# MOLECULAR PATHOLOGY OF HTLV-1

EDITED BY : Umberto Bertazzoni, Vincenzo Ciminale and Maria Grazia Romanelli PUBLISHED IN : Frontiers in Microbiology

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# MOLECULAR PATHOLOGY OF HTLV-1

Topic Editors:

Umberto Bertazzoni, University of Verona, Italy Vincenzo Ciminale, University of Padua, Italy Maria Grazia Romanelli, University of Verona, Italy

Schematic representation of the two modes of HTLV-1 amplification. Image originally published in Pasquier et al. (2018), Figure 1.

Pasquier A, Alais S, Roux L, Thoulouze M-I, Alvarez K, Journo C, Dutartre H and Mahieux R (2018) How to Control HTLV-1-Associated Diseases: Preventing de Novo Cellular Infection Using Antiviral Therapy. Front. Microbiol. 9:278. doi: 10.3389/fmicb.2018.00278

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus discovered, in 1980, by Gallo and co-workers. About 5-10% of HTLV-1-infected individuals are at risk of developing either a fatal malignancy, adult T-cell leukemia (ATL), or a chronic neuroinflammatory syndrome, HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Both diseases are incurable at present.

Many issues concerning HTLV-1's life cycle and pathobiology are still unsolved or controversial, and new approaches for prognostic stratification of patients and eradication of HTLV-1 infection are in high demand.

In this Research Topic, the focus has been centered on discussing two main themes: the functional analysis and oncogenic potential of HTLV-1 regulatory proteins and the control of HTLV-1-associated diseases. The 22 articles in this eBook cover many different aspects of HTLV-1 infection and pathogenesis, providing new perspectives and groundwork for future studies.

Citation: Bertazzoni, U., Ciminale, V., Romanelli, M. G., eds. (2019). Molecular Pathology of HTLV-1. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-750-2

# Table of Contents

*05 Editorial: Molecular Pathology of HTLV-1* Umberto Bertazzoni, Vincenzo Ciminale and Maria Grazia Romanelli

#### I. FUNCTION ANALYSIS AND ONCOGENIC POTENTIAL OF HTLV-1 REGULATORY PROTEINS

Stefania Fochi, Simona Mutascio, Umberto Bertazzoni, Donato Zipeto and Maria G. Romanelli

Norbert Donhauser, Stefanie Heym and Andrea K. Thoma-Kress

*97 Mitochondrial Proteins Coded by Human Tumor Viruses* Ilaria Cavallari, Gloria Scattolin, Micol Silic-Benussi, Vittoria Raimondi, Donna M. D'Agostino and Vincenzo Ciminale

### II. CONTROLLING HTLV-1 ASSOCIATED DISEASES

*122 How to Control HTLV-1-Associated Diseases: Preventing* de Novo *Cellular Infection Using Antiviral Therapy*

Amandine Pasquier, Sandrine Alais, Loic Roux, Maria-Isabel Thoulouze, Karine Alvarez, Chloé Journo, Hélène Dutartre and Renaud Mahieux

Fabio E. Leal, Soraya Maria Menezes, Emanuela A. S. Costa, Phillip M. Brailey, Lucio Gama, Aluisio C. Segurado, Esper G. Kallas, Douglas F. Nixon, Tim Dierckx, Ricardo Khouri, Jurgen Vercauteren, Bernardo Galvão-Castro, Rui Andre Saraiva Raposo and Johan Van Weyenbergh

# Editorial: Molecular Pathology of HTLV-1

#### Umberto Bertazzoni 1 \*, Vincenzo Ciminale2,3 \* and Maria Grazia Romanelli 1 \*

*1 Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biology and Genetics, University of Verona, Verona, Italy, 2 Department of Surgery, Oncology, and Gastroenterology, University of Padua, Padua, Italy, 3 Veneto Institute of Oncology IOV – IRCCS, Padua, Italy*

Keywords: HTLV-1, TAX 1, HBZ, ATL, HAM/TSP

**Editorial on the Research Topic**

#### **Molecular Pathology of HTLV-1**

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus discovered. It is estimated that 10–20 million people are infected worldwide by this oncogenic virus. About 5–10% of HTLV-1-infected individuals are at risk of developing either a fatal malignancy, adult T-cell leukemia (ATL), or a chronic progressive neuroinflammatory disease, called HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Both diseases are incurable at present.

#### Edited by:

*Hirofumi Akari, Kyoto University, Japan*

#### Reviewed by:

*Yuetsu Tanaka, University of the Ryukyus, Japan*

#### \*Correspondence:

*Umberto Bertazzoni<EMAIL_ADDRESS>Vincenzo Ciminale<EMAIL_ADDRESS>Maria Grazia Romanelli<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Virology, a section of the journal Frontiers in Microbiology*

Received: *19 October 2018* Accepted: *28 November 2018* Published: *12 December 2018*

#### Citation:

*Bertazzoni U, Ciminale V and Romanelli MG (2018) Editorial: Molecular Pathology of HTLV-1. Front. Microbiol. 9:3069. doi: 10.3389/fmicb.2018.03069*

Many issues concerning HTLV-1's life cycle and pathobiology are still unsolved or controversial, and new approaches for prognostic stratification of patients and eradication of HTLV-1 infection

are in high demand (Willems et al., 2017).

The present Research Topic is focused on the following themes:

#### FUNCTIONAL ANALYSES AND ONCOGENIC POTENTIAL OF HTLV-1 REGULATORY PROTEINS

Baratella et al**.** discuss the possible role of HBZ in the progression toward HTLV-1-associated diseases. One key finding of these authors is the exclusive cytoplasmic localization of HBZ in cells from HAM/TSP patients, which is in striking contrast to its nuclear localization in ATL cells. This finding suggests a correlation between HBZ's intracellular compartmentalization and the clinical outcome of HTLV-1 infection. The authors also propose that the cytoplasmic localization of HBZ in PBMC of HAM/TSP patients could be exploited as a molecular marker of disease. Kulkarni and Bangham Kulkarni and Bangham discuss the interplay between Tax and HBZ as well the influence of micro-environmental changes on latency and reactivation kinetics of the provirus. The authors present a comprehensive summary of factors that induce and inhibit plus-and minus-strand transcription of the provirus and propose that the binding of the cellular factor CTCF to the provirus produces abnormal chromatin loops, resulting in changes of cellular gene expression and possibly favoring the development of ATL. A research article by Gazon et al**.** sheds light on the long-standing hypothesis that Tax must be expressed in cycles to allow cell survival (Yoshida, 1987). By analyzing viral expression in HTLV-1 infected individuals and in sheep infected with bovine leukemia virus (BLV, a retrovirus closely related to HTLV-1 that produces B-cell neoplasias), the authors provide evidence for alternating expression of the plus- and minus proviral strands. They describe a model based on transient bursts of Tax-driven sense-strand transcription, followed by antisense RNA synthesis and silencing of plus strand synthesis. This mechanism would allow cell proliferation in the presence of a strong host immune response against Tax. Tanaka and Matsuoka review the cellular targets of HTLV infection in vivo focusing on the evidence indicating that HTLV-1 infects hematopoietic stem cells (HSC) in vivo and that HBZ influences early stages of hematopoietic cell differentiation and induces infected progenitor cells to abnormally differentiate into Treg-like T-cells. The authors also highlight the ability of HBZ to affect cell turnover by inducing genomic instability through upregulation of miR-17 and miR-21, which inhibit the expression of OBFC2A, a single-stranded DNA-binding protein that protects genome stability. Using affinity-tagged protein and shotgun proteomics, Panfil et al. identified the UBR5 E3 ubiquitin ligase as a novel binding partner of HBZ. The authors show that this interaction results in HBZ ubiquitination. Interestingly, UBR5 was found to be overexpressed in T leukemia/lymphoma cell lines and during the later stage of T-cell transformation in vitro, while loss of UBR5 decreased cellular proliferation, suggesting a role for UBR5 in maintaining the proliferative phenotype of transformed T-cells. Gazon et al**.** discuss the effects of Tax and HBZ on the transcription factors AP-1, ATF/CREB, and Maf and the contribution of these pathways to HTLV-1 induced cellular transformation. The authors dissect the intricate mechanisms of AP-1 pathway activation and propose that AP-1 is activated in HTLV-1-infected T-cells through Tax-dependent and Tax-independent mechanisms. Experimental evidence suggests that HBZ interacts with all three members of the Jun family: while sequestering c-Jun and JunB in HBZ-containing nuclear bodies, it cooperates with JunD to enhance transcription of the 3′LTR and hTERT. Several aspects of NF-κB pathway deregulation mediated by Tax and HBZ are reviewed by Fochi et al. By focusing on differences in the abilities of Tax-1/HBZ and their HTLV-2 homologs Tax-2/APH-2 to interact with host factors, the authors summarize the distinct impacts of Tax and HBZ on NF-κB deregulation and their effects in cell survival and proliferation. In an investigation of the influence of the microRNA network on the turnover of HTLV-1 infected cells, Sharma et al**.** provide evidence that HTLV-1 infection induces upregulation of miR-34a. Based on their findings, the authors propose that high miR-34a levels may provide a selective advantage to HTLV-1 infected cells in vivo that persists during the transformation process. Recent studies have shown that the p8 accessory protein of HTLV-1 induces T-cell conjugates and cellular conduits that may facilitate virus transmission. Donhauser et al**.** have developed a novel method based on flow cytometry for the automated quantitation of p8 transfer between cells. Using this method in time course experiments the authors show that p8 is rapidly transferred between Jurkat T-cells and that actin polymerization affects this process. Cavallari et al. present a review of the interactions between mitochondria and proteins coded by HTLV-1 and the human tumor viruses, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis viruses B and C (HBV and HCV), and human papillomavirus (HPV). The authors discuss how these interactions may contribute to viral replication, persistence and transformation and how these functions are connected to the crucial role of mitochondria in cellular bioenergetics, apoptosis, innate immunity, and redox balance.

### CONTROLLING HTLV-1-ASSOCIATED DISEASES

Tagaya and Gallo provide an insightful comparison of the oncogenicity of HTLV-1, other human oncoviruses and Helicobacter pylori. Interestingly, the lifetime risk to develop a malignancy is markedly higher in patients infected with HTLV-1—compared to EBV, KSHV, HBV, HCV, HPV, and H. pylori. Furthermore, the oncogenic mechanism of HTLV-1 is more direct. These striking features of HTLV-1 are often overlooked, possibly because of the low prevalence of the virus in the US and Europe. Based on these considerations the authors affirm the notion that this most carcinogenic virus deserves the "leukemia" name. Based its high oncogenic potential, Gallo et al. thus recommend to return to the virus' original name, "Human T-cell leukemia virus" in place of the more generic "Human T-cell lymphotropic virus."

Pasquier et al**.** discuss the difficulties in treating HTLV-1 patients and the current therapies available, in particular for ATL, and provide a schematic representation of the two modes of HTLV-1 transmission, i.e., through de novo by virus particles and clonal expansion of cells harboring the provirus. Using a novel in vitro assay, the authors screened an impressive number of drugs, and showed for the first time that the acyclic nucleoside phosphonates Adefovir dipivoxil and Tenofovir disporoxil are much more effective at blocking HTLV-1 transmission compared to the conventional nucleoside reverse transcriptase inhibitor AZT. In their review, Marino-Merlo et al. provide an up-todate overview of recent efforts to understand the mechanisms involved in current therapeutic regimens for ATL. Targeted biological therapies for ATL are thoroughly described in the prospective to find novel molecular targets in ATL therapy. Based on the accumulated evidence, the authors point out that the control of virus spread is a crucial aspect in ATL therapy.

Yamagishi et al. provide an overview of the multiple genetic and epigenetic aberrations that characterize in vivo HTLV-1 infection in its initial stages, when both Tax and HBZ are at work. The authors highlight the crucial, and apparent transient role of Tax in shaping the host cell genome and epigenome with the active, persistent role of HBZ, which further potentiates initiation and progression to ATL and HTLV-1 associated diseases. Queiroz et al**.** investigated the inflammatory response in HTLV-1 associated diseases and analyzed the relation between the IFNG+874A/T polymorphism and the progression of HTLV-1 infection to symptomatic disease. Patients carrying the polymorphic allele showed significantly higher levels of IFNgamma than those carrying the wild-type allele, suggesting that asymptomatic carriers with the T allele of IFNG+874A/T and a high proviral load have a high probability of developing HTLV-1 associated inflammatory diseases. Moodad et al**.** provide a comprehensive review of the mouse models of ATL and HTLV-I infection. Transgenic animals for Tax and HBZ, as well as

knock-outs for key cellular regulators provide useful models to understand the role of viral and host genes in the development of ATL. In addition, models based on xenografts and humanized immune-deficient mice provide a valuable platform to test new therapies against ATL.

An interesting new murine preclinical ATL model is presented in the original paper by Vicario et al.. The authors describe experiments in which they injected an HTLV-1-immortalized Tcell line either alone or together with normal primary fibroblasts into NGS mice, and demonstrate that fibroblasts strongly enhance HTLV-1 the growth of this cell line, which partly recapitulates the in vivo pattern of the lymphoma variant of ATL. This model may be useful to test novel therapeutic interventions for the aggressive lymphoma subtype of ATL.

Enose-Akahata et al**.** review the role of Tax and HBZ in the pathogenesis of HAM/TSP. They describe the infiltration of HTLV-1-infected lymphocytes into the CNS and their chronic release of Tax, which is capable of producing a neurotoxic effect on the long axons of corticospinal tracts involved in progressive neurological disease. It appears that HBZ expression is able to confer a pro-inflammatory phenotype to infected T-cells, a property that would position HBZ as an important factor in the HAM/TSP disease process.

Sato et al**.** describe an analysis of 453 HAM/TSP patients enrolled in the Japanese "AAM-net" registry. By measuring eight candidate biomarkers in PBMCs, serum and cerebrospinal fluid (CSF) samples, they demonstrate that the levels of neopterin and CXCL10 in CSF represent valuable markers to discriminate among rapid, slow and very slow progressors. Based on these results, the authors propose new classification criteria for disease activity which will aid in optimizing treatment.

#### REFERENCES

Willems, L., Hasegawa, H., Accolla, R., Bangham, C., Bazarbachi, A., Bertazzoni, U., et al. (2017). Reducing the global burden of HTLV-1 infection: an agenda for research and action. Antiviral Res. 137, 41–48. doi: 10.16/j.antiviral.2016.10.015

Yoshida, M. and Seiki, M. (1987). Recent advances in the molecular biology of HTLV-1: transactivation of viral and cellular genes. Annu. Rev. Immunol. 5, 541–557. doi: 10.1146/annurev.iy.05.040187.002545

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Using an integrated ex vivo approach, Leal et al**.** show that antiretroviral genes and HAM/TSP cluster in two distinct "proviral/antiviral" classes, of which the TRIM5a/TRIM22/BST2 antiviral subset is selectively up-regulated by IFN-beta signaling in HAM/TSP. These findings provide mechanistic evidence for the in vivo immunovirological effect of IFN-β treatment in HAM/TSP and identify novel biomarkers as well as possible therapeutic targets.

#### CONLUDING REMARKS

Nearly 40 years of research have yielded important insights into diverse aspects of the biology of HTLV-1 infection and pathogenesis. Nevertheless, we are still quite far from deciphering the complexity of its associated diseases. We hope that the broad range of issues addressed in this Research Topic will provide new perspectives and groundwork for future studies aimed at unraveling HTLV-1 pathogenesis and lead to the development of novel tools to eradicate the virus and its diseases.

### AUTHOR CONTRIBUTIONS

All authors UB, VC, and MGR contributed equally to the writing of the manuscript.

### ACKNOWLEDGMENTS

We warmly thank Donna D'Agostino for a critical reading of the Editorial. This work was partly supported by grants from AIRC (IG 17794), University of Verona/Veneto Institute of Oncology IOV – IRCCS Joint Project.

Copyright © 2018 Bertazzoni, Ciminale and Romanelli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# HTLV-1 HBZ Viral Protein: A Key Player in HTLV-1 Mediated Diseases

#### Marco Baratella, Greta Forlani and Roberto S. Accolla\*

Laboratories of General Pathology and Immunology "Giovanna Tosi", Department of Medicine and Surgery, School of Medicine, University of Insubria, Varese, Italy

Human T cell leukemia virus type 1 (HTLV-1) is an oncogenic human retrovirus that has infected 10–15 million people worldwide. After a long latency, 3–5% of infected individuals will develop either a severe malignancy of CD4+ T cells, known as Adult T-cell Leukemia (ATL) or a chronic and progressive inflammatory disease of the nervous system designated Tropical Spastic Paraparesis/HTLV-1-Associated Myelopathy (HAM/TSP). The precise mechanism behind HTLV-1 pathogenesis still remains elusive. Two viral regulatory proteins, Tax-1 and HTLV-1 bZIP factor (HBZ) are thought to play a critical role in HTLV-1-associated diseases. Tax-1 is mainly involved in the onset of neoplastic transformation and in elicitation of the host's inflammatory responses; its expression may be lost during cell clonal proliferation and oncogenesis. Conversely, HBZ remains constantly expressed in all patients with ATL, playing a role in the proliferation and maintenance of leukemic cells. Recent studies have shown that the subcellular distribution of HBZ protein differs in the two pathologies: it is nuclear with a speckled-like pattern in leukemic cells and is cytoplasmic in cells from HAM/TSP patients. Thus, HBZ expression and distribution could be critical in the progression of HTLV-1 infection versus the leukemic state or the inflammatory disease. Here, we reviewed recent findings on the role of HBZ in HTLV-1 related diseases, highlighting the new perspectives open by the possibility of studying the physiologic expression of endogenous protein in primary infected cells.

Keywords: HTLV-1, ATL, HAM/TSP, HBZ, Tax-1

### TAX-1 AND HBZ: THE TWO KEY PLAYERS IN HTLV-1-ASSOCIATED PATHOLOGIES

Human T-cell leukemia virus type 1 (HTLV-1) was the first oncogenic retrovirus identified in humans (Poiesz et al., 1980; Yoshida et al., 1982). It is currently estimated that HTLV-1 infects at least 10–15 million people worldwide. Large HTLV-1 endemic areas exist in Southern Japan, the Caribbean, Central and South America, the Middle East, Melanesia, and equatorial regions of Africa (Proietti et al., 2005; Gessain and Cassar, 2012). Although most HTLV-1 infected individuals remain asymptomatic carriers (AC) lifelong, about 3–5% of them develop, after many years of clinical latency, a severe malignancy of CD4+ T cells, known as Adult T-cell Leukemia (ATL).

Four clinical subtypes of HTLV-1 associated ATL are described: smoldering, chronic, lymphoma and acute (Shimoyama, 1991). Depending on the subtype and symptoms, the treatment is different and could include: 'watchful waiting,' chemotherapy, antiviral therapy, immunotherapy, allogeneic hematopoietic stem cell transplantation (alloHSCT) and targeted therapies

#### Edited by:

Umberto Bertazzoni, University of Verona, Italy

#### Reviewed by:

Jean-Marie Peloponese, UMR9004 Institut de Recherche en Infectiologie de Montpellier (IRIM), France Masao Matsuoka, Kyoto University, Japan

> \*Correspondence: Roberto S. Accolla<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Virology, a section of the journal Frontiers in Microbiology

Received: 07 November 2017 Accepted: 14 December 2017 Published: 22 December 2017

#### Citation:

Baratella M, Forlani G and Accolla RS (2017) HTLV-1 HBZ Viral Protein: A Key Player in HTLV-1 Mediated Diseases. Front. Microbiol. 8:2615. doi: 10.3389/fmicb.2017.02615

**8**

(Utsunomiya et al., 2015). However, despite the available treatments, the prognosis is still extremely poor (Nasr et al., 2017). HTLV-1 is also the causative agent of a neurological disease called tropical spastic paraparesis/HTLV-1-associated myelopathy (HAM/TSP) (Gessain et al., 1985), characterized by different neurological features such as spasticity, muscle weakness and sensory deficits. In general, the central function and cranial nerves are strongly compromised and the clinical course is progressive and without remission (Martin et al., 2014). Due to the wide spectrum of symptoms and the inefficiency of the antiviral therapies, the symptomatic treatment represents the current standard of therapy of this disorder.

The pathogenetic mechanisms at the basis of HTLV-1 infection progressing toward ATL or HAM/TSP are not clearly understood although there are sedimented indications that interactions between the viral encoded proteins and a variety of cellular targets are crucial. Two viral proteins, Tax-1 and HTLV-1 bZIP factor (HBZ), encoded by the sense and antisense viral transcripts, respectively, are largely responsible for the malignant transformation and immortalization of the infected T cells. Tax-1 is a potent activator of viral transcription and is involved in neoplastic transformation through modulation of the expression of cellular genes and deregulation of diverse cell signaling pathways involved in cell proliferation, DNA damage repair, and apoptosis (Hall and Fujii, 2005; Matsuoka and Jeang, 2007; Chlichlia and Khazaie, 2010; Tosi et al., 2011; Forlani et al., 2013) The oncogenic properties of Tax-1 are strongly associated to its ability to constitutively activate the nuclear factor kappa B (NF-kB) pathway (Petropoulos et al., 1996; Forlani et al., 2016). It is of note that the alteration of NF-kB signaling pathway could also be involved in the inflammatory state observed in HAM/TSP (Peloponese et al., 2006). Indeed, it has been suggested that pro-inflammatory CD8+ T lymphocytes (CTL), specific for Tax-1-expressing CD4+ T cells, could infiltrate the central nervous system (CNS), kill the target cells and produce inflammatory cytokines, in particular NF-kB-inducible cytokines, that may participate to tissue damage (Asquith and Bangham, 2000). The high immunogenicity of Tax-1 renders it the major target of effectors CTL, and this event may be responsible for the loss of Tax-1 expression during the long clinically latent period leading to overt ATL (Asquith et al., 2005). Genetic and epigenetic modification in the proviral genome are also responsible of Tax-1 silencing (Takeda et al., 2004). Indeed only 40% of ATL patients can express Tax-1 mRNA suggesting that this viral factor is dispensable for the maintenance of leukemia (Matsuoka and Jeang, 2007). Similar data were reported for the inflammatory diseases, in which it was found that 50% of HAM/TSP patiens can express Tax-1 mRNA. (Usui et al., 2008; Saito et al., 2009; Andrade et al., 2013). Interestingly, our recent findings in a restricted sample of patients demonstrated that, at least at protein level, Tax-1 is found in 75% of HAM/TSP cases and in 100% of HTLV-1 AC, but not in ATL cases (Baratella et al., 2017).

At variance with Tax-1, HBZ mRNA (Matsuoka and Green, 2009) and HBZ protein (Raval et al., 2015) are constantly expressed all ATL cases and in HTLV-1 infected individuals, indicating that HBZ is essential not only for cellular transformation but also for the maintenance of leukemic state. HBZ expression increases the proliferation of HTLV-1 infected T cells in culture, and more importantly induces both T-cell lymphomas and systemic inflammation in mice (Satou et al., 2006, 2011). Several studies have suggested a crucial function of HBZ also in HTLV-1-associated inflammatory disorders as it is always found at both mRNA and protein levels in TSP/HAM patients (Andrade et al., 2013). Interestingly, and at variance with Tax-1, most AC do express HBZ mRNA (Saito et al., 2009; Andrade et al., 2013) but not the protein (Baratella et al., 2017). The fact that HBZ protein is scarcely produced in infected cells may explain why is not, or only marginally, discriminated by HBZ specific CTLs in HTLV-1-infected individuals and in HAM/TSP patients (Hilburn et al., 2011; Rowan et al., 2014). The lower immunogenicity of HBZ compared to Tax-1, and the ability of HBZ to inhibit most of Tax-1 activity, could favor the virus immune escape, thus promoting the spreading of infection and the persistence of viral latency. Several studies have indicated a key function of HBZ in supporting and/or maintaining the proliferation of HTLV-1 infected cells and by this, the initiation and persistence of ATL. It has been reported that HBZ promotes ATL cell proliferation by inhibiting apoptosis through different mechanisms: it impairs the binding of AFT3 to p53, thus affecting the activation of p53-mediated apoptosis signaling (Hagiya et al., 2011); it inhibits the transcriptional activation of pro-apoptotic genes as Bim and Fas Ligand, by interfering with FoxO3 (Tanaka-Nakanishi et al., 2014). It was also reported that HBZ impairs anti-viral immunity responses: it binds to NFAT and inhibits the production of Th1 cytokines (particularly IFN-γ); it induces the expression of TIGIT on the cell surface in ATL (Yasuma et al., 2016) and promotes cells migration and proliferation by enhancing CCR4 expression on T-cell surface (Sugata et al., 2016).

Interestingly, several studies have demonstrated that HBZ exerts opposite effects with respect to Tax-1 on signaling pathways. Tax-1 activates while HBZ selectively inhibits the classical NF-κB pathway by affecting the binding of p65-RelA to its consensus DNA sequence and promoting its degradation. This inhibition leads to the activation of the alternative NF-κB pathway (Zhao et al., 2009). It suppresses Tax-1-mediated viral transactivation by interacting with the KIX domain of p300/CBP and impairing the binding of these cellular factors to Tax-1 (Clerc et al., 2008). HBZ suppresses, while Tax-1 activates, Wnt pathway by interacting with the disheveled-associating protein with a high frequency of Leucine residues (DAPLE) (Ma et al., 2013).

Taken together, all these data emphasize the critical role of HBZ in promoting cellular proliferation and the persistence of the viral infection.

### HBZ: BIOCHEMICAL ASPECTS AND DISTINCT SUBCELLULAR DISTRIBUTION OF ENDOGENOUS PROTEIN IN ATL AND HAM/TSP

Since its discovery in 2002, HBZ has become a crucial hotspot in HTLV-1 research. HBZ gene is encoded by the minus

strand of the HTLV-1 RNA genome (Gaudray et al., 2002) and transcribed by a functional promoter, contained in the U5 sequence of the 30 Long Terminal Repeat (LTR) (Yoshida et al., 2008).

Three major HBZ transcriptional isoforms have been described: the unspliced (usHBZ) form and two alternative spliced form (SP1 and SP2) (Cavanagh et al., 2006; Murata et al., 2006). Regarding the two spliced HBZ isoforms, although both of them have been found in HTLV-1 infected cells, the SP2 variant occurred less frequently than SP1 (Cavanagh et al., 2006). The SP1 spliced and unspliced HBZ transcripts are translated into polypeptides of 206 and 209 amino acids, respectively, and they have almost identical sequences except for a stretch of seven amino acids at the N-terminus of the protein (MAAS for SP1 HBZ and MVNFVSA for usHBZ). However, the halflife of usHBZ protein is much shorter than that of SP1 HBZ (Yoshida et al., 2008) and the expression level of SP1 HBZ is four times higher than that of usHBZ in ATL cells (Usui et al., 2008). Nevertheless, it was reported that the two HBZ protein variants exhibit similar functions (Ma et al., 2016), as they were characterized by conserved functional domains: an N-terminal activation domain (AD), a central domain (CD) and a C-terminal basic ZIP domain (bZIP). HBZ contains three nuclear localization signals (NLS) responsible for its nuclear localization (Hivin et al., 2005; Zhao and Matsuoka, 2012) and two functional nuclear export signals (NES) within its N-terminal region (Mukai and Ohshima, 2014). Most of the reported sub-cellular localizations, biochemical interactions and functional aspects related to HBZ have been assessed in cells overexpressing tagged HBZ. Through its bZIP domain, HBZ was reported to interact with CREB/CREB-2, and this association was instrumental to suppress Tax-mediated HTLV-1 viral transcription (Gaudray et al., 2002). Similar experiments have shown that HBZ binds to different proteins of the JUN family via its bZIP domain. Upon binding to HBZ, JunB, and cJun were recruited in nuclear bodies and degraded, thus HBZ reduces the cJun/JunB-mediated transcriptional activation of a series of genes (Basbous et al., 2003; Thébault et al., 2004). Conversely, the association of HBZ to JunD did not inhibit the JunD-mediated transcriptional activation of target genes; indeed HBZ-JunD complex was reported to increase HBZ gene expression (Thébault et al., 2004; Gazon et al., 2012).

Most of the reported HBZ interactions, however, were assessed by artificial overexpressing systems (transfected cells) and thus it has been hard to extrapolate these results to the real situation encountered in infected cells or in leukemic cells from patients. Only recently, the availability of an anti-HBZ monoclonal antibody (mAb), 4D4-F3, isolated in our laboratory, has made it possible to assess endogenous HBZ expression, localization and interaction in vivo in HTLV-1 infected and in ATL patient (Raval et al., 2015). Indeed, endogenous HBZ interacts and co-localizes with p300 and JunD. Partial colocalization was observed also for CBP and CREB2 (Raval et al., 2015). By using the 4D4-F3 mAb we were able to quantify the HBZ protein demonstrating that the amount of HBZ in ATL patients is around 0.45 × 10−2 pg/cell corresponding to 17.461 molecules/cell, that is 20- to

Leukemia (ATL), HAM/TSP and asymptomatic carriers (AC). HBZ protein localizes in distinct subcellular compartments and with different percentage in leukemic cells of ATL patients and PBMC of HAM/TSP patients. HBZ is expressed in the nucleus of 90% and in the cytoplasm of 20% of cells isolated from ATL or HAM/TSP patients, respectively. AC do not express detectable levels of HBZ.

50-fold lower than the amount expressed in HBZ transfected cells. Similar results were recently obtained by another group (Shiohama et al., 2016). Interestingly, our recent data generated by immunofluorescence with the 4D4-F3 mAb and careful confocal microscopy studies have demonstrated that HBZ protein is expressed in 80 to 100% of ATL cells, in 0.4 to 11% of PBMC of HAM/TSP cells, and very rarely, if any, in PBMC of asymptomatic HTLV-1 carriers (Raval et al., 2015; Baratella et al., 2017).

These studies have also demonstrated that endogenous HBZ protein is localized in the nucleus of ATL cells (Raval et al., 2015; Shiohama et al., 2016) with a similar speckle-like distribution as the one observed in cells transfected with tagged HBZ protein (Gaudray et al., 2002). However, the HBZ nuclear aggregates found in cells overexpressing tagged-HBZ, in particular GFP-HBZ, were shown to be artifacts of chimeric proteins. Although the composition of these nuclear structures containing HBZ is still unknown it has been demonstrated that HBZ-specific nuclear bodies did not overlap with Promyelocytic Leukemia (PML) nuclear bodies (Raval et al., 2015), indicating that HBZ did not co-localize with PML protein. Of note, PML bodies were shown to act as co-activators of Tax-1, without binding to or co-localizing with the viral transactivator (Ariumi et al., 2003)

One of the most important finding of our studies was the demonstration of the distinct subcellular localization of HBZ protein in HAM/TSP as compared to ATL cells. Until now, HBZ has been always assumed to have an exclusive nuclear localization. Thus, it was relatively unexpected to find that HBZ is exclusively localized in the cytoplasm of PBMC of HAM/TSP patients (**Figure 1**). This cytoplasmic localization was not affected by the presence of leptomycin B, a nuclear export inhibitor, indicating that the viral factor did not shuttle in and out of the nucleus (Baratella et al., 2017). Furthermore, cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment, very rarely (less than 1%) in CD8+ T cells, and never in B cells or NK cells. Interestingly, the CD4+ HBZ cells, did not expressed the CD25 T cell activation marker

(Baratella et al., 2017), thus suggesting that they were either not in rapid proliferation or, if resting, not included in the classical regulatory T cell compartment. This goes in line with recent findings showing that the HBZ-specific humoral immune response correlated with reduced CD4+ T cell activation in HAM/TSP patients (Enose-Akahata et al., 2013). Within this context, the HBZ cytoplasmic localization in HAM/TSP patients may not be appropriate to the generation of peptides that can efficiently bind MHC class I molecules for presentation to, and scrutiny by CTLs. Of further interest was the finding that, at least in the patients' sample analyzed in our studies, the expression of HBZ and Tax-1 was mutually exclusive (Baratella et al., 2017).

Taken together, these observations let us to propose the cytoplasmic localization of HBZ protein in PBMC of HAM/TSP patients as the first molecular marker of disease, since until now the only associative parameters with the neurological disease were either clinical correlates or the rather nonspecific high proviral load (Nagai et al., 1998). Recently, it has been reported that HBZ-transfected cell lines, including the T cell line Jurkat, may partially segregate HBZ in the cytoplasm as result of interaction with THEMIS (Kinosada et al., 2017). With the limitations expressed above on the physiological correlates of HBZ in overexpressing systems, THEMIS may represent a potential HBZ interactor contributing to the cytoplasmic segregation of HBZ also in HAM/TSP patients' cells. Future studies in patient settings should clarify this point.

### CONCLUDING REMARKS AND FUTURE PERSPECTIVES: THE GROWING RELEVANCE OF HBZ IN THE ONSET OF HTLV-1 ASSOCIATED DISEASES

Although several studies have been focussed at clarifying the role of HBZ in HTLV-1-associated diseases, much research is still required to clearly define the molecular and cellular basis of the distinct outcome of viral infection, whether evolving versus the leukemia/lymphoma or versus the neuroinflammatory disorders. In this review, we have summarized the basic molecular aspects of HBZ expression and, importantly, the new findings related to the distinct subcellular localization of HBZ in HTLV-1 infected individuals, AC, affected by ATL or by HAM/TSP neurological disease.

A new paradigm is emerging that suggests a possible roadmap in the evolution of infection based on these new findings (**Figure 2**). We may think that primary HTLV-1 infection is characterized by the expression of Tax-1 (this stage may be observed in PBMC of still asymptomatic patients), followed by the possible transient coexpression of HBZ (yet to be demonstrated). As we have no specific demonstration of this intermediate step, we do not know whether HBZ protein can localize in the nucleus, cytoplasm or both cellular compartments at these early stages of infection. Immediately after, a compartmentalization of HBZ in the cytoplasm can take

place in case of evolution of the infection toward HAM/TSP. At this stage, the evolving pathology can either present a concomitant Tax-1 expression in the same cells (rare), or a mutually exclusive expression of Tax-1 and HBZ in different cells, as we observed in our patients. Conversely, in case of evolution toward the leukemic stage, HBZ will be expressed always and only in the nucleus with or without the coexpression of Tax-1. This hypothetical scheme is useful to test several additional hypotheses and investigate previously unforeseen mechanisms. For example, can we pinpoint a stage in which HBZ is compartmentalized in the cytoplasm still without clinical signs of neurological disease and use this parameter also as a prognostic marker of future development to HAM/TSP? Moreover, what are the molecular mechanisms that drive the distinct nuclear or cytoplasmic localization of the HBZ protein? Are there specific interactors that retain HBZ in the two different cellular compartments? Are these mechanism related to the onset and/or the persistence of the distinct pathologies? These and other fascinating questions' are now, we believe, experimentally amenable to scrutiny.

### AUTHOR CONTRIBUTIONS

MB, GF, and RA participated in the conception and design of the review and revised the manuscript. All the authors read, critiqued, and approved the final manuscript.

### FUNDING

This work was supported by University of Insubria intramural grant FAR 2014-2016 to RA and GF; by The Institutional Grant 2017, University of Insubria, to RA; by European Community FP7 project HepaVAC Grant Number 602893 to RA (www.hepavac.eu).

#### REFERENCES

#### ACKNOWLEDGMENTS

The authors wish to thank Dr. Goutham Raval, Dr. Alessandra Tedeschi, and Dr. Farah Bou Nasser Eddine for their participation to several experiments on HBZ localization that were the ground for the results discussed in this review.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Baratella, Forlani and Accolla. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# HTLV-1: Regulating the Balance Between Proviral Latency and Reactivation

#### Anurag Kulkarni and Charles R. M. Bangham\*

Section of Virology, Division of Infectious Diseases, Department of Medicine, Imperial College London, London, United Kingdom

HTLV-1 plus-strand transcription begins with the production of doubly-spliced tax/rex transcripts, the levels of which are usually undetectable in freshly isolated peripheral blood mononuclear cells (PBMCs) from HTLV-1-infected individuals. However, the presence of a sustained chronically active cytotoxic T-cell response to HTLV-1 antigens in virtually all HTLV-1-infected individuals, regardless of their proviral load, argues against complete latency of the virus in vivo. There is an immediate burst of plus-strand transcription when blood from infected individuals is cultured ex vivo. How is the HTLV-1 plus strand silenced in PBMCs? Is it silenced in other anatomical compartments within the host? What reactivates the latent provirus in fresh PBMCs? While plus-strand transcription of the provirus appears to be intermittent, the minus-strand hbz transcripts are present in a majority of cells, albeit at low levels. What regulates the difference between the 50 - and 30 -LTR promoter activities and thereby the tax-hbz interplay? Finally, T lymphocytes are a migratory population of cells that encounter variable environments in different compartments of the body. Could these micro-environment changes influence the reactivation kinetics of the provirus? In this review we discuss the questions raised above, focusing on the early events leading to HTLV-1 reactivation from latency, and suggest future research directions.

#### Edited by:

Umberto Bertazzoni, University of Verona, Italy

#### Reviewed by:

Luc Willems, Fonds National de la Recherche Scientifique, Belgium Donna M. D'Agostino, Università degli Studi di Padova, Italy

#### \*Correspondence: Charles R. M. Bangham

<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Virology, a section of the journal Frontiers in Microbiology

Received: 22 December 2017 Accepted: 27 February 2018 Published: 19 March 2018

#### Citation:

Kulkarni A and Bangham CRM (2018) HTLV-1: Regulating the Balance Between Proviral Latency and Reactivation. Front. Microbiol. 9:449. doi: 10.3389/fmicb.2018.00449 Keywords: HTLV-1, latency, virus, metabolism, hypoxia, glucose, epigenetic regulation, transcription

## INTRODUCTION

Human T-lymphotropic virus type 1 (HTLV-1), also known as Human T-cell leukemia virus type 1, is a retrovirus that mainly infects CD4+ T-cells in vivo. In ∼5–10% of the infected individuals, HTLV-1 infection leads to either an aggressive T-cell malignancy, adult T-cell leukemia/lymphoma (ATL), or a chronic progressive neuro-inflammatory condition called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Both these conditions have limited treatment options, and ATL in particular carries a very poor prognosis.

Initial infection is asymptomatic and occurs through breast milk, blood, or semen of infected individuals (Bangham and Matsuoka, 2017). HTLV-1 replicates in the host through two distinct routes: (i) Infectious spread: this mode of spread involves productive replication from the integrated provirus followed by the transfer of newly produced virions through the virological synapse (Igakura et al., 2003; Pais-Correia et al., 2010). This is the major route of viral spread in the initial stages of infection when the proviral load – the percentage of infected PBMCs – is low (Bangham et al., 2014). Infectious spread results in the formation of distinct T-cell clones,

each clone carrying a single-copy HTLV-1 provirus integrated in a unique genomic location within the host genome. (ii) Mitotic spread: proliferation of HTLV-1-infected host cells results in passive replication of the integrated HTLV-1 provirus within their genome. The two daughter cells resulting from mitosis of an HTLV-1-infected parent cell carry the provirus in the same genomic integration site. In contrast to infectious spread, the contribution of this mode to the proviral load in infected individuals may be small in the early stages of infection but gradually increases during the chronic stage of infection (Bangham et al., 2014). Although the proviral load in each host can fluctuate by a small factor (2- to 5-fold) over time, the proviral loads can vary between infected individuals by over 1000-fold (Nagai et al., 1998; Demontis et al., 2013). Individuals with a higher proviral load are at greater risk of developing either ATL or HAM/TSP (Matsuzaki et al., 2001; Iwanaga et al., 2010). An infected individual typically carries about 104 to 105 different T-cell clones, each with a unique proviral integration site (Bangham et al., 2014). The extent of proliferation of HTLV-1-infected T-cell clones, and thus their respective contribution to an individual's proviral load, both vary greatly from one clone to another.

A peculiar characteristic of HTLV-1 is the absence of detectable cell-free virions in infected individuals (Demontis et al., 2015). HTLV-1 was previously considered to be latent in infected individuals because there are no detectable plusstrand viral structural RNA or protein products in the peripheral blood mononuclear cells (PBMCs) freshly obtained from HTLV-1 infected individuals. Also, HTLV-1 is genetically stable, with minimal sequence variation over evolutionary time (Gessain et al., 1992), suggesting that de novo viral replication, which generates sequence variation, contributes little to the longterm persistence of HTLV-1 in vivo. However, a sustained chronically active CTL response to HTLV-1 can be observed in virtually all infected individuals (Jacobson et al., 1990; Bangham, 2009). This observation suggests that the host immune system routinely encounters viral antigens in vivo. Since the HTLV-1 plus-strand appears to be latent in peripheral blood, the implication is that plus-strand expression occurs either in intermittent bursts, or outside the circulation, or both. Thus it is necessary to understand the determinants of HTLV-1 latency and reactivation in vivo (**Figure 1**) to devise effective preventive and therapeutic approaches against HTLV-1-associated diseases such as HAM/TSP and ATL.

#### Proviral Genomic Integration Site

HTLV-1 cannot be looked at in isolation because it is an inseparable part of the chromatinized host genomic DNA. Can we predict the behavior of the integrated provirus in the context of its flanking host genome? HTLV-1 integration favors transcriptionally active regions of the host genome (Melamed et al., 2013). Recently, the host enzyme Protein Phosphatase 2A (PP2A) was identified as a major host co-factor for the HTLV-1 integrase, which could influence the selection of genomic integration sites (Maertens, 2016). Furthermore, plus-strand transcription is silenced when the viral DNA is integrated downstream of a host gene promoter in the same-sense orientation, possibly by transcriptional interference (Melamed et al., 2013). Similarly, the presence of the SWI/SNF-associated ATPase BRG-1 (identified by chromatin immunoprecipitation) upstream of the integrated HTLV-1 provirus is associated with silencing of plus-strand transcription, but its presence downstream of the HTLV-1 sequence is associated with proviral plus-strand expression (Melamed et al., 2013). Recently, it was reported that integration of HTLV-1 in the vicinity of cancer driver genes causes either premature interruption of transcription or antisense-dependent cis-perturbation of these genes, perhaps contributing to leukemogenesis (Rosewick et al., 2017).

The discovery of a functional CTCF binding site within the pX region of the HTLV-1 genome adds a new dimension to the importance of the flanking genome to proviral transcription (Satou et al., 2016). CTCF is a critical host protein which binds to an insulator motif in the DNA and appears to limit the spread of epigenetic modifications (Merkenschlager and Nora, 2016). Consistent with this function, the CTCF binding region in HTLV-1 is associated with the presence of a sharp epigenetic border in several histone and DNA modifications. It is possible that the epigenetic border formed by CTCF binding within the HTLV-1 provirus allows unhindered minus-strand transcription while reversibly inhibiting the plus-strand activity. Another central function of CTCF is the formation of chromatin loops, which regulate contacts between enhancers and promoters. It is thus possible that the CTCF bound to the provirus produces abnormal chromatin loops, resulting in aberrant cellular gene expression (Cook et al., 2017). Experiments are now in progress to test these hypotheses. Ultimately, this aberrant gene expression may be an important factor in the development of ATL.

#### The Tax-HBZ Interplay

Gene transcription in HTLV-1 progresses in both the sense and anti-sense directions, governed by promoters in the identical long terminal repeats (LTRs) situated at each end of the genome.

#### Plus-Strand Transcription

This is regulated by the TATA-box-containing inducible promoter in the 50 -LTR (Miyazato et al., 2016). TATA boxes are frequently observed in promoters of cellular genes which are highly regulated and responsive to stress and extracellular signals (Basehoar et al., 2004; Bahrami and Drablos, 2016). The 50 -LTR contains three tandem 21-bp imperfect nucleotide repeats called tax-responsive elements (TREs) which bind to the ATF/CREB family of proteins to activate HTLV-1 plus-strand transcription (Armstrong et al., 1993). The transactivator protein Tax, encoded by the pX region of the HTLV-1 genome by a double-splicing mechanism, is the most potent activator of plus-strand transcription and initiates a positive-feedback loop which induces transcription and promotes replication (Beimling and Moelling, 1992; Anderson and Dynan, 1994). Tax enhances the binding of CREB to the TRE, thereby promoting the transactivation of the proviral plus-strand (Suzuki et al., 1993). In addition to ATF/CREB family, the 50 -LTR also has binding sites for several major transcription factors, namely NF-κB, SRF and Sp1, which play direct or indirect roles in

activating plus-strand transcription (Armstrong et al., 1993). In addition to Tax, the HTLV-1 pX region encodes another regulatory protein, Rex, which regulates the stabilization of unspliced and singly spliced viral mRNAs, their nuclear export and subsequent effective translation of the viral proteins (Nakano and Watanabe, 2012). The other HTLV-1 pX region plus-strand products play accessory roles in vivo: p30 and p13 promote infectivity in a rabbit model (Bartoe et al., 2000; Silverman et al., 2004; Hiraragi et al., 2006), and p30 and p12/p8 enhance persistent infection in macaques (Valeri et al., 2010; Pise-Masison et al., 2014). p13 increases the production of reactive oxygen species, selectively killing transformed cells, and may favor persistence in vivo (Silic-Benussi et al., 2010). However, these genes are not essential for viral replication or T-cell immortalization (Derse et al., 1997; Robek et al., 1998), and may not be required for transmission (Furukawa et al., 2004).

#### Minus-Strand Transcription

This is initiated at the 30 -end of the provirus and progresses in the anti-sense direction (Gaudray et al., 2002). It is governed by the TATA-less promoter in the 30 -LTR (Yoshida et al., 2008), which contains three TREs and three Sp1 binding sites (Yoshida et al., 2008; Ma et al., 2016). sHBZ is the major transcript driven by the 3 0 -LTR.

The interplay between the plus-strand and minus-strand transcription is a critical and poorly understood aspect of the HTLV-1 life cycle. We infer that Tax expression in vivo is intermittent and highly regulated as it is a very immunogenic protein (Jacobson et al., 1990; Parker et al., 1992). There is no detectable plus-strand gene expression in freshly isolated PBMCs from HTLV-1 infected individuals (Kinoshita et al., 1989; Rende et al., 2011). HBZ, in contrast, is poorly immunogenic and is expressed at a very low level; however, the presence of a detectable cytotoxic T-lymphocyte (CTL) response to HBZ is associated with a lower proviral load (MacNamara et al., 2010). This constant, low-level hbz mRNA expression is thought to be necessary for the persistence of HTLV-1 in vivo: hbz expression can be observed in all HTLV-1 infected individuals, including those with HAM/TSP and ATL (Yoshida et al., 2008). Tax and HBZ have strong effects on a number of cellular processes and signaling cascades (Ma et al., 2016); the effects of the two proteins frequently act in opposition. Certain transcription factors including TCF1 and LEF1 inhibit plus-strand transcription from the 50 -LTR while simultaneously enhancing minus-strand transcription from the 3 0 -LTR (Ma et al., 2015). In contrast, the histone deacetylase inhibitor (HDACi) valproate activates plus-strand transcription while inhibiting expression of the minus-strand (Lezin et al., 2007; Belrose et al., 2011; Olindo et al., 2011). It is also known that Tax activates minus-strand transcription through binding of the TREs in the 30 -LTR (Landry et al., 2009). Recently, it was shown by single-molecule RNA-FISH that, at a single-cell level, both plus- and minus-strand transcription occurs in bursts, albeit at different times and intensities. HTLV-1 plus-strand mRNA transcription is enhanced in the absence of minus-strand transcripts, while high-level plusstrand transcription promotes transcription from the minusstrand (Billman et al., 2017), generating a putative negative feedback inhibition of plus-strand expression. Until now, it was believed that all HTLV-1-infected cells express hbz mRNA at all times; however, this study showed that, in

naturally-infected T-cell clones isolated by limiting dilution from HTLV-1-infected individuals, not all HTLV-1-infected cells in each clone are hbz positive (Billman et al., 2017) at a given time. Many questions remain unanswered. Most hbz transcripts were found in the nuclei of cells, consistent with previous observations (Rende et al., 2011) and with the low level of protein expression of HBZ (Shiohama et al., 2016). HBZ protein inhibits tax expression by competing for binding of the transcription co-factor CREB (Gaudray et al., 2002; Lemasson et al., 2007; Clerc et al., 2008). However, since HBZ protein is expressed at such a low level in naturally-infected cells, the importance of this mechanism is uncertain (Shiohama et al., 2016).

These observations raise important questions: does hbz RNA also inhibit tax expression? What are the factors that regulate minus-strand transcriptional bursts? It has been shown that Sp1 co-operates with JunD to activate minus-strand transcription, but how and when this happens in the context of the cell cycle and viral replication are still unknown (Gazon et al., 2012). Recently, Billman et al. (2017) showed at the single cell level, in naturally-infected HTLV-1 positive T-cell clones, that cells in S and G2/M phase of the cell cycle have elevated tax and hbz mRNA levels, suggesting that the cell cycle plays a role in regulating HTLV-1 transcription. It is now becoming clear that the results of population-level analyses can mask strong single-cell heterogeneity within the population.

### Epigenetic Regulation of HTLV-1 Transcription

Small-molecule inhibitors targeting epigenetic enzymes have been widely used in HIV latency research. Indeed, reactivation of the latent HIV-1 provirus occurs in response to HDACi [e.g., suberanilohydroxamic acid (SAHA)], DNA methylation inhibitors (e.g., 5-azacytidine) and histone methyltransferase inhibitors (e.g., the EZH1/2 inhibitor GSK343) (Spina et al., 2013; Tripathy et al., 2015). Similarly, in the case of HTLV-1, the HDACi valproate and DNA methyltransferase inhibitor 5-azacytidine have been shown to activate the latent HTLV-1 provirus (Koiwa et al., 2002; Belrose et al., 2011). These observations suggest that epigenetic modulations in the promoters, enhancers and gene bodies of the integrated provirus play an important role in proviral transcription. Cytosine methylation of the 50 -LTR of HTLV-1 is associated with silencing of plus-strand transcription; the degree of this 5 0 -LTR methylation varies from individual to individual, whereas the 30 -LTR is unmethylated in most individuals (Taniguchi et al., 2005), consistent with the constitutive activation of the promoter in the 30 -LTR. However, it is unclear how dynamic the DNA methylation pattern of the provirus is. It is unlikely that the highly regulated promoter in the 50 -LTR of HTLV-1 would rely primarily on DNA demethylation for its reactivation. Data are needed on the extent of DNA methylation during bursts of gene expression from the plus-strand and minusstrand, and whether this methylation is a cause or an effect of promoter activation. Activation of plus-strand transcription is associated with Tax-mediated recruitment of pCREB and CBP/p300, resulting in histone acetylation and nucleosomal remodeling through BRG-1-containing SWI/SNF complexes (Sharma and Nyborg, 2008; Easley et al., 2010). Similarly, HTLV-1 plus-strand transcription is associated with a significant increase in the activatory histone epigenetic modifications H3K4me3 and H3K36me3 in different regions of the HTLV-1 provirus (Kulkarni et al., 2017). It is likely that, as in host gene expression, many epigenetic modifications are a cause as well as consequence of viral reactivation. Most of the epigenetic studies carried out so far have been in in vitro cellular models where Tax expression is the primary mechanism of 50 -LTR activation through a positive feedback loop. However, evidence from freshly isolated PBMCs from HTLV-1-infected individuals indicates that there is little or no Tax protein expression in vivo at steady state. Hence, it is important to examine the earliest epigenetic changes that occur at the provirus in freshly isolated PBMCs from HTLV-1-infected individuals to provide clues to the changes that occur in vivo. It is still unclear what primarily reactivates plus-strand transcription in ex vivo PBMCs from HTLV-1-infected individuals. The answer to this conundrum may elucidate the key question – what regulates the rapidly reversible silencing of plus-strand transcription in vivo?

### Metabolic Regulation of HTLV-1 Transcription

The metabolic and functional activities of circulating PBMCs depend directly on their micro-environment. What effects do metabolic changes have on the integrated HTLV-1 provirus within these cells? We recently showed that HTLV-1 reactivation from latency is severely limited in the absence of glucose in the surrounding medium (Kulkarni et al., 2017). In support of this conclusion, we found that PBMCs treated with glycolysis inhibitors – and to a lesser extent with mitochondrial electron transport chain (ETC) inhibitors – show a significant impairment in HTLV-1 plus-strand transcription when they are cultured ex vivo. The importance of glucose in the HTLV-1 life cycle is emphasized by the fact that the glucose receptor GLUT-1 is a cellular receptor for HTLV-1 (Manel et al., 2003). Surprisingly, drugs that inhibit or stimulate the TCA cycle do not alter HTLV-1 transcription (Kulkarni et al., 2017). Indeed, it is known that different subsets of T-cells use distinct metabolic pathways to satisfy their energy requirements (Dimeloe et al., 2017). How glucose metabolism influences HTLV-1 reactivation from latency is not known. A plausible explanation is that inhibition of glycolysis or the mitochondrial ETC reduces the availability of Acetyl CoA and ATP. Acetyl CoA is an important acetyl group donor in the process of histone acetylation, which is associated with transcriptional activation. Similarly, the SWI/SNF family of chromatin-remodeling complexes such as BAF and PBAF rely on energy in the form of ATP for their nucleosomal remodeling function. It has been shown before that HTLV-1 plus-strand activation is intimately linked with histone acetylation and chromatin remodeling at the 5 0 -LTR promoter (Easley et al., 2010; Nyborg et al., 2010). Another important question that arises from these results is

the possible impact of high blood glucose levels on HTLV-1 replication. Do HTLV-1-infected individuals with poorly controlled diabetes have a higher proviral load of HTLV-1, resulting in a significantly higher risk of HTLV-1-associated diseases such as HAM/TSP or ATL? Further work is needed to clarify the cause and effect relationships between metabolic activity and proviral activation in HTLV-1-infected primary CD4+ T-cells.

#### Anatomical Regulation of HTLV-1 Transcription

Lymphocytes are constantly exposed to highly variable stresses during circulation through different body compartments. As mentioned above, HTLV-1 appears to be latent in freshly isolated PBMCs from the peripheral blood of HTLV-1 infected individuals. The presence of a chronically active CTL response to HTLV-1 in all infected individuals raises the possibility that HTLV-1 expression is more frequent in different compartments like the bone marrow, lymph, and lymph nodes (Bangham, 2008). Also, Yasunaga et al. (2016) found that tax transcription in the bone marrow of STLV-1 infected Japanese macaques was significantly higher than that in other tissues. One factor that differs significantly between these compartments is the oxygen tension. While the lymph, lymph nodes, and bone marrow are highly hypoxic (∼1% oxygen), PBMCs in peripheral venous blood are exposed to significantly higher levels of oxygen (∼6–10%) (Tsai et al., 2004; Hangai-Hoger et al., 2007). We recently reported that, in primary PBMCs isolated from HTLV-1 infected individuals, physiological hypoxia (∼1% oxygen) enhances plus-strand HTLV-1 transcription (Kulkarni et al., 2017). Again, further work is needed to identify the molecular mechanism of this effect, and the consequences of this observation on the establishment and spread of viral infection within and between individuals.

#### REFERENCES

#### SUMMARY

The transcriptional regulation of the plus- and minus-strands of HTLV-1 is affected by a number of virus-specific and host-specific factors. Advances in single-cell genomics and transcriptomics have made it possible to study HTLV-1 infection at the singlecell level. We anticipate that these approaches will shed light on the effect of cellular heterogeneity on viral replication and help identify novel mechanisms that were missed by earlier studies which depended on population-level analyses. Precise identification of the molecular mechanisms involved in HTLV-1 reactivation from latency would help us design and test novel therapeutic strategies to reduce the disease burden associated with this infection.

#### AUTHOR CONTRIBUTIONS

Both authors have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

This work was supported by a Project Grant from the Medical Research Council, United Kingdom (MR/K019090/1), a Senior Investigator award from the Wellcome Trust, United Kingdom (WT100291MA) to CB, and the Imperial National Institute for Health Research Biomedical Research Centre (https://imperialbrc.nihr.ac.uk/).

#### ACKNOWLEDGMENTS

We thank Jocelyn Turpin and Goedele Maertens for comments on the manuscript.

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model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog. 9:e1003834. doi: 10.1371/journal.ppat.1003834

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Kulkarni and Bangham. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# How Does HTLV-1 Undergo Oncogene-Dependent Replication Despite a Strong Immune Response?

Hélène Gazon1,2, Pradeep Chauhan1,2, Malik Hamaidia1,2, Clotilde Hoyos1,2, Lin Li1,2 , Roghaiyeh Safari1,2 and Luc Willems1,2 \*

1 National Fund for Scientific Research, Molecular and Cellular Epigenetics, Interdisciplinary Cluster for Applied Genoproteomics, Liège, Belgium, 2 Molecular Biology, TERRA, Gemboux Agro-Bio Tech, Gembloux, Belgium

In 1987, Mitsuaki Yoshida proposed the following model (Yoshida and Seiki, 1987): "... T-cells activated through the endogenous p40x would express viral antigens including the envelope glycoproteins which are exposed on the cell surface. These glycoproteins are targets of host immune surveillance, as is evidenced by the cytotoxic effects of antienvelope antibodies or patient sera. Eventually all cells expressing the viral antigens, that is, all cells driven by the p40x would be rejected by the host. Only those cells that did not express the viral antigens would survive. Later, these antigen-negative infected cells would begin again to express viral antigens, including p40x, thus entering into the second cycle of cell propagation. These cycles would be repeated in so-called healthy virus carriers for 20 or 30 years or longer...." Three decades later, accumulated experimental facts particularly on intermittent viral transcription and regulation by the host immune response appear to prove that Yoshida was right. This Hypothesis and

#### Edited by:

Umberto Bertazzoni, University of Verona, Italy

#### Reviewed by:

Charles R. Bangham, Imperial College London, United Kingdom Roberto S. Accolla, University of Insubria, Italy

> \*Correspondence: Luc Willems<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Virology, a section of the journal Frontiers in Microbiology

Received: 21 November 2017 Accepted: 26 December 2017 Published: 15 January 2018

#### Citation:

Gazon H, Chauhan P, Hamaidia M, Hoyos C, Li L, Safari R and Willems L (2018) How Does HTLV-1 Undergo Oncogene-Dependent Replication Despite a Strong Immune Response? Front. Microbiol. 8:2684. doi: 10.3389/fmicb.2017.02684 Theory summarizes the evidences that support this paradigm.

#### Keywords: HTLV, BLV, microRNA, cytotoxic T cells, long non-coding RNA, Leukemia

### INTRODUCTION

At least 20 million people worldwide are infected with human T-cell leukemia virus type 1 (HTLV-1) (Gessain and Cassar, 2012; Bangham, 2017; Watanabe, 2017). This retrovirus is prevalent in southwestern Japan, sub-Saharan Africa, the Caribbean islands, South America, the Middle East and Austro-Melanesia.

Although frequently neglected in the field, bovine leukemia virus (BLV) is a useful model to address specific questions that cannot be answered in the HTLV-1 system (Rodríguez et al., 2011; Polat et al., 2017). Both viruses are indeed closely related δ-retroviruses that induce hematological diseases. In the bovine species, the most prevalent clinical manifestation in about one-third of infected animals is persistent lymphocytosis, a benign accumulation of infected B-lymphocytes (Gutierrez et al., 2014). In a minority of cases (about 5–10%), BLV infection can progress to fatal leukemia/lymphoma whose most dramatic consequence is spleen hypertrophy and disruption consecutive to tumor formation. BLV typically persists in less than 1% of peripheral blood cells, leading to an asymptomatic infection in the majority of infected animals. BLV is transmitted

horizontally by direct contact, iatrogenic procedures or insect bites upon transfer of infected cells from milk, blood, and body fluids from heavily infected dams (Barez et al., 2015).

### THE PARADOX: HTLV-1 REPLICATION IS DRIVEN BY ONCOGENIC PROTEINS THAT EXPOSE THE INFECTED CELL TO THE HOST IMMUNE RESPONSE

According to currently most accepted model, two viral proteins (Tax and HBZ, HTLV-1 bZIP) are hypothesized to have the highest impact on viral replication and cell transformation (Matsuoka and Jeang, 2007; Carpentier et al., 2015). The modes of action of Tax and HBZ are remarkably pleiotropic and involve a variety of cell signaling pathways (CREB, NF-κB, AKT, and TNF) (Twizere et al., 2003; Boxus et al., 2008). Tax inhibits tumor suppressors (p53, Bcl11B, and TP53INP1) and activates cyclin-dependent kinases (CDKs), both of these mechanisms leading to accelerated cell proliferation. Experimental evidence further shows that Tax drives tumor formation in transgenic mouse models, supporting its oncogenic potential. Through an interaction with the helicase complex (mcm2-7), Tax accelerates S phase progression by initiating additional replication origins. By promoting unscheduled cell growth, Tax also induces genomic instability and generates somatic alterations. Another viral protein, HBZ also favors cell proliferation by inhibiting apoptosis/senescence and modulating the cell cycle (Ma et al., 2016). In fact, the HBZ protein counteracts a series of Tax-activated viral and cellular pathways (such as NF-κB, Akt, and CREB). Transgenic expression of HBZ in CD4 + T cells induced T-cell lymphomas and systemic inflammation in mice, resembling diseases observed in HTLV-1 infected individuals (Satou et al., 2011).

A major issue of expression of viral proteins is initiation of the host immune response. Indeed, Tax induces a strong immune response that would be harmful to infected cells (Nagai et al., 2001; Kannagi et al., 2005; Rowan et al., 2014). In comparison, HBZ triggers a less efficient immunity that is consistent with low expression low expression throughout HTLV-1 infection (MacNamara et al., 2010). How does HTLV-1 persist despite a strong immune response against viral oncogenes that promote infected cell replication? The next paragraph lists the experimental evidence pertaining to this paradox.

### EXPERIMENTAL EVIDENCE AND INTERPRETATIONS

#### Evidence 1: Infected Cells Proliferate Faster to Undergo Clonal Expansion

The BLV model has been instrumental to quantify the dynamics of cell turnover in vivo (Florins et al., 2007). Experiments based on intravenous injection of bromodeoxyuridine (BrdU) and carboxyfluorescein diacetate succinimidyl ester (CFSE) demonstrate that B-lymphocytes are proliferating significantly faster in BLV-infected sheep than in healthy controls (Debacq et al., 2002, 2006). Excess of proliferation is compensated by an increase in cell death, thereby maintaining homeostasis. Increased cell proliferation is also reported in HTLV-induced HAM/TSP using a similar strategy based on incorporation of deuterated glucose (Asquith et al., 2007). In vivo, BLV and HTLV infection is thus characterized by an increased cell turnover, likely driven by viral oncogenes such as Tax and HBZ.

### Evidence 2: 50 LTR Directed Transcription Is Extremely Low in Situ

The amount of viral RNA transcribed from the 50 LTR promoter is extremely low in vivo. In primary tumor cells, only very sensitive techniques such as RT-PCR can identify viral RNA transcribed from the 50 LTR promoter (Lagarias and Radke, 1989; Rovnak and Casey, 1999; Shimizu-Kohno et al., 2011; Demontis et al., 2015). It was initially concluded that the provirus is silent. However, in situ experiments showed that rare cells expressed large amounts of viral transcripts (Lagarias and Radke, 1989).

### Evidence 3: Sense Transcription Can Be Activated by Various Stimuli

The main regulatory element of the 50 LTR promoter that is activated by Tax is a 21 bp enhancer that interacts with CREB/ATF transcription factors (Suzuki et al., 1993; Adam et al., 1996). This complex activates sense transcription when cells are isolated ex vivo from HTLV-1 carriers or BLV-infected sheep. This reactivation can further be increased by various stimuli such as polyclonal activators, HDAC inhibitors or oxygen deprivation (Kerkhofs et al., 1996; Achachi et al., 2005; Tajima and Aida, 2005; Lezin et al., 2007; Olindo et al., 2011; Kulkarni et al., 2017).

### Evidence 4: Immunity against Most Viral Antigens Is Extremely Efficient While HBZ Is Poorly Immunogenic

Persistent infection by BLV and HTLV-1 is characterized by a permanent and vigorous immunity against viral antigens (Kannagi et al., 2005; Burbelo et al., 2008; Bangham et al., 2009; Kattan et al., 2009). This immune response efficiently controls viral replication in vivo as demonstrated in the BLV model (Florins et al., 2006, 2009, 2011; Gillet et al., 2013). Among viral proteins, Tax is the immunodominant HTLV-1 antigen in the T-cell response (Kannagi et al., 1991; Goon et al., 2004). In contrast, the HBZ protein is very poorly immunogenic and expressed at very low levels in infected cells (Suemori et al., 2009; Enose-Akahata et al., 2013; Rowan et al., 2014; Raval et al., 2015; Baratella et al., 2017). Humoral immunity against HBZ protein is indeed particularly weak (Raval et al., 2015; Shiohama et al., 2016). However, cytotoxic T cells specific to HBZ but not to the immunodominant Tax are the most effective in the control of HTLV-1 (MacNamara et al., 2010). These results thus focus attention on the extremely low expression and low immunogenicity of HBZ.

### Evidence 5: Antisense Transcripts and microRNAs Are Abundantly and Permanently Expressed in Tumors

In contrast to 50 LTR directed transcription, antisense RNA synthesis initiating at the 30 LTR is consistently identified in primary ATL cells and BLV tumors (Usui et al., 2008; Saito et al., 2009; Durkin et al., 2016). In contrast to HTLV-1, BLV also abundantly transcribes a cluster of microRNAs from internal pol III promoters (Kincaid et al., 2012; Rosewick et al., 2013; Van Driessche et al., 2016). These microRNAs are required for efficient viral replication and induction of pathogenesis (Gillet et al., 2016).

### Evidence 6: HBZ and AS RNAs Are Mainly Localized in the Nucleus Suggesting a Role in Epigenetics

The HBZ RNA is mainly localized in the nucleus, consistent with a low rate of translation (Hivin et al., 2005; Rende et al., 2011; Shimizu-Kohno et al., 2011; Li et al., 2012; Raval et al., 2015). Although the function of the HBZ protein has been clearly evidenced, the dominant nuclear localization of the HBZ RNA thus suggests other regulatory roles such as for example epigenetic modulation of gene expression. In BLV, the antisense transcripts are not predicted to be translated but are rather primarily retained in the nucleus, hinting at a lncRNA-like role (Durkin et al., 2016).

#### Evidence 7: Untranslated HBZ RNA Has a Function

The oncogenic role of the HBZ RNA was revealed by an untranslatable HBZ RNA (i.e., devoid of initiation codon) able to induce the proliferation of a human IL-2-dependent T-cell line (Kit225) (Satou et al., 2006). Microarray expression analysis reveals that HBZ RNA and protein differentially modulate the transcription of host genes. HBZ RNA activates the transcription of survivin and cell-cycle related genes (Mitobe et al., 2015). Thus, the HBZ gene has bimodal functions in two different molecular forms as a polypeptide and a ribonucleic acid. Notwithstanding important activities as protein, the scarcity of the HBZ polypeptide in tumor cells contrasts with the expression of the HBZ RNA. Therefore, main functions of HBZ are exerted by its RNA form. Although the level of expression of HBZ protein is unquestionably very low, the protein must nevertheless play an essential role in the life-cycle of the virus because the coding sequence has been conserved during evolution in presence of a protective host immune response.

## Evidence 8: Alternate Transcription of Sense and Antisense Transcription

Single cell analysis shows that the Tax RNA is expressed in bursts and is exported from the nucleus, whereas the majority of hbz RNA is retained (Billman et al., 2017). Time-lapse imaging of destabilized enhanced green fluorescent protein indicates that Tax expression is transient, fluctuates between on/off states and is detected only in HBZ-negative

cells (Jasunaga and Matsuoka, 2017; Mahgoub et al., 2017). Viral persistence is thus characterized by successive cycles of sense/antisense transcription. Evidence from single-molecular RNA-FISH nevertheless indicates a more complex relationship between expression of the two strands that are not transcribed in strict alternation (Billman et al., 2017).

### A MODEL FOR VIRAL PERSISTENCE UNDER IMMUNE CONTROL

These experimental evidences are consistent with the model presented in **Figure 1** and support Yoshida's paradigm.

BLV/HTLV proviruses are transcribed in both orientations. Sense transcription from the 50 LTR (red arrow) generates genomic and spliced subgenomic RNAs (e.g., Tax). Translation of these RNAs into oncogenic proteins (Tax) triggers cell

proliferation, mitosis, and clonal expansion of the infected cell. Synthesis of structural (gag, env) and enzymatic (protease, reverse transcriptase, and integrase) proteins are required for assembly of the viral particle (red hexagon) that will further colonize new target cells. The provirus is also transcribed in the antisense orientation from the 30 LTR (green arrow) yielding HBZ and AS RNAs for HTLV and BLV, respectively. HBZ is very poorly translated while the coding potential of AS is ambiguous (hatched in red). Furthermore, the immunogenicity of HBZ is weak compared to all other viral antigens. BLV, but not HTLV, also encodes RNA polymerase III driven microRNAs (hatched green arrow). RNA synthesis from the 50 LTR is mostly silent but burst of sense transcription (red line on the time graph) transiently exposes infected cells to the immune response (e.g., cytotoxic, humoral, and innate). The only option that allows survival is to silence sense transcription, for example by initiating antisense RNA synthesis (graph on the right). This simplified model is nevertheless incomplete because the infected cell is also exposed to the CTL response to HBZ providing that the protein is expressed. Experimental evidence indicates that the HBZ RNA is mostly nuclear and is therefore not translated. Single cell kinetics of RNA and protein expression would answer to this still unsolved question. Another issue is the role of the intrinsic immunity operating within virus-infected cells, i.e., restriction factors (RFs) inhibiting Tax function or reverse transcription of viral genome (Tosi et al., 2011; Bai and Nicot, 2015; Forlani et al., 2016).

#### CONCLUSION

Yoshida predicted that the Tax oncogene should be expressed in cycles to allow cell survival. Recent reports describing bursts between sense and antisense transcriptions are consistent with this hypothesis. Regular switches between 50 and 30 transcription indeed allows transient Tax expression and fast

#### REFERENCES

silencing of viral expression. This mechanism would allow Tax-driven cell proliferation and synthesis of viral particles in presence of the host immunity. This model thus illustrates the dynamic equilibrium between a virus attempting to proliferate under a tight control exerted by the immune response.

#### AUTHOR CONTRIBUTIONS

All authors have contributed equally to this work. LW drafted the manuscript. All authors corrected, edited, and approved the paper.

#### FUNDING

This work was supported by the "Fonds National de la Recherche Scientifique" (FNRS), the Télévie, the Interuniversity Attraction Poles (IAP) Program "Virus-host interplay at the early phases of infection" BELVIR initiated by the Belgian Science Policy Office, the Belgian Foundation against Cancer (FBC), the "Centre Anticancéreux près ULg" (CAC), the "Subside Fédéral de Soutien à la Recherche and AgricultureIsLife" projects of Gembloux Agrobiotech (GxABT), the "ULg Fonds Spéciaux pour la Recherche" and the "Plan Cancer" of the "Service Public Fédéral." HG and RS (Télévie), CH (FNRS research fellow), and LW (Research Director) are members of the FNRS. PC was a Marie Curie COFUND postdoctoral fellow supported by European Union. LL and MH previously received support from AgricultureIsLife. MH and PC are presently fellows of the FBC. Nathalie Renotte and Jean-Rock Jacques provided skillful technical help.

#### ACKNOWLEDGMENTS

The authors thank the GIGA technology platforms.

(HTLV-I) pX gene products by human CD8 + cytotoxic T cells directed against HTLV-I-infected cells. Int. Immunol. 3, 761–767. doi: 10.1093/intimm/3.8.761

in B-cell leukemia/lymphoma. Proc. Natl. Acad. Sci. U.S.A. 110, 2306–2311. doi: 10.1073/pnas.1213842110

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Gazon, Chauhan, Hamaidia, Hoyos, Li, Safari and Willems. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# HTLV-1 Alters T Cells for Viral Persistence and Transmission

Azusa Tanaka1 and Masao Matsuoka2,3 \*

1 Department of Drug Discovery Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 2 Department of Hematology, Rheumatology and Infectious Diseases, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan, 3 Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan

Human T-cell leukemia virus type 1 (HTLV-1) was the first retrovirus to be discovered as a causative agent of adult T-cell leukemia-lymphoma (ATL) and chronic inflammatory diseases. Two viral factors, Tax and HTLV-1 bZIP factor (HBZ), are thought to be involved in the leukemogenesis of ATL. Tax expression is frequently lost due to DNA methylation in the promoter region, genetic changes to the tax gene, and deletion of the 50 long terminal repeat (LTR) in approximately half of all ATL cases. On the other hand, HBZ is expressed in all ATL cases. HBZ is known to function in both protein form and mRNA form, and both forms play an important role in the oncogenic process of HTLV-1. HBZ protein has a variety of functions, including the suppression of apoptosis, the promotion of proliferation, and the impairment of anti-viral activity, through the interaction with several host cellular proteins including p300/CBP, Foxp3, and Foxo3a. These functions dramatically modify the transcriptional profiling of host T cells. HBZ mRNA also promotes T cell proliferation and viability. HBZ changes infected T cells to CCR4+TIGIT+CD4+ effector/memory T cells. This unique immunophenotype enables T cells to migrate into various organs and tissues and to survive in vivo. In this review, we summarize how HBZ hijacks the transcriptional networks and immune systems of host T cells to contribute to HTLV-1 pathogenesis on the basis of recent new findings about HBZ and tax.

#### Keywords: HTLV-1, HBZ, tax, viral oncogenesis, Regulatory T Cell

### INTRODUCTION

The burden of viral infection in cancer is high, with estimates having more than 20% of cancer cases caused by infection (Bouvard et al., 2009). One oncogenic virus, human T-cell leukemia virus type 1 (HTLV-1), was identified in the United States and Japan almost 40 years ago (Poiesz et al., 1980; Yoshida et al., 1982). Thereafter, HTLV-1 was found to be a causative agent of adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Hinuma et al., 1981; Gessain et al., 1985; Gallo, 2005). Today, it is estimated that HTLV-1 infects approximately 10 million people worldwide (Gessain and Cassar, 2012). HTLV-1 transmits through cell-to-cell contacts (Igakura et al., 2003; Pais-Correia et al., 2010), while the free virus shows poor infectivity with the exception of dendritic cells (DCs) that can be infected without cell-to-cell contact (Derse et al., 2001; Jones et al., 2008; Mazurov et al., 2010; Alais et al., 2015). Accordingly, HTLV-1 increases its copy number primarily by triggering the proliferation of infected cells to facilitate its transmission (Etoh et al., 1997; Cavrois et al., 1998).

#### Edited by:

Vincenzo Ciminale, Università degli Studi di Padova, Italy

#### Reviewed by:

Renaud Mahieux, École Normale Supérieure de Lyon, France Andrea K. Thoma-Kress, Universitätsklinikum Erlangen, Germany

#### \*Correspondence:

Masao Matsuoka<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Virology, a section of the journal Frontiers in Microbiology

Received: 25 January 2018 Accepted: 27 February 2018 Published: 20 March 2018

#### Citation:

Tanaka A and Matsuoka M (2018) HTLV-1 Alters T Cells for Viral Persistence and Transmission. Front. Microbiol. 9:461. doi: 10.3389/fmicb.2018.00461

These properties distinguish HTLV-1 from another well-known human retrovirus, human immunodeficiency virus type 1 (HIV-1). The chemokine receptors CXCR4 and CCR5 function as co-receptors for HIV-1 to infect host CD4+ T cells. Unlike HIV-1, HTLV-1 can infect a variety of cell types, but more than 90% of infected cells are CD4+ memory T cells in vivo (Richardson et al., 1990; Rizkallah et al., 2017).

#### EXPRESSION AND FUNCTION OF TAX

The HTLV-1 provirus is 9 kb in length and contains multiple coding regions for Gag, Pol, Env, p12, p30, p13, Rex, Tax, and HBZ. Among the viral proteins of HTLV-1, Tax can activate various signal pathways including the NF-κB and AP-1 pathways (Grassmann et al., 2005; Gazon et al., 2018). It also can induce T-cell leukemia or lymphoma in vivo (Grossman et al., 1995; Hasegawa et al., 2006). However, Tax expression is frequently undetectable in ATL cases due to genetic and epigenetic aberrations (Takeda et al., 2004). Importantly, nonsense mutations in the tax gene are often observed not only in ATL cases but also in infected cells of asymptomatic HTLV-1 carriers (Furukawa et al., 2001; Fan et al., 2010). Tax was initially discovered as the viral trans-activator for HTLV-1 RNA transcription from a promoter located in the 50 LTR (Felber et al., 1985), thus its expression is essential for viral replication. However, it is a major target antigen recognized by cytotoxic T lymphocytes (CTL) (Kannagi et al., 1991). Therefore, Tax expression is tightly controlled for the survival of HTLV-1 infected cells in order to evade host immunosurveillance. Tax expression is usually silenced in ATL cells, but a single cell transcript analysis has revealed that Tax expression is not completely suppressed and that a small percentage of MT-1 cells transiently express Tax (Mahgoub et al., 2018). Because Tax expression is necessary for de novo infection, it may play a key role in the spreading of HTLV-1. Taken together, Tax expression is usually suppressed in order to escape from CTL, but at the same time, Tax is transiently expressed to maintain and expand HTLV-1 infected cells. These findings suggest that another key regulator may contribute to the onset of ATL.

#### HBZ AND ITS ROLE IN THE ONCOGENESIS

The HTLV-1 bZIP factor (HBZ) was first identified in 2002 as a novel viral protein that contains a N-terminal transcription activation domain and a leucine zipper motif in its C-terminus (Gaudray et al., 2002). It has been reported that HBZ mRNA is expressed in all ATL cases. Its mRNA form promotes the proliferation of T-cells, and its protein form induces the development of T-cell lymphomas in transgenic mice, indicating that HBZ is critical for the proliferation of ATL cells and leukemogenesis (Satou et al., 2006, 2011).

### THE LOCALIZATION OF HBZ AND ITS FUNCTION IN THE NUCLEUS AND CYTOPLASM

HTLV-1 bZIP factor contains nuclear localization signals in its central/bZIP domain and nuclear export signals in its N terminus (Hivin et al., 2005). HBZ is mainly localized in the cytoplasm of peripheral blood mononuclear cells (PBMCs) in HAM/TSP patients, suggesting cytoplasmic HBZ as a possible biomarker of the disorder (Baratella et al., 2017). In addition, cytoplasmic HBZ interacts with GADD34 to suppress GADD34 function and positively regulate the mechanistic target of rapamycin (mTOR) signaling pathway (Mukai and Ohshima, 2014) (**Figure 1**). Finally, the cytoplasmic localization of HBZ protein in T cells depends on the host factor THEMIS (Kinosada et al., 2017). Since THEMIS is expressed only in T cells, this function might explain why HTLV-1 promotes the proliferation of T cells.

Compared to cytoplasmic HBZ, far more studies have focused on the function of nuclear HBZ. HBZ in the nucleus makes a complex with several important transcription factors including p300, p65, LEF1, AP-1 transcription factors and forkhead family proteins (Basbous et al., 2003; Thebault et al., 2004; Satou et al.,

regulators and modulates cellular function.

2011; Zhao et al., 2011; Ma et al., 2013; Tanaka-Nakanishi et al., 2014) (**Figure 1**). For example, HBZ binds to p65 to diminish the p65 affinity for DNA and thus suppresses the classical pathway of NF-κB. Also, the interaction with c-Jun leads to a transcriptional repression of AP-1 regulated genes. Overall, nuclear HBZ has various functions necessary for the onset of ATL.

### TARGET CELLS OF HTLV-1 INFECTION

HTLV-1 provirus is mainly detected in T cells in vivo. In particular, most provirus (∼90%) was found in CD4+effector/memory T cells, while the remaining 10% of provirus was detected in CD8+ T cells (Yasunaga et al., 2001). HTLV-1 infected CD4+ T cells tend to express cell adhesion molecule 1 (CADM1), C-C chemokine receptor type 4 (CCR4), T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD45RO, and sometimes CD25, indicating that these cells are effector/memory T cells (Bangham and Matsuoka, 2017). These findings suggest two scenarios for the transmission of HTLV-1. First, HTLV-1 preferentially infects these subpopulations of CD4+ T cells. It has been reported that Tax induces the expression of CCL22, one ligand of CCR4 (Hieshima et al., 2008), which might attract CCR4+ T cells and transmit the virus in the periphery. Second, HTLV-1 infection itself induces the differentiation of T cells or modifies the immunophenotype to a specific phenotype. To answer this question, we focus on HBZ below.

#### HTLV-1 INFECTION IN HEMATOPOIETIC STEM CELLS

Tax is essential for de novo infection since transcription of the sense-strand of the provirus, which is responsible for the generation of the viral genome and viral proteins such as Gag, Env, and Pol, is Tax dependent. Tax expression in the bone marrow cells of HAM/TSP patients was reported (Levin et al., 1997), suggesting de novo infection occurs in this area. However, it remains unknown whether hematopoietic stem cells (HSCs) are truly infected with HTLV-1 or not. Recently, Furuta et al. (2017) reported that identical integration sites were detected in multiple hematopoietic lineage cells. This result indicates that HTLV-1 infects HSCs in vivo and that HTLV-1 infected HSCs differentiate into diverse hematopoietic cell lineages (**Figure 2**). Since HTLV-1 infected cells possess a similar immunophenotype, viral factors are implicated to acquire the phenotype. Because HBZ is the only viral gene that is consistently expressed in infected cells and ATL cells, we consider its role.

#### HBZ INDUCES EFFECTOR/MEMORY PHENOTYPE

Here we summarize how HBZ influences hematopoietic cell differentiation to effector/memory CD4+ T cells by modulating the function of key regulators (**Figure 2**).

### Foxp3

As mentioned above, although HTLV-1 can infect many types of cells including HSCs, most ATL cells and HTLV-1 infected cells are CD4+CD45RO+ conventional memory T cells. It is speculated that HBZ influences the differentiation of host T cells. For instance, HBZ affects the expression and function of Foxp3, a critical regulator for the development of Treg cells. In HBZ-Tg mice, the number of CD4+Foxp3+ T cells is increased, suggesting that HBZ induces Foxp3 expression (Satou et al., 2011). HBZ promotes Foxp3 expression by activating the TGF-β/Smad pathway (Zhao et al., 2011). Although HBZ induces Foxp3 expression, HBZ directly interacts with Foxp3 to interfere with Foxp3 DNA binding activity and function. In addition, HBZ increases the number of induced Treg cells, in which Foxp3 expression is unstable, and converts them to Foxp3− T cells with increased interferon-γ production (Yamamoto-Taguchi et al., 2013). These results suggest that HBZ induces Foxp3+ T cells but hijacks their transcriptional network, which leads to inflammatory disease in the host.

### CCR4

It is well known that CCR4 is a hallmark of ATL cells (Yoshie et al., 2002; Ishida et al., 2003; Iqbal et al., 2010). CCR4 is not only a marker of Th2 cells, but it is also involved in the suppressive function of Treg cells (Yuan et al., 2007). CCR4 mutations were frequently observed in ATL samples, with most mutations being nonsense or frame-shift (Nakagawa et al., 2014; Kataoka et al., 2015). A truncated mutant of the cytoplasmic

region of CCR4 enhances PI3K/Akt signaling, the migration and proliferation of ATL cells, but still many questions remain about the mechanisms for the increased expression of CCR4 and enhanced proliferation of cells even in cells expressing wild-type CCR4. Sugata et al. (2016) studied the mechanisms and effects of CCR4 expression on ATL cells. They found that both HBZ RNA and HBZ protein increased the expression of CCR4 through the induction of GATA3 expression, thereby activating transcription of the CCR4 gene promoter. Upregulated CCR4 expression is associated with enhanced T cell migration and proliferation, which are implicated in the infiltration and proliferation of HTLV-1 infected cells. They further showed that wild-type CCR4 also promotes the proliferation of T cells and that a CCR4 antagonist inhibits cell migration and proliferation effectively. Thus, CCR4 induced by HBZ is implicated in proliferation and migration of infected cells.

### TIGIT AND THEMIS

One gene upregulated by HBZ is the co-inhibitory receptor T cell immunoglobulin and ITIM domain (TIGIT) (Yasuma et al., 2016). ATL cells and HTLV-1 infected cells highly express TIGIT on their surfaces. Enhanced expression of TIGIT induces IL-10 production, leading to the suppression of host immune responses.

Although TIGIT is a co-inhibitory receptor that suppresses T-cell activation, it does not inhibit the proliferation of ATL cells or HTLV-1 infected cells (Kinosada et al., 2017). TIGIT exerts inhibitory signal through the intracytoplasmic immunoreceptor tyrosine inhibitory motif (ITIM) that interacts with a complex of SHP-2, Grb2, and THEMIS. HBZ interacts with THEMIS and hinders the inhibitory signal from THEMIS. Cytoplasmic HBZ almost disappears after knock-down of THEMIS expression while HBZ protein exists in the nucleus, suggesting that THEMIS is responsible for cytoplasmic localization of HBZ. THEMIS is a T cell-specific protein, this finding could explain why HTLV-1 induces the proliferation of only T cells despite the fact that HTLV-1 can infect many cell types.

Taken together, these studies suggest that HTLV-1 might influence earlier stages of hematopoietic cell differentiation and induce infected HSCs or other infected progenitor cells to abnormally differentiate into CD4+ Treg-like cells.

### ROLE OF TAX IN T CELL DIFFERENTIATION

No direct evidence has associated Tax with T-cell differentiation. Indeed, several HTLV-1 associated molecules, including Foxp3, CCR4, and TIGIT, are not induced in Tax transgenic mice, indicating that HBZ is closely linked to the expression of these molecules (Satou et al., 2011). HTLV-1 targets CD4+ effector/memory T cells. As described above, HBZ is mainly responsible for immunophenotypes of HTLV-1 infected cells, but Tax has also been implicated. Two T cell specific proteins, TCF-1 and LEF-1, are highly expressed in thymocytes. It has been reported that TCF1 and LEF1 perturb Tax function (Ma et al., 2015), leading to the inhibition of HTLV-1 replication in the thymus. On the other hand, TCF-1/LEF-1 expression is suppressed in peripheral T cells. Therefore, Tax is fully functional in peripheral T cells.

### GENETIC INSTABILITY INDUCED BY HBZ

Some individuals infected with HTLV-1 succumb to ATL after a long latency period (Grassmann et al., 2005). This effect suggests that along with the viral proteins Tax and HBZ, the accumulations of genetic or epigenetic alterations are required for ATL onset. Indeed, gain-of-function alterations were frequently observed in TCR/NF-κB signaling from integrated genetic and transcriptomic analysis (Kataoka et al., 2015). Recently, it has been reported that HBZ induces genomic instability in an oncogenic miRNA-dependent manner. HBZ increases the expression of miR17 and miR21, and these oncomiRs suppress the expression of OBFC2A, which codes a single-stranded DNA-binding protein that protects genome stability (Vernin et al., 2014).

### HBZ INTERFERES WITH THE HOST TRANSCRIPTIONAL AND TRANSLATIONAL MACHINERY

Detailed analysis of RNA-seq data has revealed that host genes undergo frequent and provirus-dependent transcription termination. The expression level of host-genome exons located downstream of HTLV-1 was halved on average (Rosewick et al., 2017). At the same time, systematic interaction between the HTLV-1 antisense transcript corresponding to HBZ and host genes located upstream of the provirus is observed. This fusion is classified into four types depending on the location and the direction of viral insertion (genic or intergenic integration, and concordant or discordant gene-provirus orientation). In this way, HBZ transcription perturbs host gene transcription around HTLV-1 integration sites.

Both RNA and DNA viruses are heavily dependent on the host translational machinery to produce the polypeptides that are necessary for replication. Most viral mRNAs recruit 40S ribosomal subunit to initiate their translation. For example, RPS25, a component of 40S, is essential for the internal ribosome entry sites (IRESs) of hepatits C virus (Landry et al., 2009). This recruitment not only promotes viral protein synthesis, it also impedes the host innate defenses that inhibit viral protein production. It has been reported that HBZ disrupts the host translational mechanism by negatively modulating RPS25.  There is a functional

difference between the two isoforms. JunD-FL is the more potent transcriptional activator, but only 1JunD induces the proliferation and transformation of cells. HBZ induces the loss of RPS25 protein, which leads to the down-regulation of JunD-FL and up-regulation of 1JunD. As a result, HBZ shifts the function of JunD from growth suppressor to tumor promoter, a condition advantageous for HTLV-1 infected cells to proliferate. Thus, HBZ influences host transcriptional and translational machinery.

#### HBZ SUPPRESSES APOPTOSIS

To increase the number of infected cells, both HBZ and Tax play a key role in maintaining clonal longevity. One mechanism for this maintenance is achieved by inhibiting the apoptosis of HTLV-1 infected cells. HBZ protein perturbs the localization and function of FOXO3a to down-regulate the pro-apoptotic genes Bim and FasL (Tanaka-Nakanishi et al., 2014). Similarly, the N-terminal region of HBZ interacts with IRF-1 to inhibit IRF-1 DNA binding/transcriptional activity and reduce the number of cells undergoing apoptosis (Chopra et al., 1990; Panfil et al., 2016). Finally, in addition to HBZ protein, HBZ mRNA inhibits apoptosis by upregulating the expression of survivin by enhancing its promoter activity (Mitobe et al., 2015).

#### CONCLUSION AND FUTURE DIRECTION

HTLV-1 bZIP factor affects many aspects of host immunity to promote the survival and proliferation of the infected cells

#### REFERENCES

through its mRNA form and protein form. In this review, we mainly focused on recent findings about the functions of HBZ. As described above, HBZ positively or negatively regulates various host transcription factors, modulates multiple signaling pathways and perturbs T cell function and differentiation. One of the characteristics of HBZ is that it functions both in protein form and RNA form, both of which play crucial roles in cell proliferation and cell survival. Additionally, HBZ activates oncomiRs to induce genetic instability, a hallmark of cancer.

Owing to the effort of many researchers around the world, the mystery of HBZ has been gradually uncovered. Yet, it is still unknown why most ATL cells are CD4 positive memory T cells and whether HBZ contributes to this phenomenon. Answering these questions will contribute to new therapeutic targets.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### FUNDING

This work was supported by JSPS KAKENHI (15J04853 to AT and JP16H05336 to MM), the Project for Cancer Research and Therapeutic Evolution (P-CREATE) (17cm0106306h0002 to MM), the Research Program on Emerging and Re-emerging Infectious Diseases (17fk0108227h0002 to MM), and a grant from The Naito Foundation to AT.

infection with recombinant virus vectors. J. Virol. 75, 8461–8468. doi: 10.1128/ JVI.75.18.8461-8468.2001

of adult T-cell leukemia cells. Oncogene 32, 4222–4230. doi: 10.1038/onc.201 2.450

instability by activating OncomiRs. Cancer Res. 74, 6082–6093. doi: 10.1158/ 0008-5472.CAN-13-3564

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Tanaka and Matsuoka. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Stability of the HTLV-1 Antisense-Derived Protein, HBZ, Is Regulated by the E3 Ubiquitin-Protein Ligase, UBR5

Amanda R. Panfil1 , Jacob Al-Saleem1 , Cory M. Howard1 , Nikoloz Shkriabai2 , Mamuka Kvaratskhelia2 and Patrick L. Green1,3 \*

1 Department of Veterinary Biosciences, Center for Retrovirus Research, The Ohio State University, Columbus, OH, United States, 2 Division of Infectious Diseases, School of Medicine, University of Colorado Denver, Aurora, CO, United States, 3 Comprehensive Cancer Center and Solove Research Institute, The Ohio State University, Columbus, OH, United States

#### Edited by:

Vincenzo Ciminale, Università degli Studi di Padova, Italy

#### Reviewed by:

Yorifumi Satou, Kumamoto University, Japan Roberto S. Accolla, University of Insubria, Italy

> \*Correspondence: Patrick L. Green<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Virology, a section of the journal Frontiers in Microbiology

Received: 31 October 2017 Accepted: 12 January 2018 Published: 30 January 2018

#### Citation:

Panfil AR, Al-Saleem J, Howard CM, Shkriabai N, Kvaratskhelia M and Green PL (2018) Stability of the HTLV-1 Antisense-Derived Protein, HBZ, Is Regulated by the E3 Ubiquitin-Protein Ligase, UBR5. Front. Microbiol. 9:80. doi: 10.3389/fmicb.2018.00080 Human T-cell leukemia virus type 1 (HTLV-1) encodes a protein derived from the antisense strand of the proviral genome designated HBZ (HTLV-1 basic leucine zipper factor). HBZ is the only viral gene consistently expressed in infected patients and adult T-cell leukemia/lymphoma (ATL) tumor cell lines. It functions to antagonize many activities of the Tax viral transcriptional activator, suppresses apoptosis, and supports proliferation of ATL cells. Factors that regulate the stability of HBZ are thus important to the pathophysiology of ATL development. Using affinity-tagged protein and shotgun proteomics, we identified UBR5 as a novel HBZ-binding partner. UBR5 is an E3 ubiquitin-protein ligase that functions as a key regulator of the ubiquitin proteasome system in both cancer and developmental biology. Herein, we investigated the role of UBR5 in HTLV-1-mediated T-cell transformation and leukemia/lymphoma development. The UBR5/HBZ interaction was verified in vivo using over-expression constructs, as well as endogenously in T-cells. shRNA-mediated knockdown of UBR5 enhanced HBZ steady-state levels by stabilizing the HBZ protein. Interestingly, the related HTLV-2 antisense-derived protein, APH-2, also interacted with UBR5 in vivo. However, knockdown of UBR5 did not affect APH-2 protein stability. Co-immunoprecipitation assays identified ubiquitination of HBZ and knockdown of UBR5 resulted in a decrease in HBZ ubiquitination. MS/MS analysis identified seven ubiquitinated lysines in HBZ. Interestingly, UBR5 expression was upregulated in established T lymphocytic leukemia/lymphoma cell lines and the later stage of T-cell transformation in vitro. Finally, we demonstrated loss of UBR5 decreased cellular proliferation in transformed T-cell lines. Overall, our study provides evidence for UBR5 as a host cell E3 ubiquitin-protein ligase responsible for regulating HBZ protein stability. Additionally, our data suggests UBR5 plays an important role in maintaining the proliferative phenotype of transformed T-cell lines.

Keywords: HTLV-1, HBZ, UBR5, ubiquitin, proliferation, T-cell

## INTRODUCTION

fmicb-09-00080 January 29, 2018 Time: 16:34 # 2

Categorized as a tumorigenic virus, human T-cell leukemia virus type 1 (HTLV-1) is a deltaretrovirus that infects and transforms CD4+ T-cells. Approximately 10–15 million people worldwide are infected, with areas of endemic infection in southwestern Japan, Africa, South America, and the Pacific Islands (DeThe and Bomford, 1993; Matsuoka and Jeang, 2005; Gessain and Cassar, 2012). HTLV-1 is responsible for a highly aggressive and chemotherapy-resistant peripheral T-cell malignancy called adult T-cell leukemia/lymphoma (ATL) (Uchiyama et al., 1977; Poiesz et al., 1980; Yoshida et al., 1982) and a chronic progressive neurodegenerative disease termed HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Gessain et al., 1985; Osame et al., 1986). As a blood borne pathogen, HTLV-1 is primarily spread through breastfeeding, blood transfusions, and sexual contact. Not all HTLV-1 carriers will go on to develop disease. Disease penetrance is roughly 5% for the lifetime of an infected individual (Ishitsuka and Tamura, 2014). The discrepancy between individuals with disease development and lifelong healthy carriers is still not well understood.

Studies have identified at least two viral genes, tax and hbz, that are linked to oncogenic transformation and involved in the pathogenic process (Cheng et al., 2012; Ma et al., 2016). Derived from the sense strand of the proviral genome, Tax serves as a transcriptional activator of both viral and cellular gene expression (Bex and Gaynor, 1998). In addition, it deregulates the cell cycle, which ultimately leads to the accumulation of cellular genetic mutations (Marriott and Semmes, 2005). Although essential for viral transformation, Tax is frequently absent from ATL tumor cells through epigenetic silencing, 50 LTR deletion, or abortive protein mutations in the tax gene (Takeda et al., 2004).

Conversely, a viral transcript that is consistently found in ATL cells is the antisense-derived hbz transcript (Satou et al., 2006). Hbz transcription initiates in the mostly epigenetically unmodified 30 LTR (Larocca et al., 1989; Satou et al., 2006). Viral cAMP-responsive elements (CRE) and several SP1 binding sites help regulate transcription of hbz (Yoshida et al., 2008). Hbz mRNA exists in both a spliced and unspliced transcript variant (Satou et al., 2006). The proteins encoded by these transcripts have nearly identical amino acid sequence (with the exception of the first several amino acids) and demonstrate several functional differences in cells (Yoshida et al., 2008). Spliced HBZ is more abundant in infected cells (Usui et al., 2008) and therefore most research to date has focused on this isoform. The spliced hbz transcript encodes a 206-amino acid nuclear protein comprised of 3 domains: an N-terminal activation domain, a central basic region, and a C-terminal bZIP domain (Gaudray et al., 2002; Zhao and Matsuoka, 2012). Within the activation domain are two well-characterized LXXLLlike motifs. These motifs have been shown to bind the KIX domain of CBP/p300 and are also required for HBZ to activate TGF-β signaling (Clerc et al., 2008; Zhao et al., 2011). Through its bZIP domain, HBZ is able to hetero-dimerize with cellular bZIP proteins and affect their binding to DNA recognition sites (Matsuoka and Green, 2009).

Deletion of HBZ expression in the context of the virus has been studied using an HTLV-1 infectious molecular clone with a premature stop codon in HBZ, termed HTLV-1 1HBZ (Arnold et al., 2006). HBZ knockout had little effect on viral infectivity and transformation of T-cells in cellular immortalization assays in vitro. However, when the HTLV-1 1HBZ virus was injected in rabbits, there was a significant decrease in antibody response to the virus and proviral load, suggesting a positive role of HBZ in viral infectivity or proliferation of infected cells in vivo. In monkeys, the HBZ-KO clone gradually reverts to wild-type during prolonged infection (Valeri et al., 2010), confirming the importance of HBZ in viral infectivity and persistence in vivo.

A popular theory within the HTLV-1 field has been that Tax is responsible for initiating transformation, while HBZ provides the maintenance or cell survival signals later during transformation. However, HBZ expression is found both in early and late stages of viral infection (Li et al., 2009). In addition, it has been shown that HBZ is able to sustain proliferative cellular signaling, evade growth suppressors, resist cell death, cause genomic instability and mutations, enable replicative immortality, induce tumorpromoting inflammation, and has low immunogenicity (Ma et al., 2016). Therefore, the view is shifting to appreciate the role of HBZ throughout HTLV-1-mediated oncogenesis.

To date, little is known regarding the regulation of HBZ protein expression. We therefore sought to identify cellular proteins that regulate HBZ stability using affinity capture coupled with shotgun proteomics. These experiments have identified UBR5 as a novel HBZ-interacting partner. UBR5 is an E3 ubiquitin-protein ligase that functions as a key regulator of the ubiquitin proteasome system in both cancer and developmental biology. Through shRNA-mediated knockdowns and cycloheximide pulse chase experiments, we found that UBR5 regulates HBZ protein stability. MS/MS analysis enabled us to discover ubiquitination at seven lysine residues throughout the HBZ protein. Interestingly, UBR5 protein is over-expressed in T-cell lymphoma cell lines and using our HTLV-1 mediated model of T-cell transformation, we determined this dysregulation occurs late during the transformation process. In addition, we found UBR5 positively regulates cellular proliferation in T-cell lymphoma cell lines. Taken together, our data identifies UBR5 as an E3 ubiquitin-protein ligase responsible for the regulation of HBZ protein stability. In addition, it suggests UBR5 may play a role in maintaining the proliferative phenotype of transformed T-cell lines.

#### MATERIALS AND METHODS

#### Cell Lines and Culture

HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, Broderick, CA, United States), 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL). PBL-ACH (early passage HTLV-1-immortalized human T-cells) were maintained in RPMI 1640 supplemented with 20% FBS, 20 U/mL recombinant human interleukin-2 (rhIL-2; Roche Applied Biosciences, Indianapolis, IN,

United States), 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL). SLB-1 cells (HTLV-1-transformed T-cell line) were maintained in Iscove's medium supplemented with 10% FBS, 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL). C8166, MT-1, MT-2, Hut-102 (HTLV-1-transformed T-cell lines), Hut-78, Jurkat (HTLV-1-negative transformed T-cell lines), TL-Om1, ATL-43T, and ATL-ED cells (ATL-derived T-cell lines) were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL). ATL-55T (ATL-derived T-cell line) were maintained in RPMI 1640 supplemented with 10% FBS, 20 U/mL rhIL-2, 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL). The parental 729.B (uninfected) and derivative 729.ACH (HTLV-1 producing) cell lines were maintained in Iscove's medium supplemented with 10% FBS, 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL). All cells were grown at 37◦C in a humidified atmosphere of 5% CO2 and air. Human PBMCs were isolated using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Pittsburgh, PA, United States) and naïve T-cells were enriched using a Pan T-Cell Isolation Kit (Miltenyi Biotec., Inc., Gaithersburg, MD, United States), while memory CD4 T-cells were isolated using a Memory CD4+ T Cell Isolation Kit (Miltenyi Biotec, Inc.)

### Plasmids and Cloning

Plasmid DNA was purified on maxi-prep columns according to the manufacturer's protocol (Qiagen, Valencia, CA, United States). The S-tagged APH-2 and HBZ expression vectors and the pME-APH-2 and HBZ expression vectors were generated and described previously (Arnold et al., 2006; Yin et al., 2012; Panfil et al., 2016). pCMV-Tag2B-EDD (Flag-UBR5) was a gift from Darren Saunders & Charles Watts (Addgene plasmid #37188) (Henderson et al., 2002). Flag-tagged ubiquitin was provided by F. Nina Papavasiliou (The Rockefeller University, New York, NY, United States) (Delker et al., 2013; Chesarino et al., 2014). S-tagged-BR, bZIP, and BR/bZIP were cloned by PCR amplifying the respective fragments from pME-HBZ and inserting them into pTriExTM-4 Neo (Millipore Sigma, Burlington, MA, United States) using BamHI and EcoRI restriction sites. HBZ plasmids S-tagged-AD, AD-BR2, AD-BR2-BR1, AD-BR2-BR1-BR3, and 1bZIP were constructed using PCR-based site-directed mutagenesis to create stop codons with the following primer sets: HBZ AD forward (50 - C GCATCGTGATCGGTAGCGACGGGCTGAGGAG -30 ) and reverse (50 - CTCCTCAGCCCGTCGCTACCGATCACGATG CG -30 ); HBZ AD-BR2 forward (50 - GAGCGGGAGAAATAGGA GGAAAAGCAGATTG -30 ) and reverse (50 - CAATCTGCTTT TCCTCCTATTTCTCCCGCTC -30 ); HBZ AD-BR2-BR1 forward (50 - GTCGCCAGGAGAAAGTAGGAAGAGCAGGAG CGC -30 ) and reverse (50 - GCGCTCCTGCTCTTCCTACT TTCTCCTGGCGAC -30 ); HBZ-AD-BR2-BR1-BR3 forward (50 - GCAGGAGTTGGGGTAGGATGGCTATACTAGACAGTTGG -30 ) and reverse (50 - CCAACTGTCTAGTATAGCCATCCTACCC CAACTCCTGC -30 ); and HBZ-1bZIP forward (50 - GGAAG GCGAGGTGTAGTCCTTGGAGGCTG -30 ) and reverse (50 - CA GCCTCCAAGGACTACACCTCGCCTTCC -30 ). HBZ cDNA

was cloned into the pCDH-EF1-MCS-T2A-copGFP lentiviral expression vector (SBI, Mountain View, CA, United States) to create an HBZ-GFP chimeric protein and used to generate the Jurkat-HBZ cell line.

## S-Tag Affinity Pulldown Assays

HEK293T cells were transfected with the indicated expression vectors using Lipofectamine R 2000 (Life Technologies, Carlsbad, CA, United States) according to the manufacturer's instructions. Twenty-four hours post-transfection, cells were treated with 10 µM MG-132 (Sigma–Aldrich, St. Louis, MO, United States) for 20 h. Cells were washed with 1× PBS and lysates were prepared with NP-40 lysis buffer in the presence of protease inhibitor (Roche Applied Biosciences) and 25 mM N-Ethylmaleimide (Sigma–Aldrich). Cells were centrifuged at maximum speed for 10 min at 4◦C. S-tag purification was performed by rocking cell lysates with S beads (Millipore Sigma) overnight at 4◦C. The S beads were washed twice with NP-40 lysis buffer. An equal volume of 2× SDS-sample buffer was added and proteins were extracted by heating at 95◦C for 10 min.

### Mass Spectrometry and Proteomic Analysis

The pulled-down proteins were subjected to SDS-PAGE and visualized by GelCode Blue staining. For identification of cellular binding partners of S-tagged HBZ and S-tagged APH-2 entire lanes were excised and cut into small pieces. For identification of ubiquitination sites the prominent S-tagged HBZ bands were excised. The gel pieces were then de-stained with 50% acetonitrile and then subjected to in-gel proteolysis using sequencing grade modified trypsin (Promega, Madison, WI, United States). The resulting peptides were extracted in acetonitrile by vortexing for 10 min and then desiccated in Eppendorf R Vacufuge R Plus Vacuum Concentrator. The samples were run on Thermo Scientific Q Exactive mass spectrometer and analyzed with Mascot software (Matrix Science, Boston, MA, United States). The data were visualized using Scaffold Viewer (Proteome Software, Inc., Portland, OR, United States) using 1.0% FDR Protein Threshold, 2 Min. #Peptides, and 1.0% Peptide Threshold.

### Flag Immunoprecipitation Assays

HEK293T cells were transfected with the indicated expression vectors using Lipofectamine R 2000 (Life Technologies) according to the manufacturer's instructions. Twenty-four hours post-transfection, cells were treated with 10 µM MG-132 (Sigma–Aldrich) for 20 h. Cells were washed with 1× PBS and then incubated with gentle rocking at 4◦C for 30 min in 0.5% NP-40 lysis buffer (150 mM NaCl, 0.5% NP-40, 50 mM Tris-HCl, pH 8.0) in the presence of protease inhibitor (Roche Applied Biosciences) and 25 mM NEM (Sigma–Aldrich). Cells were centrifuged at maximum speed for 10 min at 4◦C. The lysates were then diluted to 0.1% NP-40 lysis buffer. Flag immunoprecipitation was performed by rocking cell lysates with ANTI-FLAG R M2 Affinity Gel (Sigma–Aldrich) overnight at 4◦C. The FLAG resin was washed twice with 0.1% NP-40 lysis buffer. An equal volume of 2× SDS-sample buffer was added and proteins were extracted by heating at 95◦C for 10 min.

#### Immunoprecipitation Assays

fmicb-09-00080 January 29, 2018 Time: 16:34 # 4

Lymphocytes were treated with 10 µM MG-132 (Sigma–Aldrich) for 20 h. Cells were washed with 1× PBS and then incubated with gentle rocking at 4◦C for 30 min in NP-40 lysis buffer in the presence of protease inhibitor (Roche Applied Biosciences) and 25 mM NEM (Sigma–Aldrich). Cells were centrifuged at maximum speed for 10 min at 4◦C. Antibody (1 to 2 µg or no antibody for the direct load) was added to each sample, and the samples were rocked overnight at 4◦C. The antibodies used were as follows: control rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, United States), rabbit anti-HBZ antiserum, and rabbit anti-UBR5 (Bethyl Laboratories, Inc., Montgomery, TX, United States). Protein G Dynabeads (Fisher Scientific, Hampton, NH, United States) were added and the mixture was rocked at 4◦C for 2 h. The beads were washed twice in NP-40 lysis buffer. An equal volume of 2× SDS-sample buffer was added and proteins were extracted by heating at 95◦C for 10 min.

#### Immunoblotting

Cell lysates were harvested in NP-40 lysis buffer containing protease inhibitor cocktail (Roche Applied Bioscience) and quantitated using a Pierce bicinchoninic acid protein assay kit (Fisher Scientific). Equivalent amounts of protein were separated in Mini-Protean TGX precast 4 to 20% gels (Bio-Rad Laboratories, Hercules, CA, United States) and transferred to nitrocellulose membranes. Membranes were blocked in phosphate-buffered saline (PBS) containing 5% milk and 0.1% Tween 20 and incubated with primary antibody. The following antibodies were used: anti-S-tag (1:1000; Abcam, Cambridge, MA), anti-UBR5 (1:1000; Cell Signaling Technology, Danvers, MA, United States), anti-HBZ (1:1,000), anti-APH-2 (1:1,000), anti-FLAG clone M2 (1:1,000; Agilent Technologies, Santa Clara, CA, United States), anti-ubiquitin (1:250; Santa Cruz Biotechnology), anti-β-actin (1:5,000; Sigma–Aldrich), and antiα-Tubulin (1:250; Santa Cruz Biotechnology). The secondary antibodies used were horseradish peroxidase-labeled goat antirabbit and goat anti-mouse immunoglobulin antibodies (1:5,000; Santa Cruz Biotechnology). The blots were developed using an ECL Western Blotting Substrate (Fisher Scientific). Images were taken using an Amersham Imager 600 imaging system (GE Healthcare Life Sciences), and densitometric data were calculated using the ImageQuant TL program (GE Healthcare Life Sciences).

#### Co-culture Immortalization Assays

Long-term immortalization assays were performed as detailed previously (Green et al., 1995). Briefly, 2 × 106 freshly isolated human PBMCs were co-cultivated at a 2:1 ratio with lethally irradiated cells (729.B uninfected parental; 729.ACH HTLV-1 producing) in 24-well culture plates (media was supplemented with 10 U/mL rhIL-2).

### Quantitative RT-PCR