Oligonucleotides were purchased from Integrated DNA Technologies (Iowa). Hexahistidine-tagged p66/p66 RT homodimers derived from HIV-1 (HXB2 (13
)) and SIV (SIVMNECL8 (15
)), or RT monomers of MuLV (16
), were isolated from bacterial overexpression systems described in our previous studies. AMV RT and KF were purchased from Stratagene (La Jolla, CA) and New England Biolabs, Inc. (Beverly, MA), respectively.
Single nucleotide incorporation assay: Four different 19-mer DNA templates containing sequence variations (N) at the 5′ end nucleotide (5′-NTGGCGCCCGAACAGGGAC-3′) were individually annealed to an 18-mer DNA primer (5′-GTCCCTGTTCGGGCGCCA-3′), 32P-labeled at its 5′ end (template:primer, 4:1). The nucleotide at the 5′ end of the primer determines the dNTP to be measured. The primer (160 pmole in 80 μl) was labeled using 40 units of T4 polynucleotide kinase (New England BioLabs, Inc., MA) with 80 μCi α-32P-ATP (Amersham, NJ) for 30 min at 37°C. An additional 40 units of T4 polynucleotide kinase was added for an additional 30 min period of labeling. After heat inactivation (95°C, 10 min), the labeled primer was split into four separate tubes annealed with each of the four 19-mer templates (160 pmole) with addition of 10×STE (100 mM NaCl and 5 mM EDTA for 1×) in a final volume of 50 μl. The template-primer mixture was incubated for 10 min at 95°C, 5 min at 55°C, 5 min at 22°C and on ice until used.
The template/primer pairs (T/Ps) were extended using RT proteins in a standard dNTP assay reaction. Each assay reaction (20 μl) contained 0.2 pmole T/P (10nM, primer concentration), 2 μl appropriate dNTPs (Amersham, NJ) or extracted cellular dNTPs, 25 mM Tris-HCl (pH 8.0), 100 mM KCl, 2 mM DTT, 5 mM MgCl2
, 5 μM (dT)20
, 0.1 mg/ml bovine serum albumin (New England BioLabs). Reactions were initiated by addition of excess RT proteins (60 nM) in relation to [dNTP] (0.2~6.4 nM or 4~128 fmole in 20 μl), and incubated at 37°C for 5 min. Reactions were terminated with 10 μl of 40 mM EDTA, 99% formamide. Reaction products were immediately denatured by incubating at 95°C for 5 min and 4 μl of each 30 μl final reaction mixture was quantitated by PhosphorImager analysis (PerkinElmer, MA) of 14% polyacrylamide-urea denaturing gels (SequaGel, National Diagnostics, GA; Model S2 Sequencing Gel Electrophoresis Apparatus, Labrepco, PA). Note that in our purification protocol, approximately 30–60% of the purified RT protein is active as determined by our pre-steady state kinetic assay using a similar 18-mer/40-mer primer/RNA template (17
). The reaction with less RT (i.e. 10 nM), which is still a higher concentration than the highest dNTP concentration used (6.4 nM), gave a similar incorporation profile under the assay condition.
Standard curves for single nucleotide incorporation: The gels obtained from the incorporation assay () were subjected to PhosphorImager analysis (see above). The percent of primer extension in each reaction was calculated by determining the ratios of extended versus total (extended + unextended) primers. Each signal for the extended products was normalized using the background signal in the control reactions incubated without RT (lane C in ). The calculated percentage primer extension was plotted from triplicate reactions, as a function of the dNTP quantity used, generating standard curves for all four dNTPs. The percentage primer extension determined in triplicate reactions containing extracted cellular dNTPs was then extrapolated to the standard curves in order to determine the dNTP contents present in the cellular samples.
Primary cell dNTP content determination by the HIV-1 RT-based dNTP assay.
Primer extension by RT proteins:
The primer extension assay was modified from a previously described misincorporation assay (18
). Briefly, an RNA template-primer (T/P) was prepared by annealing a 38-mer RNA (5′-AAGCUUGGCUGCAGAAUAUUGCUAGCGGGAAUUCGGCGCG-3′, Dharmacon Research, CO) to the 17-mer A primer (5′-CGCGCCGAATTCCCGCT-3′; template:primer, 2.5:1, Invitrogen, CA) 32
P-labeled at the 5′ end by T4 polynucleotide kinase (New England BioLabs, MA). Assay mixtures (20 μl) contained 10 nM T/P, the RT protein concentrations specified in the individual figure legends, 4 dNTPs at concentrations indicated in the figure legends under the condition described in the dNTP assay above. Reactions were incubated at 37°C for 5 min and terminated for analysis as described in the dNTP assay. Concentrations of RTs (i.e. 1X and 4X) and dNTPs (i.e. 4 μM and 0.04 μM) used in each primer extension experiment were described in each figure legend. This reaction condition allows multiple rounds of primer extension. We also performed the single round primer extension. In this reaction, RTs were preincubated with the 32
P-labeled 17-mer/38-mer RNA template (10nM), and the reaction was initiated by adding a mixture of dNTP (0.04 μM) and a molar excess of cold T/P (1 μM) for 3 min at 37°C. For the trap control, RTs were preincubated with a mixture of the 32
P-labeled T/P and the cold T/P, and the reaction was initiated by adding dNTP (0.04 μM). We also employed a 120nt long RNA template annealed to a 32
P-labeled 20-mer primer (19
). This T/P was also extended by different concentrations of RTs and dNTP for 10 min at 37°C (see figure legend for ).
Steady state kinetic analysis of dNTP incorporation:
The steady-state kinetic assay protocol described by Boosalis et al. (20
) was modified to determine DNA polymerization efficiencies of DNA polymerases with the 18-mer/19-mer template/primers used in the dNTP assay (see above). Reaction conditions were the same as those described in the dNTP assay except for DNA polymerase (0.5~2 nM) concentrations. For the analysis of dNTP incorporation kinetics, four different templates (5′ template nucleotide T, C, G, or A for dATP, dGTP, dCTP and dTTP incorporation, respectively) annealed to the 32
P-labeled 18-mer primer were used (see above). The amounts of RTs and incubation time were adjusted to yield extension of 30~60% of the total labeled primer (0.8 pmole) at the highest dNTP concentrations, and the reactions were repeated with 8 decreasing concentrations of each dNTP (for HIV-1 RT, 5, 10, 20, 40, 80, 160, 320 and 720 nM dNTPs and for MuLV RT, 1, 2, 4, 8, 16, 32, 64, and 128 μM). We also measured the steady state kinetic values using the processive reverse transcription with the 38-mer RNA annealed to the 32
P-labeled 17mer. In this analysis, the different concentrations of the all four dNTP mixtures were used. Products (single nucleotide incorporated primer or all extended products) were resolved in 14% polyacrylamide-urea gel and quantitated by PhosphorImager analysis using the OptiQuant software (PerkinElmer). The kcat
values were determined from the Michaelis-Menten equation.
dNTP extraction from cells:
The protocol for dNTP extraction from cells was similar to that previously described (21
). Briefly, cell pellets (5×104
cells) were washed twice with 1 X DPBS (Mediatech, VA), and resuspended in 100 μl of ice-cold 60% methanol. Samples were vortexed vigorously to lyse the cells and then heated at 95°C for 3 minutes, prior to centrifugation at 12,000×g for 30 s. The supernatants were collected and completely dried under vacuum, using a SpeedVac (Savant, NY) with medium heat. The dried pellets were subsequently resuspended in dNTP buffer (50 mM Tris-HCl, pH 8.0 and 10 mM MgCl2
; 100 μl for 1×106
HeLa cells, and 10 μl for 1×106
primary cells) and usually 1~2 μl of the extracted dNTP samples were used for each 20 μl single nucleotide incorporation reaction (see above). The proper dilutions of the dNTP samples were prepared for the assay in order to make the primer extension values lie within the linear ranges of the dNTP incorporation (2~32% primer extension). The extracted dNTP samples were stored at −70 until used. Several different volumes of the extracted dNTP samples were also used to confirm the linearity of the primer extension. In addition, the dNTP samples were prepared from different cell numbers, depending on the recovery efficiency of the primary cells from each blood sample (see below). However, the dNTP content of each cell type was normalized by pmole/1×106
Isolation and culture of human T cells and monocyte-derived macrophages:
Human macrophages and CD4+ T cells were isolated from human buffy coats (Blood Research Institute, MA) as described (22
). Peripheral blood mononuclear cells were harvested from Ficoll density gradients (Lymphoprep, Axis-Shield PoC AS, Oslo, Norway), and monocytes were then purified using immunomagnetic selection with anti-CD14 antibody conjugated magnetic beads, following the manufacturer’s recommendations (Miltenyi Biotech, CA). CD4+ lymphocytes were isolated from monocyte-depleted buffy coats by immunomagnetic selection using anti-CD4 antibody conjugated magnetic beads (Miltenyi Biotech). The purified human monocytes were incubated in 10 cm dishes in RPMI 1640 (Mediatech, VA) containing 20% Human AB Serum (Sigma, MO) for 4 days in the presence of 5 ng/ml human recombinant GM-CSF (R & D Systems, MN) and then incubated for an additional 3 days in the absence of GM-CSF, to allow differentiation into macrophages. The purified resting T cells were maintained in RPMI 1640 medium supplemented with 10% autologous human serum, while activated T cells were propagated in RPMI 1640 medium supplemented with 10% autologous human serum and 5 ng/ml PHA (Sigma) for 24h, and then incubated in RPMI-1640 medium with 10% human serum and 5ng/ml human, recombinant IL-2 (Sigma) for 3 days. These cell preparations were to prepare dNTP extracts and for the analysis of cell volumes using confocal microscopy.
Pseudotyped Virus Cloning and Preparation:
pHCMV-VSVG envelope vector (23
) and pD3-GFP transfer vector, which encodes the HIV-1 NL4-3 genome with the eGFP gene in place of HIV-1 env, were used for preparing pseudotyped HIV-1 The V148I and Q151N mutations were cloned into pD3-GFP (pNL4-3 based) vector using the overlapping PCR with proper primers designed based on the pNL4-3 RT sequence. The created mutations were confirmed by sequencing with the 3305 Primer (5′–GCACTATAGGCTGTACTGTCC-3′), and correct Swa
I religation was confirmed with D3-GFP SEQ Swa
I F (5′-CAGGCCATATCACCTAG-3′) and D3-GFP SEQ Swa
I R (5′-TCTAAC TGGTACCATAAC-3′) primers. 293FT cells (Invitrogen, CA) were grown to about 90% confluency with DMEM, 10% FBS, and 500 μg/ml geneticin in T-175 flasks and transfected with 5 μg envelope vector and 25 μg transfer vector using Lipofectamine 2000 (Invitrogen) as per the manufacturer’s recommendations (in the absence of geneticin). Cells were split in half into two new T-175 flasks 16 hours post-transfection. Supernatant was collected 48 and 72 hours post-infection and stored at 4°C. Virus was harvested by ultracentrifugation for 2 hours at 4°C and 22,000 rpm with the Beckman SW28 Rotor. Virus was titered by infection of HeLa cells [obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Dr. Richard Axel (24
)] in the presence of polybrene and infection monitored by fixation and flow cytometry analysis for GFP expression 48 hours post-infection. p24 levels were also measured using the HIV-1 p24 Antigen ELISA Test System (Beckman Coulter, CA).
As a control, transfections were also performed in the absence of the pHCMV-VSV-G vector and this virus was titered on HeLa cells revealing no detectable GFP expression. In addition, controls were performed by pre-treating HeLa cells for 2-hours prior to and during infection with 0.5mM cyclohexamide (CHX), which is a general translation inhibitor. Cells were fixed and analyzed by flow 48 hours post-transfection. Results showed that very little protein was carried over with the pseudotyped virus preparations as evidenced by less than 1% GFP positive cells with plus CHX infections compared to >20% GFP positive cells in minus CHX infections (data not shown).
Infection of Primary CD4+ T cells and monocyte-derived macrophage: T cells and macrophage were purified essentially as described above.
T cell infections: After 5 days of T cell stimulation with PHA and IL-2 (see above) 2×105 stimulated T cells were infected in the presence of polybrene with an MOI of 100 based on HeLa cell titering. For QN virus infections an MOI of 65 was used due to trouble generating high titer QN virus. These infections reflect addition of viral stocks containing 200–300 ng of p24. Infected cells were incubated at 37°C and 5%CO2 in a 100 μl total volume. Two hours post-infection 100 μl RPMI with 10% heat-inactivated human AB Serum and 4ng/μl IL-2 was added and infections were incubated for an additional 46 hours. Cells were visualized and photographed by fluorescent microscopy in order to detect GFP fluorescence by infected cells. Percent infection was determined after cells were prepared for flow analysis by fixation with 0.5% formaldehyde.
Macrophage infections: After initial magnetic bead purification, 2×106 CD14+ cells were plated per well into 6-well plates and cultured in RPMI with 2% heat-inactivated Human AB Serum and 5ng/ml GMCSF for 48 hours followed by 48-hour incubation in RPMI with 20% heat-inactivated Human AB Serum and 5ng/ml GMCSF. Cells were then incubated for an additional 8 days in the absence of GMCSF with fresh media added after the initial 4 days. Macrophages were infected with pseudotyped virus at an MOI of 40 based on HeLa cell titering and assuming 1×106 cells per well in the presence of polybrene. These infections reflect the addition of viral stocks containing 80–130 ng of p24. Initial infections were in 250 μl volume for the first 2 hours at 37°C and 5%CO2 followed by addition of 2ml RPMI with 20% heat-inactivated human AB serum. Incubations were continued for up to 6 days and cells were periodically examined for GFP fluorescence by fluorescent microscopy. Cells were fixed in 0.5% formaldehyde after disassociation from the plates by 30 min incubation with 2mM EDTA and gentle scraping. Percent infection was determined by flow cytometry.