EL4-Gax cell was established by transducing with an MLV-based retrovirus vector expressing the GFP-fused Tax (Gax), in which the U3 region of the 3' LTR was replaced by that of HTLV-1 to ensure the Tax-dependent transcriptional control of HTLV-1 (Fig. 1A), and the characteristics of Gax protein as a transactivator were shown to be retained as previously reported 30. Expression of Gax gene under the control of HTLV-1 LTR in EL4-Gax cells grown in the peritoneal cavity of mice and cultured in vitro was directly monitored by the intensity of GFP fluorescence using a fluorescent-activated cell sorter (FACS). This demonstrated the in vivo -specific suppression of Tax expression (Fig. 1B) 30.

Immunoblot analysis confirmed that in vivo protein expression of Gax was abolished in cells, and the expression was reactivated in ex vivo culture (Fig. 1C, Gax). The reduction of Gax protein is not simply due to a severe growth condition inducing cell death since the proteolytic cleavage of poly(ADP-ribose) polymerase, which is known to be a sensitive marker of apoptosis 31 and necrosis 32, was not observed (Fig. 1C, PARP).

The expression of Gax mRNA was analyzed using quantitative reverse transcription polymerase chain reaction (RT-PCR) to determine whether the suppression of Tax expression was controlled at the level of transcription (Fig. 1D). The transcriptional suppression in vivo is specific for the Gax gene because no suppression was observed in the expression of cellular genes such as EF1-a, GAPDH, β-actin, 18S-ribosomal RNA and endogenous retrovirus. On the contrary, gene expression of CD4 was upregulated in vivo, while it was silenced in EL4-Gax cells grown in vitro. Real-time PCR analysis of Gax cDNA prepared from total RNA in EL4-Gax cells demonstrated that the expression of Gax mRNA was reduced in vivo and recovered after ex vivo culturing to a level comparable with that before peritoneal inoculation of the cells. Thus, Gax expression in vivo was suppressed transcriptionally.

Because complete- or hyper- methylation of cytosine residues at the CpG sites in the promoter region of the HTLV-1 5'-LTR is associate with transcriptional suppression in infected cell lines, the level of CpG methylation in the LTR U3 region at 5' site of Gax-reporter genome was examined in EL4-Gax cells. There are 11 possible CpG methylation sites in the U3 region of HTLV-1, but only low levels of methylation were observed in four independent experiments. Although one case (experiment 1 in Fig. 2) showed heavy methylation at a single CpG site in EL4-Gax cells in vivo, little or no methylation was detected at this site in the other experiments. In the other three experiments, less methylation was observed in EL4-Gax cells in vivo (where Gax expression was suppressed) than in cells grown in vitro or ex vivo. Thus, no CpG methylation specific and consistent with that in the in vivo cells was detected (Fig. 2). These results indicate that the suppression of Gax gene expression in vivo is not explained by CpG methylation in the enhancer sequence, suggesting the involvement of other mechanism(s). This is consistent with a previous analysis in which no or partial methylation was associated with silencing in the peripheral blood cells of HTLV-1 carriers, as well as in significant number of ATL cases, whereas transcriptional suppression of HTLV-1 in ATL cell lines and some ATL leukemic cells was explained by hypermethylation of the 5'-LTR 18.

CREB has been implicated as the primary player in both basal and Tax-activated HTLV-1 transcription 24. CRE-dependent transcription is generally explained by the recruitment of histone acetylating proteins, CBP/p300, to the enhancer region of genes through an interaction with CREB protein, which binds to the CRE sequence, and acetylation of histones, opening the chromatin and providing access to basic transcriptional factors including RNA polymerase. Thus, since the reduction of recruitment of either factor to the promoter region might result in the suppression of transcription, a chromatin immunoprecipitation (ChIP) assay was used to analyze the binding of these factors to the U3 region of the 5'-LTR.

Enhancer binding of CREB and pCREB was first examined in EL4-Gax cells either in vivo (b) or under in vitro (a) culture conditions. As shown in Figure 3B (lanes 7–10), no significant difference was observed in the amount of CREB or pCREB in complex with the enhancer DNA at the 5'-LTR of the provirus. CBP functions as a cofactor by being tethered to DNA through either pCREB or CREB, in association with Tax, to acetylate histone proteins. Binding of CBP to the HTLV-1 enhancer was observed but showed a similar intensity of protein binding (Fig. 3B, lanes 11, 12).

As Gax is expressed in EL4-Gax cells in vitro, it was of interest whether Gax is associated with the enhancer DNA. ChIP assay was performed with antibody against GFP, which recognizes the Gax protein. Consistent with the protein expression, Gax was associated with the enhancer DNA in EL4-Gax cells grown in vitro (lane 15) but not in in vivo cells (lane 16), where the expression of Gax protein was decreased. Tax recruits CBP to the HTLV-1 enhancer by tethering with CREB at the CRE sequence; however, the enhancer binding of CBP remained unchanged in the absence of Tax (Fig 3B, lane 11, 12). In this respect, it is noteworthy that phosphorylation of CREB protein, which leads to a complex formation between CREB and CBP, is increased in EL4-Gax cells grown in vivo (Fig 3C). Thus, pCREB seems to be involved in the sustained enhancer binding of CBP in the absence of Tax.

Although the amount of pCREB was increased in EL4-Gax cells grown in vivo, no significant difference was observed in the amount of pCREB binding to the enhancer DNA in cells either in vivo or under in vitro culture conditions. Since pCREB has been demonstrated to preferentially bind to the enhancer sequence of HTLV-1 in a complex with Tax 33, Tax might have selectively incorporated pCREB in the complex.

Activated transcription is associated with histone acetylation in the chromatin of the respective genes; thus, histone acetylation at the promoter region of the provirus was analyzed using a ChIP assay, with antibodies against acetylated histones H3 and H4. Unexpectedly, this analysis revealed that histones at the LTR of the HTLV-1 provirus were equally acetylated in EL4-Gax cells (Fig. 3D, lanes 11–14), either in vivo (b) and in vitro (a), whereas RNA expression from the HTLV-1 promoter in these cells differed substantially (Fig. 1). Methylation of histone H3 at the lysine residue was also analyzed, because this methylation is closely linked with transcriptional activation. However, no change was observed in the methylation of histone H3 in the promoter region of the provirus (Fig. 3D, lanes 15–16). Thus, the in vivo -specific transcriptional repression of the HTLV-1 promoter was not associated with an altered level of chromatin modification. These results are consistent with the previous finding that gene silencing of HTLV-1 in an ATL case was observed regardless of hyperacetylation of histones H3 and H4 in the promoter 18.

The recruitment of RNA polymerase II (Pol-II) and TFIIB, a key general transcription factor in forming and stabilizing the early initiation complex 34, was analyzed to determine whether suppression was present in the formation of the transcription initiation complex in the 5'-LTR promoter. Although binding of TFIIB (Fig. 3D, lanes 9, 10) and Pol-II (Fig. 3D, lanes 7,8) to the constitutive promoter of the EF-1a gene as positive controls was observed equally in the ChIP assay, a substantial reduction in the binding of these factors to the provirus promoter sequence was detected under condition of suppressed Gax expression in comparison with EL4 -Gax cells in the in vitro culture (37 ± 5% for TFIIB and 47 ± 5% for Pol-II). These results suggest that the loss of recruitment of basal transcription factors is associated, at least in part, with the suppression of Gax expression in vivo, regardless of the constitutive binding of CREB-CBP/p300 to the enhancer DNA.

In addition to the CREB-CBP/p300 pathway, another family of CREB cofactors, TORCs, has been recently identified as activating CREB-dependent, but pCREB-independent, transcription 2526, including that of HTLV-1, with or without Tax 2728. Thus, we next examined the involvement of TORCs in transcriptional control in vivo.

The TORC family consists of three proteins, TORC1, TORC2, and TORC3; expression of these proteins in EL4-Gax cells was assessed by immunoblot analysis using antibodies against each. All three TORC proteins were detected in the cell lysate prepared from EL4-Gax cells, at molecular weights of 75, 77/82, and 75 kDa respectively. Consistent with previous reports, all TORC proteins appeared to migrate as multiple bands, likely because of they are phosphorylated. In particular, TORC2 protein was composed of two distinct bands, of which the slower migrating band was previously shown to be a phosphorylated form of the faster migrating species. In fact, alkaline phosphatase treatment of cellular lysate from EL4-Gax cells reduced the intensity of the slower migrating band and resulted in the increase of the faster migrating band (Fig. 4D).

When expression of the TORC proteins in EL4-Gax cells grown in vitro, in vivo, and ex vivo was compared, the amounts of TORC1 and TORC2 were reduced significantly under in vivo growth conditions, and they recovered to some extent upon their ex vivo culturing (Fig. 4A, B). In contrast, the expression of TORC3 increased little, if any, in in vivo or ex vivo conditions (Fig. 4C). Because a previous report demonstrated that the suppression of TORC1, TORC2 or TORC3 expression by siRNA resulted in reduced transcription from the HTLV-1 LTR 27, it seems likely that reduced expression of TORC1 and/or TORC2 is involved in the suppression of Gax gene expression in in vivo conditions. It is noteworthy that the reduction of the unphosphorylated TORC2 protein was more significant than that of the phosphorylated form, because the former is an active form of TORC2 retained in the nucleus.

As TORC proteins are recruited to enhancer DNA in combination with CREB protein to activate CRE-dependent transcription of HTLV-1, a ChIP assay was used to analyze whether these proteins are associated with the U3 region of the 5'-LTR in EL4-Gax cells grown in vitro and in vivo. As shown in Figure 4E, recruitment of TORC2 and TORC3 proteins to the enhancer sequence was demonstrated in EL4-Gax cells in vitro and both of the bindings were substantially reduced in in vivo cells, where little or no binding of TORC1 to enhancer DNA was observed. As judged by densitometoric analysis, TORC2 appears to be the main TORC protein that is associated with the enhancer sequence of HTLV-1 in EL4-Gax cells, and the reduced enhancer binding of TORC2 in cells grown in vivo was in good agreement with the transcriptional suppression of Gax in vivo.

To investigate which TORC protein functioned dominantly in EL4-Gax cells, we analyzed Gax expression after the knock-down of the three TORC genes by transducing the cells with a retrovector for siRNA against each TORC genes. Expression of siRNA resulted in the reduction of the respective gene product by more than 50% (Fig. 5B) but a significant reduction of Gax protein expression was only observed in cells with the siRNA to the TORC2 RNA (Fig. 5A, B). We, thus, concluded that TORC2 is primarily involved in the transcriptional control of the HTLV-1 promoter in EL4-Gax cells. Together, these results suggest that the reduced TORC2 expression in EL4-Gax cells in vivo is closely associated with the silencing of Gax gene expression in vivo.

Phosphorylation of TORC2 protein by cellular kinases, such as AMPK (AMP-activated protein kinase) kinase, induces the translocation of TORC2 from the nucleus to the cytoplasm, thereby suppressing CREB-dependent transcription. In fact, the unphosphorylated form of the TORC2 protein in vivo appeared to be reduced more significantly than the phosphorylated form, when compared in vitro or ex vivo by Western blotting (Fig. 4B). Therefore, activity of AMPK was examined by measuring the phosphorylation at Thr172, which is required for AMPK activation 35. The results shown in Figure 6C clearly demonstrate the activation of AMPK activity in EL4-Gax cells in vivo and its reduction in cells cultured ex vivo.

Subsequently, the subcellular localization of TORC2 in EL4-Gax cells in vitro and in vivo was examined using immunostaining (Fig. 6A). Consistent with the Western blotting, expression of the TORC2 protein in in vivo cells was greatly reduced in comparison with that in the in vitro cultured cells, and the expression was restored after ex vivo culture (Fig. 6A, "TORC2", and Fig. 6B). Furthermore, the subcellular localization of TORC2 was restricted to the cytoplasm of in vivo cells (Fig. 6A, "TORC2 + DAPI"), whereas the protein was primarily expressed in the nucleus in cells cultured in vitro and ex vivo (Fig. 6A, B).

Because cytoplasmic retention of TORC2 results in its degradation by proteasomes, it appears that some in vivo -specific cellular signal(s) may induce the cytoplasmic translocation, and thereby the degradation of the TORC2 protein, resulting in the suppression of HTLV-1 transcription in EL4-Gax cells.

The establishment of the EL4-Gax mouse model system was previously reported 30. Briefly, 1 × 10⁶ EL4-Gax cells were injected into the peritoneal cavity of 6-week-old male C57BL/6J mice (Charles River Laboratories Japan, Kanagawa, Japan) in 0.1 mL phosphate -buffered saline (PBS). After three weeks, cells in ascitic fluids (in vivo cells) were recovered and cultured in culture medium for 48 h (ex vivo culture). Gax expression was monitored using a FACS (Becton Dickinson, NJ) as the intensity of GFP fluorescence. The mice were maintained under standard pathogen-free conditions in the animal facility of Kansai Medical University, and the protocol for the experiments was approved by the Institutional Animal Care and Use Committee of Kansai Medical University.

Total RNA was extracted from cells using the Trizol reagent (Invitrogen, Carlsbad, CA) and treated with RNase-free DNase I (GIBCO BRL) at 37°C for 30 min, followed by phenol-chloroform extraction and ethanol precipitation. Total RNA (1 μg) was subjected to reverse transcription using RivaTra Ace (Toyobo, Osaka, Japan) with random 9 mer oligonucleotides (Takara, Kyoto, Japan) as a primer.

Real-time PCR was performed in a 25-μL reaction mixture consisting of 2-fold SYBR Green mastermix (Applied Biosystems, UK); 50 pg or 10 ng cDNA for 18S-rRNA or Gax, respectively; 0.4 μM primer; and the BioRad-iQ-Real-Time PCR Detection System. The cycling conditions for all amplifications were 95°C for 15 min; 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with a single fluorescence measurement melting curve program (60–95°C with a heating rate of 0.2°C per second and a continuous fluorescence measurement); and finally a cooling step to 24°C. Each individual sample was run in triplicate wells; the cycle threshold (Ct) of each well was recorded at the end of the reaction. The Ct was manually set up at the level that reflected the best kinetic PCR parameters, and melting curves were analyzed. The average and standard deviation (SD) of the three Cts was calculated, and the average value was accepted if the SD was less than 0.38. The 2^ΔΔCt method was used to analyze the relative changes in Gax gene expression in in vivo or ex vivo samples with those in vitro.

The primers used for PCR were as follows: for the Gax gene, 5'-ACGCCTATGATTTCCGGGCC-3' and 5'-GGATATTTGGGGCTCATGGTCA-3'; for CD4, 5'-CAGAGCCTGACCCTGACCTTG-3' and 5'-CATCACCACCAGGTTCACTTCC-3'; for endogenouse mouse retroviral env, 5'-GAAGGTCCAGCGTTCTCAAAAT-3' and 5'-CACGTGATTTCACTTCTTCTGG-3'47; for elongation factor (EF)-1α, 5'-TCTGGTTGGAATGGTGACAACATGC-3' and 5'-CCAGGAAGAGCTTCACTCAAAGCTT-3'; for GAPDH, 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'; for β-actin: 5'-GAGATCTGCCGATCCGCCGCCCG-3', and 5'-GCTCGAGGTGTGCACTTTTATTCAACTGG-3'; for 18S-rRNA, 5'-CTCGATTCCGTGGGTGGTGGTG-3' and 5'-GGCCGATCCGAGGGCCTCAC-3'.

Cells were lysed and sonicated in cell lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1%Triton X-100, Cell Signaling Technology, MA) plus complete proteinase inhibitor cocktail and phosphatase inhibitor mixture (Roche Applied Science, Indianapolis, IN). Aliquots containing 5–30 μg of protein were separated by SDS/polyacrylamide gel electrophoresis; transferred onto PVDF membrane (Immobilon-P, Millipore, Billerica, MA); and detected by the ECL or ECL plus system (GE Healthcare, Buckinghamshire, UK). Used antibodies were TORC2 rabbit polyclonal antibody (#ST1099, Calbiochem, La Jolla, CA), TORC1 rabbit polyclonal antibody (#2501), TORC3 rabbit polyclonal antibody (#2768), phospho-AMPKα (Thr172) rabbit monoclonal antibody (40H9, #2535), AMPKα rabbit monoclonal antibody (23A3, #2603, Cell Signaling Technology); CREB1 (#06–863), phospho-CREB (#06–519, Upstate Technology, NY); PARP-1 mouse monoclonal antibody (#611038, BD. Biosciences, San Jose, CA) or β-actin mouse monoclonal antibody (clone AC-5, Sigma-Aldrich, MO) and HRP-conjugated protein A for rabbit primary antibodies or anti-mouse IgG-HRP (GE Healthcare) for mouse monoclonal primary antibodies.

1 × 10⁶ EL4-Gax cells were lysed in 250 μl of cell lysis buffer (mentioned above, Cell Signaling Technology) and 1× complete protease inhibitor cocktail (Roche) with or without phosphatase inhibitor cocktail (2.5 mM sodium pyrophosphate; 1 mM β-glycerophosphate; 1 mM Na₃VO₄; 50 mM sodium fluoride), plugged into liquid nitrogen and thawed on ice immediately. Lysate was clarified by centrifugation at 14,000 rpm for 10 minutes at 4°C. After measuring protein concentration, 20 μg protein was dispended to 1× rAPid Alkaline Phosphatase buffer with or without phosphatase inibitor cocktail, treated with or without 20 units of rAPid Alkaline Phosphatase (Cat No.04898133001, Roche) for 60 minutes at 37°C in a 100 μl reaction volumes. The 1/3 volume of samples was then separated by SDS-PAGE.

Methylation of the cytosine residue at CpG sites was analyzed using the bisulfite genomic sequencing method 16 with minor modifications. Briefly, 1 μg genomic DNA was used for bisulfite treatment. The DNA sample in 0.2 N NaOH was denatured at 37°C for 10 min, followed by incubation at 50°C overnight in 2.6 M sodium bisulfite and 0.5 mM hydroquinone (Sigma-Aldrich) solution. The solution was then sealed with 200 μL mineral oil and kept in the dark. Sample DNA was purified using the Wizard DNA Clean-Up system (Promega, Madison, WI) and treated with 0.3 N NaOH. DNA was precipitated with ethanol and dissolved in 50 μL H₂O. Then 30% of this solution was subjected to PCR.

PCR was performed in 50-μL reaction mixtures containing 1 μL Acuprime Taq DNA polymerase and its buffer (Invitrogen) and a primer set at 0.2 μM. The primer sequences to amplify the 5'-LTR HTLV-1 U3 region were sense primer (complementary to the modified cellular flanking region), 5'-CCTCCTATCAAAACTAACTTTAAC-3', and antisense primer (complementary to the modified HTLV-1 U3 region), 5'-AAGTTTTTTGAGGTGAGGGGT-3'.

The cycling conditions for all amplifications were 94°C for 2 min; 40 cycles of 94°C for 30 s, 56°C for 30 s, and 68°C for 30 s; and a final extension at 68°C for 5 min. The amplified PCR products, purified with the QIA quick PCR kit (QIAGEN, Germantown, MD), were subcloned into a pGEM-T easy vector (Promega), and the nucleotide sequences of at least 13 clones were determined.

ChIP assays were performed according to the manufacturer's protocol (Upstate Biotechnology, Lake Placid, NY) with some modifications. Briefly, 2 × 10⁶ cells were fixed with 1% formaldehyde for 5 min at room temperature and sonicated (Biorupter UCD-200T; CosmoBio, Tokyo, Japan) to obtain on average 600 -bp length of soluble chromatin. The chromatin solutions were pre-cleared with 80 μL 50% Protein G Sepharose slurry (Amersham) preabsorbed with 0.2 mg/mL sonicated salmon sperm DNA and 0.05% BSA. After a 10-fold dilution with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl), 2 mL pre-cleared complex was combined with 2–15 μg antibodies against CREB1 (06–863), phosphor-CREB (06–519), acetylated H3 at Lys-9, 14 (06–599), acetylated H4 at Lys-5, 8, 12, 16 (06–598, Upstate Technology), CBP (sc369), TFIIB (sc-225), Pol-II (sc899, Santa Cruz Biotechnology, CA), methylated H3 Lys-4 (AB8580, Abcam Ltd., Cambridge, UK), GFP (mouse monoclonal antibody, clones 7.1 and 13.1, Boehringer Mannheim), or normal mouse IgG (I-5381, Sigma), rabbit IgG (011-000-002, Jackson immunoresearch Laboratories, West Grove, PA) and rotated at 4°C overnight. The immune complexes were collected by incubating with 80 μL of protein G slurry. DNA-protein cross-linking was reversed by incubation at 65°C overnight, and the samples were treated with RNase A and then with proteinase K. After phenol extraction and ethanol precipitation, the DNA was finally dissolved into 30 μL of water.

The primers used for PCR were as follows. For the 5'-LTR enhancer, sense primer, complementary to the 5'flanking cellular region, 5'-AAGCACAGAAGACACCTTGTGCAC-3' and antisense primer, complementary to 5'-LTR HTLV-1 U3 region, 5'-AAGTTTTTTGAGGTGAGGGGTTGTCG-3'. 5'-LTR promoter region, sense primer, complementary to HTLV-1 U3/MLV U3 junction region, 5'-CATGGCACGCATATGTCTAGAGAACC-3', antisense primer, complementary to MLV gag region, 5'-TCTCCCGATCCCGGACGAGCC-3'. The following sequences of primers were used for other control gene promoters: for EF1a, f (-20): 5'-CGAGGGTGGGGGAGAACGGTAT-3' and r (152): 5'-AGCTAATCCCCGCCGACGACAG-3'; for β-globin, f (-117): 5'-ACCGAAGCCTGATTCCGTAGAGC-3' and r (+133): 5'-CTCACCACCAACTTCATCGGAGTT-3'.

All PCRs were performed in 20-μL reaction mixes consisting of 1–10 μL of the ChIP product, 1 U Ex Taq polymerase (Takara), 200 mM each of deoxynucleoside triphosphates, and a primer set at 0.4 μM. The cycling conditions for all amplifications were 94°C for 5 min; 33–35 cycles of 94°C for 30 s, 62–68°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min.

Cells were grown in suspension or harvested from ascites and were then fixed by adding an equal volume of 4% paraformaldehyde diluted in PEM/PBS(-) buffer (1/5 volume of PEM [see below] and 4/5 volume of PBS(-)) for 30 min at 4°C, collected by centrifugation (300g, 3 min), washed and resuspended in PBS(-), and then cytospun (Shandon Cytospin3; 20,000 cells/slide, 700 rpm, 1 min) onto 1 N HCl and 95% ethanol-washed slide glasses (Matsunami, Osaka, Japan). The cells were washed twice with PEM buffer (80 mM potassium PIPES [piperazine-N, N'-bis(2-ethanesulfonic acid)] [pH 6.8], 5 mM EGTA [pH 7.0], 2 mM MgCl2) and permeabilized by incubation in PEM/PBS(-) buffer containing 0.1% Triton X-100 for 30 min at room temperature and blocked with goat serum (1:20 dilution in TBS containing 0.1% Tween-20) for 30 min at room temperature. After blocking, the cells were incubated with anti-TORC2 antiserum ((#ST1099, Calbiochem) or normal rabbit IgG for 3 h at room temperature or overnight at 4°C in TBS containing 0.1% Tween-20 (TBS-T). Thereafter, the cells were washed three times with TBS-T and incubated with Cy5-conjugated AffiniPure donkey anti-rabbit IgG (#711-175-152, Jackson Immuno Research Laboratories) in TBS-T. After 1 h, the cells were washed three times in TBS-T. To visualize the nucleus, we counterstained the cells with DAPI (4', 6'-diamidino-2-phenylindole; Nacalai Tesque, Kyoto, Japan; final concentration 0.4 μg/mL, 5 min incubation at room temperature) and washed them once with PBS(-), then mounted them in mounting media and examined them with a Zeiss LSM510 META microscope.

TORC siRNA hairpin expression vectors (shTORC1 retroviral vector, #NM-001004062; shTORC3 retroviral vector, #XM-344915; shTORC2 lentiviral vector, #NM-02881) were purchased from Open Biosystems (Huntsville, AL). Recombinant retroviruses for shTORC1 and shTORC3 were generated by transfecting respective retroviral vector plasmids into BOSC23 cells, and recombinant lentivirus expressing shTORC2 was generated in HEK293T cells by the cotransfection of the lentiviral vector plasmid with expression vectors for Gag/Pol (pGag/Pol), Rev (pRev), and VSV-G protein (pVSV-G) using the Lipofectamine 2000 (Invitrogen) transfection reagent. Viral supernatant was collected 48 h after transfection and added to EL4-Gax cells for infection in the presence of polybrene (8 μg/mL). Infected cells were selected with puromycin (4 μg/mL), and mixtures of 40–200 clones of each virus were used for further analysis.