Analysis of the transcriptional activity of TaxYFP fusion proteins. (A and B) HeLa cells were cotransfected either with 0.1 μg of pLTR_HTLV-1-Luc (A) or 1 μg of EP180 and 0.75 μg of pCMV-E47 (B) reporter constructs together with a control plasmid (bar 1) or the pCMV-Tax (bar 2), pHR-TaxYFP (bar 3), or pHR-K88ATaxYFP (bar 4) plasmids (30 ng). The luciferase activities were normalized with tk-luc activities and are presented relative to those of cells transfected with pCMV-Tax (A) or with pCMV-E47 alone (B). The values shown are those obtained in triplicate from two experiments ± standard deviations. (C) Western blot analysis of Tax expression. Equal amounts of whole-cell extracts of HeLa cells transfected as indicated above were analyzed by Western blotting using a polyclonal Tax antibody. The blots were reprobed for actin as a loading control.

Comparative analysis of the effects of Tax, TaxK88A, and Id3 on pTα gene transcription in MOLT-4 cells. Cells were transduced as described in Materials and Methods with either the Id3-IRES-GFP retrovirus or the pHR-TaxYFP or pHR-TaxK88AYFP lentiviruses. After 7 days of culture, GFP⁺ or YFP⁺ cells were sorted, and the cytoplasmic RNAs were isolated and reverse transcribed. The cDNA samples were subjected to qPCR using primers specific for pTα and normalized for the amount of cDNA, using actin as an internal control. Standard deviations are from at least two determinations performed in duplicate.

Tax down-regulates pTα gene transcription in human immature thymocytes. Human immature (CD34⁺ CD3⁻ CD4 ISP) thymocytes were isolated and nucleofected either with a pHR-YFP control vector (A) or with a pHR-TaxYFP vector (B). They were incubated in complete RPMI containing interleukin-7 and stem cell factor. Twenty-four hours later, the YFP⁻ and YFP⁺ cells were sorted from both cultures. The RNAs were isolated and reverse transcribed. Twofold-diluted cDNA samples from YFP⁻ cells (lanes 1 and 2) and from YFP⁺ cells (lanes 3 and 4) were subjected to PCR (A and B, lower panels) and to qPCR (C) using primers specific for the indicated genes.

Activation of the regulatory sequences of the pTα gene in the presence of E47 and/or Tax. (A) E47 enhances the activity of the enhancer and promoter sequences of the pTα gene. Above, schematic representation of the reporter plasmids encoding the luciferase gene under the control of enhancer and promoter sequences of the pTα gene containing either intact (▪) E-boxes (EP180), deleted E-boxes in the promoter (EP180ΔE), or base-substituted E-boxes in the enhancer (EmP180). HeLa cells were transfected with 1 μg of EP180, EP180ΔE, or EmP180 with (black bars) or without (white bars) 0.75 μg of pCMV-E47. Luciferase activities were normalized with tk-luc activities and are presented relative to those in cells transfected with EP180 and pCMV-E47 constructs. The values are those obtained in triplicate, from one representative experiment (out of three experiments) ± the standard deviation. (B) The HTLV-1 Tax protein represses E47-mediated transcriptional activation of the regulatory sequences of the pTα gene. HeLa cells were transfected with 1 μg of EP180 with 0.75 μg of pCMV-E47 and increasing concentrations (50 to 500 ng) of pCMV-Tax plasmid. Luciferase activities were normalized with tk-luc activities and are presented relative to those in cells transfected with EP180 and pCMV-E47 constructs, in the absence of Tax. Eighteen hours after transfection, the cells were harvested and an aliquot of each cell extract was resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The same membrane was probed successively with a polyclonal anti-Tax or monoclonal anti-E47 and anti-actin antibodies. (C) The p300 coactivator releases Tax-induced inhibition of E47-dependent transcription of the pTα gene. HeLa cells were cotransfected with 1 μg of EP180, 0.75 μg of pCMV-E47, and 0.5 μg of pCMV-Tax, and with increasing amounts of the p300 expression plasmid (50 to 250 ng). Luciferase activities were normalized with tk-luc activities and are presented relative to those in cells transfected with EP180 and pCMV-E47 constructs, in the absence of Tax and p300.

Transcription of the pTα gene in TaxYFP- and control-transduced MOLT-4 cells. (A) Cells were transduced by either pHR-TaxYFP at an MOI of 2 (experiment 1) or 5 (experiment 2) or pHR-YFP at an MOI of 5. They were cultured for 7 days and analyzed by flow cytometry and by RT-qPCR for the determination of pTα mRNA levels. (B) Extracts of cells transduced by pHR-YFP or pHR-TaxYFP (experiment 2) were immunoprecipitated using a Tax antibody and then immunoblotted to determine the expression of the TaxYFP fusion protein. (C) cDNA samples from experiment 2 (undiluted, lanes 1 and 4; 1/5 diluted, lanes 2 and 5; 1/10 diluted, lanes 3 and 6) from pHR-YFP-transduced cells and from pHR-TaxYFP-transduced cells were subjected to semiquantitative PCR using primers specific for Tax, pTα, and actin.

Characterization of the immature and mature human thymocytes. (A) Two-color flow cytometry analysis of CD34⁺ and CD4 ISP cells recovered from human thymus. Single-cell suspensions were prepared and analyzed for the expression of the indicated surface antibodies. Numbers in the dot plots represent percentages of dots in each quadrant. (B) Two-color flow cytometry analysis of CD3⁺ thymocytes. (C) Quantification of pTα gene expression in immature and mature thymocytes by qPCR. The values for pTα mRNA expression relative to those of actin mRNAs were calculated from a standard curve of cDNA obtained from MOLT-4 cells.