Janet F. Duvall

Inactivation of p53 by Human T-Cell Lymphotropic Virus Type 1 Tax Requires Activation of the NF-κB Pathway and Is Dependent on p53 Phosphorylation

ABSTRACT p53 plays a key role in guarding cells against DNA damage and transformation. We previously demonstrated that the human T-cell lymphotropic virus type 1 (HTLV-1) Tax can inactivate p53 transactivation function in lymphocytes. The present study demonstrates that in T cells, Tax-induced p53 inactivation is dependent upon NF-κB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-κB but not p300 binding or CREB transactivation. The Tax-induced p53 inactivation can be overcome by overexpression of a dominant IκB mutant. Tax-NF-κB-induced p53 inactivation is not due to p300 squelching, since overexpression of p300 does not recover p53 activity in the presence of Tax. Further, using wild-type and p65 knockout mouse embryo fibroblasts (MEFs), we demonstrate that the p65 subunit of NF-κB is critical for Tax-induced p53 inactivation. While Tax can inactivate endogenous p53 function in wild-type MEFs, it fails to inactivate p53 function in p65 knockout MEFs. Importantly, Tax-induced p53 inactivation can be restored by expression of p65 in the knockout MEFs. Finally, we present evidence that phosphorylation of serines 15 and 392 correlates with inactivation of p53 by Tax in T cells. This study provides evidence that the divergent NF-κB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels, including posttranslational modification of p53.

A 36-kilodalton cellular transcription factor mediates an indirect interaction of human T-cell leukemia/lymphoma virus type I TAX1 with a responsive element in the viral long terminal repeat.

The human T-cell leukemia/lymphoma virus type I (HTLV-I) trans activator, TAX1, interacts indirectly with a TAX1-responsive element, TRE-2, located at positions -117 to -163 in the viral long terminal repeat. This report describes the characterization of a 36-kilodalton (kDa) protein identified in HeLa nuclear extract which mediates the interaction of TAX1 with TRE-2. Purification of the protein was achieved by zinc chelate chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The renatured 36-kDa protein bound specifically to a TRE-2 oligonucleotide but not to nonfunctional base substitution mutant probes in a gel retardation assay. Renatured proteins of differing molecular weights were unable to form this complex. In addition, the 36-kDa protein specifically activated transcription from the HTLV-I promoter in vitro. Purified TAX1 protein formed a complex with the TRE-2 oligonucleotide in the presence of the 36-kDa protein, suggesting that indirect interaction of TAX1 with the viral long terminal repeat may be one of the mechanisms by which HTLV-I transcription is regulated.

Indirect binding of human T-cell leukemia virus type I tax1 to a responsive element in the viral long terminal repeat.

Several laboratories have demonstrated that tandem copies of the human T-cell leukemia virus type I 21-base-pair (bp) repeat cloned upstream of either a homologous or heterologous promoter increase transcription in the presence of tax1 protein. In this report, we provide evidence for a second tax1-responsive sequence in the viral long terminal repeat. Analysis of human T-cell leukemia virus type I promoter deletion mutants and plasmids containing cloned oligonucleotide motifs demonstrated that this 47-bp sequence, located between -117 and -163, confers responsiveness to tax1. We further demonstrated that proteins present in HeLa nuclear extracts bind specifically to this tax1-responsive sequence. Mutants that affected in vivo activity also decreased in vitro binding. Using an in vitro binding assay, we demonstrated that tax1 interacts indirectly with the 47-bp sequence, most likely through protein-protein interaction. Thus, while tax1 does not bind directly to DNA to enhance transcription, it may influence sequence-specific responses by interacting with the primary DNA-protein complex.

Interaction of the human T-cell lymphotropic virus type 1 tax transactivator with transcription factor IIA.

The Tax protein of human T-cell lymphotropic virus type 1 (HTLV-1) is a 40-kDa transcriptional activator which is critical for HTLV-1 gene regulation and virus-induced cellular transformation. Tax is localized to the DNA through its interaction with the site-specific activators cyclic AMP-responsive element-binding protein, NF-kappaB, and serum response factor. It has been suggested that the recruitment of Tax to the DNA positions Tax for interaction with the basal transcriptional machinery. On the basis of several independent assays, we now report a physical and functional interaction between Tax and the transcription factor, TFIIA. First, Tax was found to interact with the 35-kDa (alpha) subunit of TFIIA in the yeast two-hybrid interaction system. Importantly, two previously characterized mutants with point mutations in Tax, M32 (Y196A, K197S) and M41 (H287A, P288S), which were shown to be defective in Tax-activated transcription were unable to interact with TFIIA in this assay. Second, a glutathione-S-transferase (GST) affinity-binding assay showed that the interaction of holo-TFIIA with GST-Tax was 20-fold higher than that observed with either the GST-Tax M32 activation mutant or the GST control. Third, a coimmunoprecipitation assay showed that in HTLV-1-infected human T lymphocytes, Tax and TFIIA were associated. Finally, TFIIA facilitates Tax transactivation in vitro and in vivo. In vitro transcription studies showed reduced levels of Tax-activated transcription in cell extracts depleted of TFIIA. In addition, transfection of human T lymphocytes with TFIIA expression vectors enhanced Tax-activated transcription of an HTLV-1 long terminal repeat-chloramphenicol acetyltransferase reporter construct. Our study suggests that the interaction of Tax with the transcription factor TFIIA may play a role in Tax-mediated transcriptional activation.

Cell Cycle-Regulated Transcription by the Human Immunodeficiency Virus Type 1 Tat Transactivator

ABSTRACT Cyclin-dependent kinases are required for the Tat-dependent transition from abortive to productive elongation. Further, the human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents proliferation of infected cells by arresting them in the G2phase of the cell cycle. These findings suggest that the life cycle of the virus may be integrally related to the cell cycle. We now demonstrate by in vitro transcription analysis that Tat-dependent transcription takes place in a cell cycle-dependent manner. Remarkably, Tat activates gene expression in two distinct stages of the cell cycle. Tat-dependent long terminal repeat activation is observed in G1. This activation is TAR dependent and requires a functional Sp1 binding site. A second phase of transactivation by Tat is observed in G2 and is TAR independent. This later phase of transcription is enhanced by a natural cell cycle blocker of HIV-1,vpr, which arrests infected cells at the G2/M boundary. These studies link the HIV-1 Tat protein to cell cycle-specific biological functions.

Insights into the molecular mechanism of p53 inhibition by HTLV type 1 Tax.

The p53 protein plays a pivotal role in transmitting signals from many forms of genotoxic stress to genes and factors that control aspects of the cell cycle and death. Although mutated in approximately 60% of all human cancers, only a minority of human T-lymphotropic virus type 1 (HTLV-1)-transformed cells carry p53 mutations. Nevertheless, the p53 protein in HTLV-1-transformed cells is functionally inactive. We have previously demonstrated that the HTLV-1 Tax protein can inhibit p53 trans-activation function. Tax does not accomplish this by directly binding to p53, but rather by a unique mechanism that includes constitutive phosphorylation of p53 at Ser-15 and Ser-392. Analysis of Tax mutants in lymphocytes demonstrates that Tax-induced p53 inhibition correlates with the ability of Tax to activate NF-kappaB, but not p300 binding or CREB trans-activation. Consistent with these results, expression of the I-kappaBalpha(S32,36A) mutant that blocks NF-kappaB activation blocks Tax-mediated p53 inhibition. We further demonstrate the importance of Tax activation of NF-kappaB in p53 inhibition, using p65 knockout (KO) mouse embryo fibroblasts (MEFs). In the absence of p65 Tax could not inhibit p53. Tax does activate IKKbeta in the p65 KO MEFs, indicating that prenuclear events of NF-kappaB activation are not sufficient for Tax-mediated p53 inhibition, but rather NF-kappaB transcriptional activation is critical. Importantly, using phosphospecific antibodies, we demonstrate that phosphorylation of p53 at Ser-15 and Ser-392 correlates with Tax-mediated inhibition. In addition, mutation of p53 at Ser-15 and Ser-392 to alanines renders p53 resistant to Tax inhibition. This report reviews p53 inhibition by Tax and presents our current model.

Regulation of Viral Transcription Units by SV40 T-Antigen

We have investigated the ability of SV40 T-antigen to trans-activate the SV40 late and the Adenovirus E2 promoters. Transcriptional control signals required for T-antigen trans-activation of the SV40 late promoter include T-antigen binding site II and the SV40 72-bp repeats. In vivo competition with recombinant plasmids containing the entire SV40 late regulatory region and promoter sequences (mp 5171–272) results in quantitative removal of limiting trans-acting factors required for late gene expression in COS-1 cells. Insertion of increasing lengths of DNA sequences between the T-antigen binding sites and the 72-bp repeats dramatically reduces the competition efficiency, suggesting a physical interaction between proteins binding to the separate regulatory domains. Transfection experiments have been performed in ts2 COS cells, which express the ts 1609 SV40 T-antigen. Transfection at the non-permissive temperature (40°C) resulted in a 5- to 10-fold reduction in SV40 late promoter activity compared to the permissive temperature (33°C), suggesting that trans-activation of the SV40 late promoter requires continued expression of T-antigen.

The Role of Cis- and Trans-Acting Functions in Simian Virus 40 Gene Regulation

Simian virus 40 (SV40) is a small DNA tumor virus, the circular genome [5243 base pairs (bp)] of which encodes at least two early proteins and four late gene products (Tooze, 1980). The lytic cycle of this virus is expressed over a temporal course in permissive African green monkey kidney cells. Early viral gene expression is predominant in the first 10-12 hr postinfection. The early viral program appears to be represented by a single transcription unit that gives rise to one primary messenger RNA (mRNA). This transcript is then differentially spliced into one of two mRNAs that encode the two early SV40 proteins, large tumor (large-T) and small tumor (small-t) antigens. All subsequent events in the lytic cycle appear to depend on the presence of a functional large-T antigen, which, through binding to three large-T-antigen-binding sites near the origin for DNA replication: (1) modulates downward the level of early gene transcription through a repressorlike function, (2) stimulates the initiation of viral DNA replication, and (3) both directly and indirectly activates the SV40 late transcriptional program (see Fig. 1). A set of late mRNA molecules Open image in new window Figure 1 Genomic map and control region of SV40. The diagram presents the control region for expression of the SV40 early genes (large-T and small-t antigens) and late genes (VP1, 2, and 3). The origin for viral DNA replication (or) is flanked by the early transcriptional control sequences, including the Goldberg-Hogness box (AT), the three 21-bp repeats (each containing two copies of a GC-rich hexanucleotide), and the tandem 72-bp enhancer element. The early transcripts are initiated predominantly at position E early in infection and shift to position L after DNA replication. The late viral transcripts have heterogeneous 5′ ends, indicated by dots. Reprinted from Hamer and Khoury (1983) with permission. encodes the structural proteins of SV40, namely, VP1, VP2, and VP3. In addition, a 61-amino-acid polypeptide, the agnoprotein, is made late in the lytic cycle. While the function of this protein is not entirely clear, it appears to play a role in encapsidation or assembly of the mature viral particles. Furthermore, the late agnoprotein may play a role in regulation of late gene expression.