Interaction of HTLV-1 Tax with minichromosome maintenance proteins accelerates the replication timing program

Abstract

The Tax oncoprotein encoded by the human T-cell leukemia virus type 1 plays a pivotal role in viral persistence and pathogenesis. Human T-cell leukemia virus type 1-infected cells proliferate faster than normal lymphocytes, expand through mitotic division, and accumulate genomic lesions. Here, we show that Tax associates with the minichromosome maintenance MCM2-7 helicase complex and localizes to origins of replication. Tax IntroductionHuman T-cell leukemia virus-1 (HTLV-1) is a retrovirus that infects approximately 20 million people worldwide. 1 Although the majority of infected people remain lifelong asymptomatic carriers, 2% to 5% develop either adult T-cell leukemia (ATL) or chronic inflammatory diseases, such as HTLV-1-associated myelopathy/tropical spastic paraparesis. 1 Two major hallmarks of HTLV-1-infected cells are: preferential proliferation compared with normal lymphocytes 2 and tendency to accumulate genomic lesions, a prerequisite for oncogenesis. 3,4 Although the molecular mechanisms underlying T-cell transformation remain to be elucidated, it is generally accepted that the virally encoded Tax protein plays a central role. Tax indeed displays pleiotropic activities that cooperate to drive cell cycle progression and perturb genome stability. 4,5 For example, Tax shortens G 1 phase and accelerates transition into S phase by manipulating positive and negative G 1 phase regulators (ie, cyclin D/CDK complex, Rb and CDK inhibitors). [6][7][8] In addition, Tax is thought to indirectly generate DNA damage by suppressing DNA repair processes and competing with cellular DNA damage response (DDR). 4,5 Recent data also support a direct role for Tax-induced reactive oxygen species in the occurrence of DNA lesions. 9 Conceptually, Tax activities are achieved through proteinprotein interactions in both nuclear and cytoplasmic cellular compartments. 10 In the nucleus, Tax accumulates within foci called Tax speckled structures (TSS), 11 which overlap with the cellular machinery required for transcription, splicing, post-transcriptional modifications of proteins, DNA repair, and checkpoint control. 12 In this report, we further demonstrate that Tax recruits the MCM2-7 replicative helicase within the TSS and modulates the program of replication origin firing. Notably, this mechanism favors cell cycle progression and directly compromises genome stability. Methods Plasmids, antibodies, yeast 2-hybrid, and GST pulldownSee supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Cell cultureHeLa, Rat-1, and 293GP2 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and 2mM l-glutamine and penicillin/ streptomycin. Jurkat, HUT78 and JPX9 (Jurkat cell lines stably transfected either with tax-1 gene driven by a metallothionein-inducible promoter) were kept in RPMI 1640 supplemented with 10% FBS, 2mM l-glutamine, and penicillin/streptomycin. Expression of the viral protein was induced in JPX9 cells by adding 120 M zinc chloride...

Tax impairs DNA replication forks and increases DNA breaks in specific oncogenic genome regions

“…We think that MT4 cells have adapted to constitutive expression of Tax. Our data is in agreement with the previously reported effects of Tax on the minichromosome maintenance complex and increased origin firing [30]. Our results show that Tax induction leads to multiple replication defects, partly compensated by activation of back-up origins.…”

“…Tax has also been shown to target multiple G1/S cell cycle checkpoints to enhance proliferation of HTLV-I leukemic cells. Finally, Tax prematurely activates the anaphase promoting complex [40], inhibits nucleotide excision repair [41], and alters topoisomerases [42] and beta-polymerases [43], and the mini-chromosome maintenance, MCM2-7, helicase [30]. Consequently, Tax expression is associated with increased genomic and genetic instability.…”

Reducing the global burden of HTLV-1 infection: An agenda for research and action

“…-genes promoting viral replication and persistence (Tax and HBZ) (Matsuoka and Jeang, 2007); -modes of viral entry and replication (infectious and mitotic cycles) (Ghez et al, 2010;Boxus et al, 2012;Gillet et al, 2011Gillet et al, , 2013Sibon et al, 2006); -genomic and epigenetic mechanisms leading to ATL (oncogenic stress, driver mutations) (Kataoka et al, 2015;Yamagishi et al, 2012); -the role of the immune response in the control of infection (Asquith and Bangham, 2008); -the pathogenic potential of HTLV-2, -3 and -4 (Calattini et al, 2006).…”

HTLV-I Tax-Mediated Inactivation of Cell Cycle Checkpoints and DNA Repair Pathways Contribute to Cellular Transformation: “A Random Mutagenesis Model”

“…In addition, Tax associates with the mini-chromosome maintenance (MCM) helicases MCM2-7 and localizes to origins of replication. In the human Jurkat T-cell line, Tax was recruited to the origin of replication in G1 and early S phase, resulting in supplementary replication clusters at the onset of S phase and the accumulation of double-strand breaks [48]. In a separate study, molecular combing techniques were used to study the effect of HTLV-I Tax on DNA replication in human T cells that constitutively express Tax as well as cells stably transfected with an inducible Tax expression vector to account for confounding effects associated with potential cellular adaptation [49].…”