Tax and overlapping rex sequences do not confer the distinct transformation tropisms of human T-cell leukemia virus types 1 and 2

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are distinct oncogenic retroviruses that infect several cell types but display their biological and pathogenic activity only in T cells. Previous studies have indicated that in vivo HTLV-1 has a preferential tropism for CD4+ T cells, whereas HTLV-2 in vivo tropism is less clear but appears to favor CD8+ T cells. Both CD4+ and CD8+ T cells are susceptible to HTLV-1 and HTLV-2 infection in vitro, and HTLV-1 has a preferential immortalization and transformation tropism of CD4+ T cells, whereas HTLV-2 immortalizes and transforms primarily CD8+ T cells. The molecular mechanism that determines this tropism of HTLV-1 and HTLV-2 has not been determined. HTLV-1 and HTLV-2 carry the tax and rex transregulatory genes in separate but partially overlapping reading frames. Since Tax has been shown to be critical for cellular transformation in vitro and interacts with numerous cellular processes, we hypothesized that the viral determinant of transformation tropism is encoded by tax. Using molecular clones of HTLV-1 (Ach) and HTLV-2 (pH6neo), we constructed recombinants in which tax and overlapping rex genes of the two viruses were exchanged. p19 Gag expression from proviral clones transfected into 293T cells indicated that both recombinants contained functional Tax and Rex but with significantly altered activity compared to the wild-type clones. Stable transfectants expressing recombinant viruses were established, irradiated, and cocultured with peripheral blood mononuclear cells. Both recombinants were competent to transform T lymphocytes with an efficiency similar to that of the parental viruses. Flow cytometry analysis indicated that HTLV-1 and HTLV-1/TR2 had a preferential tropism for CD4+ T cells and that HTLV-2 and HTLV-2/TR1 had a preferential tropism for CD8(+) T cells. Our results indicate that tax/rex in different genetic backgrounds display altered functional activity but ultimately do not contribute to the different in vitro transformation tropisms. This first study with recombinants between HTLV-1 and HTLV-2 is the initial step in elucidating the different pathobiologies of HTLV-1 and HTLV-2.

FIG. 1.

Organization of the HTLV genome and expanded coding region. (A) The complete proviral genome is shown schematically. LTRs are depicted with their U3, R, and U5 regions. The locations of the gag, pro, pol, env, tax, and rex genes and their corresponding reading frames are indicated, along with orf-I and orf-II of HTLV-1. Numbers below the genome indicate kilobases. (B) The genome containing the two tax/rex coding exons has been expanded, and the general locations of the ORFs (Tax, Rex, Env, Pol, and Orf-II) based on the nucleotide sequence of the proviral clones pH6neo (HTLV-2) and Ach (HTLV-1) are presented. Vertical arrows indicate the locations of protein start sites (ATG) and relevant restriction enzyme sites (SphI, Tth111I, and EcoRV). Numbers below the Tax and Rex ORFs indicate amino acid numbers (Tax-2 and Tax-1 are 331 and 353 amino acids, respectively; Rex-2 and Rex-1 are 170 and 189 amino acids, respectively).

FIG. 2.

Reciprocal exchange of HTLV-1 and HTLV-2 tax/rex sequences alters p19 Gag production from recombinant proviruses. 293T cells (2 × 10⁵) were transfected with 2 μg of wt HTLV-1, wt HTLV-2, HTLV-1/TR2, and HTLV-2/TR1 proviral DNAs. At 72 h posttransfection, p19 Gag production was measured in the supernatant by ELISA. The values, which represent p19 Gag levels for three independent experiments, are normalized for transfection efficiency. Error bars indicate standard deviations. The data indicate that Tax/Rex is functional in both recombinant proviral clones and suggest that Tax/Rex-1 is more active than Tax/Rex-2 irrespective of the viral backbone.

FIG. 3.

Tax transcriptional activation of LTR-1- and LTR-2-linked gene expression. 293T cells (2 × 10⁵) cells were cotransfected with 2 μg of LTR-1-CAT or LTR-2-CAT, 1 μg of CMV-luciferase, and 5 μg of tax expression plasmids or a negative control (C). After 48 h cells were harvested, and lysates were normalized for luciferase activity and assayed for CAT activity. The numbers represent the average fold activation over control values for three independent experiments. The data suggest that the HTLV-1 LTR can be transactivated to a greater extent than the HTLV-2 LTR and that Tax-1 displays a greater capacity to transactivate HTLV-1 or HTLV-2 LTR-linked gene expression.

FIG. 4.

Rex transactivation of RxRE-I and RxRE-II reporter genes. 293T cells (2 × 10⁵) T cells were cotransfected with pctat, pCgagRxRE-I (RxRE-I), or pCgagRxRE-II (RxRE-II), CMV-luciferase, and rex wt (Rex-1 and Rex-2) or rex recombinant (Rex-1.2 and Rex-2.1) mutant expression vectors or vector control alone (C) as indicated. At 48 h posttransfection, cells were harvested and assayed for p24 Gag protein as described in Materials and Methods. Lysates were assayed for luciferase activity to control for transfection efficiency. The numbers, which represent p24 Gag for three independent experiments, are averaged. Error bars indicate standard deviations. The significance of differences from the values for wt Rex and its respective response element was determined by the Student t test; values that are statistically different (P < 0.0001) are indicated by an asterisk. These data indicate that Rex-1 activity is partially impaired on RxRE-II and that at least two functional domains contribute to this impairment.

FIG. 5.

p19 Gag expression in permanent transfectants. Three stable 729 transfectants were isolated for wt HTLV-1 (Ach) and the two recombinants (HTLV-1/TR2 and HTLV-2/TR1) as described in Materials and Methods. Our well-characterized 729pH6neo clone was used as our wt HTLV-2 stable producer cell line. Forty-eight-hour culture supernatant was tested for p19 Gag production by ELISA. As expected, p19 production from different cell clones varied. Clones indicated by asterisks, which produce similar quantities of p19 Gag, were used in transformation assays.

FIG. 6.

Growth curve for HTLV T-lymphocyte transformation assay. Human PBMCs were isolated with Ficoll-Paque and cocultivated with irradiated (10,000 rads) 729 producer cells (729-wt HTLV-1, 729-wt HTLV-2, 729-HTLV-1/TR2, and 729-HTLV-2/TR1) or 729 uninfected control cells as indicated. PBMCs (2 × 10⁶) were cocultured with irradiated donor cells (1 × 10⁶) in 24-well plates. Cells were fed once per week with RPMI 1640 supplemented with 20% FCS. (A) Cell viability was determined by trypan blue exclusion staining at 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 weeks postcocultivation. The mean and standard deviation for each time point were determined from three independent samples. (B) The presence of HTLV gene expression was confirmed by detection of structural Gag protein in the culture supernatant by p19 Gag ELISA at 3, 4, 5, 6, 7, 8, and 9 weeks postcocultivation. The mean and standard deviation for each time point were determined from three independent samples.

FIG. 7.

Cell surface phenotypes of HTLV-transformed cells. Transformation assays were performed as described in the legend to Fig. 6. Wells containing transformed T cells, defined as cells with continuous growth 9 weeks postplating in the absence of IL-2, were stained with anti-CD3 antibody-FITC, anti-CD4 antibody-PE, and anti-CD8 antibody-PE-Cy5 and analyzed on a Coulter Epics Elite flow cytometer. The percentages of transformed CD4⁺ and CD8⁺ cells in individual wells for wt HTLV-1 (n = 25), wt HTLV-2 (n = 25), HTLV-1/TR2 (n = 28), and HTLV-2/TR1 (n = 34) are plotted. Mean values for CD4⁺ and CD8⁺ viral transformants are indicated.