Human T-cell leukemia virus type 1 Tax transactivates the human proliferating cell nuclear antigen promoter.

The human T-cell leukemia virus type 1 (HTLV-1) transforming protein, Tax, is a potent transactivator of both viral and cellular gene expression. The ability of Tax to transform cells is believed to depend on its transactivation of cellular-growth-regulatory genes. Expression of proliferating cell nuclear antigen (PCNA) is intimately linked to cell growth and DNA replication and repair. By testing a series of PCNA promoter deletion constructs, we have demonstrated that the PCNA promoter can be transactivated by Tax. The smallest construct that was activated did not include the ATF/CRE binding site at nucleotide -50, and mutations in the ATF/CRE element in the context of a larger promoter were still activated by Tax. In addition, a Tax mutant that is defective for activation of the CRE pathway retained the ability to activate the -397 promoter construct. When a series of linker scanner mutations that span the region from nucleotide -45 to -7 were assayed, mutations in and around a repeat sequence were found to abolish Tax transactivation. Multimerized copies of either half of the repeat were Tax responsive. A single protein complex was shown to bind specifically to the Tax-responsive region, and the binding of this complex was enhanced in the presence of Tax. These results demonstrate that the PCNA promoter contains a Tax-responsive element located between nucleotides -45 and -7 whose sequence is different from those of other, previously identified Tax-responsive elements. The ability of Tax to activate the PCNA promoter may play an important role in cellular transformation by HTLV-1.     

Regulation of the Epstein-Barr virus C promoter by AUF1 and the cyclic AMP/protein kinase A signaling pathway

EBNA2 is an Epstein-Barr virus (EBV)-encoded protein that regulates the expression of viral and cellular genes required for EBV-driven B-cell immortalization. Elucidating the mechanisms by which EBNA2 regulates viral and cellular gene expression is necessary to understand EBV-induced B-cell immortalization and viral latency in humans. EBNA2 targets to the latency C promoter (Cp) through an interaction with the cellular DNA binding protein CBF1 (RBPJk). The EBNA2 enhancer in Cp also binds another cellular factor, C promoter binding factor 2 (CBF2), whose protein product(s) has not yet been identified. Within the EBNA2 enhancer in Cp, we have previously identified the DNA sequence required for CBF2 binding and also determined that this element is required for efficient activation of Cp by EBNA2. In this study, the CBF2 activity was biochemically purified and microsequenced. The peptides sequenced were identical to the hnRNP protein AUF1. Antibodies against AUF1 but not antibodies to related hnRNP proteins reacted with CBF2 in gel mobility shift assays. In addition, stimulation of the cellular cyclic AMP (cAMP)/protein kinase A (PKA) signal transduction pathway results in an increase in detectable CBF2/AUF1 binding activity extracted from stimulated cells. Furthermore, the CBF2 binding site was able to confer EBNA2 responsiveness to a heterologous promoter when transfected cells were treated with compounds that activate PKA or by cotransfection of plasmids expressing a constitutively active catalytic subunit of PKA. EBNA2-mediated stimulation of the latency Cp is also increased in similar cotransfection assays. These results further support an important role for CBF2 in mediating EBNA2 transactivation; they identify the hnRNP protein AUF1 as a major component of CBF2 and are also the first evidence of a cis-acting sequence other than a CBF1 binding element that is able to confer responsiveness to EBNA2.     

Transcription factor AP-4 participates in activation of bovine leukemia virus long terminal repeat by p34 Tax.

Three 21bp repeats can be found in the bovine leukemia virus long terminal repeat, which are crucial for the LTR directed gene expression by the trans activator protein Tax. Previous studies demonstrated that the major target of the Tax directed activation are the CRE-like elements in the center of these repeats. In this work we report that another motif of the 21bp repeats is also required for the Tax activation. Gel retardation--with the wild type or mutant 21bp repeats--revealed that cellular factors from HeLa cells were specifically bound to the center (CRE-like element) and the 3'' region of the repeats, which contains a CAGCTG consensus AP-4 binding site. In vivo analysis using the synthetic 21bp repeats indicated that beyond the consensus CRE-like motif, the AP-4 site is also essential for Tax activation. To determine the role of AP-4 in BLV Tax trans activation, we used the AP-4 cDNA in antisense transient assays. In the in vivo experiments the antisense AP-4 RNA resulted in strongly decreased Tax activation. On the basis of these results we conclude that AP-4 is a good candidate of cellular factors involved in BLV Tax trans activation.