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Transformation and Oncogenesis

Immortalization of CD4+ and CD8+ T Lymphocytes by Human T-Cell Leukemia Virus Type 1 Tax Mutants Expressed in a Functional Molecular Clone

Michael D. Robek, Lee Ratner
Michael D. Robek
Departments of Medicine, Pathology, and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110
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Lee Ratner
Departments of Medicine, Pathology, and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110
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DOI: 10.1128/JVI.73.6.4856-4865.1999
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  • Fig. 1.
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    Fig. 1.

    Construction of ACH Tax mutants. The ACH.pcTax, ACH.M22, and ACH.M47 constructs used in this study contain the taxgene of the HTLV-1 strain C91/PL between the AccI site at position 7346 and the SmaI site at position 8307 in the ACH molecular clone. The M22 mutation results in a Thr130Leu131-to-Ala130Ser131substitution, while the M47 mutation is a Leu319Leu320-to-Arg319Ser320substitution.

  • Fig. 2.
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    Fig. 2.

    Viral particle production by ACH Tax mutants. Human 293T cells were transfected with the ACH.pcTax, ACH.M22, and ACH.M47 clones or empty vector, and viral particle production was measured by p19 antigen ELISA. The error bars indicate the standard deviations of three replicate transfections. Whereas the ACH.M22 clone produces viral particles at amounts similar to wild-type levels, the ACH.M47 clone produces much lower levels of virus.

  • Fig. 3.
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    Fig. 3.

    Immortalization of PBMC by ACH Tax mutants. PHA-IL-2-activated human PBMC were transfected by electroporation with ACH.wt (one replicate), ACH.pcTax (two replicates), ACH.M47 (two replicates), and ACH.M22 (three replicates), and cellular viability was monitored by MTT conversion assays. Like the clones containing wild-type Tax, the ACH.M47 clone retains immortalizing activity. However, the M22 mutant fails to immortalize the infected cells.

  • Fig. 4.
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    Fig. 4.

    Cell surface phenotype of immortalized cell lines. ACH.pcTax- and ACH.M47-immortalized cells were stained with anti-human CD3, CD25, CD4, and CD8 and analyzed by flow cytometry. Although all cell lines express CD3 and CD25, expression of CD4 and CD8 is variable, with a tendency for CD4 expression by ACH.pcTax-transfected cells and CD8 expression by cells immortalized with the ACH.M47 clone. The open histogram on each plot corresponds to unstained cells, while the shaded histogram corresponds to cells stained with the indicated antibody.

  • Fig. 5.
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    Fig. 5.

    Lack of reversion of M47 mutation. (A) The Tax ORF was amplified by PCR from genomic DNA and digested with the restriction endonuclease BglII. Whereas the PCR products from all three ACH.pcTax-immortalized cell lines lack the BglII site, the site is present in the Tax fragment amplified from the ACH.M47-immortalized cells. Lanes 1, 3, 5, 7, 9, and 11 are undigested (u), while lanes 2, 4, 6, 8, 10, and 12 are digested withBglII (c [cut]). (B) The PCR-amplified taxgenes from ACH.pcTax- and ACH.M47-immortalized cells were cloned into a CMV expression vector and cotransfected with HTLV-LTR-luciferase reporter construct in 293T cells. As positive and negative controls, the wild-type and M47 mutant tax genes were amplified from the ACH.pcTax and ACH.M47 clones. The data are expressed as fold increase in luciferase activity relative to the HTLV LTR-luciferase construct transfected alone, and the error bars represent the standard deviations of four replicate transfections.

  • Fig. 6.
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    Fig. 6.

    Activation of HTLV-1 LTR and Tax expression in immortalized cells. (A) ACH.pcTax- and ACH.M47-immortalized cells were transfected with 20 μg of HTLV LTR-luciferase and 5 μg of CMV-CAT. Luciferase activity was measured in 30 μg of whole-cell extract, and CAT activity was measured in 3 μg of extract. The data are expressed as relative light units per 1% conversion CAT activity. The error bars indicate the standard deviations of three replicate transfections. (B) Tax expression in whole-cell extracts prepared from PCTAX and M47 cell lines. For negative and positive controls. Tax expression was also examined in PHA-IL-2-stimulated PBMC and the HTLV-1-infected cell line MT-2.

  • Fig. 7.
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    Fig. 7.

    Replication and immortalization by ACH.M47 virus. (A) Five hundred thousand PCTAX-2- or M47-3-immortalized cells were lethally gamma irradiated (6,000 rads) and cocultured with 5 × 106 PHA-activated uninfected PBMC. Viral replication was determined by p19 antigen ELISA. The standard deviation of two replicate infections is indicated by the error bars. (B) Results of microtiter infectivity-immortalization assay. Ten thousand PHA-IL-2-activated PBMC were cocultured with 103, 102, or 101 gamma-irradiated PCTAX-2-, PCTAX-3-, M47-2-, or M47-3-immortalized cells in replicates of 20 in 96-well plates. The cultures were examined at 8 weeks postcoculture for the presence of viable cells, and the number of cultures which were immortalized at each dilution was determined.

Tables

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  • Table 1.

    Activity of Tax mutants and summary of immortalization assays

    CloneActivity (%)aNo. of immortalized cultures/no. of transfections (no. of donor PBMC)
    HTLV LTR-luciferaseHIV LTR-CAT
    ACH.wtNDND4/5 (5)
    ACH.pcTax1001005/6 (4)
    ACH.M228070/6 (4)
    ACH.M4721385/5 (4)
    Empty vector000/8 (7)
    • ↵a Luciferase or CAT activity above empty vector cotransfection normalized to 100% for the wild-type ACH.pcTax clone. A 100% activity corresponds to an approximately 20-fold increase in luciferase activity for HTLV LTR-luciferase and a 2-fold increase in CAT activity for HIV LTR-CAT. ND, not done.

  • Table 2.

    Cell surface phenotype of immortalized PBMC from transfections and infections

    Status and cloneNo. of cultures with phenotype/total cultures
    CD4−CD8−CD4+CD8− cCD4−CD8+ cMixed CD4+CD8+
    Transfecteda
     ACH.wt0/44/40/40/4
     ACH.pcTax1/43/40/40/4
     ACH.M470/51/52/52/5
    Infectedb
     PCTAX-20/66/60/60/6
     PCTAX-30/63/60/63/6
     M47-20/60/63/63/6
     M47-30/60/62/64/6
    • ↵a Immortalized cell cultures which were derived from electroporation of PBMC with ACH molecular clones. The tabulation includes the data shown in Fig. 4.

    • ↵b Immortalized cell cultures created from microtiter infectivity-immortalization assay.

    • c Cultures were considered to be CD4+ CD8− or CD4−CD8+ if >90% of cells expressed CD4 or CD8, respectively.

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Immortalization of CD4+ and CD8+ T Lymphocytes by Human T-Cell Leukemia Virus Type 1 Tax Mutants Expressed in a Functional Molecular Clone
Michael D. Robek, Lee Ratner
Journal of Virology Jun 1999, 73 (6) 4856-4865; DOI: 10.1128/JVI.73.6.4856-4865.1999

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Immortalization of CD4+ and CD8+ T Lymphocytes by Human T-Cell Leukemia Virus Type 1 Tax Mutants Expressed in a Functional Molecular Clone
Michael D. Robek, Lee Ratner
Journal of Virology Jun 1999, 73 (6) 4856-4865; DOI: 10.1128/JVI.73.6.4856-4865.1999
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