Human immunodeficiency virus type 1 genome activation induced by human T-cell leukemia virus type 1 Tax protein is through cooperation of NF-kappaB and Tat

Abstract

For productive replication of human immunodeficiency virus type 1 (HIV-1) in host cells, the viral genome-encoded transactivator Tat and several cellular transcription factors are required for efficient viral gene transcription. However, it remains unclear how the viral genome initiates transcription before Tat is transcribed or when Tat is at suboptimal levels. Here, we utilized the human T-cell leukemia type 1 Tax protein as a molecular tool to investigate the mechanism of viral gene transcription that initiates the early phase of infection of HIV-1. Tax alone does not significantly increase the activity of HIV-1 long terminal repeat (LTR) in T lymphocytes, but it markedly enhanced the replication of an infectious HIV-1 provirus with a truncated nef gene. This enhancement is preferentially mediated by the cooperation of Tax and Tat which is dependent on TAR and duplicated kappaB cis elements of the HIV-1 LTR as well as the NF-kappaB activation domain of Tax. Furthermore, phorbol myristate acetate and membrane-targeted HIV-1 Nef also enhanced the LTR activity in the presence of Tat in the TAR- and kappaB cis element-dependent manner. These data suggest that activated NF-kappaB can functionally interact with a suboptimal amount of Tat and the HIV-1 LTR for efficient initiation of viral gene transcription.

FIG. 1

Tax enhances replication of the HIV-1 provirus with a truncated nef gene in human T lymphocytes. (A) Schematic representation of the truncated-nef infectious provirus of HIV-1 and the HIV-1 LTR-SEAP reporter. The truncated-nef HIV-1 provirus (pNL4-3neo) is derived from pNL4-3 in which a neo gene fragment was inserted into the XhoI site at the nef locus of pNL4-3 at amino acid position 35. The nef gene is truncated and encodes only the first N-terminal 35 amino acids of the Nef protein. The complete LTR of HIV-1 was derived from pNL4-3 and was constructed by PCR with Pfu DNA polymerase. The SEAP reporter gene (14) is linked to the full-length LTR. (B) Flag-tagged wild-type Tax (Flag-Tax) and frameshift mutant (Flag-Tax_FS) were constructed in a phEFneo vector in which the human elongation factor promoter controls the expression of Tax. Tax (1 μg) or Tax_FS (1 μg) was cotransfected with or without pNL4-3neo (0.1 μg), together with pHIV-1 LTR-SEAP (0.2 μg) into Jurkat TAg cells by using DMRIE-C liposome transfection reagent. SEAP activity was quantified by using chemiluminescent substrate. The basal activity of the LTR was set at 1. The data are representative of at least two independent experiments.

FIG. 2

Tax cooperates with Tat to enhance HIV-1 LTR activity. (A) The transient cotransfection was performed with LTR-SEAP (0.4 μg), Flag-Tax (1 μg), Tat (0.05 μg), and Vpr (0.1 μg) either alone or in combination as indicated in the figure. The DNA amount used for transfection was standardized to a total of 2 μg with phEFneo vector. The data are representative of two independent experiments. (B) The dose-response activation to the LTR by costimulation of Jurkat T cells with Tax and Tat was measured by cotransfection of Tat (0.1 μg), LTR-SEAP (0.4 μg), and various amounts of Tax plasmid as indicated in the figure.

FIG. 3

The cooperative transactivation activity of Tax requires the TAR element. (A) Schematic representation of the wild-type HIV-1 LTR and the mutants with various cis element deletions. The nucleotide acid number is based on the numbering of the complete sequence of pNL4-3 derived from GenBank. LTR272-773 flanks a full-length promoter, but the upstream cis sequences immediately before the USF element were deleted. LTR296-773 covers the complete promoter region with the upstream sequence deletion immediately after the USF site. LTR329-773 contains the entire promoter but the upstream region immediately before the duplicated κB sites was deleted. LTR1-536 covers the entire promoter and enhancer, but the cis elements immediately after the TAR sequence and HS4 region were deleted. LTR1-477 (LTRΔTAR) includes the complete upstream modulatory region and the core region but the TAR sequence was deleted. The wild-type LTR and all LTR mutants were linked to the SEAP gene. (B) The transfection of Jurkat TAg cells was performed using Tax (0.6 μg) and Tat (0.1 μg) as well as various LTR constructs (0.2 μg/each) as indicated in the figure. The data are representative of two independent experiments.

FIG. 4

Tax’s transactivation to the HIV-1 LTR is κB element dependent. (A) Structures of wild-type LTR (LTRwt) and LTRΔκB. The wild-type HIV-1 LTR contains a complete upstream modulatory region as well as the core region and the TAR sequence. LTRΔκB was constructed by deleting all upstream modulatory region including the duplicated κB elements, but the core and the TAR regions are reserved. Both LTR fragments were linked to the SEAP gene. (B) Tax or Tax_FS and Tat together with the wild-type LTR (LTRwt) or LTRΔκB reporter plasmid were transiently cotransfected into Jurkat TAg cells. The SEAP activity was quantified 48 h after transfection. The data shown below represent two independent experiments.

FIG. 5

The NF-κB activation domain of Tax is required for the cooperative transactivation of the HIV-1 LTR. (A) Flag-tagged Tax mutants with mutations in the NF-κB activation domain (M22) and in the transcriptional activation domain (M318). M22 contains amino acid substitutions Ser130 Ala131 for Thr130 Leu131 within the NF-κB activation domain of the Tax protein. M318 has a carboxyl-terminal deletion in the transcriptional activation domain of Tax. Expression of Tax was detected by immunoblotting analysis. The Tax constructs were transiently transfected into 293T cells by using Lipofectamine reagent. The cellular extract prepared from the transfected cells was directly analyzed by immunoblotting with anti-HTLV-I Tax or with anti-Flag. (B) The wild-type Tax or the Tax mutants including M22, M318, and Tax_FS (1 μg/each) were cotransfected with the LTR-SEAP reporter plasmid (0.2 μg) and Tat (0.1 μg) into Jurkat T cells. The SEAP assay was performed as described above. The data are representative of at least three independent experiments.

FIG. 6

T-cell mitogens cooperate with Tat, but not Tax, to enhance the HIV-1 LTR activity in the TAR- or κB element-dependent manner. (A) Tax has no cooperative effect with either PMA or PHA. The LTR-SEAP was cotransfected with or without Tax into Jurkat TAg cells. Twenty-four hours after transfection, the transfected cells were left unstimulated or stimulated with either PMA (50 ng/ml) or PHA (2 μg/ml) for another 24 h. The SEAP activity was quantified as described above. (B) PMA or PHA synergistically activates the HIV-1 LTR in the presence of Tat. The experiment was performed as described above except that the Tat plasmid (0.1 μg) was used to replace Tax. The data are representative of two independent experiments.

FIG. 7

Membrane-targeted Nef activates HIV-1 LTR in cooperation with Tat in Jurkat T cells. (A) The CD8-Nef chimera was described previously (4). The truncated CD8 molecule (CD8ET) that encodes only extracellular and transmembrane domains was used as a control. CD8-Nef (1 μg) or CD8-ET (1 μg) was cotransfected with or without Tat (0.05 μg) together with the wild-type LTR (LTRwt)-SEAP reporter vector (0.2 μg) into Jurkat TAg cells. The SEAP activity was measured 48 h after transfection. (B) The cooperative effect of the CD8-Nef requires the κB and TAR elements. The transient cotransfection was performed by using CD8-Nef and Tat together with the LTRwt-SEAP, LTRΔκB-SEAP, or LTRΔTAR-SEAP in Jurkat cells. The data are representative of two independent experiments.