The human retrovirus HTLV-1 is the etiologic agent of adult T-cell leukemia. The viral transforming protein, Tax, plays a critical role in viral replication and transformation by regulating the expression of viral and cellular genes. Previous studies have demonstrated that Tax interacts with the cellular CREB protein and facilitates the binding of the coactivator and histone acetyltransferase CBP and PCAF, forming a multimeric complex on the CRE-like sites in the HTLV-1 promoter. The studies presented in this report focus on the ability of Tax to interact with cellular transcription factors and chromatin modifying proteins to regulate the CREB transcription pathway, which is essential for viral gene expression. We recently demonstrated that arginine methyltransferase CARM1, which methylates histone H3 and p300/CBP, is involved in the regulation of Tax transactivation. Over expression of CARM1 wild type, but not a methyltransferase mutant, resulted in increased Tax transactivation of the HTLV-1 long terminal repeat in CARM1 (+/+) and (-/-) cells. Consistent with these results, general methyltransferase inhibitor adenosine dialdehyde (AdOx) inhibited Tax transactivation. Moreover, CARM1 synergistically activated Tax transcription of the HTLV-1 LTR with the transcriptional coactivator CBP as well as PCAF. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro glutathione S-transferase (GST)-Tax pulldown, in vivo co-immunoprecipitation and confocal microcopy experiments. Histone methyltransferase assays demonstrated that Tax increased the methyltransferase activity of CARM1 on histone H3. Our data provide the first experimental evidence that CARM1 enhances Tax transcription of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of H3 in vitro and in vivo. Unexpectedly, we found that AdOx potently inhibited the growth of HTLV-1-transformed cells. Further investigation revealed that AdOx also inhibited the Tax-activated NF-kappaB pathway, resulting in reactivation of p53 and induction of p53 target genes. Analysis of the NF-kappaB pathway demonstrated that AdOx treatment resulted in degradation of the IkappaB kinase complex and inhibition of NF-kappaB through stabilization of the NF-kappaB inhibitor IkappaBalpha. Our data further demonstrated that AdOx induced G(2)/M cell cycle arrest and cell death in HTLV-1-transformed but not control lymphocytes. These studies demonstrate that protein methylation plays an important role in NF-kappaB activation and survival of HTLV-1-transformed cells as well as mediating viral transcriptional activation. Mechanisms through which Tax interacts and communicates with RNA polymerase II and cyclin dependent kinases are not clearly understood. We have recently demonstrated that Tax recruits P-TEFb, a critical cellular elongation factor, to the viral promoter. The recruitment likely involves protein-protein interactions since Tax associates with P-TEFb in vitro as demonstrated by GST protein pull-down assays and in vivo as shown by co-immunoprecipitation assay. Functionally, a siRNA directed toward CDK9 inhibited Tax transactivation in transient assays. Consistent with these findings, depletion of CDK9 from nuclear extracts inhibited Tax transactivation in vitro. Reconstitution of the reaction with WT P-TEFb, but not a kinase dead mutant, recovered HTLV-1 transcription. Addition of the CDK9 inhibitor flavopiridol blocked Tax transactivation in vitro and in vivo. Our studies further demonstrate that Tax regulates CDK9 kinase activity through a novel autophosphorylation pathway by inducing autophosphorylation of threonine 29, the first experimental evidence of an inhibitory phosphorylation site in CDK9. Cellular P-TEFb is found in two major complexes, the inactive form which is associated with inhibitory subunits 7SK snRNA and HEXIM1 and the active form which is associated with, at least in part, Brd4. Our recent results suggest the possibility that Tax may compete and functionally substitute for Brd4 in P-TEFb regulation. In vitro binding studies demonstrate that Tax and Brd4 compete for binding to P-TEFb through direct interaction with cyclinT1. Tax interacts with cyclinT1 amino acids 426-533 which overlaps the region responsible for Brd4 binding. In vivo, overexpression of Tax decreased the amount of 7SK snRNA associated with P-TEFb and stimulates Serine 2 phosphorylation of the RNA Pol II CTD, suggesting Tax regulates the functional activity of P-TEFb. Overexpression of Brd4 repressed Tax transactivation of the HTLV-1 LTR in a dose dependent manner. In this study, we analyzed the effect of Brd4 on human T-lymphotropic virus type 1 (HTLV-1) transcription. Overexpression of Brd4 repressed Tax transactivation of the HTLV-1 LTR in a dose-dependent manner. In vitro binding studies suggest that Tax and Brd4 compete for binding to P-TEFb through direct interaction with cyclin T1. Tax interacts with cyclin T1 amino acids 426 to 533, which overlaps the region responsible for Brd4 binding. In vivo, overexpression of Tax decreased the amount of 7SK snRNA associated with P-TEFb and stimulates serine 2 phosphorylation of the RNA polymerase II carboxyl-terminal domain, suggesting that Tax regulates the functionality of P-TEFb. Our results suggest the possibility that Tax may compete and functionally substitute for Brd4 in P-TEFb regulation and provides a molecular target for antiviral therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC005254-27
Application #
7732865
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
27
Fiscal Year
2008
Total Cost
$583,273
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Dasgupta, Arindam; Jung, Kyung-Jin; Jeong, Soo-Jin et al. (2008) Inhibition of methyltransferases results in induction of g2/m checkpoint and programmed cell death in human T-lymphotropic virus type 1-transformed cells. J Virol 82:49-59
Bonome, Tomas; Levine, Douglas A; Shih, Joanna et al. (2008) A gene signature predicting for survival in suboptimally debulked patients with ovarian cancer. Cancer Res 68:5478-86
Chevalier, Sebastien Alain; Ko, Nga Ling; Calattini, Sara et al. (2008) Construction and characterization of a human T-cell lymphotropic virus type 3 infectious molecular clone. J Virol 82:6747-52
Cho, Won-Kyung; Zhou, Meisheng; Jang, Moon Kyoo et al. (2007) Modulation of the Brd4/P-TEFb interaction by the human T-lymphotropic virus type 1 tax protein. J Virol 81:11179-86
Zhou, Meisheng; Lu, Hanxin; Park, Hyeon et al. (2006) Tax interacts with P-TEFb in a novel manner to stimulate human T-lymphotropic virus type 1 transcription. J Virol 80:4781-91
Gegonne, Anne; Weissman, Jocelyn D; Zhou, Meisheng et al. (2006) TAF7: a possible transcription initiation check-point regulator. Proc Natl Acad Sci U S A 103:602-7
Ohyama, Manabu; Terunuma, Atsushi; Tock, Christine L et al. (2006) Characterization and isolation of stem cell-enriched human hair follicle bulge cells. J Clin Invest 116:249-60
Chevalier, S A; Meertens, L; Pise-Masison, C et al. (2006) The tax protein from the primate T-cell lymphotropic virus type 3 is expressed in vivo and is functionally related to HTLV-1 Tax rather than HTLV-2 Tax. Oncogene 25:4470-82
Minn, Alexandra H; Pise-Masison, Cynthia A; Radonovich, Michael et al. (2005) Gene expression profiling in INS-1 cells overexpressing thioredoxin-interacting protein. Biochem Biophys Res Commun 336:770-8
Jang, Moon Kyoo; Mochizuki, Kazuki; Zhou, Meisheng et al. (2005) The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol Cell 19:523-34

Showing the most recent 10 out of 18 publications