Introduction

RNA interference (RNAi) can be used as a therapeutic strategy to target human pathogenic viruses. RNAi is induced by double-stranded RNA, resulting in the sequence-specific degradation of homologous single-stranded RNA^1,2. The effector molecules of this evolutionarily conserved mechanism, which are produced by a ribonuclease named Dicer, are short interfering RNAs (siRNAs) of 22 nt^3,4. Transfection of siRNAs into cells has proven to be a powerful tool to suppress gene expression transiently⁵. Stable expression of short hairpin RNAs (shRNAs), which are processed into effective siRNAs by Dicer, can result in long-term gene suppression^6,7. We have demonstrated long-term inhibition of human immunodeficiency virus type 1 (HIV-1) replication in human T cells stably expressing shRNAs that target the viral Nef gene (shRNA^Nef)^8,9. Thus, RNAi shows potential as a means to achieve intracellular immunization against HIV-1.

We previously described an HIV-1 variant that replicates conditionally in the presence of doxycycline (dox)^10,11. In this HIV-rtTA virus, the Tat–TAR regulatory mechanism controlling viral gene expression and replication was inactivated and functionally replaced by the Tet-on system for inducible gene expression¹². The rtTA gene encoding the new transcriptional activator replaces the accessory Nef gene, and the Tet operator (tetO) DNA elements were introduced in the viral LTR promoter. Dox induces a conformational change in the rtTA protein such that it can bind to tetO and subsequently activate viral transcription. The initial HIV-rtTA construct has been significantly improved through virus evolution^13,14,15. Efficient dox-dependent replication has been demonstrated in vitro in T cell lines and primary cells and ex vivo in human lymphoid tissue¹⁶.

The HIV-rtTA virus is under development as a conditional-live virus vaccine. In this study, we have modified the HIV-rtTA virus such that it becomes a replicating vector for the efficient delivery of anti-HIV shRNAs to those cells that are susceptible to HIV-1 infection. Replication of this therapeutic virus with dox results in virus spread to HIV-susceptible cells. Subsequent dox withdrawal generates cells containing a silent integrated provirus with a constitutively active shRNA expression cassette. The HIV-rtTA-encoded shRNA^Nef inhibitor does not target the vector itself because the rtTA gene has replaced Nef sequences, but it does efficiently inhibit HIV-1 replication in transduced cells.

Results

Construction of the therapeutic HIV-rtTA-shNef virus

We previously described the incorporation of the Tet-on system in the HIV-1 genome¹⁰. The shRNA^Nef expression cassette under the control of a polymerase III promoter was inserted in the 3' U3 region of this HIV-rtTA construct (Fig. 1). Upon transduction of cells, the 3' U3 domain will be copied in the 5' LTR, thus yielding a provirus with two shRNA^Nef expression cassettes. We constructed HIV-rtTA variants with the cassette in forward (F-shNef) or reverse (R-shNef) orientation and two corresponding controls with an empty F or R cassette. The target sequence of shRNA^Nef is absent in HIV-rtTA-shNef but present in the Nef gene of wild-type HIV-1 variants (Fig. 1).

Figure 1.

Construction of the therapeutic HIV-rtTA-shRNA^Nef virus. Schematic of the HIV-rtTA genome (top) and wild-type HIV-1 LAI (bottom). In HIV-rtTA, we inserted the rtTA gene in place of the Nef gene and tetO binding sites in the LTR promoter. Upon administration of doxycycline (dox, ) rtTA can bind to tetO and activate transcription of the viral genome, thus inducing viral gene expression and replication. The polymerase III promoter-shRNA^Nef expression cassette is inserted into the 3' LTR (EcoRV site) in a forward (F) or reverse (R) orientation. Upon virus replication, the cassette is inherited in both LTRs. The shRNAs are processed by Dicer into siRNAs, which target the Nef^wt sequence in wild-type HIV-1 RNA. ShRNA^Nef does not self-target the Nef-minus HIV-rtTA genome. The mutant Nef^R2 sequence was selected in an shRNA^Nef-resistant HIV-1 variant and contains an 11-nt deletion in the target sequence⁹. In the U3-HIV-rtTA variants, the 5' U3 sequences upstream of the tetO elements were deleted.

Full figure and legend (144K)

A unique feature of this shNef-expression vector is that it replicates in a dox-dependent manner and that it can spread to all HIV-susceptible cells. We first tested the replication capacity of the four HIV-rtTA vectors in the SupT1 T cell line. All constructs replicated efficiently (Fig. 2A) and similar to the parental HIV-rtTA virus¹⁵. Most importantly, replication is strictly dox-dependent. We cultured the viruses for a longer period by passage on fresh SupT1 cells. We PCR amplified the U3 region of integrated proviruses at each passage and analyzed the products by agarose gel electrophoresis. All variants show loss of sequences after multiple passages (HIV-rtTA-R-shNef is shown as an example in Fig. 2B, left). Sequence analysis revealed the exact deletion of the shRNA^Nef expression cassette (310 bp), which resulted in restoration of the original U3 sequence. A similar deletion and reversion to the original HIV-rtTA was observed with the other variants (results not shown). The wild-type U3 sequences are likely inherited from the 5' LTR through an unusual recombination event during reverse transcription¹⁷. To prevent this route, we minimized the 5' U3 region by deletion of nonessential promoter sequences in all viral constructs (U3-HIV-rtTA-shNef, see also Fig. 1). These U3 variants are indeed more stable during replication in SupT1 cells, but deletions occurred eventually (Fig. 2B, right). Sequence analysis of the U3 R-shNef progeny revealed deletions larger than the shRNA^Nef expression cassette (371 and 423 bp). All U3 variants eventually deleted a variable part of the shRNA^Nef cassette and/or the flanking U3 sequences (results not shown). Thus, although we could not completely block deletion of the shRNA^Nef cassette during virus evolution, we could improve the genetic stability of the HIV-rtTA variants by blocking the dominant deletion route in which the wild-type 5' U3 sequence was copied. We therefore considered these U3-HIV-rtTA variants for their capacity to deliver the antiviral shRNA^Nef to T cell cultures.

Figure 2.

Replication and genetic stability of HIV-rtTA-shRNA^Nef. (A) The different HIV-rtTA variants (5 g) were transfected into SupT1 T cells. Cells were cultured with (1 g/ml) or without dox and virus replication was monitored by determining the level of CA-p24 in the culture supernatant. No replication was scored in any of the cultures without dox. (B) Infected cells were collected at several passages. The complete shRNA^Nef expression cassette was PCR amplified from integrated proviral DNA as an 847-bp fragment. The PCR products were analyzed by gel electrophoresis. Sequence analysis of the PCR-amplified fragments demonstrated that at day 18 the HIV-rtTA-R-shNef virus contained a 310-bp deletion, which exactly removes the complete polymerase III cassette. The U3-HIV-rtTA-R-shNef virus contained a deletion of either 371 or 423 bp at day 29.

Full figure and legend (201K)

Expression of shRNA^Nef by the HIV-rtTA vectors suppresses target genes

We tested the expression of shRNA^Nef from these U3-HIV-rtTA constructs by Northern blot analysis of transfected C33A cells. Upon expression, the shRNA molecules will be processed into siRNA molecules by Dicer. We indeed detected 19-nt siRNA^Nef effector molecules when the cells were transfected with F-shNef or R-shNef, but not when transfected with the control F or R construct (Fig. 3A). The C33A cells do not support HIV-1 replication because they lack the receptors for viral entry. This allows us to determine the effect of dox addition and induced polymerase II transcription on polymerase III transcription. The siRNA^Nef level was slightly reduced when transcription from the proviral LTR promoter was activated with dox (Fig. 3A). This observation indicates that polymerase II-driven viral transcription can interfere with polymerase III-driven shRNA^Nef expression. Most importantly, these results demonstrate that the antiviral shRNA can be efficiently expressed from the proviruses with the shRNA^Nef cassette in the forward or reverse orientation (F-shNef and R-shNef, respectively).

Figure 3.

Expression of shRNA^Nef and efficient suppression of target genes. (A) Northern blot analysis of shRNA^Nef expression in C33A cells transfected with U3 HIV-rtTA constructs, cultured with or without dox. The expressed shRNAs are efficiently processed into the 19-nt siRNA^Nef effector molecules. C33A cells transfected with a pBluescript plasmid carrying the shRNA^Nef expression cassette (pBS-shRNA-Nef⁹) are included as a positive control. (B) C33A cells were cotransfected with HIV-1 LAI (500 ng) and the indicated U3-HIV-rtTA-shNef or control variant (100 ng). When indicated, cells were cultured in the presence of dox. Virus production in the culture supernatant was measured at day 2 by CA-p24 ELISA. The level of HIV-1 production with the control F and R constructs was set at 100%. (C) The U3-HIV-rtTA-shNef constructs were cotransfected into C33A cells with reporter plasmids containing either the intact shRNA^Nef target (Nef^wt) or a shRNA^Nef-resistant version (Nef^R2) downstream of the luciferase gene. The level of luciferase expression measured with the controls F and R was set at 100%.

Full figure and legend (113K)

To demonstrate that the shRNA^Nef expressed from the viral constructs is functional, we tested the capacity of these constructs to inhibit the expression of target genes. First, we cotransfected the U3-HIV-rtTA constructs in a 1 to 5 ratio with the HIV-1 LAI molecular clone into cells and measured virus CA-p24 production in the supernatant. In the absence of dox, the U3-HIV-rtTA LTR promoter is silent and only HIV-1 LAI will produce CA-p24. Even in the presence of dox, the U3-HIV-rtTA constructs will only slightly contribute to the total CA-p24 production due to the 1:5 ratio in the transfection mix. HIV-1 production was inhibited more than 80% upon cotransfection with F-shNef in comparison with the empty F control (Fig. 3B, left). Similar results were obtained with R-shNef versus the R control (Fig. 3B, right). We observed a similar level of shRNA^Nef-mediated inhibition in the presence of dox, indicating that the slightly reduced siRNA^Nef level observed upon activation of viral polymerase II-driven transcription (Fig. 3A) is sufficient to inhibit HIV-1 production. As a second test, we cotransfected the U3-HIV-rtTA constructs with a firefly luciferase-reporter construct with a downstream Nef-target sequence⁹. Both F-shNef and R-shNef potently inhibit luciferase expression compared with the controls F and R (Fig. 3C). We also tested a luciferase-reporter construct with a mutant Nef sequence in which the target sequence was partially deleted (Nef^R2 in Fig. 1). This mutant Nef sequence was selected in an HIV-1 variant that escaped from shRNA^Nef-mediated RNAi in long-term evolution studies^8,9. This luciferase-Nef^R2 reporter is not inhibited by F-shNef or R-shNef, demonstrating the strict sequence specificity of this RNAi-mediated silencing.

Protecting cells against HIV-1 replication

We next tested whether U3-HIV-rtTA-shNef viruses can be used as a replicating vector for delivery of shRNA^Nef to cells that are susceptible to HIV-1 infection. This therapeutic strategy is illustrated in Fig. 4. Dox-dependent replication of the therapeutic virus results in spread to the HIV-1 target cells. Withdrawal of dox results in transduced cells with a silent integrated provirus, but with an active expression cassette for shRNA^Nef, which may protect these cells against subsequent HIV-1 replication.

Figure 4.

Protecting HIV-1-susceptible cells with HIV-rtTA-shNef. A spreading infection of HIV-rtTA-shNef in all susceptible cells is triggered by transient dox treatment. Dox withdrawal prohibits HIV-rtTA-shNef replication and silences the integrated provirus. However, shRNA^Nef is constitutively expressed from the polymerase III expression cassette. This shRNA^Nef may target the incoming RNA genome²⁹ (route 1) and/or the de novomade RNA transcripts (route 2) and thus prevent HIV-1 replication.

Full figure and legend (207K)

To test this strategy, we started a spreading infection of the U3-HIV-rtTA-F-shNef on SupT1 cells in the presence of dox (Fig. 5A). As a control, we similarly established a spreading infection with the U3-HIV-rtTA-F control virus (Fig. 5B). When HIV-induced cytopathic effects indicated a high infection rate (day 5), we washed the cells and cultured them without dox. We challenged the cells at day 38 with wild-type HIV-1 LAI virus (HIV^wt) or the shRNA^Nef-resistant HIV^R2 variant⁹, in which the shRNA^Nef target sequence was partially deleted (Fig. 1). Whereas the F-transduced control cells supported replication of both HIV^wt and HIV^R2, the F-shNef-transduced cells supported replication of only the RNAi-resistant HIV^R2 variant and resisted HIV^wt replication (Figs. 5A and 5B). These results demonstrate that HIV^wt is efficiently inhibited through shRNA^Nef-induced RNAi and not through unrelated events such as superinfection interference¹⁸. To verify the stable presence of proviruses with an intact polymerase III expression cassette in U3-HIV-rtTA-infected cells, we PCR-amplified the viral U3 region and analyzed the products by agarose gel electrophoresis (Fig. 5C). Both the F- and the F-shNef-infected cells stably maintained the polymerase III expression cassette during multiple cell passages. Consistent with this result, we obtained similar HIV-1 inhibition results when the cultures were challenged at day 95 (results not shown).

Figure 5.

Harnessing SupT1 cells against HIV-1 infection. (A, B) U3-HIV-rtTA-F-shNef (A) and the F control (B) constructs (5 g) were transfected into SupT1 T cells. The cells were cultured in the presence of 1 g/ml dox to initiate a spreading infection. Dox was washed away at day 5 and the cells were cultured for over a month without dox. The cultures were challenged at day 38 with HIV^wt or HIV^R2 (200 pg of CA-p24/ml) and virus replication was monitored by determining the level of CA-p24 in the culture supernatant. (C) Integrated proviral DNA was PCR amplified at days 17, 31, and 48 with primers that amplify the complete F-shRNA^Nef cassette as an 847-bp fragment and the F empty control cassette as a 789-bp fragment. The PCR amplification product was analyzed by agarose gel electrophoresis.

Full figure and legend (248K)

Although these experiments demonstrate that F-shNef infection of cells does inhibit replication of HIV^wt, this inhibition is not absolute, as a low level of replication was apparent when a high HIV^wt virus input was used (results not shown). This incomplete protection may have resulted from an incomplete spread of the therapeutic virus. To obtain pure F-shNef-containing cells, we performed limiting dilution of the F-shNef-infected cells and selected 38 cell clones. We tested these clones by PCR for the presence of the shRNA^Nef expression cassette and challenged them with HIV^wt. Clones positive for F-shNef (left half of circle is gray in Fig. 6A) are expected to resist HIV^wt replication (right half of circle is white in Fig. 6A) and cell clones lacking the F-shNef provirus (left half is white in Fig. 6A) are expected to be susceptible to HIV^wt replication (right half is black in Fig. 6A). This limiting dilution experiment reveals that approximately 63% of the cells (24/38) were infected with F-shNef (Fig. 6B). Apparently, this level of spread is sufficient to obtain a high degree of protection against HIV^wt infection (Fig. 5A), but it also explains the breakthrough replication with high input challenge virus. Of the 24 shRNA^Nef-positive clones, 79% (19 cultures) were protected against HIV^wt replication. The other 5 shNef-positive clones were susceptible to HIV^wt. We tried to induce HIV-rtTA-F-shNef replication in these cells by dox administration, but failed to reactivate the integrated provirus in 4 of the 5 clones. This might indicate transcriptional silencing of the integrated therapeutic provirus, which may affect both the polymerase II LTR promoter and the polymerase III shRNA^Nef cassette and thus explain the susceptibility of these cells for HIV^wt. The majority (86%; 12/14) of the shRNA^Nef-negative control cells are susceptible to HIV^wt replication (Fig. 6B). The two clonal cultures that do not support HIV^wt replication may spontaneously have lost an essential cofactor such as the CD4 receptor. We obtained similar results when we performed the viral challenge on the clones 4 weeks later (results not shown). These results confirm the stable presence and shRNA expression of the therapeutic provirus.

Figure 6.

HIV-rtTA-shNef-containing cell clones resist HIV-1 replication. (A) SupT1 cells with a silent U3-HIV-rtTA-F-shNef provirus (obtained from the experiment shown in Fig. 5A, isolated at day 60) were serially diluted to obtain clones. A total of 38 clones were PCR screened for the presence or absence of U3-HIV-rtTA-F-shNef provirus, indicated in the left half of the circle (gray, present; white, absent). The clones were challenged with HIV^wt virus (750 pg CA-p24/ml) and virus replication was monitored, indicated in the right half of the circle (black, replication; white, no replication). (B) Pie chart summary. U3-HIV-rtTA-F-shNef-positive cell clones (middle chart, gray) are predominantly HIV^wt resistant (left chart, white). U3-HIV-rtTA-F-shNef-negative cells (middle chart, white) are mainly susceptible to HIV-1 infection (right chart, black).

Full figure and legend (136K)

Discussion

We have used the conditional-live HIV-rtTA virus to create a replicating vector for the efficient delivery of shRNA^Nef to cells that are susceptible to HIV-1 infection. The shRNA^Nef targets the wild-type HIV-1 RNA genome but not the viral vector. The dox-dependent replication of this therapeutic virus allows one to control its spread. Upon dox withdrawal the HIV-rtTA-shNef-infected cells stably maintain the silent provirus and the constitutively active shRNA^Nef expression cassette harnesses these cells against subsequent infection with the wild-type virus.

We previously demonstrated that shRNA^Nef-resistant escape variants can be selected with a variety of mutations in the viral target sequence⁹. The use of combination shRNA therapy in which multiple conserved viral RNA sequences are targeted by multiple shRNAs at the same time may block the emergence of RNAi-resistant variants¹⁹. The introduction of multiple shRNA cassettes into the HIV-rtTA genome may pose several problems. First, we targeted Nef sequences because this gene is deleted in the HIV-rtTA vector. There are a few additional options to target HIV-1 sequences that are absent or no longer required in HIV-rtTA, e.g., in the U3 domain of the LTR promoter or in Tat. When targets are chosen that are also encoded in the HIV-rtTA genome, one could try to modify these vector sequences, e.g., by silent codon changes. A second problem may be imposed by the presence of multiple similar polymerase III units in a single retroviral vector. Recombination on repeated sequences may occur during reverse transcription¹³, thus seriously affecting the genetic stability of these viruses. However, the use of different polymerase III promoters may avoid such problems (ter Brake and Berkhout, unpublished results).

The HIV-rtTA virus is under development as a novel approach to an HIV vaccine. Live attenuated viruses have shown promise as vaccine candidates that elicit both a humoral and a cellular immune response²⁰. However, attenuated HIV-1 causes a persistent infection, maintains a high genetic instability, and can thus evolve into a pathogenic HIV-1 variant²¹. HIV-rtTA replication can be restricted by transient dox administration to the extent needed for induction of the immune system. As a result, chronic virus replication and restoration of virulence are prevented. Safety of this drug-controlled virus can be improved further by addition of other restrictions, e.g., T20-dependence^22,23. Addition of the therapeutic shRNA^Nef expression cassette, which maintains full activity when virus replication is stopped, may prevent HIV-1 infection of those cells that are not efficiently protected by the elicited immune response. Similarly, the HIV-rtTA-shNef vector may possibly be used to protect HIV-1 target cells in HIV-1-infected individuals. Transient replication of the therapeutic virus with dox would result in a population of cells that can no longer be infected by the patient's virus. This strategy seems particularly suitable for patients infected with a multidrug-resistant virus that can no longer be suppressed by the current antivirals. Several safety concerns remain for the use of this therapeutic virus in humans. For instance, this virus will insert at a random position in the human genome and may trigger deregulation of neighboring genes. Another problem may relate to off-target effects of the anti-HIV shRNA, but one could use inducible systems, preferably a polymerase promoter II unit that is under control of the HIV-1 Tat protein²⁴ such that the shRNA is expressed exclusively in virus-infected cells. The proposed therapy could provide a unique combination of vaccination with a conditional-live virus and intracellular immunization by means of RNAi.

Materials and methods

Cells and viruses

SupT1 T cells were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 units/ml streptomycin at 37°C and 5% CO₂. SupT1 cells were transfected with the variant HIV-rtTA molecular clones by electroporation. Briefly, 5 10⁶ cells were washed in RPMI 1640 medium with 20% FCS and mixed with 5 g of DNA in 250 l of RPMI 1640 medium with 20% FCS. Cells were electroporated in 0.4-cm cuvettes at 250 V and 975 F and subsequently resuspended in RPMI 1640 medium with 10% FCS and 1 g/ml doxycycline (Sigma). Cells were split 1 to 5 twice a week.

For the virus evolution experiments, virus replication was maintained by passage of the cell-free culture supernatant of massively infected cells (as apparent from the presence of large syncytia) onto uninfected SupT1 cells. Cells and supernatant samples were isolated and stored at -80°C.

SupT1 cells transduced with the HIV-rtTA-shNef variants (2.5 10⁵ cells in 1 ml medium) were infected with HIV^wt or HIV^R2 (200 ng of CA-p24), and viral replication was monitored by determining the CA-p24 level in the supernatant by ELISA as described²⁵.

C33A cervix carcinoma cells were grown as a monolayer in Dulbecco's modified Eagle's medium supplemented with 10% FCS and minimal essential medium nonessential amino acids at 37°C and 5% CO₂. For the production of virus stocks, C33A cells were transfected by the calcium phosphate method. Briefly, cells were grown in 3 ml of culture medium in 10-cm² wells to 60% confluence. HIV^wt or HIV^R2 DNA (5 g) in 110 l water was mixed with 125 l 50 mM Hepes (pH 7.1)–250 mM NaCl–1.5 mM Na₂HPO₄ and 15 l 2 M CaCl₂, incubated at room temperature for 20 min, and added to the culture medium. The culture medium was changed after 16 h and viruses were harvested at 2 days posttransfection.

Cotransfection of HIV-1 and HIV-rtTA-shNef variants was performed in C33A cells grown in 1 ml culture medium in 2-cm² wells at 60% confluence. A total of 1 g DNA was mixed in 15 l water (500 ng HIV^wt or HIV^R2, 100 ng U3-HIV-rtTA-shNef variants, 400 ng pBluescriptII KS(⁺) (Stratagene). The DNA was mixed with 25 l 2 HBS and 10 l 0.6 M CaCl₂, incubated at room temperature for 20 min, and added to the culture medium. The culture medium was changed after 16 h and virus was harvested at 3 days posttransfection. Virus was quantitated by CA-p24 ELISA.

DNA constructs

The pRetro-SUPER-shNef vector^8,26, which expresses shRNA^Nef under control of the H1 RNA polymerase III promoter, and the empty pRetro-SUPER-vector were digested with EcoRI and XhoI and the polymerase III expression cassette (310 and 249 bp, respectively). The strands were completed with Klenow DNA polymerase in the presence of dNTPs and cloned into the EcoRV site of pBlue 3'LTRext-U3-rtTA_{F86Y A209T}-2tetO¹⁵, which had been digested with EcoRV and subsequently treated with shrimp alkaline phosphatase to prevent self-ligation. Sequence analysis was performed by Big Dye terminator cycle sequencing to identify shRNA^Nef and control inserts (empty polymerase III units) in both orientations (F, forward, and R, reverse). The BamHI–BglI fragment of these shuttle vectors was exchanged with the BamHI–BglI fragment in HIV-rtTA, resulting in the HIV-rtTA-F-shNef and HIV-rtTA-R-shNef therapeutic vectors and the empty controls HIV-rtTA-F and HIV-rtTA-R. The HIV-rtTA version used in this study, HIV-rtTA_{F86Y A209T}-2tetO, carries the inactivating Y26A mutation in the Tat gene, five nucleotide substitutions in the TAR (trans-acting response region) hairpin motif, the improved rtTA_{F86Y A209T} gene¹⁵ in place of the Nef gene, and the optimized 2tetO promoter configuration^13,14 in the 5' and 3' LTRs. In the U3-HIV-rtTA constructs the 5' U3 sequences from position -333 up to -159 (with position +1 representing the transcription initiation site²⁷) were deleted through substitution of the 5' LTR-tetO promoter region of the HIV-rtTA variants with the corresponding fragment of the HIV-rtTA-dNF construct in which the NF-B sites and upstream U3 sequences had been deleted (-333/-159; A. T. Das et al., manuscript in preparation).

The full-length molecular HIV-1 clone LAI²⁸ was used to produce the wild-type HIV-1 virus. Construction of the HIV^R2 molecular clone with an 11-nt deletion in the shRNA^Nef target sequence and the firefly luciferase expression vectors pGL3-Nef^wt and pGL3-Nef^R2, containing an 250-bp Nef fragment (+8448 to +8698) downstream of the luciferase gene, were described previously⁹. The plasmid pRL-CMV (Promega) expresses Renilla luciferase under control of the CMV promoter. The pBluescriptII-based (KS+) (Stratagene) plasmid pBS-shRNA-Nef, which contains the shRNA-Nef expression cassette, was previously described⁹.

Viral RNA isolation

C33A cells (25 cm²; 65% confluent) were transfected with 10 g U3-HIV-rtTA DNA construct using Lipofectamine (Invitrogen). Dox (1 g/ml) was added to the culture medium 6 h after transfection when indicated. Cells were lysed 2 days after transfection and RNAs were isolated using the mirVana miRNA isolation kit (Ambion).

Northern blotting

Gel electrophoresis of 5 g RNA was performed on a 15% acrylamide Novex TBE–urea gel (Invitrogen) at 180 V in 1 TBE buffer (90 mM Tris, 90 mM boric acid, and 2 mM EDTA, pH 8.3). RNA was transferred onto a positively charged nylon membrane (Boehringer Mannheim) for 2 h at 80 V and crosslinked to the membrane with a UV crosslinker (Stratagene). A 19-nt LNA molecule (Eurogentec) with a sequence similar to the shRNA^Nef target sequence (5'-GTGCCTGGCTAGAAGCACA-3'; locked nucleotides are underlined) was used as a probe. The probe was 5' end labeled using the kinaseMax kit (Ambion) in the presence of 1 l of [-³²P]ATP (0.37 MBq/l; Amersham Biosciences) and purified over a MicroSpin G-25 column (GE Healthcare). Prehybridization and hybridization were done in ULTRAhyb buffer (Ambion) at 42°C for 30 min and 18 h, respectively. The membrane was washed twice at 42°C with low-stringency buffer (2 SSC, 0.2% SDS). Images were obtained using the Typhoon Trio phosphorimager (Amersham Biosciences).

Proviral DNA analysis

HIV-rtTA-infected cells were pelleted by centrifugation at 5000 rpm for 5 min and washed with phosphate-buffered saline. DNA was solubilized by resuspending the cells in 10 mM Tris–HCl (pH 8.0)–1 mM EDTA–0.5% Tween 20, followed by incubation with 200 g/ml proteinase K at 56°C for 30 min and at 95°C for 10 min. Proviral DNA sequences were PCR-amplified from total cellular DNA with the 5' primer tTA-tetO-1-AD (annealing to rtTA sequences 54–30 nt upstream of the 3' U3) and the 3' primer CN1 (annealing to U5 sequences 124–152 nt downstream of U3).

Analysis of clones

SupT1 cells transduced with U3-HIV-rtTA-F-shNef were used for limiting dilution to obtain cell clones. Serial dilutions (fivefold) were prepared in 96-well plates. Fresh medium was added every 5 days. The presence of the U3-HIV-rtTA-F-shNef provirus was determined by PCR, and RNAi-mediated inhibition of HIV-1 was determined in an HIV^wt challenge (500 ng of CA-p24).

Luciferase assay

C33A cells were grown in 1 ml culture medium in 2-cm² wells to 60% confluence and transfected by the calcium phosphate method. One hundred nanograms of pGL3-Nef^wt or pGL3-Nef^R2 was mixed with 0.5 ng of pRL-CMV, 100 ng of the U3-HIV-rtTA-shNef variants, and 800 ng of pBluescriptII (1 g total DNA) in 15 l of water. The DNA was mixed with 25 l of 2 HBS and 10 l of 0.6 M CaCl₂, incubated at room temperature for 20 min, and added to the culture medium. The culture medium was refreshed after 16 h. After another 24 h the cells were lysed in 150 l of Passive Lysis Buffer (Promega) by shaking for 20 min at room temperature. The cell lysate was centrifuged and 10 l of the supernatant was used to measure firefly and Renilla luciferase activities with the Dual-Luciferase Reporter Assay System (Promega). The expression of Renilla luciferase in transfected cells allowed us to correct for variation in transfection efficiency.

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Acknowledgements

We thank Stephan Heynen for CA-p24 ELISA. This research was sponsored by The Netherlands Organisation for Health Research and Development (ZonMW; VICI grant) and The Netherlands Organisation for Scientific Research (NWO-CW; TOP grant).