Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich, all restriction enzymes were obtained from New England BioLabs, and all cell culture products were purchased from Gibco. Sources for the other reagents were as follows: DuraScribe T7 transcription kit (Epicentre Biotechnologies), Silencer siRNA Labeling kit (Ambion), Hoechst 33342 (nuclear dye for live cells) (Molecular Probes, Invitrogen), random primers (Invitrogen), SuperScript III RT kit (Invitrogen), and Bio-Spin 30 Columns (Bio-Rad). CHO-Env transfectants (CHO-WT and CHO-EE), the HIV-1 NL4-3 and HIV-1 BaL viruses, and clinical isolates (X4-strain: HIV-1 92UG021; R5-strain: HIV-1 RU570 and HIV-1 98CN009) were obtained from the AIDS Research and Reference Reagent Program. Normal mouse serum was obtained from Jackson ImmunoResearch Laboratories.
Generation of aptamer-siRNA chimera RNA (Ch A-1) by in vitro transcription
The design, synthesis, and in vitro efficacies of the aptamer A-1 and aptamer-siRNA conjugates have been described in detail (18
). siRNA Dicer substrate sense, antisense strand RNAs, and DNA oligonucleo-tides were purchased from Integrated DNA Technologies: tat
Site I 27-mersiRNA, 5′-GCGGAGACAGCGACGAAGAGCUCAUCA-3′ (sense) and 5′-UGAUGAGCUCUUCGUCGCUGUCUCCGCdTdT-3′ (antisense); aptamer A-1, 5′-GGGAGGACGAUGCGGAAUUGAGGGACCACGCGCUGCUUGUUGUGAUAAGCAGUUUGUCGUGAUGGCAGACGACUCGCCCGA-3′; chimera A-1, 5′-GGGAGGACGAUGCGGAAUUGAGGGACCACGCGCUGCUUGUUGUGAUAAGCAGUUUGUCGUGAUGGCAGACGACUCGCCCGAUUGCGGAGACAGCGACGAAGAGCUCAUCA
-3′ (sense); chimera A-5, 5′-GGGAGGACGAUGCGGGAAACUAGUUUGAAUAAUGGUGUAGAGGAGGGUCAAUAGUUUCGUUGGUGCAGACGACUCGCCCGAUUGCGGAGACAGCGACGAAGAGCUCAUCA
-3′ (sense) and 5′-UGAUGAGCUCUUCGUCGCUGUCUCCGCdTdT-3′ (antisense). The sense strands of the chimeras are underlined. The italic UU
is the linker between the aptamer and siRNA portions. The assembly of these chimeric constructs was described previously (17
), and a schematic is presented in .
Serum stability assay
Five micrograms of refolded Ch A-1 was incubated at 37°C in 100 μl of RPMI 1640 medium (Mediatech) containing 50 or 5% (v/v) mouse serum (normal mouse serum; Jackson ImmunoResearch Laboratories), resulting in an RNA concentration of 50 ng/μl. At 0 min to 72 hours, 10-μl aliquots of the reaction were withdrawn and 10 μl of 2× loading buffer containing 8 M urea, 120 EDTA, and 0.1% SDS was added to each sample. The mixtures were heated at 95°C for 5 min and then stored at −80°C until all incubations were completed. Each mixture (20 μl) was analyzed by electrophoresis in an 8% denaturing polyacrylamide gel, and the RNAs were visualized after ethidium bromide staining with an ultraviolet transilluminator.
Generation and HIV-1 infection of humanized Rag2−/−γc−/− mice (RAG-hu mice)
mice were prepared as described (20
) with human fetal liver–derived CD34+
cells. Briefly, neonatal mice were conditioned by irradiating at 3.5 Gy and then injected intrahepatically with 0.5 × 106
to 1 × 106
cells. About 12 weeks after reconstitution, mice were screened for human cell engraftment. Blood was collected by tail bleeds, and red blood cells were lysed with the Whole Blood Erythrocyte Lysing Kit (R&D Systems). The white blood cell fraction was stained with antibodies against the human panleukocyte marker CD45 (Caltag) and FACS (fluorescence-activated cell sorting)–analyzed as described (20
). To infect human cell–reconstituted RAG-hu mice, we injected HIV-1 NL4-3 (1.2 × 105
IU) in a 100-μl volume intraperitoneally at least 12 weeks after cell engraftment. Viral loads were examined weekly, and viremia was established in all the mice by 3 weeks. Treatment was done by intravenous injection on the last day of week 4 with 0.25 nmol of experimental RNAs [tat
siRNA (0.15 mg/kg) or Ch A-1 (0.38 mg/kg)] in a 40-μl volume, followed by another the next day. Later, the injections were continued on a weekly basis for 4 weeks. In the second in vivo treatment experiment, the same amounts as described above of mutant chimera Ch A-5, aptamer A-1, or chimera Ch A-1 in a 40-μl volume were administered at 5 weeks after infection as above and continued for only three weekly injections.
Measurement of viral load in plasma
To quantify cell-free HIV-1 by qRT-PCR, we extracted RNA from 25 to 50 μl of EDTA-treated plasma with the QIAamp Viral RNA kit (Qiagen). cDNAs were produced with SuperScript III reverse transcriptase (Invitrogen) with a primer set specific for the HIV-1 long terminal repeat (LTR) sequence, and qPCR was performed with the same primer set and an LTR-specific probe with Supermix UDG (Invitrogen) as described (20
). If there was no detectable viral RNA, we established this as a value of 1 (100
) to allow for the use of logarithmic values on the y
Whole blood was collected and red blood cells were lysed as reported (20
). Peripheral blood cells were stained by hCD3-PE and hCD4-PECy5 (Caltag) antibodies and analyzed with a Coulter EPICS XL-MCL FACS analyzer (Beckman Coulter). CD4+
T cell levels were calculated as a ratio of the entire CD3 population (CD4+
). To establish baseline CD4+
T cell ratios, we analyzed all mice before infection. Each individual mouse was bled two times before HIV-1 infection and averaged within treatment groups to establish a baseline CD4-CD3 level.
Detection of tat/rev siRNA
At 5, 7, and 12 weeks after infection (1, 3, and 9 weeks after treatment), blood samples were collected and small RNAs were isolated with MirVana miRNA isolation Kit (Applied Biosystems) according to the manufacturer's instruction. The siRNA quantification was performed with TaqMan MicroRNA Assay according to the manufacturer's recommended protocol (Applied Biosystems). Small RNA (10 ng), 0.2 μM stem-loop RT primer, RT buffer, 0.25 mM deoxynucleotide triphosphates, MultiScribe reverse transcriptase (3.33 U/ml), and ribonuclease inhibitor (0.25 U/ml) were used in 15-μl RT reactions for 30 min at 16°C, 30 min at 42°C, and 5 min at 85°C with the TaqMan MicroRNA RT kit (Applied Biosystems). For real-time PCR, 1.33 μl of cDNA, 0.2 mM TaqMan Probe, 1.5 mM forward primer, 0.7 mM reverse primer, and TaqMan Universal PCR Master Mix were added in 20-μl reactions for 10 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C. All real-time PCR experiments were done with an iCycler iQ system (Bio-Rad). Primers were as follows: site I looped RT primer, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGCG-3′; site I forward primer, 5′-GCTGATGAGCTCTTCGTCG-3′; site I reverse primer, 5′-GTGCAGGGTCCGAGGT-3′; site I probe primer, 5′-6-FAM-TCGCACTGGATACGACACAGCGACGA-BHQ1-3′. In this case, a synthetic 27-mer duplex RNA was used as positive control.
Cell culture conditions
Human embryonic kidney (HEK) 293 and CEM cells were purchased from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM) and RPMI 1640 supplemented with 10% fetal bovine serum (FBS). CHO-WT and CHO-EE cells were obtained through the AIDS Research and Reference Reagent Program and grown in modified Glasgow minimal essential medium (GMEM-S) supplement with 400 μM methionine sulfoximine (MSX). Cells were cultured in a humidified 5% CO2 incubator at 37°C. PBMCs were obtained from healthy donors from the City of Hope National Medical Center. They were isolated from whole blood by centrifugation through a Ficoll-Hypaque solution (Histopaque-1077, Sigma). CD8 cells (T cytotoxic/suppressor cells) were depleted from the PBMCs by CD8 Dynabeads (Invitrogen) according to the manufacturer's instructions. CD8+ T cell–depleted PBMCs were washed twice in phosphate-buffered saline (PBS) and resuspended in culture media [RPMI 1640 with 10% FBS, 1× penicillin-streptomycin, and IL-2 (100 U/ml)]. Cells were cultured in a humidified 5% CO2 incubator at 37°C.
HIV-1 challenge and p24 antigen assays
CEM cells or human PBMCs were infected with HIV strains IIIB, NL4-3, or BaL for 5 days [multiplicity of infection (MOI), 0.001 or 0.005]. For clinical isolates, human PBMC-CD4+ cells were infected for 3 days (MOI, 0.001 or 0.0005). Before RNA treatments, the infected cells were gently washed with PBS three times to remove free virus.Next, 2 × 104 infected cells and 3 × 104 uninfected cells were incubated with refolded RNAs at 400 nM final concentration in 96-well plates at 37°C. The culture supernatants were collected at different times after treatment (3, 5, 7, and 9 days or 11 days). Here, the p24 antigen analyses were performed with a Coulter HIV-1 p24 Antigen Assay (Beckman Coulter) according to the manufacturer's instructions.
Determination of HIV-1 tat/rev expression
Human PBMCs were obtained from treated mice at 5 and 7 weeks after infection (1 and 3 weeks after treatment began), and total RNAs were isolated with STAT16 60 (Tel-Test “B”) according to the manufacturer's instructions. Residual DNA was digested with a DNA-free kit per the manufacturer's instructions (Ambion). cDNA was made with 2 μg of total RNA. RT was carried out with Moloney murine leukemia virus reverse transcriptase and random primers in a 15-μl reaction according to the manufacturer's instructions (Invitrogen). Expression of the tat/rev coding RNAs was analyzed by qRT-PCR with 2× iQ SYBRGreen MasterMix (Bio-Rad) and specific primer sets at a final concentration of 400 nM. gapdh expression was used for normalization of the qPCR data. Primers were as follows: NL4-3 or IIIB tat/rev, 5′-GGCGTTACTCGACAGAGGAG-3′ (forward) and 5′-TGCTTTGATAGAGAAGCTTGATG-3′ (reverse); BaL tat/rev, 5′-GAAGCATCCAGGAAGTCAGC-3′ (forward) and 5′-TGCTTTGATAGAGAAACTTGATGA-3′ (reverse); gapdh, 5′-CATTGACCTCAACTACATG-3′ (forward) and 5′-TCTCCATGGTGGTGAAGAC-3′ (reverse).
5′-RACE PCR assay to detect in vivo RNAi-mediated target mRNA cleavage
Total RNA was isolated from PBMCs of treated mice with STAT-60 as described above. Residual DNA was digested with the DNA-free kit per the manufacturer's instructions (Ambion). Subsequently, total RNAs (5 mg) were ligated to a GeneRacer adaptor (Invitrogen) without previous treatment. Ligated RNA was reverse-transcribed with a gene-specific primer 1 (GSP-Rev 1: 5′-CCACTTGCCACCCATCTTATAGCA-3′) and SuperScript III reverse transcriptase according to the manufacturer's instructions (Invitrogen). To detect cleavage products, we performed PCR and nested PCRs with primers complementary to the RNA adaptor (5′-cDNA primer: 5′-GGACACTGACATGGACTGAAGGAGTA-3′) and gene-specific primers (GSP-Rev 2: 5′-CCCAGAAGTTCCACAATCCTCGTT-3′; GSP-Rev 3: 5′-TGGTAGCTGAAGAGGCACAGGCTC-3′; GSP-Rev4: 5′-CGCAGATCGTCCCAGATAAGTGCTAA-3′). Amplification products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. The specific PCR products were recovered with a QIAquick Gel purification kit and then cloned into 17 TOPO TA cloning vector pCR 2.1-TOPO vector (Invitrogen). Individual clones were identified by DNA sequencing.
Total RNA was isolated from PBMCs of treated mice with STAT-60. Expression of mRNAs encoding p56 (CDKL2) and OAS1 were analyzed by qRT-PCR with 2× iQ SYBRGreen MasterMix (Bio-Rad) as described above and specific primer sets for these genes at final concentrations of 400 nM. Primers were as follows: p56 (CDKL2), 5′-TCAAGTATGGCAAGGCTGTG-3′ (forward) and 5′-GAGGCTCTGCTTCTGCATCT-3′ (reverse); OAS1, 5′-ACCGTCTTGGAACTGGTCAC-3′ (forward) and 5′-ATGTTCCTTGTTGGGTCAGC-3′ (reverse). gapdh expression was used for normalization of the qPCR data. In addition, 25 to 50 μl of EDTA-treated plasma was collected 2 and 24 hours after treatment. As a positive control for IFN-α induction, mice were injected with 5 μg of poly I:C (Sigma) intravenously in a 50-μl volume. IFN-α levels were evaluated by Human IFN-α1 ELISA Ready-SET-Go! (eBioscience).
PBMCs were obtained from treated mice 3, 4, and 5 weeks after Ch A-1 injection, and total RNAs were isolated with STAT-60 (Tel-Test) according to the manufacturer's instructions. Residual DNA was removed with the DNA-free kit per the manufacturer's instructions (Ambion). cDNA was synthesized with 1 to 5 μg of total RNA. RT was carried out with SuperScript III reverse transcriptase and an oligo(dT)20 primer in a 20-μl reaction according to the manufacturer's instructions (Invitrogen). To detect viral RNAs, we performed PCR and nested PCRs using gene-specific primers (NL4-3 primer S1-F: 5′-TACAATGAATGGACACTAGAG-3′; NL4-3 primer S1-R: 5′-TTCTAGGTCTCGAGATACTG-3′; NL4-3 primer S2-F: 5′-AGGCGTTACTCGACAGAGGA-3′; NL4-3 primer S2-R: 5′-TGGCGAATAGCTCTATAAGC-3′; NL4-3 S3-F: 5′-ATGAGAGTGAAGGAGAAGTAT-3′; NL4-3 S3-R: 5′-AAGAGTAAGTCTCTCAAGCG-3′; NL4-3 S4-F: 5′-TCAGCACTTGTGGAGATGGG-3′; NL4-3 S4-R: 5′-TGGTGAATATCCCTGCCTAA-3′). Amplification products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. The specific PCR products were recovered with a QIAquick Gel purification kit. Illumina deep sequencing was carried out by the City of Hope DNA sequencing core, and data analyses were performed by the City of Hope Bioinformatics Core facility. As an alternative approach, gel-purified PCR products were also cloned into the TOPO TA cloning vector pCR 2.1-TOPO vector and individual clones were identified by DNA sequencing.
Illumina deep sequencing and data analysis
Treatment and control libraries of the target region of HIV-1 NL4-3 (from 5830 to 8785) were pooled into a single lane of a flow cell with an Illumina multiplexing sample preparation oligonucleotide kit. Each sample was labeled with a different six-base oligo as a bar code. Deep sequencing on the mixed sample was carried out with an Illumina Genome Analyzer (GA) II by running 43 cycles (36 cycles for sample read, 6 cycles for bar code, and 1 extra cycle for bar code offset estimation). Raw data from Illumina GA II were processed with the Solexa software pipeline v. 1.5.1 into 36–base pair shotgun reads. Sequences were aligned back to the HIV-1 NL4-3 genome [National Center for Biotechnology Information (NCBI): AF324493] with CLC Genomics Workbench, and sequences that mapped to more than one position with similar alignment scores were removed. Variations were detected in both treatment and control samples with Neighborhood Quality Standard (NQS) algorithm (33
). Because of the experiment design, the mutation ratio of the variation caused by treatment could be very low. To distinguish these low-frequency mutations from the background noise (such as sequencing error, alignment bias, and system error), we used two thresholds. First, 1% variation rate or 100-fold variation coverage (coverage refers to the number of overlapping sequences used to build a region of the assembly) was used as quality control threshold for variation detection. This threshold makes it possible to detect all possible variations at each position and maintains reasonable quality of the variation. Second, a variation-to-noise ratio was used if there were multiple variations at the same location. We defined the variation-to-noise ratio as the ratio between the majority variation ratio and the sum of rest variations observed at this location. Here, the variation-to-noise ratio threshold was 10, and this threshold filtered out those variations that were caused by experimental background noise. Variations above the threshold were compared with the variations observed in controls to identify the mutations caused by treatment.
The mouse viral loads and CD4-CD3 T cell ratios were plotted with a LOWESS (locally weighted scatterplot smoothing) smoother across values. For larger sample sizes, some type of linear model such as linear regression, multivariate analysis of variance (MANOVA), or generalized estimating equations are used. However, each of these requires a larger number of observations per group for stable variance estimation. Our approach transformed the data into a test of mean areas under the curve (AUCs), which may be compared using a t
test, despite the small samples. Viral loads were first log-transformed before smoothing and then anti-transformed for plotting. Missing values were imputed with a last observation carried forward scheme. To measure the differences between mouse treatment groups, we considered a primary endpoint evaluating longitudinal behavior over time. Our method was to calculate a cumulative AUC for each mouse, and then compare aggregate mean AUCs between mouse groups. For any sequential time points (xi
), and their corresponding endpoints (yi
), the AUC was calculated with the area of a trapezoid: 0.5 × (xj
) × (yi
). The cumulative AUC of a single observation for the duration of the experiment was the cumulative sum of trapezoids. The Kruskal-Wallis rank sum ANOVA analog was used for the groupwise cumulative AUC comparisons. We compared pairwise group mean AUCs using t
tests and exact permutation tests under the Hothorn and Hornik exactRankTests package for the R language (34
). Details for deriving the permutation P
values in general are discussed by Streitberg and Röhmel (38