Retroviral reverse transcriptases (RTs) convert a single-stranded viral RNA genome into a double-stranded DNA (
4). Among the crucial events in the process of reverse transcription is initiation of minus-strand DNA synthesis to generate minus-strand strong-stop DNA, selective degradation of genomic RNA by RNase H, minus-strand DNA transfer, initiation of plus-strand DNA synthesis, formation of plus-strand strong-stop DNA, plus-strand DNA transfer and additional minus- and plus-strand DNA synthesis to complete the formation of viral DNA.
Several questions regarding the complex nature of human immunodeficiency virus type 1 (HIV-1) reverse transcription in cells remain unanswered; these questions include the efficiency of DNA synthesis initiation and strand-transfer events, the rates of RNA- and DNA-dependent DNA synthesis, and preferential inhibition of minus- or plus-strand DNA synthesis by RT inhibitors. Studies using purified RT and template (
5,
7,
9,
12,
18) as well as endogenous reverse transcription reactions using permeabilized virions (
1,
19,
23) have provided insights into these questions. Additionally, recent application of real-time PCR technology (
2,
24) has greatly facilitated the analysis of reverse transcription in cell-based assays; however, like all PCR methods, the real-time PCR technique cannot distinguish between the two DNA strands and a system for quantitative strand-specific analysis of reverse transcription during the course of viral infection has not been available.
We have now developed a novel strand-specific amplification (SSA) assay for site-specific amplification and quantification of each strand during HIV-1 reverse transcription and used it to measure the relative abundance of HIV-1 reverse transcription products generated at distinct steps over the time course of viral infection. These studies have allowed us to measure the kinetics of minus-strand DNA synthesis in 293T cells as well as human primary CD4+ T cells, one of the target cells of HIV-1 infection. We have also measured the kinetics of plus-strand DNA synthesis and the efficiencies of minus- and plus-strand DNA initiation and transfer in 293T cells. Finally, we have used SSA to analyze the effects of RT inhibitors on minus- and plus-strand DNA synthesis, which provide insights into their mechanism of inhibition.
MATERIALS AND METHODS
Plasmids and mutagenesis.
HIV-1-based retroviral vector pHDV-EGFP, which expresses HIV-1 Gag-Pol and the enhanced green fluorescent protein (EGFP) from the Nef open reading frame and does not expresses HIV-1 Env, was kindly provided by Derya Unutmaz (Vanderbilt University Medical Center, Nashville, TN) (
22). pHCMV-G expresses vesicular stomatitis virus G envelope (VSV-G) (
26). Site-directed mutagenesis of the central polypurine tract (cPPT) in pHDV-EGFP was performed using the QuikChange XL site-directed mutagenesis kit (Stratagene, Inc.). The wild-type cPPT sequence (5′-AAAAGAAAAGGGGGG-3′) was modified by introducing six silent mutations (5′-AA
GCG
CAA
GGG
CGG
C-3′; the substitutions are underlined). A restriction fragment (SbfI-SalI) containing the cPPT was subcloned into the pHDV-EGFP plasmid to generate pHIV-GFP-cPPT
− and sequenced to confirm the presence of the desired mutations and the absence of the undesired mutations.
Preparation of virus particles.
For most experiments, virus was prepared from a 293T-based cell line HIV-GFP2, which contains an undetermined number of integrated proviruses derived from pHDV-EGFP. To generate virus, HIV-GFP2 cells were plated at 2 × 10
6 cells per 100-mm-diameter dish and pretreated for 2 days before transfection with 1 μM 3′-azido-3′-deoxythymidine (AZT) for 10 h to prevent possible reinfection of transfected cells with produced virus. Calcium phosphate transfection (CalPhos transfection kit; Clontech) was performed using 4 μg of VSV-G-expressing plasmid (
26). After 7 h, DNA-containing transfection solution with 1 μM AZT was removed by washing cells once with AZT-free medium, and fresh medium was then added to the cells. The virus was collected 17 h later and concentrated 10-fold by ultracentrifugation at 20,000 ×
g for 1 h. After resuspension, the virus was treated with DNase I (30 units/ml, 10 mM final concentration of MgCl
2) for 1 h at room temperature, divided into 1-ml aliquots, and frozen at −80°C.
For some experiments, virus was produced following transient transfection of 293T cells with pHDV-EGFP or pHIV-GFP-cPPT− and VSV-G-expressing plasmid, using the MBS mammalian transfection kit (Stratagene). The treatment of virus produced from the transfected cells with DNase I (105 U/ml, 10 mM MgCl2, 3 h at room temperature) resulted in very low background levels, indicating little contamination with transfected DNA.
Time course for analyzing reverse transcription products in 293T cells.
To study the kinetics of HIV-1 reverse transcription, a time course of infection with HIV-1 HDV-EGFP virus was performed. For infection, 2 × 106 or 6 × 105 293T cells were plated per 100-mm- or 60-mm-diameter dish, respectively, the day before infection. A virus preparation concentrated 10-fold by centrifugation was diluted 20-fold in Dulbecco's modified Eagle's medium. Two or 0.66 ml of diluted virus solution was used to infect 293T target cells plated on a 100-mm- or 60-mm-diameter dish, respectively. After infection for 30 min, the virus was removed, cells were washed twice with phosphate-buffered saline to remove any residual virus, and fresh medium was added to the plate. At the end of each time point, cells were washed twice with phosphate-buffered saline, resuspended in 2 ml of phosphate-buffered saline, pelleted, and frozen until DNA isolation. In a typical time-course experiment, one plate of cells was collected at either 30-min or 1-h intervals for up to 6 h postinfection. Total cellular DNA was isolated using the QiaAmp DNA blood kit (QIAGEN) and resuspended in 250 to 400 μl of water. Real-time PCR or SSA analysis was performed four times for each primer-probe set using 4 μl of this extracted DNA.
To monitor the efficiency of infection, the percentage of GFP-expressing cells was measured 48 h postinfection using flow cytometry (FACscan and Cell-Quest Software; Bectin Dickinson). In most experiments, 10 to 30% of the infected cells expressed GFP (multiplicity of infection < 1).
To analyze the effects of RT inhibitors on reverse transcription, 293T cells were pretreated with the inhibitors for 24 h and maintained in the presence of the drugs during and after the infection. The following concentrations of inhibitors were used: efavirenz (EFV), 9 nM; 2′,3′-dideoxyinosine (ddI), 50 μM; 3′-deoxy-2′,3′-didehydrothymidine (d4T), 4 μM; AZT, 1 μM. For each inhibitor, infection efficiencies were reduced 97 to 99% relative to a minus-inhibitor control infection, as determined by flow cytometry 36 h after infection.
Time course for analyzing reverse transcription products in primary CD4+ T cells.
Activated CD4
+ T cells were purified from human peripheral blood mononuclear cells using the CD4 positive isolation kit (Dynal Biotech) according to the manufacturer's instructions. Cells were infected with HDV-EGFP virus by spinoculation as previously described; briefly, the cells were incubated for 2 h at 1,200 ×
g at 10°C, followed by incubation at 37°C for 1 h (
16). The cells were then washed four times to remove residual virus and placed back at 37°C. The cell samples for each time point during a time course were collected and processed as described above for 293T cells.
Oligonucleotides.
All padlock probes, primers, and dual-labeled probes were synthesized by Integrated DNA Technologies, Inc. Padlock probes containing a 5′ phosphate group were chemically synthesized by standard phosphoramidite chemistry and purified by polyacrylamide gel electrophoresis by the manufacturer. All of the probes used in this study were 83-mers containing the following common spacer region of 49 bases: 5′-TTGCGACTCGTCATGTCTGAACTCTAGTGATCTTAGTGTCAGGATAGCT-3′. The target arms were directed against various minus- and plus-strand sites in pHDV-EGFP.
Amplification of ligated probes was performed using RCA-23 (5′-ACTAGAGTTCAGACATGACGAGT-3′) as the forward primer and REV-21 (5′-GATCTTAGTGTCAGGATAGCT-3′) as the reverse primer. These primers were chemically synthesized with a 5′ OH. Dual-labeled probes used for real-time quantitative PCR were end-labeled with 5′ 6-carboxyfluoescein (FAM) and 3′ 6-carboxytetramethylrhodamine (TAMRA).
SSA assay.
During the first step of SSA, a padlock probe was hybridized to a denatured target DNA and circularized by treatment with thermostable Taq DNA ligase (New England Biolabs). The 10-μl reaction mix contained 1× Taq DNA ligase buffer, 109 molecules of padlock probe, 12 units of Taq DNA ligase, and 4 μl of total cellular DNA extracted from infected cells as described above. After an initial incubation at 95°C for 5 min to denature the target DNA, ligation was performed during 20 cycles of denaturing at 95°C for 1 min and probe annealing and ligation at 50°C for 4 min, followed by a final incubation at 50°C for 10 min. Negative control reaction mixtures were prepared as described above except that either target DNA or Taq DNA ligase was omitted. For each of the 12 padlock probes, the ligation reaction mixtures were performed using serial dilutions of linearized pHDV-EGFP DNA and 25 ng of uninfected 293T cell DNA to generate a standard curve to calculate the copy numbers of target DNA samples.
Detection of ligated padlock probe products was monitored by first subjecting dilutions of the ligation reaction mixture to a modified RCA method using two primers, followed by quantitative real-time PCR using dual-labeled probes. Ligation reaction mixtures were diluted to 100 μl with water, and 5-μl aliquots were added to a reaction mixture (25 μl, final volume) containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 200 μM concentrations of deoxynucleoside triphosphates, 500 nM (each) RCA-23 (forward) and REV-21 (reverse) primers, 100 nM dual-labeled probe, 1 unit of Platinum Taq DNA polymerase (Invitrogen), and 2.4 units of Bst DNA polymerase (large fragment; New England Biolabs). An initial incubation was performed at 63°C for 8 min for the two-primer RCA reaction, followed by 2 min of incubation at 94°C, which effectively denatured Bst DNA polymerase while simultaneously activating Platinum Taq DNA polymerase. Samples were then subjected to 40 cycles of PCR (94°C for 15 s and 60 to 63°C for 1 min). Fluorescence data were collected during the extension step using an ABI PRISM 7700 Sequence Detection System. Copy numbers of target DNA were calculated using the ABI PRISM 7700 Sequence Detection System software based on standard curves generated with serial dilutions of pHDV-EGFP in the ligation reaction mixtures.
To normalize for the amount of input DNA added to an SSA reaction, samples were measured by conventional real-time PCR with a primer-probe set specific for the cellular porphobilinogen deaminase (PBGD) gene (GenBank accession number M95623) (
27) as previously described (
25). The forward primer was 5′-AGGGATTCACTCAGGCTCTTTCT-3′, the reverse primer was 5′-GCATGTTCAAGCTCCTTGGTAA-3′, and the probe was 5′-FAM-TCCGGCAGATTGGAGAGAAAAGCCTG-TAMRA-3′. Final copy numbers after PBGD normalization and subtraction of 0-h copy numbers are expressed as percentages of the 6-h copy number.
The kinetics of minus-strand DNA transfer and initiation of plus-strand DNA synthesis at the polypurine tract (PPT) were determined by using probes 1, 2, and 7, which detect reverse transcription products derived from the long terminal repeats (LTRs). During reverse transcription, the 3′ LTR is first synthesized soon after minus-strand DNA transfer and the 5′ LTR is synthesized after plus-strand DNA transfer. The kinetics of accumulation of LTR products over the 6-h time course represents synthesis of both LTRs; analysis of the kinetics of accumulation of LTR-specific probes suggested that most of the synthesis of the 3′ LTR is completed within the first 3 h after infection. Therefore, the kinetics of minus-strand DNA transfer and plus-strand DNA synthesis initiation at the PPT were analyzed over a time course of 3 h instead of 6 h. Finally, the copy numbers of the minus-strand DNA products detected by probe 4, which are complementary to the central flap on the plus-strand DNA, were similar to those detected with probe 1.
DISCUSSION
The novel SSA assay described here should be applicable to a wide range of molecular studies in which quantification of specific strands of nucleic acids is desirable, including replication of other viruses (hepadnaviruses, adenoviruses, herpesviruses, etc.) and leading- and lagging-strand synthesis in organisms with double-stranded genomes. The SSA assay now further advances the quantitative PCR technology that has been applied to the measurements of HIV-1 viral load (
3,
17), integrated DNA copies (
2), and reverse transcription kinetics (
2,
8,
24) by providing a tool for analyzing the strand-specific aspects of viral replication.
To our knowledge, these studies have provided the first measurements of several key steps during HIV-1 replication in cells, including the rate of minus-strand DNA synthesis (∼68 to 70 nt/min). Previous measurements based on in vitro assays using either purified HIV-1 RTs (
5,
7,
9,
12,
18) or endogenous reactions using components from permeabilized virions (
1,
19,
23) varied greatly from 30 to 5,000 nt/min, presumably because the rates of DNA synthesis can be influenced by the assay conditions. The kinetics of HIV-1 replication probably also depend on intracellular conditions (
15) and may exhibit cell-specific differences. Interestingly, the rates we obtained here for 293T cells and activated human primary CD4
+ T cells were very similar, suggesting that intracellular conditions such as deoxynucleoside triphosphate concentration are similar in these two cell types. However, we noted a delay in the kinetics for both probes 3 and 6 in human primary CD4
+ T cells, suggesting a possible delay in viral entry and early postentry steps, such as uncoating, delay in initiation of minus-strand DNA synthesis, or a delay in minus-strand DNA transfer (Fig.
2A). The SSA assay can also be used to determine the effects of specific mutations in viral proteins (RT, NC, Vif, Vpr, Nef, etc.) as well as
cis-acting elements (PBS, PPT, cPPT, CTS, etc.) on the kinetics of various steps in HIV-1 replication.
The mechanism(s) that might contribute to the regional differences in the rate of minus-strand DNA synthesis are not known but could involve template RNA structures or greater availability of nucleocapsid protein later in replication, leading to more efficient denaturation of template structures, thereby increasing the rate of reverse transcription. The observation that plus-strand DNA transfer (∼26 min) is much slower than minus-strand DNA transfer (∼4 min) suggests that tRNA primer removal might be a slow and rate-limiting step. The observation that plus-strand initiation at the cPPT (∼28 min) was much slower than at the PPT (∼8 min) suggests that differences in sequences surrounding the two identical PPTs can influence the kinetics with which these plus-strand initiation sites are utilized.
Analysis of the cPPT
− mutant of HIV-1 and the similar kinetics with which plus-strand products separated by long distances accumulated has provided strong evidence for multiple sites of plus-strand DNA synthesis initiation during replication in cells, as previously reported (
10,
13). It is unclear at this time how many additional sites are used to initiate plus-strand DNA synthesis during HIV-1 replication, whether they represent specific sequences or RNA fragments that remain randomly associated with the minus-strand DNA, and how they avoid being displaced by DNA synthesis initiated at upstream sites.
Our observation that probe 10 products specifically detected two- to threefold-higher copy numbers of the central flap sequence, which has been implicated in the nuclear transport of HIV-1 preintegration complexes (
20,
28), indicates that the SSA assay can provide a quantitative method for the detection of the central flap and could be used to analyze the efficiency of displacement synthesis in other regions of the HIV-1 genome.
In these studies, the inhibition of viral replication by AZT, d4T, and ddI was similar (97 to 99%), representing inhibition of minus- and plus-strand DNA synthesis. For AZT and d4T, most of the inhibition of viral replication could be attributed to the observed 95% inhibition of minus-strand DNA synthesis. However, ddI treatment resulted in 55% inhibition of minus-strand DNA synthesis, suggesting that ddI also inhibited viral replication during plus-strand DNA synthesis.
In summary, the SSA method should provide a widely applicable technology for strand-specific analysis of nucleic acids. The SSA assay described here can be used to address a variety of questions about the process of HIV-1 replication, elucidate the mechanism of action of antiretroviral agents, and facilitate development of novel antiviral agents that interfere with specific steps in HIV-1 reverse transcription.
Acknowledgments
We thank Anne Arthur for expert editorial help. We also thank Rebekah Barr and Hongzhan Xu for technical assistance and Krista Delviks-Frankenberry and Eric Freed for critical readings of the manuscript.
This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.