HIV-1 RT and RT inhibitors
Recombinant HIV-1BH10 RT p66 produced from E. coli was obtained from University of Alabama at Birmingham, Center for AIDS Research, Gene Expression core Facility through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. AZTTP, d4TTP, ddCTP and 3TCTP were purchased from ChemCyte Inc (San Diego, CA, USA). AZT, d4T, ddC, 3TC, ddI, ABC, FTC and bis-POM PMPA were purchased from Sigma-Aldrich (St. Louis, MO, USA). NVP, DLV and EFV were purchased from Research Toronto Chemicals (North York, ON, Canada). Template/primer mix poly (rA)-oligo (dT)12-18, poly (rI) and oligo (dC)12-18 were purchased from Midland Certified Reagent Company (Midland, TX, USA). [α-32P] dTTP and [α-32P] dCTP were purchased from PerkinElmer Life And Analytical Sciences, Inc. (Waltham, MA, USA). Transfection reagent Fugene 6 was purchased from Roche Applied Science (Indianapolis, IN, USA). All RT inhibitors were first made as 100 mM stock solutions. d4T, bis-POM PMPA, NVP, DLV, EFV were dissolved in pure DMSO and other chemicals were dissolved in water. Template/primer mix poly (rI)-oligo (dC)12-18 was made by mixing poly (rI) and oligo (dC)12-18 according to Cheng et al .
Synthetic human L1 ORF2 sequences were created by following a codon optimization procedure as previously described , and were ligated into pBluescript KS(-) (Stratagene, Santa Clara, CA, USA) to make pWA112.
To make pWA195, pCEP puro was first made by replacing the hygromycin resistance cassette pCEP4 (Invitrogen, Carlsbad, CA) with a 1.7 kb Sal I fragment of puromycin resistance cassette from pPGKpuro (a gift from Dr. Peter Laird). Plasmid pCEPpurosmL1 was made by replacing the pCEP4 backbone with pCEPpuro using NotI/BamHI sites. Plasmid pWA195 was made by replacing smL1 in pCEPpurosmL1 with the synthetic human L1 coding sequence from pBSshL1.
Plasmid pLD48 was constructed by PCR amplification of pWA112 with the primers JB11578 (5'-CCGGATCCCGCATCAAGAACCTGACCCAGAGCC-3') and JB11584 (5'-ACGCGTCGACTTAGTAGATCTGCTTCAGCTCGTTGTAG-3'), digestion of the product with BamHI and SalI, and cloning of the product into BamH I and Sal I sites of pMal-c2x (NEB, Ipswich, MA, USA). Human L1 ORF2 amino acid 238-1061 was inserted behind MBP gene in frame, and a "TAA" stop codon was introduced at the end of the insert. Plasmid pMal-ORF1 was constructed by cloning full-length human L1 ORF1 into the BamHI and SalI sites of pMal-c2x plasmid. Both constructs were confirmed by sequencing.
pQH1 was constructed by subcloning a DNA fragment containing the last 115 amino acids of Ty1 integrase and full-length RT-RH from pGEX-4T-3  into the NdeI and PstI sites of the pCold I plasmid (Takara, Japan).
Expression and purification of human L1 and Ty1 RTs
MBP tagged L1 RT was overexpressed and purified according to the protocol provided by NEB. Plasmid pLD48 was transformed into E. coli BL21 cell and plated on LB with 100 μg/ml carbenicillin. 200 ml LB medium (0.2% glucose, 100 μg/ml carbenicillin) was inoculated with a 2 ml overnight culture and grown at 37°C with shaking to an OD600 of 0.3, followed by induction with 0.5 mM IPTG for 3 h at 37°C. Cells were harvested by centrifugation at 4000 × g for 20 minutes and the supernatant was discarded. The cells were resuspended in 5 ml column buffer (20 mM Tris-Cl pH 7.4, 200 mM NaCl, 1 mM EDTA) and lysed on ice with sonication with five bursts of 15 sec. The lysate was centrifuged at 9,000 × g for 30 mins and the supernatant was loaded onto 1 ml amylose resin, which was washed with 10 ml column buffer and then the fusion protein was eluted with column buffer with 10 mM maltose. The majority of protein eluted in the first two fractions, which were pooled and concentrated using a Centricon YM-100. Glycerol was added to a final concentration of 50% and enzyme was stored at -20°C.
pQH1 was co-transformed with chaperone plasmid pG-Tf2 (Takara, Japan) into E. coli BL21 cell. Cells were grown at 37°C in LB medium containing 50 μg/ml of carbenicillin, 34 μg/ml chloramphenicol and 1 ng/ml tetracycline until A
600 reached 0.4. After 30 minutes incubation at 15°C, expression of recombinant Ty1 RT was induced with 0.5 mM IPTG. The cells were further cultured for 24 hours at 15°C, harvested by centrifugation and stored at -80°C. Recombinant Ty1 RT was sequentially purified on a HisTrap chelating column, a desalting column, a cation exchange column, a Superdex 200 gel filtration column, and again on a HisTrap chelating column with an Äkta FPLC system (GE Healthcare, Piscataway, NJ, USA). Eluted protein from the last step of purification was dialyzed overnight into the dialysis buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM EDTA, and 0.1 mM TCEP), concentrated to 6 mg/ml as measured by Bio-Rad Bradford assay, and stored at -80°C in 50 μl aliquots.
In vitro RT assay
The RT assays for HIV-1 and L1 RT were performed in a 20 μl reaction mixtures containing 50 mM Tris-Cl (pH 8.0), 50 mM KCl, 5 mM MgCl2, 10 mM DDT, 0.01 U template/primer, 1 μl [α-32P] dTTP or [α-32P] dCTP (final concentration 0.17 μM, 3000 Ci/mmol, 10 mCi/ml) at 37°C for 30 min. Then the mixture was spotted on DE81 paper and washed three times with 2×SSC buffer for a total time of 30 min. The DE81 paper was dried and counted by scintillation counter Beckman LS6000SC. RT assay for Ty1 RT was done under the same buffer condition except that 20 mM MgCl2 was used and the reaction mixture was incubated at room temperature. In each reaction the amounts of L1, HIV-1 and Ty1 RTs with the same specific activity were added. To test the inhibition of the RT inhibitors, 1 μl inhibitor was included in the mixture to obtain the desired concentration. In testing the effect of NVP, DLV and EFV, 1 μl pure DMSO was added in the positive control reaction. All assays were done at least in triplicate.
Kinetic analysis of NRTIs
The kinetic analysis was performed under the same conditions described above but with various concentrations of substrates and inhibitors as indicated in the text. Poly (rA)-oligo (dT)12-18 and [α-32P] dTTP were used to analyze AZTTP and d4TTP. Poly (rI)-oligo (dC)12-18 and [α-32P] dCTP were used to assay ddCTP and 3TCTP.
Cell culture and L1 retrotransposition assay
The cell-based retrotransposition assay was conducted as described . HeLa cells (a gift from Dr. John Moran, University of Michigan) were seeded in 6-well dishes in DMEM (2×105 cells/well). The next day cells were transfected with Fugene 6 according to the manufacturer's manual. Each transfection consisted of 96 μl Opti-Mem, 1 μg pWA195 DNA and 3 μl Fugene 6. Sixteen hours after transfection, the cells were trypsinized and transferred to a 6 cm plate in DMEM with 2.5 μg/ml puromycin and the RT inhibitors. Three days after puromycin selection, dead cells were removed and puromycin resistant cells were trypsinized and counted with a hemocytometer. The puromycin resistant cells were plated on a 10 cm plate (1×104 cells/plate) in 10 ml DMEM with 500 μg/ml G418. The RT inhibitors were added to the same concentration as in the puromycin selection. For assays of d4T, bis-POM PMPA, NVP, EFV and DLV, the same amount of DMSO (final conc <0.01%) was added to the control plate. Two weeks later, G418 resistant cells were fixed to the plate and stained with 0.1% crystal violet. The number of G418 resistant colonies was counted to calculate retrotransposition frequency. Six independent assays were done for each RT inhibitor.
Cell cytotoxicity of RT inhibitors
Untransfected Hela cells were plated on 10 cm plate (500 cells/plate) with and without RT inhibitors. Ten days later, cells were fixed to the plate and stained with 0.1% crystal violet. The number of colonies was counted to calculate colony formation ability. Colony formation ability of control assay without inhibitors was considered as 1.0 and colony formation ability in the presence of inhibitors was indicated as relative efficiency with respect to the control.
The RT sequences of HIV-1, HIV-2, L1 and Ty1 RTs were aligned automatically by Clustal X  and manually adjusted according to Shaharabany et al  and Wilhelm et al .