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

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.

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.

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.

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.

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.