT levels in pVA-HIV-3-TAR+RRE transfectants reached a maximum at day 23(4.9 0.47 c.p.m./105 ml) and the peak level of RT pro-duction was only 30% of the peak levels seen in the othercell populations. Thus, TAR+RRE in the pVA-HIV-3cas-sette is the most effective inhibitor of HIV infection. Asthe level of mRNA expression is greatest in the pVA-HIV-3-TAR+RRE population (9.6 times higher than thatin the pVA-HIV-TAR+RRE population), the levels of inhi-bition with these decoys directly correlates with levels ofdecoy mRNA.

DiscussionThe use of RNA decoys and ribozymes specific to HIVregulatory proteins Tat and Rev has been proposed as apossible therapeutic method for inhibition of HIV repli-cation. In this study, we have examined the ability of sev-eral RNA decoys corresponding to TAR and RRE regionsof HIV RNA and a ribozyme specific for tat mRNA toinhibit HIV-1 replication. These inhibitory moleculeswere stably produced in a human T lymphoid cell line(CEM) using three different plasmid expression vectorsin order to examine which expression cassette yielded thehighest levels of inhibitor RNA and whether expressionlevel correlated with degree of inhibition of HIV infec-tion.

Rbztat, TAR, RRE, RWZ6, TAR+RWZ6 and TAR+RRE

inhibitors were generated and tested for inhibition ofHIV-1 infection. The TAR region from HIV-2 was usedsince both HIV-1 and HIV-2 Tat are able to bind andtransactivate the HIV-2 LTR.25 Hence the HIV-2 TARdecoy should be an effective inhibitor of both HIV-1 andHIV-2 infection. While others have shown that HIV-1TAR and multiple TAR decoys are effective in inhibiting

HIV infection,21,55

our studies with the HIV-2 TAR decoyshow that it acts as an inhibitor when used on its own.However, we also show that it is most effective whenused in combination with the RRE decoy. In biochemicalassays, small Rev-binding sequences, RWZ2 and RWZ6,have been demonstrated to cooperatively bind severalmolecules of Rev protein.46 We sought to determine ifthese subunits are more effective in inhibiting HIV infec-tion than the entire RRE. In Figure 1c, our results showthat RWZ6 is slightly less or equal in effectiveness toRRE, and RWZ2 possesses no HIV inhibiting activity(data not shown). Hence the entire RRE sequence wasused for quantitative expression and inhibition studies.Finally, the Rbztatinhibitor was found to be only as active

as the RRE or RWZ6 decoys when examining CEM celltransfectants infected at a very low MOI of HIV-1(0.0001).

To enable intracellular immunization strategies to besuccessful therapies for in vivo anti-HIV treatment, mol-ecular inhibitors must be expressed at high levels indefi-nitely in virally susceptible cells, chiefly T cells. Thehuman CD2 expression cassette containing the CD2 LCRpromotes such high level, long-term expression in T cells.In vivo studies have shown that the CD2 LCR conferschromosomal site position-independent, copy number-dependent expression of exogenous genes in T cells.49

Hence, the CD2 LCR offers great advantage over conven-tional expression cassettes for long-term therapeutic pur-poses. Therefore we used an expression cassette contain-ing the CD2 gene regulatory elements, 3UTR and LCR.To promote higher level expression in HIV-infected cellswe also generated a Tat-inducible expression cassettecontaining the HIV-2 LTR promoter sequences, the CD23UTR and the CD2 LCR. Since Tat-mediated transactiv-ation has been shown to increase transcription signifi-cantly from this promoter, we sought to determinewhether higher expression would yield greater inhibitionafter HIV infection. Finally, a third expression cassettewas used in which the 3UTR of CD2 was replaced withthe 3UTR of the -globin gene so as to stabilize mRNAs.Our studies demonstrate that the pVA-HIV-3expressioncassette yields the highest levels of inhibitor transcrip-tion. In a comparison between pVA-CD2 and pVA-HIV

directed expression, a five- to six-fold increase inexpression was observed for Rbztat with the pVA-HIVvector. The ability of the pVA-HIV expression cassette todirect high level T cell specific transcription in vivo hasbeen recently tested in transgenic mice. In preliminaryresults we have found lymphoid-specific basal and highlevel Tat-inducible expression (D Abraham, personalcommunication). Moreover, a 15.8-fold increase inTAR+RRE expression was revealed when the pVA-HIVcassette was modified with the -globin 3 UTR, asdetermined after normalization to the transgene copynumber (Table 1). Thus, higher levels of steady-statemRNA can be obtained with basal expression from theHIV promoter. Additionally the inducibility of the HIV

promoter to highly transactivate transcription of HIV


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F igure5 Increase of TAR+RRE inhibitor expression in pVA-HIV vector in presence of Tat protein. CEM cells stably transfected with pVA-HIV-TAR+RRE and pVA-HIV-3-TAR+RRE were resuspended at 106 cell/ml in culture medium in the absence or presence of 5 g/ml Tat peptide (aa 172). After a 3-day culture, RNA was extracted and expression of RRE RNA decoy was determined by semi-quantitative RT-PCR, Southern blottingand Phosphorimager analysis (see Figure 4b).

F igure6 Inhibition of HIV replication in CEM transfectant populationscontaining the tat ribozyme. CEM cells stably transfected with pVA-CD2-Rbz tat ( ) and pVA-HIV-Rbz tat () were infected with HIV-1IIIB/LAIat an MOI of 0.002. Cell-free supernatants were tested every 34 daysfor the presence of reverse transcriptase activity (RT). HIV replicationwas compared with that in control infected CEM cells (). To ensure thateach transfectant population was equally infectable by HIV, CD4 levelsand percentages were determined by FACScan analysis. Before infection,pVA-CD2-Rbz tat, pVA-HIV-Rbz tat and control CEM cells were 99%,97% and 91% CD4-positive, respectively, and the intensity of CD4 stain-ing between each of these populations was equivalent. Uninfected CEMcells () were used as the negative control. Triplicate samples were ana-lyzed for all time-points and average of the mean is shown. Values of RTactivity (c.p.m./105 ml) at day 9 after infection are 15.1 0.7 for controlCEM cells, 14.61.1 for pVA-CD2-Rbz tat and 2.5 0.2 for pVA-HIV-

Rbz tat cells.

inhibitory genes in the presence of Tat has been shown5557

and is confirmed for the constructs used in this study.Quantification of steady-state mRNA from pVA-HIV vec-tors after Tat-mediated induction in the presence of extra-cellular Tat peptide shows a two- to four-fold increase ofexpression of RRE in cells carrying pVA-HIV-3-TAR+RRE and pVA-HIV-TAR+RRE, respectively. Hencetaken together (1) the 5.4-fold increase of Rbz tatexpression in pVA-HIV as compared with pVA-CD2; (2)

the 15.8-fold increase of TAR+RRE expression in pVA-HIV-3 compared with pVA-HIV; and (3) the two- tofour-fold increase of RNA decoy expression in the pres-ence of extracellular Tat, the inducible pVA-HIV-3vectorcompared with the pVA-CD2 vector yields levels ofsteady-state inhibitor RNA increased by at least 170- to340-fold. This pVA-HIV-3 expression cassette shouldfacilitate in vivo application of molecular-based antiviraltreatments.

While it appears intuitive that higher expression ofmolecular inhibitors should result in greater inhibition ofviral infection, we sought to determine this specificallyfor the expression vectors and inhibitors described in thisstudy. In initial experiments using MOIs of 0.002 and

0.0004, either no or minimal inhibition was observed forpCD2-TAR, RWZ6 and Rbztat transfectant cells. Only ata very low MOI of 0.0001 and in the pVA-HIV cassettewas significant inhibition seen for these inhibitors. In allcases the degree of inhibition of HIV infection is greaterwhen the molecular inhibitor is expressed in the pVA-HIV cassette. This is directly demonstrated in Figure 6 forRbztat. As RbztatRNA is found in the pVA-HIV transfec-tants at a five- to six-fold higher level than in the pVA-CD2-Rbztat transfectants, significant inhibition resultedonly with pVA-HIV-Rbztat at an MOI of 0.002. No inhi-bition resulted with pVA-CD2-Rbztat. Thus, theexpression of the ribozyme, and similarly the RNAdecoys in the pVA-CD2 cassette, may be too low to cleave

tat mRNAs or sequester Tat or Rev proteins efficiently.


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1673Only with higher basal expression from the HIV pro-moter or Tat-induced expression from this promoter arefunctionally effective levels of these inhibitors present.

This is more dramatically demonstrated by the com-parison of steady-state mRNA levels and inhibition ofinfection (MOI 0.002) in transfectant populations carryingpVA-HIV-TAR+RRE and pVA-HIV-3-TAR+RRE

(Figure 7a). The 9.6-fold increase in steady-state mRNAin the pVA-HIV-3 transfectants leads to a much pro-longed period of low level RT production, as well as pro-tecting the cells from producing peak levels of RT activityas seen for the pVA-HIV transfectants. The replacementof the CD2 3 UTR with the -globin 3 UTR results inclear evidence that the inhibition of HIV replication isdependent upon the level of the RNA inhibitor. It is inter-esting that these same cells infected at a high MOI of 0.01(5000 TCID50/ml) show even greater inhibition of HIVinfection (Figure 7b). While the peak of infection in thecontrol cells is slightly earlier than in the control cellsinfected at an MOI of 0.002, the highest level of RTactivity in the pVA-HIV-3-TAR+RRE transfectants is

found at day 2023 and is only 20% of maximal levels ofcontrol infected cells. Hence, infection with a higher viraldose may provide an initial and added amount of Tat toallow transactivation of transcription from the pVA-HIV-3-TAR+RRE inhibitor construct. At this time it is unclearin these experiments whether all the pVA-HIV-3-

Figure 7 Inhibition of HIV replication is related to the level of expression of the inhibitor TAR+RRE. (a) Cells stably transfected with pVA-HIV-RRE(), pVA-HIV-TAR+RRE () and pVA-HIV-3-TAR+RRE () were infected with HIV-1IIIB/LAIat an MOI of 0.002. Cell-free supernatants were testedevery 34 days for the presence of reverse transcriptase activity (RT). HIV replication was compared with that in control infected CEM cells (). Toensure that each transfectant population was equally infectable by HIV, CD4 levels and percentages were determined by FACScan analysis. Beforeinfection, pVA-HIV-RRE, pVA-HIV-TAR+RRE, pVA-HIV-3-TAR+RRE and control CEM cells were 93%, 92%, 93% and 91% CD4-positive, respect-ively, and the intensity of CD4 staining between each of these populations was equivalent. Triplicate samples were analyzed for all time-points andaverage of the mean is shown. Values of RT activity (c.p.m./105 ml) at day 9 after infection are 15.1 0.7 for control CEM cells, 11.2 4.8 for pVA-

HIV-RRE, 0.71 0.08 for pVA-HIV-TAR+RRE and 0.06 0.007 for pVA-HIV-3-TAR+RRE cells. (b) pVA-HIV-TAR+RRE () and pVA-HIV-3TAR+RRE () cells were also infected with HIV-1 IIIH/LAIat MOI of 0.01. Cell-free supernatants were tested every 34 days for the presence of reversetranscriptase activity (RT). At each time of sample collection, cells were counted using trypan blue and replated in fresh medium at 5 105 cell/ml; nodifference in cell proliferation and viability was observed between the two transfectant populations. HIV replication was compared with that in controlCEM cells () and with that in CEM cells transfected with control pVA-CD2 () and pVA-HIV () plasmid vectors. Before infection, pVA-HIV-TAR+RRE and pVA-HIV-3-TAR+RRE transfectant cells were 94% and 92% CD4-positive. Control pVA-CD2 and pVA-HIV transfected cells andnon-transfected CEM cells were 91%, 96% and 95% CD4-positive, respectively. Uninfected CEM cells () were used as the negative control. Triplicatesamples were analyzed for all time-points and the average of the mean is shown. Values of RT activity (c.p.m./10 5 ml) at day 9 after infection are15.0 1.8 for control CEM cells, 16.2 0.5 for pVA-CD2 and 12.2 1.6 for pVA-HIV control transfected cells, and 0.97 0.13 for pVA-HIV-TAR+RRE

and 0.16 0.02 for pVA-HIV-3-TAR+RRE cells.

TAR+RRE cells are infected and producing low levels ofHIV, or whether a few cells are highly infected. In futureexperiments we will sample the cultures at various timesafter infection and analyze CD4 and gp120 levels on thesurface of infected cells. Also in situ hybridization withprobes detecting other RNAs encoded by HIV willfurther clarify this point.

Successful inhibition of HIV infection has beenobtained in CD4+ cells from noninfected individualstransduced with vectors expressing anti-tat ribozymes,tat antisense molecules and TAR RNA decoys.39 Sup-pression of HIV replication and prolonged survival werealso observed in CD4+ cells from HIV-positive subjects,ex vivo transduced with anti-HIV genes expressing ananti-U5 ribozyme.36 Hence, to prove efficacy of the RNAdecoys and the tat-ribozyme described further, experi-ments should be carried out to determine whether theseanti-HIV molecules are protective against different lab-oratory strains and against primary field isolates of HIV,and whether they can inhibit HIV infection in primarycells.

Finally, the antiviral approach taken in these studieswill require an efficient method to deliver thesesequences to the appropriate cells (the first choice deliv-ery is to hematopoietic stem cells) for therapeutic appli-cation. While viral,2 as well as nonviral2,55,58 delivery sys-tems have been proposed and tested, there is at this time


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1674no method that is highly reliable. As these methodsbecome improved, the specificity, prolonged nature andefficiency of expression of therapeutic molecules willbecome important. We have shown in the studiespresented here that high levels of molecular inhibitorscan be expressed in human T cells using the HIV pro-moter. The use of the CD2 LCR will ensure long-term

position-independent expression, as found in vivo inmouse transgenic models.59 Together with the increase insteady-state mRNA due to the use of the -globin 3UTRin pVA-HIV-3 and the Tat-induced increase in HIV-mediated transcription during HIV infection, these stud-ies suggest the efficacy of this HIV-inducible vector todirect the in vivo expression of the combination decoyTAR+RRE in possible therapeutic applications.

Materials and methods

Cells and virusCEM cells were grown in RPMI 1640 (Gibco BRL, LifeTechnologies, Paisley, UK) containing 5% heat-inacti-vated fetal calf serum (FCS), and supplemented in anti-biotics and glutamine, at 37C 5% CO2(culture medium).

HIV-1IIIB/LAI strain was kindly provided by the MRCAIDS Reagent Project. Virus stocks were prepared byinfecting CEM cells. The virus titer of cell-free super-natant from infected CEM cells was determined to be 106

tissue culture 50% infective dose (TCID50) per ml.

Generation of ribozyme and RNA decoys constructsThe ribozyme specific to the tat RNA (Rbztat) wasdesigned so that it cleaves the tat RNA at the first GUCat position 49 (Figure 1b). The Rbztat was cloned intoEcoRI and SmaI sites of the pVA-CD2 expression cassetteand the equivalent pVA-HIV vector where the human

CD2 promoter BglII-EcoR721 fragment has been substi-tuted by the HIV-2 LTR BglII-blunted SpeI fragment(Figure 1a). The HIV-2 TAR sequence (Figure 1b) wascloned into the EcoRI site of the pVA-CD2 cassette. TheRNA decoy corresponding to the HIV-1 RRE sequence(Figure 1b) was cloned into EcoRI and SmaI sites of thepVA-HIV cassette. The RWZ6 sequence in a EcoRV-SmaIfragment (kindly provided by R Zemmel and J Karns)(Figure 1b) was cloned into the SmaI site of pVA-CD2and pVA-HIV. The combination TAR+RRE RNA decoywas cloned into the EcoRI and SmaI sites of pVA-HIVand the equivalent pVA-HIV-3where the 3untranslatedregion (UTR) of the human CD2 gene has been substi-tuted by the 3 UTR of the human -globin gene

(Wrighton et al, manuscript in preparation). For gener-ation of TAR+RWZ6 expression vector, the EcoRV-SmaIRWZ6 fragment was cloned into the SmaI-digested pVA-CD2-TAR plasmid DNA.

Electroporation of CEM cellsCEM cells were washed twice with cold RPMI 1640 andresuspended at 107 cell/ml in cold RPMI 1640. For eachsample, cells were mixed with 2030g of XbaI-lin-earized plasmid DNA and placed into disposableelectroporation cuvettes (0.4 cm gap width, Bio-Rad,Richmond, CA, USA) for 10 min on ice. Electroporationswere then performed with the BioRad Gene Pulser at500F and 300 V for 20 ms. After electroporation the

cells were maintained in the cuvettes for 10 min on ice

and then transferred in 15 ml of RPMI 1640 15% FCS.Two days later, debris and dead cells were removed bydensity gradient (Lymphoprep; Nycomed, Oslo,Norway) and viable cells were resuspended in RPMI1640 15% FCS containing 0.5 g/ml puromycin (Sigma,St Louis, MO, USA).

Tat-inducible RNA decoy expressionCEM cells stably transfected with pVA-HIV-TAR+RREand pVA-HIV-3-TAR+RRE Tat-inducible vectors werewashed and resuspended at 106 cell/ml in culturemedium containing 8 g/ml polybrene, in absence orpresence of 5g/ml Tat peptide (aa 172) (kindly pro-vided by Dr E Blair, Wellcome, Beckenham, UK). After 3days, RNA was extracted and analyzed by RT-PCR forexpression of RRE (see below)

HIV-1 challengeControl and stably transfected CEM cells were grownwithout puromycin at least 3 days before infection. Thecells were challenged at 5 105 cell/ml with HIV-1IIIB/LAI

viral doses from 50 to 5000 TCID50/ml (MOI from 0.0001to 0.01) for 24 h at 37C. Cells were washed, resuspendedin fresh medium and incubated in triplicate in 12-wellplates (1 ml per well). Cell-free supernatants were col-lected every 34 days and tested for the presence ofreverse transcriptase (RT) or the presence of the HIV-1core p24 protein using a commercial ELISA (Dupont, Bos-ton, MA, USA). At the time of sample collection, cellswere counted and replated in fresh medium at 5 105

cell/ml.

Southern blot analysisFor determination of copy number, cellular DNA (10 g)was digested with BamHI andXhoI, separated on a 0.8%agarose gel, transferred on to a nylon membrane(Hybond-N+, Amersham, Amersham, UK), andhybridized with a 32P-labeled 1.4 kb XhoI puro-specificprobe. A 3.4 kb BamHI human -globin fragment frompLCR plasmid DNA containing a 20 kb -globin LCRfragment was also used as an internal loading control.Increasing amounts of pVA-HIV plasmid DNA digestedwithBamHI andXhoI were also added to negative controlgenomic DNA from untransfected CEM cells and ana-lyzed simultaneously as copy number controls. Quanti-fication was performed by Phosphorimager (MolecularDynamics, Sunnyvale, CA, USA) analysis.

RNA analysisRNA samples were extracted with LiCl/Urea or RNA

isolation reagent (Ultraspec; Biotecx, Houston, TX, USA).To remove potentially contaminating DNA, all the RNAsamples were digested with DNase. Fifty micrograms ofRNA was mixed with 4 units of RNase-free DNase I(RQ1; Promega, Madison, WI, USA), 50 mm Tris pH 7.5,10 mmMgCl2in 200l reaction, incubated at 37C for 30min. DNase-treated RNA was reverse transcribed. Twomicrograms of RNA were incubated with 1 l ofoligo(dT) in 20 l reaction, incubated at 100C for 2 minand stored on ice. Ten microliters of the reaction wasreverse transcribed in the presence of reverse tran-scriptase (RT) buffer (Enzyme Biotechnologies, Cam-bridge, UK), 1 mm dNTPs, 0.5l (28 900 U/ml) RNAguard (Pharmacia, Milwaukee, WI, USA), 1 l (21 U/l)

RT (Enzyme Biotechnologies) in 20 l reaction, incubated


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1675at 37C for 90 min, or at 50C for 20 min, and storedat 20C before PCR. Control reactions lacking RT wereperformed to confirm that contaminating DNA waseliminated by DNase treatment.

For semi-quantitative RT-PCR, three four-fold (RRE)and two-fold (Rbztat), and five four-fold (-actin) dilutedcDNA samples were amplified. A 50 l reaction mixture

containing 1 l cDNA, 10 l PCR buffer (Tris-HCl300 mm, NH4SO4 75 mm, MgCl2 17.5 mm, pH 9.5) forRbztat and RRE primers (TatS1: 5-ATGGAGCCAGTA-GATCCTAG-3, TatS2: 5-CTCCGCTTCTTCCTGCCA-3;RRE-S: 5-GAGCAGTGGGAATAGGAGC-3, RRE-AS: 5-GGAGCTGTTGATCCTTTAGG-3), or 5 l SuperTaqbuffer (Enzyme Biotechnologies) for human -actinprimers (-actin1: 5-ATGGATGATGATATCGCCGC-3,-actin2: 5-GCGCTCGGTGAGGATCTT-3), 200 mmdNTPs, 0.1 unit SuperTaq (Enzyme Biotechnologies)polymerase and 200 ng each appropriate primer wasused for amplification as follows; 94C 3 min, one cycle;94C 45 s, 58C 45 s, 72C 45 s, 30 cycles; 72C 10 min,one cycle. RT-PCR products were electrophoresced on

1.5% (-actin) and 2.5% (Rbztat, RRE) agarose gels andanalyzed by Southern blot hybridization using a nylonmembrane (Hybond-N+; Amersham). Rbztat, RRE and -actin DNA probes were 32P-labeled by random primingmethod using Klenow enzyme (Boehringer, Mannheim,Germany) and -32P-dATP (Amersham). Relative levelsof specific RNAs were quantified by Phosphorimager(Molecular Dynamics) analysis and normalized to the -actin internal control. The dilutions of cDNA weredetermined so that quantification values after hybridiz-ation fell within a linear range.

Acknowledgements

We thank Professor ADME Osterhaus (EUR, Rotterdam,

NL) for access to the category-3 facilities in the Depart-ment of Virology (EUR, Rotterdam, NL). The HIV-1IIIB/LAIstrain was obtained from the MRC AIDS Reagent Projectand was kindly provided by Dr R Daniels and Dr C Vella(NIMR, London. UK). We thank Dr E Blair (WellcomeLaboratories, Beckenham, UK) for the Tat peptide, Dr DKioussis (NIMR, London, UK) for the pVA-CD2 vectorand Dr J Karn and Dr R Zemmel (MRC Laboratory ofMolecular Biology, Cambridge, UK) for the RWZ6sequence. We thank Dr D Abraham, Dr D Drabek andother members of the laboratory for advice and criticalcomments. CF was supported by a grant from Therexsys.

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