Susan J. Marriott

Impact of HTLV-I Tax on cell cycle progression and the cellular DNA damage repair response

Human T-cell lymphotropic virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL), a rapidly progressing, clonal malignancy of CD4+ T lymphocytes. Fewer than one in 20 infected individuals typically develop ATL and the onset of this cancer occurs after decades of relatively symptom-free infection. Leukemic cells from ATL patients display extensive and varied forms of chromosomal abnormalities and this genomic instability is thought to be a major contributor to the development of ATL. HTLV-I encodes a regulatory protein, Tax, which is necessary and sufficient to transform cells and is therefore considered to be the viral oncoprotein. Tax interacts with numerous cellular proteins to reprogram cellular processes including, but not limited to, transcription, cell cycle regulation, DNA repair, and apoptosis. This review presents an overview of the impact of HTLV-I infection in general, and Tax expression in particular, on cell cycle progression and the repair of DNA damage. The contribution of these activities to genome instability and cellular transformation will be discussed.

Cellular transformation by the HTLV-I Tax protein, a Jack-of-All-Trades

The human T-cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that is responsible for adult T-cell leukemia and a neurological disease, HTLV-I-associated myelopathy/tropical spastic paraparesis. HTLV-I encodes an oncogenic protein, Tax, which affects a variety of cellular functions prompting it to be referred to as a jack-of-all trades. The ability of Tax to both transcriptionally regulate cellular gene expression and to functionally inactivate proteins involved in cell-cycle progression and DNA repair provide the basis for Tax-mediated transformation and leukemogenesis. This review will concentrate on the effects of Tax on the dysregulation of the G1/S and G2/M checkpoints as well as the suppression of DNA damage repair leading to cellular transformation.

Ancient, independent evolution and distinct molecular features of the novel human T-lymphotropic virus type 4.

BackgroundHuman T-lymphotropic virus type 4 (HTLV-4) is a new deltaretrovirus recently identified in a primate hunter in Cameroon. Limited sequence analysis previously showed that HTLV-4 may be distinct from HTLV-1, HTLV-2, and HTLV-3, and their simian counterparts, STLV-1, STLV-2, and STLV-3, respectively. Analysis of full-length genomes can provide basic information on the evolutionary history and replication and pathogenic potential of new viruses.ResultsWe report here the first complete HTLV-4 sequence obtained by PCR-based genome walking using uncultured peripheral blood lymphocyte DNA from an HTLV-4-infected person. The HTLV-4(1863LE) genome is 8791-bp long and is equidistant from HTLV-1, HTLV-2, and HTLV-3 sharing only 62–71% nucleotide identity. HTLV-4 has a prototypic genomic structure with all enzymatic, regulatory, and structural proteins preserved. Like STLV-2, STLV-3, and HTLV-3, HTLV-4 is missing a third 21-bp transcription element found in the long terminal repeats of HTLV-1 and HTLV-2 but instead contains unique c-Myb and pre B-cell leukemic transcription factor binding sites. Like HTLV-2, the PDZ motif important for cellular signal transduction and transformation in HTLV-1 and HTLV-3 is missing in the C-terminus of the HTLV-4 Tax protein. A basic leucine zipper (b-ZIP) region located in the antisense strand of HTLV-1 and believed to play a role in viral replication and oncogenesis, was also found in the complementary strand of HTLV-4. Detailed phylogenetic analysis shows that HTLV-4 is clearly a monophyletic viral group. Dating using a relaxed molecular clock inferred that the most recent common ancestor of HTLV-4 and HTLV-2/STLV-2 occurred 49,800 to 378,000 years ago making this the oldest known PTLV lineage. Interestingly, this period coincides with the emergence of Homo sapiens sapiens during the Middle Pleistocene suggesting that early humans may have been susceptible hosts for the ancestral HTLV-4.ConclusionThe inferred ancient origin of HTLV-4 coinciding with the appearance of Homo sapiens, the propensity of STLVs to cross-species into humans, the fact that HTLV-1 and -2 spread globally following migrations of ancient populations, all suggest that HTLV-4 may be prevalent. Expanded surveillance and clinical studies are needed to better define the epidemiology and public health importance of HTLV-4 infection.

Damaged DNA and Miscounted Chromosomes: Human T Cell Leukemia Virus Type I Tax Oncoprotein and Genetic Lesions in Transformed Cells

Genetic instability is a recurring theme in human cancers. Although the molecular mechanisms mediating this effect commonly observed in transformed cells are not completely understood, it has been proposed to involve either the loss of DNA repair capabilities or the loss of chromosomal stability. The transforming retrovirus human T cell leukemia virus type I (HTLV-I) encodes a viral oncoprotein Tax, which is believed to cause the genomic instability characteristic of HTLV-I-infected cells. This review focuses on the ability of HTLV-I Tax to disrupt the cellular processes of DNA repair and chromosomal segregation. The consequences of these effects as well as the evolutionary advantage this may provide to HTLV-I are discussed.

Suppression of DNA repair by HTLV type 1 Tax correlates with Tax trans-activation of proliferating cell nuclear antigen gene expression.

The human T cell leukemia virus type 1 (HTLV-1) viral oncoprotein Tax acts as a transcriptional trans-activator affecting viral as well as cellular gene expression. To understand how Tax induces transformation, the consequences of its ability to alter expression of cellular genes must be examined. We have previously demonstrated that Tax activates expression of the cellular gene, proliferating cell nuclear antigen (PCNA), and that Tax suppresses DNA repair. In this study we tested the ability of previously described Tax mutants to activate PCNA gene expression and their ability to interfere with DNA repair. The results revealed a strong correlation between Tax trans-activation of PCNA gene expression and its ability to inhibit DNA repair via the nucleotide excision repair (NER) pathway. Thus, a consequence of activated PCNA gene expression appears to be reduced DNA repair capacity. These effects of Tax are likely to play important roles in its transforming activity.

HTLV-1 Tax protein sensitizes cells to apoptotic cell death induced by DNA damaging agents

Transient HTLV-1 Tax expression suppresses cellular nucleotide excision repair, and this effect correlates with Tax transactivation of the proliferating cell nuclear antigen promoter. The inability to repair DNA damage typically induces apoptotic cell death. Therefore, we investigated the effect of Tax-mediated suppression of DNA repair on apoptosis in stable Tax-expressing cells. Constitutive Tax expression reduced cellular nucleotide excision repair activity compared with parental and control cells. Tax-expressing cells were also more sensitive to apoptosis induced by DNA damaging agents than control cells. Even though Tax-expressing cells displayed reduced DNA repair, they showed increased DNA replication following UV damage. These results suggest that Tax suppresses the cells ability to repair DNA damage and stimulates DNA replication even in the presence of damage. The inability to repair DNA damage is likely to stimulate apoptotic cell death in the majority of Tax-expressing cells while the ability to promote DNA replication may also allow the survival of a small population of cells. We propose that together these effects contribute to the monoclonal nature and low efficiency of HTLV-1 transformation.

Human T-Cell Leukemia Virus Type 1 Tax Attenuates the ATM-Mediated Cellular DNA Damage Response

ABSTRACT Genomic instability, a hallmark of leukemic cells, is associated with malfunctioning cellular responses to DNA damage caused by defective cell cycle checkpoints and/or DNA repair. Adult T-cell leukemia, which can result from infection with human T-cell leukemia virus type 1 (HTLV-1), is associated with extensive genomic instability that has been attributed to the viral oncoprotein Tax. How Tax influences cellular responses to DNA damage to mediate genomic instability, however, remains unclear. Therefore, we investigated the effect of Tax on cellular pathways involved in recognition and repair of DNA double-strand breaks. Premature attenuation of ATM kinase activity and reduced association of MDC1 with repair foci were observed in Tax-expressing cells. Following ionizing radiation-induced S-phase checkpoint activation, Tax-expressing cells progressed more rapidly than non-Tax-expressing cells toward DNA replication. These results demonstrate that Tax expression may allow premature DNA replication in the presence of genomic lesions. Attempts to replicate in the presence of these lesions would result in gradual accumulation of mutations, leading to genome instability and cellular transformation.

Suppression of DNA Repair by Human T Cell Leukemia Virus Type 1 Tax Is Rescued by a Functional p53 Signaling Pathway

The Tax protein of human T cell leukemia virus type 1 is a viral transactivator and transforming protein. Tax is known to suppress cellular nucleotide excision repair (NER), and this activity has been proposed to play an important role in Tax transformation. In this study we have investigated the mechanism by which Tax suppresses NER with specific focus on the previously characterized ability of Tax to inhibit p53 function. Suppression of NER by Tax was rescued by overexpression of wild-type p53; however, a p53 transactivation-incompetent mutant did not restore NER activity. The cyclin-dependent kinase inhibitor p21, a major transcriptional target of p53, plays an important role in regulating DNA replication and repair. Overexpression of p21 reversed Tax-induced suppression of NER; however, a p21 C-terminal mutant that lacks the proliferating cell nuclear antigen binding domain did not restore NER activity. Thus, p53 and its downstream effector p21 can inhibit Tax-mediated suppression of DNA repair. These results imply that the inactivation of p53 function by Tax contributes to Tax suppression of DNA repair.

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export

BackgroundThe HTLV-I oncoprotein, Tax, is a pleiotropic protein whose activity is partially regulated by its ability to interact with, and perturb the functions of, numerous cellular proteins. Tax is predominantly a nuclear protein that localizes to nuclear foci known as Tax Speckled Structures (TSS). We recently reported that the localization of Tax and its interactions with cellular proteins are altered in response to various forms of genotoxic and cellular stress. The level of cytoplasmic Tax increases in response to stress and this relocalization depends upon the interaction of Tax with CRM1. Cellular pathways and signals that regulate the subcellular localization of Tax remain to be determined. However, post-translational modifications including sumoylation and ubiquitination are known to influence the subcellular localization of Tax and its interactions with cellular proteins. The sumoylated form of Tax exists predominantly in the nucleus while ubiquitinated Tax exists predominantly in the cytoplasm. Therefore, we hypothesized that post-translational modifications of Tax that occur in response to DNA damage regulate the localization of Tax and its interactions with cellular proteins.ResultsWe found a significant increase in mono-ubiquitination of Tax in response to UV irradiation. Mutation of specific lysine residues (K280 and K284) within Tax inhibited DNA damage-induced ubiquitination. In contrast to wild-type Tax, which undergoes transient nucleocytoplasmic shuttling in response to DNA damage, the K280 and K284 mutants were retained in nuclear foci following UV irradiation and remained co-localized with the cellular TSS protein, sc35.ConclusionThis study demonstrates that the localization of Tax, and its interactions with cellular proteins, are dynamic following DNA damage and depend on the post-translational modification status of Tax. Specifically, DNA damage induces the ubiquitination of Tax at K280 and K284. Ubiquitination of these residues facilitates the dissociation of Tax from sc35-containing nuclear foci, and stimulates nuclear export of Tax through the CRM1 pathway.

Genomic instability driven by the human T-cell leukemia virus type I (HTLV-I) oncoprotein, Tax.

The importance of maintaining genomic stability is evidenced by the fact that transformed cells often contain a variety of chromosomal abnormalities such as euploidy, translocations, and inversions. Gene amplification is a well-characterized hallmark of genomic instability thought to result from recombination events following the formation of double-strand, chromosomal breaks. Therefore, gene amplification frequency serves as an indicator of genomic stability. The PALA assay is designed to measure directly the frequency with which a specific gene, CAD, is amplified within a cells genome. We have used the PALA assay to analyse the effects of the human T-cell leukemia virus type I (HTLV-I) oncoprotein, Tax, on genomic amplification. We demonstrate that Tax-expressing cells are five-times more likely to undergo gene amplification than control cells. Additionally, we show that Tax alters the ability of cells to undergo the typical PALA-mediated G1 phase cell cycle arrest, thereby allowing cells to replicate DNA in the absence of appropriate nucleotide pools. This effect is likely the mechanism by which Tax induces gene amplification. These data suggest that HTLV-I Tax alters the genomic stability of cells, an effect that may play an important role in Tax-mediated, HTLV-I associated cellular transformation.