# **COMPARATIVE STUDIES BETWEEN HTLV-1 AND HTLV-2 FUNCTION AND PATHOBIOLOGY**

# **Topic Editors Umberto Bertazzoni and Fran**ç**oise Bex**

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**ISSN** 1664-8714 **ISBN** 978-2-88919-498-8 **DOI** 10.3389/978-2-88919-498-8

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# **COMPARATIVE STUDIES BETWEEN HTLV-1 AND HTLV-2 FUNCTION AND PATHOBIOLOGY**

# Topic Editors:

**Umberto Bertazzoni,** Università degli Studi di Verona **Françoise Bex,** Université Libre de Bruxelles

Fig. 1. Schematic representation of HTLV-1, HTLV-2, HTLV-3 and HTLV-4 genomic organization. Romanelli M.G. etal, Front. Microbiol, 9 Sept 2013, doi:10.3389/fmicb 2013.00271

Human T-cell leukemia viruses type 1 and 2 (HTLV-1 and HTLV-2) share a common genetic organization, expression strategy and ability to infect and immortalize T-cells in vitro; however, HTLV-1 and HTLV-2 are strikingly different in terms of clinical impact. HTLV-1 is recognized as the aetiological agent of adult T-cell leukemia/lymphoma (ATLL), and HTLV-associated myeolopathy/tropical spastic paraparesis (HAM/TSP), in contrast, HTLV-2 does not cause hematologic disorders and is only sporadically associated with cases of subacute myelopathy. HTLV-1 and HTLV-2 also exhibit distinct cellular tropisms in vivo: HTLV-1 is mainly found in CD4+T lymphocytes, whereas CD8+T-cells are the preferred target for HTLV-2.

The articles contributed in this Research Topic are covering all the different aspects that characterize HTLV-1 and HTLV-2, by highlighting differences in their biology that might provide clues to their distinct pathogenic properties.

# Table of Contents

Greta Forlani, Rawan Abdallah, Roberto S. Accolla and Giovanna Tosi

*74 Tax-1 and Tax-2 Similarities and Differences: Focus on Post-Translational Modifications and NF-*κ*B Activation*

Margret Shirinian, Youmna Kfoury, Zeina Dassouki, Hiba El-Hajj and Ali Bazarbachi

*87 Functional Comparison of Antisense Proteins of HTLV-1 and HTLV-2 in Viral Pathogenesis*

Benoit Barbeau, Jean-Marie Peloponese and Jean-Michel Mesnard

# *Umberto Bertazzoni\**

*Department of Life and Reproduction Sciences, Section of Biology and Genetics, University of Verona, Verona, Italy \*Correspondence<EMAIL_ADDRESS>
#### *Edited and reviewed by:*

*Akio Adachi, The University of Tokushima Graduate School, Japan*

#### **Keywords: HTLV-1, HTLV-2, expression, proteins, co-infection**

Human T-cell leukemia viruses type 1 and 2 (HTLV-1 and HTLV-2) share a common genetic organization, expression strategy and ability to infect and immortalize T-cells *in vitro*; however, HTLV-1 and HTLV-2 are strikingly different in terms of clinical impact. HTLV-1 is recognized as the aetiological agent of adult T-cell leukemia/lymphoma (ATLL), and HTLV-associated myeolopathy/tropical spastic paraparesis (HAM/TSP), in contrast, HTLV-2 does not cause hematologic disorders and is only sporadically associated with cases of subacute myelopathy. HTLV-1 and HTLV-2 also exhibit distinct cellular tropisms *in vivo*: HTLV-1 is mainly found in CD4+ T lymphocytes, whereas CD8+ T-cells are the preferred target for HTLV-2.

The articles contributed in this Research Topic are covering all the different aspects that characterize HTLV-1 and HTLV-2, by highlighting differences in their biology that might provide clues to their distinct pathogenic properties.

Emphasis was placed on the comparison of mRNA expression (Cavallari et al., 2013), the genetic organization and expression patterns (Ciminale et al., 2014), the structural and functional properties of HTLV-1 and HTLV-2 Tax proteins (Forlani et al., 2013; Ren and Cheng, 2013; Romanelli et al., 2013; Shirinian et al., 2013), the role of accessory proteins and antisense proteins in viral pathogenesis (Anupam et al., 2013; Barbeau et al., 2013), and the mechanisms of HIV-1/HTLV-1 and HIV-1/HTLV-2 co-infections (Pilotti et al., 2013).

The fine tuning of expression of HTLV-1 and HTLV-2 (Cavallari et al., 2013) was focused on the X region that codes for the regulatory and accessory proteins in partially overlapping ORFs. Expression of such compact genomes is accomplished by a combination of ribosomal frameshifting, alternative splicing and polycistronic translation as well as production of negative-strand transcripts that code for the HBZ and APH-2 proteins. Recent studies of the temporal sequence of HTLV-1 gene expression in PBMCs isolated from infected patients revealed a "two-phase" kinetics in which tax/rex mRNA expression precedes that of other viral transcripts. A similar analysis of HTLV-2 mRNA expression indicated a comparable expression pattern, although HTLV-2 is characterized by a more abundant expression of gene products that may favor viral latency (i.e., p28 and tRex).

Given the relevance of Tax in the initial steps of T-cell transformation, their key role as the main viral oncoproteins was discussed in four different articles, each one focusing on a specific aspect of Tax function. Romanelli et al. (2013), highlighted distinctive properties of Tax proteins with emphasis on the activation of the NF-κB pathways and interactions with host factors that participate in signal trasduction. Tax-1 and Tax-2, though sharing many common properties, significantly differ for the presence of a PDZ motif, which is missing in Tax-2. This motif mediates the interaction of Tax with host factors regulating the cell cycle; in addition Tax-2 is unable to activate the non-canonical NF-κB which is attributed only to Tax-1. Shirinian et al. (2013), specifically addressed the subcellular localization and trafficking of Tax-1 and Tax-2, and their effects on cellular regulatory proteins. A special attention was given to Tax-1/Tax-2 post-translational modifications, as well to NF-κB activation and protein-protein interactions involved in oncogenecity both *in vivo* and *in vitro*. Ren and Cheng (2013), discussed their recent observations and views on the differential transforming activity of Tax-1 and Tax-2 in human T cells. Both proteins are unable to immortalize CD8 cells but Tax-2 is more efficient than Tax-1 in immortalizing CD4 cells. Forlani et al. (2013), discussed the pivotal role of the class II transactivator (CIITA) in triggering the adaptive immune response against pathogens. CIITA acts as an endogenous restriction factor against HTLV-1 and HTLV-2 by targeting their viral transactivators Tax-1 and Tax-2. The authors reviewed their findings on CIITA-mediated inhibition of viral replication and discussed similarities and differences in the molecular mechanisms by which CIITA specifically counteracts the function of Tax proteins.

The function of the Rex protein was presented by Ciminale et al. (2014). Although Rex-1 and Rex-2 share a similar domain structure, the truncated forms of Rex-2 are capable of inhibiting Rex function, while their HTLV-1 homolog p21 Rex might lack this activity.

The role in viral replication and viral pathogenesis of accessory proteins HTLV-1 p30 and HTLV-2 p28 were highlighted by Anupam et al. (2013). Both p30 and p28 regulate viral gene expression at the post-transcriptional level whereas p30 can also function at the transcriptional level. Since p30 and p28 have distinct interactome profiles even though they both interact with similar proteins, they must have divergent functions during the lifecycle of the viruses.

The functions of other accessory proteins have been presented by Ciminale et al. (2014). HTLV-1 p13 and p8 are not expressed by HTLV-2 which codes for p11, whose function is still unclear and does not seem to have a homolog in HTLV-1.

The possible role played by antisense proteins HTLV-1 HBZ and HTLV-2 APH-2 in the establishment of pathologies induced by viral infection has been carefully considered by Barbeau et al. (2013). Unlike APH-2, HBZ possesses specific domains which mediate its capacity to enhance its own expression, deregulate several pathways and induce T cell proliferation, providing substantial evidence toward its implication in viral persistence and ATLL cell survival.

Finally the problem of HIV/HTLV co-infection has been discussed by Pilotti et al. (2013). During co-infection with HIV-1, HTLV-2 modulates the cellular microenvironment favoring its own propagation and inhibiting HIV-1 progression. Possible differences between HTLV-1 and HTLV-2 on innate immune mechanisms induction and a particularly impact on NK cells are becoming evident.

In conclusion, in this Research Topic, HTLV-1 and HTLV-2 have been thoroughly compared and major differences outlined and discussed. Of particular interest is that HTLV-2 infection, contrary to HTLV-1, is characterized by an oligoclonal distribution in asymptomatic hosts; this indicates that the etiology of malignant transformation by HTLV-1 cannot be uniquely attributed to this phenomenon and further investigation is needed to understand why HTLV-2 is defective in promoting leukemogenesis.

# **ACKNOWLEDGMENT**

I warmly thank Françoise Bex, Vincenzo Ciminale and Maria Grazia Romanelli for critical reading of the Editorial.

#### **REFERENCES**

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

*Received: 22 December 2014; accepted: 23 December 2014; published online: 14 January 2015.*

*Citation: Bertazzoni U (2015) Editorial to the Research Topic "Comparative studies between HTLV-1 and HTLV-2 function and pathobiology". Front. Microbiol. 5:792. doi: 10.3389/fmicb.2014.00792*

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

*Copyright © 2015 Bertazzoni. 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 and HTLV-2: highly similar viruses with distinct oncogenic properties

### *Vincenzo Ciminale1, Francesca Rende1, Umberto Bertazzoni 2 and Maria G. Romanelli 2 \**

*1 Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy 2 Department of Life and Reproduction Sciences, University of Verona, Verona, Italy*

#### *Edited by:*

*Hirofumi Akari, Kyoto University, Japan*

#### *Reviewed by:*

*Hidekatsu Iha, Oita University, Japan Fatah Kashanchi, George Mason University, USA*

#### *\*Correspondence:*

*Maria G. Romanelli, Department of Life and Reproduction Sciences, University of Verona, Strada le Grazie 8, Verona 37134, Italy e-mail<EMAIL_ADDRESS>
**INTRODUCTION**

Four types of HTLV have been described so far, the most prevalent types being HTLV-1 and HTLV-2 (Gessain et al., 2013).

HTLV-1 and HTLV-2 show considerable homology in terms of genome structure, replication pattern, and properties of the structural, regulatory, and accessory proteins. Their transmission follows the same route, by transfer of infected lymphocytes by perinatal transmission and breastfeeding and through blood transfusion, sexual contact, and use of intravenous drugs (Pique and Jones, 2012). Both viruses utilize the GLUT-1 and NRP1 cellular receptors for their entry, although HTLV-1, but not HTLV-2, is dependent on heparan sulfate proteoglycans (Jones et al., 2006). Cell-to-cell transmission is essential for virus replication and occurs through the formation of a virological synapse (Nejmeddine et al., 2005).

Despite these important analogies, HTLV-1 and HTLV-2 are strikingly different in terms of clinical impact, as only HTLV-1 is conclusively associated with neoplasia, namely adult T-cell leukemia/lymphoma (ATLL), which develops in up to 5% of infected individuals. A similar percentage of HTLV-1-infected persons develop a neurological disease termed HTLV-associated myeolopathy/tropical spastic paraparesis (HAM/TSP; Gessain and Mahieux, 2012). In contrast, HTLV-2, though persistently associated with elevated lymphocyte and platelet counts (Bartman et al., 2008) and with an increase in overall cancer mortality (Biswas et al., 2010), does not cause hematologic disorders and is only sporadically associated with myelopathy (Araujo and Hall, 2004).

The two viruses also differ in their geographical distribution. HTLV-1 is endemic in Japan, sub-Saharan Africa, South America, and the Caribbean (Gessain and Cassar, 2012), whereas HTLV-2 is prevalent among the indigenous populations in Africa and the Indian-American tribes in Central and South America as well as among drug users in Europe and North America (Zella et al., 1990; Roucoux and Murphy, 2004). Although their receptor usage

HTLV-1 and HTLV-2 share broad similarities in their overall genetic organization and expression pattern, but they differ substantially in their pathogenic properties. This review outlines distinctive features of HTLV-1 and HTLV-2 that might provide clues to explain their distinct clinical outcomes. Differences in the kinetics of viral mRNA expression, functional properties of the regulatory and accessory proteins, and interactions with cellular factors and signal transduction pathways are discussed.

**Keywords: HTLV,Tax, Rex, HBZ, APH2, clonality**

allows HTLV-1 and HTLV-2 to be quite promiscuous for different cell types *in vitro*, they exhibit distinct cellular tropisms *in vivo*: HTLV-1 is mainly found in CD4+ T lymphocytes, whereas CD8+ T cells are the preferred target for HTLV-2 (Ijichi et al., 1992).

The following sections highlight differences in the biology of HTLV-1 and HTLV-2 that might provide clues to their distinct clinical outcomes. Emphasis is placed on the comparison of the regulatory proteins, kinetics of mRNA expression, clonal distribution patterns, and interaction with cellular factors and signal transduction pathways.

# **THE COMPARATIVE ANATOMY OF THE HTLV-1 AND HTLV-2 GENOMES**

In their pioneering studies, Seiki et al. (1983) noted the presence of a region in the HTLV-1 genome between the env open reading frame (ORF) and the 3- LTR that was not present in the previously described oncogenic retroviruses. This region, termed the "X region," is also present in HTLV-2 and codes for the regulatory and accessory proteins in partially overlapping ORFs named x-I through x-V. Expression of the complex arrangement of ORFs in such a compact genome is accomplished by a combination of ribosomal frameshifting, alternative splicing, and polycistronic translation (Ciminale et al., 1992, 1995; Koralnik et al., 1992) as well as production of negative-strand transcripts that code for the HBZ (HTLV-1) and APH-2 (HTLV-2) proteins.

Like other retroviruses, the HTLVs produce an unspliced mRNA which codes for the Gag-Pol-Pro precursor protein and also serves as the viral genome and a singly spliced mRNA that codes for the Env surface glycoproteins. Peculiar to the HTLVs is the expression of the Tax and Rex regulatory proteins that play critical roles in driving expression from the 5- LTR promoter (Tax) and in enhancing the expression of partially spliced plus-strand mRNAs (Rex). The Tax and Rex ORFs (x-IV and x-III, respectively) are expressed from the same doubly spliced mRNA. In HTLV-1, all the other X region proteins are coded by individual singly or doubly spliced transcripts, while some HTLV-2 transcripts express more than one ORF.

Recent studies of the temporal sequence of HTLV-1 gene expression in peripheral blood mononuclear cells (PBMCs) isolated from infected patients (Corradin et al., 2011; Rende et al., 2011) revealed a "two-phase" kinetics with tax/rex mRNA expression preceding that of other viral transcripts. A similar analysis of HTLV-2 mRNA expression indicated a comparable pattern, although the relative abundance of some transcripts showed some intriguing differences among the two viruses (Bender et al., 2012; Cavallari et al., 2013).

The major differences between HTLV-1 and HTLV-2 gene products are summarized in **Table 1** and commented in the following sections.

### **Tax PROTEINS AND THEIR INTERACTIONS WITH CELLULAR PATHWAYS**

The principal role of Tax during viral replication is to activate transcription of the LTR promoter through a process that involves the recruitment of CREB/ATF complexes on binding sites in the U3 region. Tax-1 and Tax-2 share this mechanism and are able to cross-activate each others' LTRs (Semmes et al., 1996).

Experiments performed in the early 1990s established a key role for Tax as the main viral oncoprotein necessary for the initial steps of T-cell transformation by HTLV-1 (Tanaka et al., 1990). Transgenic mouse models have consistently demonstrated that Tax expression causes tumors, including lymphoma and leukemia (Ruddle et al., 1993; Hasegawa et al., 2006). Recent comparative studies on the transforming activities of HTLV-1 Tax (Tax-1) and HTLV-2 Tax (Tax-2) demonstrated that, in the absence of other viral proteins, both Tax-1 and Tax-2 immortalize human primary CD4+ T cells, but only Tax-2 is able to immortalize CD8+ T cells (Imai et al., 2013). Furthermore, Tax-2 immortalizes human primary CD4+ T cells more efficiently than Tax-1 (Imai et al., 2013). Although additional viral products have been demonstrated to be relevant for immortalization, the interactions between Tax and host proteins are still considered to be essential for the transformation process. Several studies of the oncogenic properties of Tax-1 highlighted its effects on DNA repair, cell cycle progression, cell death, and p53 function (reviewed by Cheng et al., 2012).

In addition to CREB/ATF proteins, Tax-1 interacts with many other host factors that influence cell proliferation and transformation, including components of the PI3K, AKT, MAPK, TGFβ, SRF, and NF-κB pathways (Romanelli et al., 2013). Tax-2 has been analyzed mainly for its effects on the NF-κB pathway, and was shown to activate the canonical pathway, but, unlike Tax-1, does not activate the noncanonical pathway (Shoji et al., 2009), suggesting that activation of this latter pathway might be a key element in HTLV-1-driven transformation *in vivo*. Although overall highly homologous, Tax-1 and Tax-2 present some distinct structural features. Tax-1 contains a PDZ binding motif (PBM) and leucine zipper domains that are absent in Tax-2. The C-terminal PBM domain is relevant for interaction of Tax-1 with host factors that regulate cell cycle progression and proliferation, such as the human homologue of the *Drosophila melanogaster* disk

large tumor suppressor protein (hDLG; Suzuki et al., 1999). The leucine zipper regions present in the central region of Tax-1 are responsible for activation of the non-canonical NF-κB pathway (Higuchi and Fujii, 2009). The nuclear localization and nuclear export signals of Tax-1 and Tax-2 present significant differences which could explain the predominant nuclear localization of Tax-1 compared to Tax-2 (Turci et al., 2006, 2009; Avesani et al., 2010).

An additional difference between Tax-1 and Tax-2 that may play a role in NF-κB activation is the absence of interactions between Tax-2 and TNF receptor-associated factor TRAF6, an E3 ubiquitin ligase that is involved in ubiquitination of effectors of the NF-κB pathway (Journo et al., 2013). Both Tax-1 and Tax-2 are ubiquitinated and SUMOylated, but they differ in the pattern of modification (Zane and Jeang, 2012). The role of ubiquitination, sumoylation, and acetylation of Tax in NF-κB activation and cellular transformation is still an open field of research (Turci et al., 2012; Xiao, 2012; Journo et al., 2013; Lodewick et al., 2013).

Activation of NF-κB by Tax is also connected to the deregulation of autophagy, an additional pathway that is altered in oncogenic signaling. Both Tax-1 and Tax-2 induce autophagosome accumulation, but their interactions with the components of the process differ. In fact, Tax-1 (but not Tax-2) directs the IKK complex to lipid rafts associated with autophagic molecules such as Beclin 1 and Bif-1 (Huang et al., 2009; Ren et al., 2013).

#### **POST-TRANSCRIPTIONAL REGULATION BY Rex**

HTLV-1 Rex (Rex-1) and HTLV-2 Rex (Rex-2) share 60% homology at the amino acid level. Rex-1 and Rex-2 are phosphorylated proteins that actively shuttle between the nucleus and the cytoplasm (Palmeri and Malim, 1996; Narayan et al., 2003) and accumulate in the nucleus and nucleoli (Nosaka et al., 1989; Ciminale et al., 1995, 1997; Nosaka et al., 1995; Narayan et al., 2003), a property that is intimately linked to their ability to enhance the nuclear export of incompletely spliced viral mRNAs.

Rex-1 and Rex-2 share a similar domain structure which includes: (i) a nuclear localization signal (NLS; Siomi et al., 1988; Nosaka et al., 1989), which mediates binding to the RXRE (Grassmann et al., 1991) located at the 3 end of all HTLV-1 transcripts and at the 5 end of the unspliced HTLV-2 mRNA (Ohta et al., 1988; Black et al., 1991); (ii) multimerization domains; and (iii) a leucine-rich sequence located near the middle of the protein (Rex-1 aa 79–99; Rex-2 aa 81–94), which functions as an activation domain (AD; Weichselbraun et al., 1992) and contains the nuclear export signal (NES; Kim et al., 1996; Palmeri and Malim, 1996) that interacts with CRM1/exportin, which mediates the nuclear export of the Rex-viral mRNA complex (Bogerd et al., 1995; Kim et al., 1996; Palmeri and Malim, 1996). In addition a C-terminal domain unique to Rex-2 is a target for serine phosphorylation and may also contribute to efficient nucleocytoplasmic shuttling (Narayan et al., 2003). Although Rex is not required for cellular immortalization *in vitro*, it is necessary for infectivity and viral persistence *in vivo* (Ye et al., 2003), as it is required for the expression of the virion-associated structural proteins.

The dependence of HTLV mRNAs on Rex function is controlled by positively and negatively acting RNA sequences present

#### **Table 1 | Structural and functional differences between HTLV-1 and HTLV-2 gene products.**

on the primary transcript. Two major types of such RNA *cis*-acting elements have been described: (i) the Rex responsive element (RXRE) which, besides binding Rex, acts as an inhibitory sequence in the absence of Rex, and (ii) *cis*-acting repressive sequences (CRS) that determine poor stability and/or inefficient nucleocytoplasmic export. HTLV-1 contains a CRS which maps at the 5end of the unspliced mRNA but is spliced out of the other transcripts (Seiki et al., 1990; King et al., 1998). An additional CRS overlaps the RXRE and acts synergistically with the 5- - CRS. In contrast to the 5- -CRS, this 3- -CRS/RXRE is present at the 3 end of all viral transcripts. Both the 5- - and 3- -CRSs were shown to act mainly as nuclear retention sequences. The 5- -CRS does not bind Rex-1 (Ballaun et al., 1991; Bogerd et al., 1991; Unge et al., 1991), and its inhibitory function is likely to

be mediated by other viral and/or cellular RNA-binding proteins. Other *cis*-acting inhibitory elements (CIEs) were mapped within the gag-pol and env regions of HTLV-1 (Saiga et al., 1997). The inhibitory effect of these regions is counteracted by binding of Rex-1 to the RXRE, although it is not clear whether they function mainly at the level of RNA stability or nucleocytoplasmic export. A 5- -CRS acting as a nuclear retention sequence was also mapped in the R-U5 region of HTLV-2 (Black et al., 1991).

#### **THE ACCESSORY PROTEINS**

The "accessory" proteins were labeled as such because their ablation does not have apparent consequences on viral replication *in vitro*. However, studies performed in animal models indicate that some of these proteins are essential for efficient infectivity *in vivo*. Among the accessory proteins, p30Tof/p28, p12/p10, and p21Rex/tRex (in HTLV-1 and HTLV-2, respectively) are considered to be homologous based on their structure and functional properties, while p13 and p8 appear to be unique to HTLV-1, and p11 is peculiar to HTLV-2.

p21Rex and tRex are truncated forms of Rex-1 and Rex-2, respectively, which lack the *N*-terminal arginine-rich NLS and are therefore incapable of binding the RXRE. HTLV-2 tRex is detected as four main isoforms of 22, 20, 18, and 17 kDa which differ in the initiation codon usage and phosphorylation status (Ciminale et al., 1997). The tRex proteins were shown to inhibit Rex-2 function (Ciminale et al., 1997), an activity that might favor latent infection. Although one study indicated that p21rex acts as a repressor of full-length Rex (Heger et al., 1999), this finding was not supported by other studies (Ciminale et al., 1997; Bai et al., 2012).

HTLV-1 p30Tof and HTLV-2 p28 are important for viral propagation in animal models (Silverman et al., 2004; Yamamoto et al., 2008; Valeri et al., 2010). Both proteins sequester the tax/rex mRNA in the nucleus, an effect that may result in reduced viral expression and latency (Nicot et al., 2004; Younis et al., 2004). p30Tof was also shown to interact with the RNA-binding domain of Rex and thereby interfere with its binding to the RXRE (Sinha-Datta et al., 2007; Bai et al., 2010) and to inhibit the expression of Toll-like receptor 4 (Datta et al., 2006), suggesting a role in the innate immune response. These two properties have not been reported for p28. By interacting with the co-activator CBP/p300 (Zhang et al., 2000, 2001), p30Tof also affects Tax-mediated viral expression and transcription of cellular genes involved in T-cell activation and apoptosis (Michael et al., 2004; Taylor et al., 2009). Interestingly, p28 does not appear to affect CBP/p300-mediated transcription. Both p30Tof and p28 are targeted to the nucleus, but only p30Tof shows evident accumulation in the nucleoli (Ciminale et al., 1995; D'Agostino et al., 1997). Interestingly, HTLV-2 p28 and tRex are expressed at higher levels compared to their HTLV-1 counterparts p30/Tof and p21Rex, suggesting a higher propensity of HTLV-2 for latency compared to HTLV-1 (Bender et al., 2012).

p13 corresponds to the C-terminal 87 amino acids of p30Tof (Koralnik et al., 1992), and it is localized mainly in the mitochondrial inner membrane (Ciminale et al., 1999; D'Agostino et al., 2002) and in part to the nucleus (Silic-Benussi et al., 2010b; Andresen et al., 2011). Using a rabbit animal model, Hiraragi et al. (2006) showed that p13 is required for viral infectivity *in vivo*. p13 increases mitochondrial permeability to K+ and activates the electron transport chain, resulting in increased mitochondrial reactive oxygen species (ROS) production (Silic-Benussi et al., 2009). While p13 has a mitogenic effect in normal resting T cells, which have low ROS levels, the protein induces death of transformed T-cells, which are characterized by a high ROS setpoint (Ciminale et al., 1999; D'Agostino et al., 2005; Hiraragi et al., 2005; Silic-Benussi et al., 2004, 2010a,b). So far no HTLV-2 homologue of HTLV-1 p13 has been identified.

HTLV-1 p12 and its HTLV-2 homologue p10 are coded by the x-I ORF. p12 localizes in the endoplasmic reticulum (ER) and in the Golgi apparatus, where it reduces the expression of the β and γc chains of the interleukin-2 receptor (IL-2R, Mulloy et al., 1996) and of MHC-I, thus hindering lysis of HTLV-1-infected cells by CTL (Johnson et al., 2001). p12 also activates STAT-5, which provides a mitogenic signal to T cells (Nicot et al., 2001) and interacts with calreticulin and calnexin (Ding et al.,2001), resulting in incresased Ca2+ release from the ER (Ding et al., 2002) and activation of NF-AT, a mitogenic pathway in T-cells (Albrecht et al., 2002; Kim et al., 2003). Within the ER, p12 is cleaved into an 8-kDa protein (p8) which trafficks to the immunological synapse and favours T-cell anergy. p8 also increases cell-to-cell viral transmission through the formation of intercellular conduits (Van Prooyen et al., 2010a,b). In analogy to p12, HTLV-2 p10 was shown to bind the MHC heavy chain; however, p10 does not bind the IL2R β chain or the 16-kDa subunit of the vacuolar H+ ATPase (Johnson et al., 2000). No homologue of p8 has been described in HTLV-2.

A recent study from Valeri et al. (2010), showed that p12 is required for *in vivo* propagation in macaques but not in rabbits.

The x-V ORF of HTLV-2 codes for p11 from a doubly spliced transcript that also codes for p10 (Ciminale et al., 1995). Aside from its ability to bind to MHC heavy chain (Johnson et al., 2000), nothing is known about the function of this protein. HTLV-1 also possesses an x-V ORF, but it does not appear to produce a p11 homologue. The function of p11 is still unclear.

#### **HBZ AND APH-2**

The structure and function of HBZ and APH-2 were recently reviewed (Barbeau et al., 2013). HBZ is a nuclear transcriptional factor (Gaudray et al., 2002), able to interact with ATF/CREB proteins through its basic zipper (bZIP) domain causing the inhibition of its DNA-binding activity (Hivin et al., 2006); this effect also influences the ability of Tax-1 to activate HTLV-1 transcription (Halin et al., 2009). APH-2 is likewise able to inhibit Tax-2-mediated transcription but its repressive activity is weaker than that of HBZ (Halin et al., 2009). This difference may be ascribed to the presence in HBZ but not in APH-2, of a transcriptional activation domain within its *N*-terminal region that mediates its interaction with the KIX domain of p300/CBP, thus competing for its binding to Tax-1 (Clerc et al., 2008).

HBZ also deregulates several cellular pathways including c-Jun and JunD, FoxP3, NF-κB, TGF-β and Wnt (Barbeau et al., 2013). In addition, HBZ, may enhance its own expression by controlling the transcriptional activity of JunD (Gazon et al., 2012). Very little is known about the regulation of APH-2 expression in infected cells. However, unlike HBZ, APH-2 enhances the transcriptional activity of c-Jun family proteins (Marban et al., 2012).

The role of HBZ in T-cell transformation is supported by the finding that while Tax-1 expression is often repressed in ATL cells and appears to be dispensable for the late stages of leukemogenesis, HBZ is constitutively expressed in most ATL cases (Satou et al., 2006). Transgenic mice expressing HBZ in CD4+ T cells develop T-cell lymphomas and systemic inflammation that are reminiscent of ATL and HAM/TSP (Satou et al., 2011).

Although HTLV-2 is not causally linked to leukemia or lymphoma, it has been associated with lymphocytosis in infected patients (Bartman et al., 2008). This is consistent with the observation that APH-2 is detected in most PBMCs of HTLV-2-infected patients (Halin et al., 2009) and that its expression is well correlated to proviral DNA load (Douceron et al., 2012). Interestingly, it was observed that the APH-2 mRNA, similarly to HBZ, accumulates in the nucleus (Bender et al., 2012). Barbeau et al. (2013) have recently proposed a model to explain the different effects of HBZ and APH-2 on T-cell proliferation. Both HBZ and of APH-2 can suppress Tax expression, thus favoring evasion of the immune response. In addition, HBZ can stimulate its own expression, inhibit Tax-1-dependent viral expression and induce T-cell proliferation, which can lead to ATL. APH-2 is unable to induce cell proliferation and only partially down-regulates Tax-2 expression.

Although dispensable for the HTLV-1 infection and immortalization of T lymphocytes *in vitro*, studies conducted on a rabbit animal model suggest that HBZ enhances HTLV-1infectivity and persistence *in vivo* (Arnold et al., 2006). Furthermore, HBZ transgenic mice develop systemic inflammation and a CD4+ T-cell neoplasm that is reminiscent of ATL (Satou et al., 2011). Interestingly, in contrast to HBZ, HTLV-2 APH-2 appears to be dispensable for viral infection and persistence in a rabbit animal model (Yin et al., 2012), suggesting a functional divergence of the *in vivo* function of the HTLV-1 and HTLV-2 antisense proteins.

#### **TROPISM AND CLONALITY**

The preferential cellular tropism of HTLV-1 for CD4+ T cells and of HTLV-2 for CD8+ T cells is still not clearly understood. It appears that the two viruses make different use of the heparan sulfate proteoglycans to enter T cells (Jones et al., 2006), although recent evidence obtained *in vivo* rabbit model indicates that the apparent tropism for CD4+ or CD8+ cells mainly reflects preferential clonal expansion of infected cells (Kannian et al., 2012). Both HTLV-1 and HTLV-2 are capable of inducing clonal expansion of infected cells *in vivo* (Wattel et al., 1995; Cimarelli et al., 1996). A detailed analysis in asymptomatic carriers, TSP/HAM and ATLL patients revealed that the genomic integration site and transcriptional orientation of the provirus are important factors for determining clonal abundance *in vivo* (Gillet et al., 2011).

A very recent study demonstrated that, contrary to previous hypotheses, HTLV-2-infected individuals have a small number of highly expanded CD8+ T-cell clones, suggesting that HTLV-2 may be subjected to a more strict clonal selection than HTLV-1 in healthy carriers. These data suggest that selective clonal proliferation is more directly responsible for determining the viral burden of HTLV-2 than it does for HTLV-1 (Melamed et al., 2014). In contrast to observations made for HTLV-1, the environment surrounding the integration sites does not seem to have a substantial impact on the expansion of HTLV-2-infected clones (Melamed et al., 2014).

The presence of few very abundant HTLV-2-infected clones is apparently reminiscent of the profile found in ATLL patients. However, the fact that HTLV-2 is not causally linked to a T-cell malignancy suggests that clonal abundance and heterogeneity may not *per se* constitute a determinant of malignant transformation and clinical outcome of HTLV infection (Melamed et al., 2014).

#### **CONCLUSIONS**

From the comparative analysis on the functional properties of HTLV-1 and HTLV-2 reported above, the following major differences can be outlined: (i) HTLV-2 is characterized by a more abundant expression of gene products that may favor viral latency (i.e., p28 and tRex); (ii) Tax-1 presents a PDZ binding motif, which is absent from Tax-2 and allows interaction with host factors regulating the cell cycle and proliferation; in addition, Tax-2 is unable to activate the non-canonical NF-κB pathway; (iii) *Cis*-acting inhibitory elements acting at the level of RNA stability are present within HTLV-1 whereas they have not been described in HTLV-2; (iv) while the truncated forms of Rex-2 were shown to inhibit Rex function, the HTLV-1 homologue p21Rex might lack this activity; (v) HTLV-1 expresses p13 and p8, which have not been described in HTLV-2; (vi) HTLV-2 expresses p11, which does not seem to have a homologue in HTLV-1; (vii) unlike APH-2, HBZ presents a basic zipper domain, as well a transcriptional activation domain, which mediate its capacity to enhance its own expression and deregulate several cellular pathways; (viii) HTLV-2 shows *in vivo* tropism for CD8+ T cells and induces expansion of a relatively small number of highly abundant clones.

In spite of the evidence accumulated so far on the similarities and differences between HTLV-1 and HTLV-2, it is not yet clear why only HTLV-1 causes a T-cell malignancy. While Tax-1 and HBZ induce T-cell lymphomas when expressed as transgenes in animal models, the *in vivo* transforming activities of Tax-2 and APH-2 have not been investigated. Aside from the NF-κB pathway, not much information is available regarding the interactions of Tax-2 with cellular pathways known to be engaged by Tax-1. The fact that HTLV-2, contrary to HTLV-1, is characterized by an oligoclonal proliferative distribution in asymptomatic hosts clearly indicates that the etiology of malignant transformation by HTLV-1 cannot be uniquely attributed to this phenomenon.

#### **ACKNOWLEDGMENTS**

The authors thank Luigi Chieco-Bianchi and Donna D'Agostino for discussions. Research on the HTLVs performed by Vincenzo Ciminale and Maria G. Romanelli was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) – Cariverona and the Universities of Padova and Verona.

#### **REFERENCES**

in vitro immortalization but functions to repress early virus replication *in vivo*. *J. Virol.* 86, 8412–8421. doi: 10.1128/JVI.00717-12

as a transcription factor and differentially modulates CREB-responsive promoters. *J. Virol.* 74, 11270–11277. doi: 10.1128/JVI.74.23.11270- 11277.2000

**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.

*Received: 26 May 2014; accepted: 15 July 2014; published online: 29 July 2014. Citation: Ciminale V, Rende F, Bertazzoni U and Romanelli MG (2014) HTLV-1 and HTLV-2: highly similar viruses with distinct oncogenic properties. Front. Microbiol. 5:398. doi: 10.3389/fmicb.2014.00398*

*This article was submitted to Virology, a section of the journal Frontiers in Microbiology. Copyright © 2014 Ciminale, Rende, Bertazzoni and Romanelli. This is an openaccess 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/-2 and HIV-1 co-infections: retroviral interference on host immune status

# *Elisabetta Pilotti 1, Maria V. Bianchi 1, Andrea De Maria 2,3,4 , Federica Bozzano 2,3 , Maria G. Romanelli 5 , Umberto Bertazzoni 5 and Claudio Casoli 1\**

*1 GEMIB Laboratory, Center for Medical Research and Molecular Diagnostics, Parma, Italy*

*2 Department of Health Sciences, University of Genova, Genova, Italy*

*3 Center of Excellence for Biomedical Research, University of Genova, Genova, Italy*

*4 IRCCS AOU San Martino-IST Genova, Genova, Italy*

*5 Department of Life and Reproduction Sciences, University of Verona, Verona, Italy*

#### *Edited by:*

*Françoise Bex, Université Libre de Bruxelles, Belgium*

#### *Reviewed by:*

*Akifumi Takaori-Kondo, Kyoto University Graduate School of Medicine, Japan Carine M. C. Van Lint, Université Libre de Bruxelles, Belgium*

#### *\*Correspondence:*

*Claudio Casoli, GEMIB Laboratory, Center for Medical Research and Molecular Diagnostics, Borgo G. Cantelli n.11, Parma 43121, Italy e-mail<EMAIL_ADDRESS>The human retroviruses HIV-1 and HTLV-1/HTLV-2 share similar routes of transmission but cause significantly different diseases. In this review we have outlined the immune mediated mechanisms by which HTLVs affect HIV-1 disease in co-infected hosts. During co-infection with HIV-1, HTLV-2 modulates the cellular microenvironment favoring its own viability and inhibiting HIV-1 progression. This is achieved when the HTLV-2 proviral load is higher than that of HIV-1, and thanks to the ability of HTLV-2 to: (i) up-regulate viral suppressive CCL3L1 chemokine expression; (ii) overcome HIV-1 capacity to activate the JAK/STAT pathway; (iii) reduce the activation of T and NK cells; (iv) modulate the host miRNA profiles. These alterations of immune functions have been mainly attributed to the effects of the HTLV-2 regulatory proteinTax and suggest that HTLV-2 exerts a protective role against HIV-1 infection. Contrary to HIV-1/HTLV-2, the effect of HIV-1/HTLV-1 co-infection on immunological and pathological conditions is still controversial. There is evidence that indicates a worsening of HIV-1 infection, while other evidence does not show clinically relevant effects in HIV-positive people. Possible differences on innate immune mechanisms and a particularly impact on NK cells are becoming evident. The differences between the two HIV-1/HTLV-1 and HIV-1/HTLV-2 co-infections are highlighted and further discussed.

**Keywords: HTLV, HIV-1, co-infection, cytokines, chemokines, JAK/STAT, miRNA, natural killer cells**

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# **INTRODUCTION**

Microbes that infect the same host may positively influence each other's replication, or fight for supremacy (Margolis, 2003; Kannangara et al., 2005). In recent years, different data have demonstrated that a microbe, in order to favor its own replication, can attenuate or support the infection of other infecting agents by altering the host immune system (Lisco et al., 2009). The role of human T lymphotropic viruses type 1 and type 2 (HTLV-1 and HTLV-2) as determinants of HIV-1 disease during co-infection has been widely studied, but is still a matter of speculation.

Since HTLV-1, HTLV-2, and HIV-1 have common modes of transmission, it is not surprising that co-infection is a frequent condition especially among people with high risk behaviors, as needle sharing and unprotected sexual contact. Though human retroviruses have worldwide distribution (Goubau et al., 1992; Vrielink and Reesink, 2004; Proietti et al., 2005), dually infected subjects have been mainly diagnosed in large metropolitan area or in endemic regions (Briggs et al., 1995; Dezzutti et al., 1998; Araujo et al., 2002; Morimoto et al., 2005; Magri et al., 2012, 2013). Because HTLV screening is not routinely performed in many countries and is not always recommended by physicians to outpatients, the seroprevalence of co-infection is underestimated (Beilke, 2012; Pinto et al., 2012).

HTLV-1, HTLV-2, and HIV-1 share similar genomic organization and tropism for immune cells, in particular CD4+ and CD8+

T cells. However, the finality of their viral cycle is different. In infected patients, HIV-1 is generally present as a virion (either cell-associated or cell-free) and the provirus is detected clearly in a minority of cells (Josefsson et al., 2011), whereas HTLVs are prevalently integrated in their target cells and the propagation of infection occurs by clonal expansion of infected cells (Bangham, 2003).

To understand whether HTLVs may accelerate or attenuate HIV-1 progression, several studies have interpreted HIV-1-associated clinical outcomes, taking into account laboratory records as CD4 mean cell count and HIV-1 viral load. However, conflicting data have emerged on both HTLV-1 and HTLV-2 interactions with HIV-1 and the debate is still open. The lack of uniformity of used criteria may explain this discrepancy, including differences in sampling, variations in outcome surveillance, with other parameters, such as HTLVs and HIV-1 seroconversion time and sex, not always defined. Furthermore, some authors believe that matching patients by immune markers is not a good strategy (Beilke et al., 2004). When not well-defined, also anti-HIV-1 therapy, considering its effect on immunologic host factors and potentially on replication of co-invading agents, could make the comparison of data coming from different studies difficult.

Concerning HTLV-1/HIV-1 co-infection, a more rapid HIV-1 disease progression and a lower mean survival time in co-infected individuals compared to HIV-1-mono-infected patients (Brites et al., 2001) was seen. Additionally, an increased risk to develop tropical spastic paraparesis/HTLV associated myelopathy (TSP/HAM) and other neurodegenerative conditions was found in dually infected subjects (Tulius Silva et al., 2009). Also, a recent work reported a higher mortality and shortened survival rate in HTLV-1/HIV-1-co-infected children compared to HIV-1-monoinfected patients irrespective of baseline CD4 cell count (Pedroso et al., 2011).

On the contrary, other researchers observed a delayed HIV-1 disease progression in HTLV-1-co-infected patients when compared to HIV-1-mono-infected individuals (Shibata et al., 1989; Page et al., 1990; Chavance et al., 1995; Beilke et al., 2004). In according to these findings, a higher survival rate was reported for HIV-1/HTLV-1-co-infected subjects (Brites et al., 2001, 2005).

It is noteworthy that the impact of HIV-1 on HTLV-1 replication was also investigated. Seroepidemiologic studies showed that HIV-1 positive subjects are more susceptible to HTLV-1 infection, and vice versa (Harrison et al., 1997). Moreover, the HIV-1 Rev protein was found to enhance HTLV-1 gene expression, by interacting with 5'-RU5 region of the HTLV-1 genome (Kubota et al., 1998). A later study, in which the quantification of HTLV-1 and HIV-1 DNA load was determined, suggested that HIV-1 coinfection does not affect HTLV-1 proviral load in peripheral blood compartments (Cesaire et al., 2001).

Similarly to HTLV-1/HIV-1, contrasting results were also reported for HTLV-2/HIV-1 co-infection. However, it is now generally accepted that HTLV-2 exerts a negative effect on HIV-1 replication. In fact, several authors have associated HTLV-2 co-infection with a better outcome for HIV-1 positive individuals (Beilke et al., 2004; Casoli et al., 2007). Earlier studies did not observe this effect and ascribed the lack of significant associations between co-infection and progression to AIDS or death to the absence of clear evidence of HTLV-2 as pathogenic agent (Visconti et al., 1993; Hershow et al., 1996).

HTLVs interfere with HIV infection by mechanisms that appear to be complex and multilayered. More specifically, HTLVs can act on HIV-1 expression directly at molecular levels or indirectly by modulating the expression of immune host factors. This in turn can be induced by HTLVs directly or through cellular activation.

Thus, the intimate relationship between the HIV-1 life cycle and the activation state of cells supporting viral replication results in a dynamic interaction between co-infecting agents and HIV-1 replication in dually infected individuals.

HTLV-1 differs from HTLV-2 in regulating cellular activation of target cells.  These divergent conditions may contribute to explain the different impact of the two types of HTLV on HIV-1 infection. In particular, for HTLV-2 it was observed that HIV-1/HTLV-2-co-infected patients showed a reduced HIV-1 replication presumably due to lower levels of T cell activation (Bassani et al., 2007).

In this review, we report and discuss recently published data on host immunomodulating factors involved in retroviral interference, and in that regard we also point at other cellular and molecular components that may be considered potentially good candidates.

## **CYTOKINES/CHEMOKINES**

The role of the cytokine/chemokine network as strategic weapon in germ warfare has been extensively discussed (Margolis, 2003). Infecting agents benefit from their ability to drive immune reactions mediated by cytokines and chemokines. This favorable condition for a microbe could be adverse or advantageous for a co-pathogen.

Thus, the perturbation of the immune system, including host cytokine synthesis, induced by HTLV-1 and HTLV-2 infections (Hollsberg, 1999) could have positive or negative impacts on HIV-1 replication. Such immune activation is mainly modulated by HTLV-1 and HTLV-2 Tax proteins (Tax-1 and Tax-2).

Regarding HTLV-1/HIV-1 co-infection, it was reported that Tax-1 up-regulates HIV-1 expression (Bohnlein et al., 1989) by activating the transcriptional factor NF-κB that recognizes two binding sites in the U3 region of the HIV-1 LTR (Leung and Nabel, 1988). In addition, Tax-1 enhances the expression of several cellular proteins, including transcription factors and cytokines as IL-2, tumor necrosis factor α (TNF-α), and others (Banerjee et al., 2007; Boxus et al., 2008). More specifically, some of these cytokines as TNF-α or IL-1β are capable of triggering HIV-1 transcription through a NF-κB-dependent mechanism (Siebenlist et al., 1994). Tax-1 is also responsible for the induction of cytokine receptor expression (Franchini and Streicher, 1995). This state of activation was frequently observed among co-infected subjects who showed a sharp drop in CD4 cell count and rapid progression of HIV-1 disease (Bartholomew et al., 1987; Page et al., 1990; Pagliuca et al., 1990; Gotuzzo et al., 1992; Schechter et al., 1994; Fantry et al., 1995).

However, other studies describe detrimental effects on HIV-1 infections (Harrison et al., 1997; Beilke et al., 2004, 2007). Recently, Abrahao et al. (2012) observed a higher production of IL-2 and IFN-γ in HIV-1/HTLV-1-co-infected individuals than in HIV-1 or HTLV-1-mono-infected individuals. Moreover, while IL-6 and IL-10 levels were similar in all infected groups, IL-4 production was lower in HTLV-1-mono-infected individuals. These findings support the notion that high levels of Th-1 cytokines in co-infected patients provide adverse conditions for HIV-1 infection, suggesting a predominant role of HTLV-1 over HIV-1. This hypothesis is supported by the evidence that IL-2 suppresses HIV-1 replication in some HTLV-1-infected cell lines by inducing APOBEC3G expression (Oguariri et al., 2013).

For what concerns HTLV-2, it was reported that PBMCs derived from HTLV-2 seropositive individuals undergo spontaneous proliferation in short-term cultures in association with the secretion of several cytokines, including TNF-α, IL-5, IL-6, and IFN-γ (Dezzutti et al., 1998).

By studying the cytokine pattern in HTLV-2/HIV-1-co-infected subjects, we determined that HTLV-2 drives immune activation to implement the secretion of cytokines as GM-CSF and IFN-γ (Pilotti et al., 2007), which are capable to induce a "protective" Th1 response against HIV-1 (Creery et al., 2004), since a dominant Th2 profile seems to favor HIV-1 progression (O'Garra, 1998).

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Other important immune correlates able to facilitate or suppress HIV-1 infection are the chemokines and their cellular receptors. *In vivo*, HIV-1 prevalently targets immune cells expressing the surface receptor CD4 that mediates virus binding and membrane fusion together with chemokine co-receptors (CCR5 and CXCR4; Kowalski et al., 1987; Lasky et al., 1987). Thus, changes in conformation status or surface availability of these molecules may, in turn, modify HIV-1 disease progression. Beside genetic modifications, the co-receptors expression is mainly affected by the binding of natural ligands. In particular three CCR5 binding chemokines, CCL3, CCL4, and CCL5, act as major HIV-1-suppressive factors.

Because three chemokines are released by both cultured T cells and primary CD8+ T cells in response to HTLV infection (Scarlatti et al., 1997), it was supposed that their up-regulation could explain HIV-1 inhibition observed during co-infection. The fact that HTLV-1-specific CD8+ cytotoxic T lymphocyte (CTL) clones derived from patients with HAM/TSP are actively producing CCL3 and CCL4 chemokines (Cocchi et al., 1995), reinforces the hypothesis that HTLV-1 can influence HIV-1 replication via chemokine expression and release.

As HTLV-2-infected cells become activated, they spontaneously proliferate and produce high levels of various cytokines and chemokines (Casoli et al., 1998, 2000; Lewis et al., 2000; Bovolenta et al., 2002b; Bassani et al., 2007). We observed that up-regulation of CCR5-binding chemokine expression occurs in cultured PBMCs from HTLV-2/HIV-1-co-infected individuals in comparison to HIV-1-single-infected individuals. In particular, we demonstrated that CCL3 secretion is responsible for anti-HIV-1 activity in PBMC cultures from co-infected subjects (Casoli et al., 2000). Lewis et al. (2000) associated the spontaneous synthesis of CCR5 binding chemokines to the ability of HTLV-2 regulatory proteins to transactivate *CCL4* and *CCL5* gene promoters. Also, we found that an isoform of CCL3, namely CCL3L1, which is considered the most potent anti-R5 HIV-1 chemokine (Visconti et al., 1993), was preferentially induced by HTLV-2 (Pilotti et al., 2007). Up-regulation of this chemokine leads to CCR5 down-modulation and subsequent receptor internalization (Townson et al., 2002). Although other groups have shown that the HIV-1 susceptibility is associated with *CCL3L1* gene dose, which is variable among individuals, we demonstrated that HIV-1 inhibition occurred in HTLV-2-co-infected subjects was independent by *CCL3L1* copy number, and that enhanced *CCL3L1* expression was presumably stimulated by Tax-2 protein at the transcriptional level (Pilotti et al., 2007). High levels of GM-CSF and IFN-γ secreted by PBMCs from HTLV-2-infected individuals were found to contribute to HIV-1 interference via CCR5 down-modulation (Pilotti et al., 2007).

Two recent works confirmed the pivotal role of Tax proteins in inducing CC-chemokine synthesis. The first paper reported that both recombinant Tax-1 and Tax-2 induce high levels of CC-chemokines which in turn cause CCR5 down-regulation in cultured PBMCs (Barrios et al., 2011), and the second article demonstrated that Tax-2 transactivates CC-chemokines production in cultured monocyte-derived macrophages (Balistrieri et al., 2013).

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# **JAK/STAT SIGNALING**

HTLV-1 and HTLV-2 can efficiently transform human T cells *in vitro* but significantly differ in pathogenicity. The Janus kinase (JAK)/signal transducer/activator of transcription (STAT) signaling pathway (JAK/STAT) is constitutively activated in HTLV-1-transformed cells. This may occur by autocrine stimulation of IL-2, IL-9, and IL-15 cytokines, and IL-2 and IL-15 receptor expression, as a result of Tax-induced NF-κB expression, which in turn stimulate lymphocyte proliferation (Migone et al., 1995; Mariner et al., 2001; Chen et al., 2008). HTLV-1 Tax protein is crucial for viral replication and for initiating malignant transformation and is able to inhibit host antiviral signaling via NF-κB-dependent induction of suppressor of cytokine signaling protein 1 (SOCS1) to evade innate immunity (Charoenthongtrakul et al., 2011). In T cells transformed *in vitro* by HTLV-1, the JAK/STAT activation correlates with the transition from an IL-2 dependent to an IL-2 independent phase of growth (Migone et al., 1995; Xu et al., 1995). In contrast to HTLV-1, the activation status of the JAK/STAT pathway is not constitutively activated in HTLV-2-transformed T cells. However, this pathway could be induced upon IL-2 treatment of the cells. Similarly, the constitutive activation of STAT1, STAT3, and STAT5, as well as the phosphorylation status of JAK kinases (JAK3 and JAK1), observed in HTLV-1-transformed T cell lines, was not detected in HTLV-2 transformed T cells (Mulloy et al., 1998). However, we showed that the ability of human CD34+ IL-3 dependent TF-1 cell line to proliferate after HTLV-2 exposure in conditions of IL-3 deprivation is following the production of the GM-CSF and IFN-γ, through the activation of the JAK/STAT pathway (Bovolenta et al., 2002b). Previously, it was demonstrated that a signature of PBMC freshly derived from HIV-1 infected individuals represents the constitutive activation of a C-terminal truncated STAT5 (STAT5-) and STAT1 (Bovolenta et al., 1999).

When analyzing the levels of STATs in HTLV-2 mono-infected and HTLV-2/HIV-1-dually-infected individuals, we observed that these factors are not activated in PBMCs of HTLV-2-monoinfected unless they are cultured *in vitro*, in the absence of any mitogenic stimuli, for at least8h(Bovolenta et al., 2002a). The emergence of STAT activation, mainly of STAT1, appears to be related to the secretion of IFN-γ. Of note, this is a characteristic feature of both HTLV-2 and HIV-1-mono-infected individuals. Surprisingly, HTLV-2/HIV-1 co-infection resulted in a low/absent STAT activation *in vivo*, thus correlating with a diminished secretion of IFN-γ in *ex vivo* cultivated PBMCs (Bovolenta et al., 2002a). These findings indicate that HTLV-2 and HIV-1 infection can prime T lymphocytes for STAT1 activation, but they also highlight that an interference is exerted by HTLV-2 on HIV-1-induced STAT1 activation. These results clearly suggest that HTLV-2 may interfere with HIV-1 infection at multiple levels. The observation that PBMCs obtained from both HIV-1- and HTLV-2-infected individuals activate STAT1 as a consequence of the spontaneous release of IFN-γ is supported by previous findings indicating an up-regulation of this cytokine following either HIV-1 or HTLV-2 infection (Dezzutti et al., 1998; Levine et al., 2002). An enhanced transcription of IFN-γ is also induced by the HTLV-2 Tax trans-activator (Nimer et al., 1989; Brown et al., 1991). A large body of evidence points to an increased level of

either IFN-γ or its correlates (such as neopterin or IP-10) in the plasma/serum of HIV-1-infected individuals (Poli et al., 1994), explaining the low but detectable constitutive STAT1 activation observed in HIV-1-infected. This correlation was not seen in HTLV-2-infected individuals (Bovolenta et al., 1999). Therefore, T cells from both HIV-1- and HTLV-2-infected individuals share a constitutive priming for IFN-γ secretion and, consequently, for STAT1 activation; in contrast, only HIV-1 infection is characterized by activation of STAT5 *in vivo* (Bovolenta et al., 1999). Because these factors are absent in both HTLV-2 mono-infected and HTLV-2/HIV-1 co-infected individuals, this would reflect the higher pathogenic potential of HIV-1 with respect to HTLV-2, but also highlights a dominant position of HTLV-2 over HIV-1 in terms of maintaining T cells in a primed but not completely STAT5 activated state. Since there is evidence that HTLV-2/HIV-1 co-infection is frequently associated with a state of long-term non-progression (LTNP) of HIV-1 disease (Giacomo et al., 1995; Magnani et al., 1995), HTLV-2 infection, and co-infection with HIV-1, represent an important model to better understand the interaction between human exogenous retroviruses and the immune system. IFN-γ-related priming for STAT1 activation may be an alarming signal that biases the immune response toward a Th1-model of containment of HTLV-2 infection, overcome by a peculiar STAT5 activation in HIV-1-infected individuals. Concerning the role of JAK/STAT in HTLV-1/HIV-1 co-infection no results have yet been reported.

#### **NATURAL KILLER CELL ACTIVITY**

Evidence has been accumulating on the specific targeting of innate immune defenses, and in particular of natural killer (NK) cells, by chronically replicating viruses (Marras et al., 2011). Successful weakening of NK cell response represents a critical step for virus persistence, since NK cells are involved in patrolling peripheral tissues for immediate defense against virus or tumor aggression as well as in cross-talking with critical components of innate immunity, including monocytes and dendritic cells, leading to relevant downstream impact on the shaping of adaptive immune responses (Vivier et al., 2011).

Natural killer cells main cytolytic and cytokine productive function is tightly controlled by a wide array of activating NK cell receptors which alone, or in combination with Toll-like- o cytokine- receptors, are responsible for triggering the NK cell functional program (Vivier et al., 2011). Inhibitory NK cells receptors (i.e., KIRs, NKG2A/CD94, CD85j, IRP60, SIGLEC-7), which are mainly, but not exclusively, HLA class I-specific, provide a negative regulatory signal that is able to override any triggering receptor signaling in the presence of the appropriate cognate receptor. Thus, proper NK cell function may occur only in the presence of activating receptor triggering, with reduced or absent overriding control by inhibirtory receptors sensing the respective ligands on target cells. For example, in the absence of MHC class I expression induced by virus down-modulation, the induction in the infected cells of ligands (e.g., MIC-A, MIC-B, ULBPs, Nectin-2, PVR, etc.) recognized by activating NK cell receptors would result in NK cell activation, cytokine/chemokine production, and cytotoxic activity. Several viruses and mycobacteria exploit altered expression of natural/cytotoxicity receptors in NK cells such as HIV-1 (De Maria et al., 2003), influenza (Arnon et al., 2001; Mandelboim et al., 2001; Gazit et al., 2006), HCV (Bozzano et al., 2011), mycobacterium tuberculosis, and Calmette–Guerin (Vankayalapati et al., 2002, 2004; Bozzano et al., 2009; Marras et al., 2012; reviewed in Bozzano et al., 2012 and in Marras et al., 2011), or skew the NK cell peripheral repertoire inducing expansion of NKG2C+ NK cell subsets as is the case for CMV infection (Guma et al., 2004, 2006a,b; Della Chiesa et al., 2011). Similar interference with inhibitory receptor expression (e.g., NKG2A, KIRs) may be induced by acute infection, or exploited through KIR:HLA class I haplotype interaction, as for example is observed during infection with CMV (Muntasell et al., 2013), HCV (Khakoo et al., 2004; Knapp et al., 2010; Vidal-Castineira et al., 2010), or Chikungunya virus (Petitdemange et al., 2011).

Very little is known on NK cell phenotype and function during HTLV infection, in particular when HTLV-2 is considered.

Characterization of NK cell triggering and inhibitory receptors has been so far poorly addressed to understand the differences underlying HTLV-1 and HTLV-2 diseases.

Early reports on the definition of NK cell receptors point towards a decreased NK cell activity against HTLV-1-infected MT-2 cell lines (Fujihara et al., 1991; Zheng and Zucker-Franklin, 1992). Lysis of cell lines infected with HTLV-1 by *in vitro* activated NK cells was subsequently shown to occur and to depend on viral gene expression, that may be absent in some adult T cell leukemia (ATL) lines (Stewart et al., 1996). HTLV-1 antigen-driven proliferation may result in a ATL form, as shown in mice transgenic for Tax-1 (Grossman et al., 1995) and in humans with expansions in hypofunctional NK cells (Loughran et al., 1997). The proportion of circulating mature NK cells (CD56+ CD16+) is decreased in patients with TSP/HAM (Wu et al., 2000; Brito-Melo et al., 2002; Ndhlovu et al., 2009) and other innate immune cells (NKT) are decreased during TSP/HAM as well. In addition, more recent evidence suggests that NK cells may be targeted and induced to expand by HTLV-1 through a viral load-associated but Tax-independent mechanism (Norris et al., 2010).

Overall, there is need for further insight into NK cell phenotype and function during HTLV-1/-2 infection. More precise characterization of possible changes or modulation of triggering and inhibitory receptor expression on NK cells during chronic infection would help to better understand the mechanism(s) that are exploited by HTLV-1 to divert innate immune responses and downstream CD4+ and CD8+ T cell function. In particular, it is possible that different NK cell regulation induces clinical divergence spanning from the lack of symptoms to TSP/HAM and transformation to ATL, similar to what was observed for HCV infected patients clearing infection (Khakoo et al., 2004; Alter et al., 2011) or for HIV-infected patients who control virus replication (Elite Controllers) or without disease progression in LTNPs (Marras et al., 2013). In addition, in view of the compelling evidence of a remarkable NK cell activation during HIV-1 infection both in untreated patients (Fogli et al., 2004) and in successfully treated combined antiretroviral therapy (ART) patients (Lichtfuss et al., 2012) though belonging to AIDS or non-AIDS clinical groups (Bisio et al., 2013), understanding NK cell activation during HIV-/HTLV-1 co-infection needs improved focusing. These

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considerations should be extended to HTLV-2 infection *in vitro* and *in vivo*, since no data have been yet published on NK cells in mono-infected patients.

### **ROLE OF MicroRNAs**

MicroRNAs (miRNAs) are small single-strand non-coding RNAs that repress gene expression by inhibiting translation and inducing mRNA degradation (Ambros, 2004; Bartel, 2004). MicroRNAs can be encoded by both cellular and viral genomes (Berezikov et al., 2005; Grassmann and Jeang, 2008). Furthermore, viral miRNAs have been described not only for DNA viruses but also for RNA viruses as HIV-1 and BLV (Bennasser et al., 2004; Du and Zamore, 2007; Klase et al., 2013). MicroRNAs have been found to regulate up to 92% of the human genes (Miranda et al., 2006), and also to modulate viral gene expression. These alterations could be considered key mechanisms by which the virus imbalances immune system.  The importance of RNA interference (RNAi) machinery in retroviral infection outcome was confirmed by recent findings that demonstrated how the miRNAs expression can be controlled by retroviruses (Sampey et al., 2012). Thus, the mechanism of RNAi mediated by miRNAs could be used by a virus to remain hidden from host immune surveillance by generating an advantageous cellular environments, and leading to adverse conditions for a co-invading agent.

MicroRNAs expression was studied during of HTLV-1 or HIV-1 infection but no data were so far reported for the HIV-1/HTLV-1 co-infection. Similarly, studies of miRNAs pattern during HTLV-2/HIV-1 co-infection have not been published up to date.

Concerning HIV-1, changes in host miRNA transcription levels have been detected in CD4+ purified cells from naive and LTNP HIV-1-mono-infected patients. The hypothesis that miR-NAs either could directly influence viral RNA sequences, or could affect cellular factors involved in HIV replication were confirmed by several findings (Houzet and Jeang, 2011). More specifically, it was demonstrated that five cellular miRNAs recognize the 3 end of HIV-1 mRNAs and are up-regulated in resting, but not activated, CD4+ T cells (Huang et al., 2007), providing evidence of a direct inhibition of HIV-1 replication by miRNAs. Two independent groups support this notion with the demonstration that HIV-1 *nef* gene contains a miR-29a targeted site that interferes with the replication of the virus (Ahluwalia et al., 2008; Nathans et al., 2009). Other authors found indirect inhibitory mechanisms miR-NAs mediated. Specifically, it was found that miRNAs suppress viral gene expression by decreasing PCAF (P300/CBP-associated factor) expression and interfering with histone acetylation, and leading to HIV-1 latency (Triboulet et al., 2007). However, the deregulation of cellular miRNA expression which was shown to correlate to HIV-1 latency may also favor virus production (Han and Siliciano, 2007; Huang et al., 2007). Other miRNAs were involved in the different monocyte or macrophage susceptibility to the HIV infection (Wang et al., 2009, 2011).

Recently, it was shown that HIV-1 and other retroviruses as bovine leukemia virus can affect the expression of both host miRNAs and small virus-derived interfering RNAs (Klase et al., 2007, 2009; Houzet et al., 2008; Ouellet et al., 2008; Althaus et al., 2012; Kincaid et al., 2012; Schopman et al., 2012).

Similarly to HIV-1, it was demonstrated that HTLV-1 infection is responsible for alteration of host miRNAs profile (Bellon et al., 2009; Rahman et al., 2012). By profiling the expression of miRNAs, known to be involved in differentiation and proliferation of hematopoietic cells (Merkerova et al., 2008), abnormal levels of miR-223, miR-181a, miR-150, miR-142.3p, and miR-155, were detected in both primary ATL cells and in HTLV-1 cell lines (Bellon et al., 2009). Furthermore, these samples showed an altered expression of miR-155, miR-125a, miR-132, and miR-146 that play a role in the regulation of immune response (Bellon et al., 2009). A forced expression of miR-155, induced by Tax with the contribution of NF-κB and AP-1, has been found to enhance the proliferation of HTLV-1-infected cells (Tomita, 2012).

A loss of miR-31 was also observed in primary ATL cells, where the expression of this miRNA is epigenetically regulated, and was correlated with the constitutively activation of NF-κB which contributes to the oncogenic transformation (Yamagishi et al., 2012).

Concerning miRNAs biogenesis, low levels of Drosha enzyme, a key processor of miRNAs synthesis, were found in HTLV-1 infected cell lines and infected primary cells. In addition, *in vitro* studies revealed a nuclear co-localization of Tax and Drosha, and that the interaction between the two proteins leads to absence of cleavage of miRNAs by Drosha (Van Duyne et al., 2012).

MicroRNAs can target complementary sequences of both HTLV-1and HIV-1 transcripts and act as a major line of defense against retroviruses, but on the other hand viral proteins can interfere with RNAi machine directly or by altering the expression of cellular transcription factors. HTLV-1 Tax also interacts with CRE binding (CREB) protein, a key factor to viral transcription, in the presence of the HATs CREB binding protein (CBP), p300, and P/CAF to activate HTLV-1 gene expression while HTLV-2 Tax specifically cooperates with CBP and p300 but not with p300 associated factor to enhance transcription from the viral promoter (Tosi et al., 2006). Another mechanism by which HTLV-1 may influence the host cell miRNA expression profile is through the activation of host transcription factors.  In particular, the NF-κB pathways activation is a hall mark of HTLV-1 infection and may be the result of direct interaction between Tax and the NF-κB regulatory subunit IKKγ (Sun and Yamaoka, 2005; Yasunaga and Matsuoka, 2011). Because numerous miRNA promoter sites are positively regulated by NF-κB, it can be inferred that the activation of NF-κB by Tax increases the expression of several host cell

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miRNAs (Li et al., 2012; Lukiw, 2012; Wang et al., 2012). One specific example is given by miR-155 which has been found to be up-regulated in HTLV-1-infected cells, as well as in a TNFα stimulated cell line through an NF-κB pathway (Bellon et al., 2009; Liu et al., 2011). Similar findings have already been described for HIV-1 Tat which activates NF-κB by acting through the same biochemical pathways used by a variety of other NF-κB inducers, reviewed in Baeuerle and Henkel (1994), and for other viral proteins, including Tax of HTLV-2, HBx, and MHBs of hepatitis B virus, and EBNA-2 and LMP of Epstein-Barr virus (Baeuerle and Henkel, 1994) and hemoagglutinin of influenza virus (Pahl and Baeuerle, 1995). Of particular interest is the role of miRNA in HTLV cellular transformation and recent findings demonstrate that alteration of the miRNA profile of infected cells leads to the development of ATL and HAM/TSP diseases (Sampey et al., 2012). In HIV-1 disease, a modification of chromatin by viral proteins and host cell miRNAs can contribute to the dysregulation of host cell miRNA expression and likely provides a key system used by the virus to modify host miRNA profiles (Bignami et al., 2012). Also, it was demonstrated that Tax-1 induces a prompt activation of chromatin remodeling factors as p300 and p/CAF (Rahman et al., 2012). The chromatin reorganization, affected by miRNAs which expression is in turns influenced by Tax proteins, drives the establishment of viral latent status (Aliya et al., 2012).

Recently, we studied miRNA profiles in CD4+ T-cells purified from HTLV-2-mono-infected patients and found evidence for a miRNA signature (miR-329, miR-337-5p, miR-379-5p, miR-503, miR-518d-3p, miR-203, miR-449a, miR-502-5p) that discriminates infectedfrom uninfected subjects, similarly to HIV-1 (**Table 1**; Bignami et al., 2012). Furthermore, by analyzing in detail some functional aspects of the miRNAs belonging to retroviral signature we identified 135 predicted target genes that, on the basis of gene ontology (GO) resources, revealed the presence of three ontology aspects (**Figure 1**). Interestingly, we found that the most significant GO terms, i.e., positive regulation of macromolecule and cellular biosynthetic process, are related to the formation of substances carried out by individual cells.

With specific reference to the deregulated host miRNAs linked to the development of the HTLV-1 oncogenic or neurodegenerative diseases (**Table 2**), only miR-155, a key regulatory component of the innate immune response, is differentially expressed in HIV-1 and HTLV-2 infection. Thus, we speculated that a functional impact of cross-talk between miRNA pattern, and the subsequent multifunctional pathways, may occur during HTLVs/HIV-1 co-infection.

The assessment of how the altered profiles of miRNA expression can influence viral replication and latency, as well as the efficiency of host defenses, may be useful for understanding the basis of the retroviral related modifications of cellular pathobiology and immunologic control.

# **CONCLUSION**

Is the retroviral interference relevant to HIV-1 infection? In this review we discussed how HIV-1/HTLVs co-infection can either positively or negatively affect the course of HIV-1 disease. In particular, co-infection with HTLV-2 seems to confer immunological benefits in patients with HIV-1. By contrast, HTLV-1 is mainly associated to HIV-1 disease progression and to an increased risk of TSP/HAM and ATL.

An overall picture of different effects of HTLVs on HIV infection is emerging. The widely diverging effect of HTLV-1 and HTLV-2 on the clinical course of HIV-1 progression is remarkable, and the findings initially reported for HTLV-2 are particularly surprising. The possibility that specific co-infections may improve the clinical course of HIV-1 infection by directly or indirectly interfering with HIV-1 cell entry, replication and spread, originally proposed by the study of HIV-1/HTLV-2-infected cells (Casoli et al., 2000) has been confirmed by subsequent work on GB virus C which is able to infect B cells and CD4+ or CD8+ T lymphocytes (George et al., 2006).

Modulation of the cytokine/chemokine network represents a major element of shift dynamics that regulates the co-existence of several infections. Thus, cytokines and chemokines might be considered strategic weapons in the bid to gain benefits to the

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*nd indicates not determined.*

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infecting agents. Since a poor Th1 response and a dominant Th2 response have been implicated in the pathogenesis and progression of HIV infection (Clerici and Shearer, 1993, 1994), HTLV-2 priming for a Th1 response via up-regulation of IFN-γ expression may contribute to the "protective" effect of HTLV-2 infection on HIV-1 disease progression. In the case of HTLV-1 co-infection, high frequencies of activated HTLV-1-infected CD4+ T cells can give a boost to HIV-1 replication.

An enhanced secretion of CC-chemokines, in particular of CCL3L1, was ascribed to the transactivating function of Tax-2 and the original studies of HTLV-2/HIV-1 co-infection proposed this as a key mechanism of retroviral interference. The CCL3L1 isoform down-regulates CCR5 co-receptor for HIV-1 entry leading to a LTNP status in co-infected individuals with high HTLV-2 proviral load (Pilotti et al., 2007).

In the same manner, GBV-C acquisition via blood transfusion increases the secretion of CCL5, CCL3, CCL4, and CXCL12 and by means of their NS5A and E2 proteins support the deregulation of co-receptors, thus inhibiting HIV entry and resulting in a reduced mortality in patients with advanced HIV-1 disease (Tillmann et al., 2001; Bhattarai and Stapleton, 2012). Similarly to what occur in HIV-1/HTLV-1 co-infection, HIV-1 disease progression is faster in individuals affected by HCV or HSV co-infection (Van Asten and Prins, 2004; Corey, 2007).

Analysis of JAK/STAT regulation during HTLV-2 infection provides some clues of intervention to interfere with HIV-1 replication by taking advantage of pathway interference instead of enzymatic inhibition of viral enzymes. The lack of knowledge of HTLV-1 mediated activation pathway interference has probably limited efforts in this direction.

Concerning innate immune responses in HIV-1 co-infection with either HTLV-1 or HTLV-2, no data are so far available for NK cell function, and little is known on other innate immune cellular mechanisms. The observed effect of proviral load, but not of Tax-1, on NK cell proliferation during HTLV-1 infection (Norris et al., 2010), is likely to impact also on HIV-1/HTLV-1 co-infected patients, leading to enhanced NK cell activation and possibly disease progression. On the contrary, when considering HTLV-2/HIV-1 co-infection, evidence of HTLV-2 interference

**Table 2 | MicroRNAs either up- or down-regulated in HTLV-1 infected cells vs HIV-1 and HTLV-2 infection.**

*ns indicates not significant; and nd not determined.*

with STAT/JAK pathways could possibly be linked to a decreased HIV-1-associated NK cell activation. Further work in this direction is needed to improve our understanding of the mechanism(s) associated with the positive effect of HTLV-2 on HIV-1 disease course, and of the underlying causes leading to AIDS progression by HTLV-1/HIV-1 co-infection and/or to HTLV-1 associated morbidity.

Since miRNAs have been correlated with viral life cycle, they represent good candidates among the top cellular factors to be used by HTLVs to favor their own replication. In the case of coinfection with HIV-1, HTLV proteins were found to interact with cellular chromatin modifying enzymes and with cellular transcription and other immune factors. The interaction of HTLV Tax proteins with cellular factors results in the alteration of miRNAs profile that in turns can activate transcription and consequently viral replication. Furthermore a likely interplay between two competing mechanisms is taking place: the ability of Tax to manipulate chromatin structure and the innate host cellular defense mechanism of RNAi to regulate pathogen gene expression.

HTLV/HIV-1 co-infection can be considered as a useful model for the study of new strategic approaches for HIV-1 vaccine development, as suggested by the finding that the exposure of HTLV-2 infected macaques to SIVmac251 was not accompanied by an exacerbation of SIVmac251 infection (Gordon et al., 2010).

#### **ACKNOWLEDGMENTS**

This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) through a 2008 AIRC-Cariverona Regional grant (Maria G. Romanelli) and by Joint project University of Verona-GEMIB laboratory of Parma (IT).

#### **REFERENCES**

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virus type 1 (HTLV-1) and HTLV-2 Tax in primary human glial cells. *J. Virol.* 81, 1690–1700. doi: 10.1128/JVI.01513-06

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type 1 tax transactivator with transcription factor IIA. *Mol. Cell. Biol.* 16, 4656–4664.

myelopathy/tropical spastic paraparesis (HAM/TSP). *J. Neurol. Sci.* 105, 99–107. doi: 10.1016/0022-510X(91)90125-Q

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binding sites and their corresponding heteroduplexes. *Cell* 126, 1203–1217. doi: 10.1016/j.cell.2006.07.031

1-transformed T lymphocytes. *Retrovirology* 5:100. doi: 10.1186/1742-4690- 5-100

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*Immunol.* 32, 3016–3026. doi: 10.1002/1521-4141(2002010)32:10<3016::AID-IMMU3016>3.0.CO;2-D

(HAM/TSP) and hyperglycemic myelitis by analyses of memory CD4 positive T cell subsets and NK cell subsets. *J. Neurol. Sci.* 177, 24–31. doi: 10.1016/S0022- 510X(00)00322-1

**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.

*Received: 31 July 2013; paper pending published: 02 September 2013; accepted: 20 November 2013; published online: 23 December 2013.*

*Citation: Pilotti E, Bianchi MV, De Maria A, Bozzano F, Romanelli MG, Bertazzoni U and Casoli C (2013) HTLV-1/-2 and HIV-1 co-infections: retroviral interference on host immune status. Front. Microbiol. 4:372. doi: 10.3389/fmicb.2013. 00372*

*This article was submitted to Virology, a section of the journal Frontiers in Microbiology. Copyright © 2013 Pilotti, Bianchi, De Maria, Bozzano, Romanelli, Bertazzoni and Casoli. 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.*

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# Differential transforming activity of the retroviralTax oncoproteins in humanT lymphocytes

# *Tong Ren1 and Hua Cheng2\**

*1 Penn State Hershey Cancer Institute, Hershey, PA, USA*

*2 Institute of Human Virology, School of Medicine, University of Maryland, Baltimore, MD, USA*

#### *Edited by:*

*Umberto Bertazzoni, University of Verona, Italy*

#### *Reviewed by:*

*Masanori Baba, Kagoshima University, Japan Edward Harhaj, Johns Hopkins School of Medicine, USA Masahiro Fujii, Niigata University Graduate School of Medical and Dental sciences, Japan*

#### *\*Correspondence:*

*Hua Cheng, Institute of Human Virology, School of Medicine, University of Maryland, 655 West Baltimore Street, Baltimore, MD 21201, USA e-mail<EMAIL_ADDRESS>Human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are two closely related retroviruses. HTLV-1 causes adultT cell leukemia and lymphoma, whereas HTLV-2 infection is not etiologically linked to human disease. The viral genomes of HTLV-1 and -2 encode highly homologous transforming proteins, Tax-1 and Tax-2, respectively. Tax-1 is thought to play a central role in transforming CD4+ T lymphocytes. Expression of Tax-1 is crucial for promoting survival and proliferation of virally infected human T lymphocytes and is necessary for initiating HTLV-1-mediated oncogenesis. In transgenic mice and humanized mouse model, Tax-1 has proven to be leukemogenic. Although Tax-1 is able to efficiently transform rodent fibroblasts and to induce lymphoma in mouse model, it rarely transforms primary human CD4+ T lymphocytes. In contrast, Tax-2 efficiently immortalizes human CD4+ T cells though it exhibits a lower transforming activity in rodent cells as compared to Tax-1. We here discuss our recent observation and views on the differential transforming activity of Tax-1 and Tax-2 in human T cells.

**Keywords: HTLV-1/-2,Tax, human T lymphocytes, immortalization, transformation**

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HTLV-1 and -2 are two highly homologous human retroviruses.  Although both HTLV-1 and -2 utilize GLUT-1, a ubiquitously expressed cell surface (SU) protein, as the receptor for viral entry (Igakura et al., 2003; Manel et al., 2003; Coskun and Sutton, 2005), they exhibit distinct cell tropism. HTLV-1 preferentially infects and transforms CD4+ T cells, whereas HTLV-2 has tropism for CD8+ T cells, which suggests that there exists a CD8+ T cell-specific co-factor for assisting viral entry and permissive replication of HTLV-2 (Ijichi et al., 1992). HTLV-1 is the etiological factor that causes adult T cell leukemia and lymphoma (Hattori et al., 1981; Hinuma et al., 1981, 1982; Yamaguchi et al., 1984). HTLV-2 was detected in CD8 T cells from a patient with hairy cell leukemia, a rare type of leukemia that affects B lymphocytes (Kalyanaraman et al., 1982), however the causative link between HTLV-2 infection and hairy cell leukemia has not been established. Thus far, HTLV-2 infection has not been etiologically linked to any human disease.

HTLV-1 viral genome is known to encode two oncogenic products, Tax-1 and HBZ (Satou et al., 2006; Satou and Matsuoka, 2012). Tax-1 is the first recognized viral component to display oncogenic potential. Evidence showed that Tax-1 plays an essential role in mediating transformation of T lymphocytes (Tanaka et al., 1990). The transforming activity of Tax-1 in lymphocytes was first demonstrated using HTLV-1 infectious molecular clone. The wild type molecular clone efficiently transformed human primary T cells, while the molecular clone with disrupted *tax* gene failed to do so, defining the role of Tax-1 as a viral transforming protein (Yamaoka et al., 1996, 1998; Akagi et al., 1997; Matsumoto et al., 1997; Iwanaga et al., 1999). However, this experiment did not rule out potential participation of other viral components in HTLV-1-mediated cell transformation. Unexpectedly, when expressed alone, Tax-1 rarely immortalizes or transforms terminally differentiated T cells derived from human peripheral blood (Bellon et al., 2010). In humanized mouse model, Tax-1-expressing human CD34+ blood progenitor cells, when transplanted into NOD/SCID mice, developed T cell lymphoma of human origin (Banerjee et al., 2010). This experimental finding suggests that in some cases, leukemic cells might be evolved from HTLV-1-infected CD34+ blood progenitor cells rather than from infected mature T cells. This assumption is yet to be validated.

A comparative study on two highly homologous Tax proteins from HTLV-1 and -2 is crucial in deciphering the molecular pathogenesis of HTLV-1-associated ATL. We expressed the Tax proteins in human T cells isolated from peripheral blood to imitate the process of HTLV-1 infection. This prospective approach would allow us to evaluate sequential oncogenic events in Tax-mediated proliferation, immortalization and transformation of human primary T cells. Normal lymphocytes isolated from healthy donors are typically at quiescent stage. Stimulation with mitogens such as PHA, recombinant IL-2 or mitogenic anti-CD3/-CD28 antibodies results in rapid proliferation of peripheral blood lymphocytes with displaying lymphoblastic morphology. Activated lymphocytes stop growing 2–3 weeks following stimulation at normal culture conditions by reaching the checkpoint of T cell senescence. HTLV-1 infection overrides replicative senescence of T cells, promotes continuous growth of HTLV-1-infected T cells and induces immortalization, ultimately resulting in IL-2-independent growth of the transformed T cells. At each step of aberrant proliferation, immortalization or transformation during HTLV-1 infection of T cells, a variety of cellular oncogenic alterations are expected to occur. Thus, studying these processes will provide crucial insights into the pathogenesis of ATL.

We previously hypothesized that in the absence of other viral proteins, Tax-1 is sufficient to immortalize human mature CD4+ T cells while Tax-2 preferentially immortalizes CD8+ T cells. We unexpectedly found that both Tax-1 and Tax-2 failed to immortalize human primary CD8+ T cells, and Tax-2 was able to immortalize human CD4+ T cells more efficiently than Tax-1 in the study with including 12 healthy blood donors. A similar finding was reported later (Imai et al., 2013). In addition, a majority of Tax-2-immortalized CD4+ T cell lines grew *in vitro* at the growth rate similar to some lymphoblastic leukemia cells (Ren et al., 2012). In contrast, Tax-1-immortalized CD4+ T cells were slow growing and exhibited spontaneous cell death at normal culture conditions (Ren et al., unpublished data). These experimental results were unlikely caused by technical variation because Tax-1 and Tax-2, in which their expressions were driven from human elongation factor promoter, were introduced into human primary T cells via VSV-G pseudotyped lentiviruses at similar efficiency.

Tax-2 appears to be more oncogenic than Tax-1 in primary CD4+ T cells when they are expressed alone, however Tax-1 is clearly oncogenic in CD4+ T cells in the context of an intact proviral clone and other expressed viral proteins. Although both Tax proteins are highly homologous, Tax-1 does exhibit distinct structural features in which Tax-2 lacks. Tax-1 contains a PDZ binding motif (PBM) in its carboxyl-terminus that is importantfor binding to DLG-1 and other PDZ containing proteins, and these interactions were thought to play an important role for cell transformation by Tax-1. In addition, Tax-1, but not Tax-2, undergoes K63-linked polyubiquitination as part of its mechanism to activate NF-κB (Shembade et al., 2007; Journo et al., 2013). It is apparent that these differences do not account for the stronger transforming capability of Tax-2 in primary CD4+ T cells. Tax-1 may acquire a full transforming ability in human CD4+ T cells in cooperation with HBZ, an HTLV-1 antisense gene product that is constitutively transcribed and remains intact in ATL cells. In fact, HBZ itself was proven leukemogenic in mouse model. 30% of HBZ-transgenic (Tg) mice developed T cell lymphoma and most of HBZ Tg mice developed spontaneous inflammatory lesions (Satou and Matsuoka, 2012). Furthermore, increasing evidences showed that the functional interaction between Tax and HBZ is crucial for HTLV-1 oncogenesis in patients. Since high levels of Tax-1 expression lead to hyper-activation of NF-κB, consequently resulting in replicative senescence or even cell death (Zhi et al., 2011), HBZ antagonizes Tax-1's toxic function to facilitate immune evasion and to promote cell cycle progression and proliferation. Interestingly, Tax-2 also induces hyper-activation of NF-κB in CD4+ T cells, and it is capable of promoting T cell proliferation in the absence of the antisense gene product encoded from HTLV-2. This suggests that unlike Tax-1, Tax-2 is a singly important viral component of the HTLV-2 genome to execute its transforming activity in CD4+ T cells.

We also found that the Tax proteins had their own preference on donor selection, and Tax-1 and Tax-2 immortalized CD4+ T cells from different blood donors. In one unusual case, Tax-1 immortalized CD4+ T cells while Tax-2 immortalized CD4+ myeloid dendritic cells (mDCs) from CD4+ cell pools of the same blood donor (Ren et al., unpublished data). These mDCs showed negative lineage markers (CD3−/TCRαβ- /TCRγδ-/CD19−/CD56−/CD16−/CD14−/CD34−) and positive myeloid/dendritic cell markers (CD11c+/CD123+/HLA-DR+/CD2+/CD71+/CD117+/CD33+). Because the number of mDCs in healthy donors is typically less than 1%, the finding that Tax-2 is able to immortalize mDCs from enriched CD4+ pools suggests that the Tax-2-mediated immortalization of blood cells is highly selective. Aside from this finding, genotyping analysis of one Tax-2-established CD4 T cell line showed that these cells were clonal population. Together, our data demonstrate that in the absence of other viral components, Tax-2 alone is sufficient to promote clonal expansion of selected subsets of human primary CD4+ T cells. The potential cellular factor that contributes to Tax's selectivity is currently unclear, which will certainly be an interesting subject for further investigation.

It is known that HTLV-1 has preferential tropismfor CD4 T cells in healthy carriers, HAM/TSP and ATL patients. In addition to utilization of GLUT1 as an entry factor for HTLV viruses, both viruses also utilize NRP1. HTLV-1 and -2 have a differential requirement for heparan sulfate proteoglycans (HSPGs). Distinct from HTLV-1, HTLV-2 is not dependent on HSPGs for cell entry (Jones et al., 2006). Because HTLV-1 utilizes a ubiquitously expressed receptor for viral entry, it is expected that this virus would have the capacity to infect cell types other than CD4+ T cells. Indeed, CD8+ lymphocytes, monocytes and B-lymphocytes are found to harbor HTLV-1 (Koyanagi et al., 1993; Eiraku et al., 1998; Nagai et al., 2001). In addition, macrophages, dendritic cells, megakaryocytes as well as glial cells (astrocytes and microglial cells) are also the cell targets for HTLV-1 infection *in vivo* (Macatonia et al., 1992; Koyanagi et al., 1993; Levin et al., 1997; Grant et al., 2002). HTLV-1 isolates can be transmitted to primary human endothelial cells and basal mammary epithelial cells *in vitro* (Ho et al., 1984; Hoxie et al., 1984). Regardless of a broad cell tropism of HTLV-1, this virus exclusively causes malignant transformation of infected CD4+ T cells, suggesting that a panel of CD4 cell-specific cellular factors is crucial in assisting HTLV-1-mediated oncogenesis. Conversely, it is also possible that other types of cells may express cellular repressors to restrict HTLV-1 oncogenesis as seen in other retroviruses. For instance, SAMHD1 is able to restrict HTLV-1 replication in myeloid cells (St. Gelais and Wu, 2011). Unlike HTLV-1, HTLV-2 preferentially infects CD8+ T cells in human. However, Tax-2, when expressed by a pseudotype form of lentivirus, executes its transforming activity in CD4+ T cells. This phenomenon of disintegrated cell tropism (in CD8+ T cells) and disease-causing capacity (in CD4+ T cells) may partially explain why HTLV-2 does not currently cause leukemia in human.

These findings may have important implications on pathogenic virus evolution. Many viruses are considered non-pathogenic, because they do not apparently cause human disease based on current data. These non-pathogenic viruses usually do not receive much attention as known pathogenic viruses do. From an evolutionary point of view, recombination and cell tropism switch among homologous pathogenic and non-pathogenic viruses may occur, potentially generating a more lethal virus. A recent study

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demonstrated that cell tropism switch could be initiated by artificially generating chimeric envelope proteins. Switch of the envelope SU domains between HTLV-1 and HTLV-2 alters the tropism of HTLV-1 from CD4 to CD8+ T cells (Kannian et al., 2013). In the case of HTLV-2, the infection rate among IV drug abusers is as high as 30%. Since no treatment is available for HTLV-2 infection and host immunity is not sufficient to eradicate this virus, it is possible that the rate of infection is going to rise due to the presence of accumulative HTLV-2 reservoirs in host CD8+ T cells. Indeed, co-infection of HTLV-2 with HTLV-1 or with HIV-1 has been documented and is increasing (Uchiyama et al., 1977; Shimoyama, 1991), probably because these viruses share similar transmission pathways and risk factors. HTLV-2 could be eventually modified in dendritic cells or monocytes that are co-infected with HTLV-1 or HIV-1 to acquire new cell tropism for CD4+ T cells, leading to its transformation of CD4+ T cells. Although naturally occurring cell tropism switch among homologous viruses may take unpredictable amount of time, this event does occur evolutionarily.

Although Tax-2 does not transform normal CD8 T cells, this viral protein exhibits its transforming activity in CD8+ T cells derived from T cell type of large granular lymphocyte leukemia (T-LGLL). T-LGL leukemia is the malignancy of CD8+ cytotoxic T cells, which usually occurs in elderly patients. The disease course is frequently associated with autoimmune diseases (Rose and Berliner, 2004). Primary T-LGL leukemia cells display the CD3+/CD8+/CD57+ SU markers, representing activated cytotoxic T lymphocytes. Despite T-LGLL cells are leukemic cells, these cells do not grow in culture. By expressing Tax-2 in these cells, long-term growth and clonal expansion of T-LGLL cells could be achieved (Ren et al., unpublished data).

In summary (see **Table 1**), Tax-1 and Tax-2 display differential transforming activities in human T lymphocytes. Tax-2 demonstrates a more potent activity than Tax-1 in immortalizing human terminally differentiated CD4+ T cells. Besides, Tax-2, not Tax-1, is able to transform CD8+ T cells from T-LGL leukemic cells with preexisting oncogenic profile. The ability of Tax-2 to establish specific subsets of T cell lines implicates its utilization in studying

#### **REFERENCES**

*Blood* 115, 2441–2448. doi: 10.1182/blood-2009-08-241117

**Table 1 | Differential activities of Tax-1 and Tax-2 in human T cells.**

a*Tax-1 recruits I*κ*B kinases into lipid raft microdomains for persistent activation of NF-*κ*B signaling, while Tax-2 activation of NF-*κ*B does not appear to involve lipid rafts (Huang et al., 2009).*

b*Tax-1 is able to immortalize human primary CD4*+ *T cells with low efficiency (2 out of 12), and Tax-1-immortalized T cells grow slowly in culture and experience spontaneous cell death.*

c*Tax-2 immortalizes human primary CD4*+ *T cells more efficiently than Tax-1 (4 out of 12). Tax-2-immortalizedT cells grow in culture at a rate comparable to some lymphoblastic leukemia cells with healthy growth appearance.*

human T cell biology and in developing leukemia model. Furthermore, the hypothesis that Tax-2 has a disease-causing capacity in CD4+ T cells needs to be validated in humanized mouse model.

#### **ACKNOWLEDGMENTS**

Research reported in this publication was supported by the National Institute of Allergy And Infectious Diseases of the National Institutes of Health under award number R01AI090113 to Hua Cheng. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

individuals. *J. Immunol.* 161, 6674– 6680.

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antigen (ATLA) in sera from patients with ATL and controls in Japan: a nation-wide sero-epidemiologic study. *Int. J. Cancer* 29, 631–635. doi: 10.1002/ijc.2910290606

human endothelial cells by human Tcell leukemia virus type I. *Proc. Natl. Acad. Sci. U.S.A.* 81, 7591–7595. doi: 10.1073/pnas.81.23.7591

leukemia. *Science* 218, 571–573. doi: 10.1126/science.6981847

*Oncologist* 9, 247–258. doi: 10.1634/theoncologist.9-3-247

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leukemia virus (ATLV or HTLV) proviral DNA for the classification of T-cell malignancies. *Rinsho Ketsueki* 25, 547–553.

**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.

*Received: 30 July 2013; accepted: 05 September 2013; published online: 23 September 2013.*

*Citation: Ren T and Cheng H (2013) Differential transforming activity of the retroviral Tax oncoproteins in human T lymphocytes. Front. Microbiol. 4:287. doi: 10.3389/fmicb.2013.00287*

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

*Copyright © 2013 Ren and Cheng. 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.*

**REVIEW ARTICLE** published: 17 September 2013 doi: 10.3389/fmicb.2013.00275

# The need to accessorize: molecular roles of HTLV-1 p30 and HTLV-2 p28 accessory proteins in the viral life cycle

# *Rajaneesh Anupam1,2†, Rami Doueiri1,2† and Patrick L. Green1,2,3,4\**

*1 Center for Retrovirus Research, The Ohio State University, Columbus, OH, USA*

*2 Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, USA*

*3 Comprehensive Cancer Center and Solove Research Institute, The Ohio State University, Columbus, OH, USA*

*4 Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH, USA*

#### *Edited by:*

*Umberto Bertazzoni, University of Verona, Italy*

#### *Reviewed by:*

*Vincenzo Ciminale, University of Padova, Italy Donna M. D'Agostino, University of Padova, Italy*

#### *\*Correspondence:*

*Patrick L. Green, Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus, OH 43210, USA e-mail<EMAIL_ADDRESS>
†*Rajaneesh Anupam and Rami Doueiri are joint first authors.*

Extensive studies of human T-cell leukemia virus (HTLV)-1 and HTLV-2 over the last three decades have provided detailed knowledge on viral transformation, host–viral interactions and pathogenesis. HTLV-1 is the etiological agent of adult T cell leukemia and multiple neurodegenerative and inflammatory diseases while HTLV-2 disease association remains elusive, with few infected individuals displaying neurodegenerative diseases similar to HTLV-1. The HTLV group of oncoretroviruses has a genome that encodes structural and enzymatic proteins Gag, Pro, and Env, regulatory proteins Tax and Rex, and several accessory proteins from the pX region. Of these proteins, HTLV-1 p30 and HTLV-2 p28 are encoded by the open reading frame II of the pX region. Like most other accessory proteins, p30 and p28 are dispensable for *in vitro* viral replication and transformation but are required for efficient viral replication and persistence *in vivo*. Both p30 and p28 regulate viral gene expression at the post-transcriptional level whereas p30 can also function at the transcriptional level. Recently, several reports have implicated p30 and p28 in multiple cellular processes, which provide novel insight into HTLV spread and survival and ultimately pathogenesis. In this review we summarize and compare what is known about p30 and p28, highlighting their roles in viral replication and viral pathogenesis.

**Keywords: HTLV-1, HTLV-2, p30, p28, accessory proteins**

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# **INTRODUCTION**

Human T-cell leukemia virus (HTLV) are complex deltaretroviruses, with HTLV-1 and HTLV-2 causing the most prevalent worldwide infections. HTLV-1 infects approximately 25 million people worldwide and is endemic in Japan, Africa, South America, Iran, and the Caribbean basin (Proietti et al., 2005; Goncalves et al., 2010; Iwanaga et al., 2012). In contrast, HTLV-2 is endemic in Central and West Africa (Goubau et al., 1992; Gessain et al., 1993; Goubau et al., 1993), in native Amerindian populations in North, Central, and South America (Hjelle et al., 1990; Lairmore et al., 1990; Heneine et al., 1991; Levine et al., 1993), and among cohorts of intravenous drug users (IVDUs) in the United States and Europe. Although few HTLV-1 infected individuals (1–5%) develop adult T-cell leukemia/lymphoma (ATL/ATLL) or HTLV-1-associated myelopathy/tropic spastic paraparesis (HAM/TSP), all infected individuals exhibit a persistent antiviral immune response that fails to clear the virus (Poiesz et al., 1980; Yoshida et al., 1982; Takatsuki, 2005; Yoshida, 2005; Lairmore et al., 2012). Furthermore, HTLV-2 infection is not associated with leukemia/lymphoma, although in rare cases it causes a neurodegenerative condition similar to HAM/TSP (Araujo and Hall, 2004).

The genome of HTLV encodes structural and enzymatic genes typical of all retroviruses. In addition, the pX region located between the *env* gene and 3 long terminal repeat (LTR) encodes four open reading frames (ORF I to IV) with a potential for encoding several proteins (Berneman et al., 1992; Ciminale et al., 1992; Koralnik et al., 1992). As a result of complex splicing, various mRNAs encode regulatory and accessory proteins. Positive regulators of viral gene expression, Tax and Rex, are encoded by a doubly spliced bicistronic mRNA from ORFs IV and III, respectively (Felber et al., 1985; Kiyokawa et al., 1985). Reverse transcription-PCR of mRNA from HTLV-1 infected cell lines and uncultured primary lymphocytes from ATL patients has shown that alternative splicing produces the accessory proteins p12, p30, and p13 (Berneman et al., 1992; Ciminale et al., 1992; Koralnik et al., 1992). A singly spliced mRNA containing ORF-I codes for the accessory protein p12 that can be cleaved to produce a smaller protein, p8 (Van Prooyen et al., 2010). p30 is encoded by a doubly spliced message in which ORF-II is linked to the Tax initiation codon located on exon II, resulting in a 241 amino acid protein. ORF-II also can be singly spliced to produce mRNA that can encode p13 from the internal initiation codon in ORF-II, which corresponds essentially to the last 87 amino acids of p30. Similar studies in the MoT cell line identified accessory proteins in HTLV-2: a bicistronic doubly spliced mRNA encodes p10 and p11 from ORF-I and ORF-V, respectively, and two distinct bicistronic singly spliced mRNAs encoded p28 from ORF II as well as the truncated ORF III isoforms of Rex (Ciminale et al., 1995). p30 and p28 share certain amino acid sequence homology, the last 50 amino acids of p30 share 70% homology with the first 50 amino acids of p28 (Ciminale et al., 1995), and both are nuclear/nucleolar proteins (Koralnik

et al., 1993; Ciminale et al., 1995; D'Agostino et al., 1997; Younis et al., 2004). In addition, newly identified proteins, HTLV-1 basic leucine zipper factor (HBZ) and anti-sense protein HTLV-2 (APH-2), are encoded from the antisense genome strand in HTLV-1 and HTLV-2, respectively (Gaudray et al., 2002; Halin et al., 2009).

Tax transactivates viral gene transcription by recruiting transcription factors p300/CREB binding protein (CBP), CREB and AP-1 to the Tax response element (TRE) in the LTR region (Seiki et al., 1986). Tax drives cellular transformation through its ability to alter cellular gene expression, signaling pathways, and cell cycle (Grassmann et al., 2005). Of the factors targeted by Tax, NFκB clearly plays a prominent role in deregulation of cellular gene expression and cellular transformation (Smith and Greene, 1990; Rosin et al., 1998; Robek and Ratner, 1999; Ross et al., 2000). Although Tax is indispensable for viral transformation, the mechanism by which the virus persists *in vivo* leading to T-cell transformation is not clearly understood (Matsuoka and Jeang, 2007). Studies suggest that HBZ and accessory proteins might play a role *in vivo* in HTLV-1 viral persistence and T-cell malignant transformation (Bartoe et al., 2000; Arnold et al., 2006; Arnold et al., 2008; Valeri et al., 2010). Rex binds to the Rex response element (RxRE) on unspliced and singly spliced viral mRNAs to facilitate their nuclear-cytoplasmic export for translation in the cytoplasm (Younis and Green, 2005). p30 and p28 mRNA species can be identified in infected cells (Li and Green, 2007) and in cells from HTLV-1 infected patients (Rende et al., 2011; Bender et al., 2012), albeit at 103–104 lower levels than *tax/rex* mRNA.

Reports identifying antibodies and cytotoxic CD8+ T-cells in infected patients with HTLV-1 (symptomatic and asymptomatic) against p30, demonstrate the importance of HTLV-1 accessory proteins in viral persistence and ultimately in the viral life cycle (Jacobson et al., 1992; Chen et al., 1997; Pique et al., 2000). However no studies to date have attempted to identify antibodies or cytotoxic CD8+ T-cells against p28 in HTLV-2 infected patients. In this review we will compare the current knowledge on p30 and p28, highlighting the differences and similarities in their roles in the HTLV life cycle.

### *IN VIVO* **ROLE OF P30 AND P28**

Initial studies to understand the role of p30 were performed by deleting either ORF-I and –II, or ORF II alone from an HTLV-1 infectious molecular clone, which showed that p30 is dispensable for viral gene expression, infectivity, replication, and T-cell immortalization *in vitro* (Derse et al., 1997; Robek et al., 1998). To examine the role of p30 in viral replication and infectivity *in vivo*, T-cell lines immortalized with a viral clone containing mutations ablating both p30 and p13 reading frames (Bartoe et al., 2000) or p30 alone (Silverman et al., 2004) were generated. It was noted that loss of p30 and p13 or p30 alone resulted in a significantly reduced antibody response and lower proviral loads in rabbits indicating that p30 and p13 are required for maintenance of high proviral loads *in vivo* (Bartoe et al., 2000; Silverman et al., 2004). In addition, when p30 alone was ablated, reversion to the wild type sequence was observed, underlining the *in vivo* importance of p30 in maintaining high level viral infection (Silverman et al., 2004). It is noteworthy that these first experiments were performed before the identification of the HTLV-1 HBZ, which is encoded from the anti-sense viral mRNA that overlaps ORFs in the pX region including p30 ORF-II (Gaudray et al., 2002). It is very likely that the mutations ablating p30 expression interfered with the expression of HBZ, which also was shown to be important for viral infectivity *in vivo* (Arnold et al., 2006). Recently, to investigate the relative contribution of HBZ and p30 to viral infectivity *in vivo*, B cell lines expressing viral mutants with specific ablation of HBZ and p30 were generated and used to infect rabbits and macaques. In contrast to the earlier *in vivo* rabbit studies (Silverman et al., 2004), it was shown that viral mutants lacking p30 had little effect on antibody response, infectivity or proviral loads. However, lack of HBZ resulted in reduced proviral loads in rabbits. In addition, there was no evidence of reversion to wild type sequence by the p30 or HBZ mutants (Valeri et al., 2010). Interestingly, in macaque infections it was noted that lack of p30 and HBZ resulted not only in reduced antibody response and infectivity but also reversion to wild type sequence indicating that p30 and HBZ are important for viral infectivity in macaques (Valeri et al., 2010). The discrepancy regarding the requirement for p30 in rabbit infections may be due to the contribution of HBZ to the viral infection. In the same study it was shown that p30 is also required for productive cellfree infection of dendritic cells *in vitro*. The requirement of p30 in terms of *in vitro* viral infection might be cell type-dependent.

On the other hand, an initial report revealed that deletion of the pX region of HTLV-2 causes a reduction in viral replication but not infectivity *in vivo*, but had no effect *in vitro* (Cockerell et al., 1996). To delineate the role of p28, Yamamoto et al. (2008) created a p28 knockout HTLV-2 molecular clone and stably transfected virus producing cells with the wild type HTLV-2 molecular clone or the HTLV-2p28 mutant. The irradiated producer cells were cocultured with human PBMCs, and viral p19 Gag production and cell survival were measured over time. No difference was observed between HTLV-2 and HTLV-2p28 indicating that p28 was not required for *in vitro* primary T lymphocyte infectivity, proliferation, and immortalization. The authors also determined the effect of p28 on viral persistence *in vivo* using a rabbit model and found that rabbits infected with HTLV-2 lacking p28 had lower antibody responses and reduced proviral load compared to those infected with wild type HTLV-2 (Yamamoto et al., 2008). The mutation to delete p28 had no effect on the APH-2 gene providing clear evidence that p28 contributes to viral persistence *in vivo*.

Collectively, it is clear that p30 (HTLV-1) and p28 (HTLV-2) are required for efficient viral replication and persistence *in vivo*. Recent findings discussed below provide various possible mechanisms to explain the role of these accessory proteins in viral infection and/or pathogenesis.

#### **VIRAL GENE EXPRESSION**

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#### **TRANSCRIPTIONAL REGULATION**

It was demonstrated that p30 is a nuclear/nucleolar localizing protein (Ciminale et al., 1992; Koralnik et al., 1992; see **Figure 1** for summary of key p30 domains). Structurally, p30 contains a bipartite nucleolar/nuclear localization signal (NoRS/NLS), which is comprised of two arginine rich domains (amino acids 73–78 and 91–98; D'Agostino et al., 1997). A later study reconfirmed these regions to be a nucleolar retention signal and also identified two other possible nuclear localization signals in the amino (N) and

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carboxy (C) termini of p30 (Ghorbel et al., 2006). The latter study also implicated the role of transcription and RNA-interaction in nucleolar/nuclear localization of p30 with the specific interaction of p30 with a 60S ribosomal protein L18a (Ghorbel et al., 2006). Similar to p30, expression of p28 cDNA resulted in nuclear localization of p28 (Ciminale et al., 1995; Younis et al., 2004).

p30 has marked homology with serine and threonine rich transcription factors Oct-1 and-2, Pit-1, and POU-M1 suggesting that p30 could function as a transcriptional regulator (Ciminale et al., 1992). The initial evidence of p30 as a transcriptional regulator was provided by the ability of p30 to differentially modulate CREB responsive promoters by acting as a transactivator when fused to the DNA binding domain of Gal4, and behaving as a repressor when expressed alone. In addition, p30 increased transcription from the TRE at a low concentration but repressed transcription at a higher concentration in the presence and absence of Tax (Zhang et al., 2000). On the other hand, in a similar Gal4 reporter assay, p28 failed to modulate Gal4 DNA binding ability, demonstrating that p28 is devoid of transcriptional activity (Younis et al., 2006). Subsequently, p30 was shown to regulate transcription by binding to CBP/p300. The interaction of p30 and CBP/p300 was evaluated by two hybrid screening, immunoprecipitation, and localization studies. The binding of p30 to p300 was mapped to a highly conserved KIX region of p300, which is also the binding site of Tax. The transcriptional repression of TRE by p30 is a result of competition between p30 and Tax for p300 binding and disruption of the assembly of Tax/CBP/P300 on the TRE (Zhang et al., 2001). The p300 binding region was mapped to amino acids 1–132 whereas the TRE transcription repression function of p30 was mapped to amino acids 100–179. This 80 bp region contains five of the six lysine residues in p30 of which lysine 106 was found to be important for repression. Unlike wild type p300, mutant p300 with impaired histone acetyltransferase activity (HAT) was unable to rescue p30-mediated LTR transcription repression. Moreover, p30 LTR repression was enhanced by histone deacetylase (HDAC-1) and inhibited by Trichostatin A (inhibitor of HDAC-1). These data suggest that the LTR transcription repression function of p30 could be regulated by acetylation on lysine or that these effects are due to global effects of HATs and HDACs on transcription (Michael et al., 2006).

p30 has the ability to transcriptionally activate numerous cellular genes. In one study it was shown that p30 binds and stabilizes the Myc/Tat-interacting protein (TIP60) transcriptional complex (Awasthi et al., 2005). Furthermore, p30 binds and recruits the 60-kDa (TIP60) to the Myc transcription complex to activate Myc responsive genes, which requires the HAT of TIP60. In addition, the transcriptional activity of p30 was dependent on transforming transcriptional activator protein (TRRAP)/p434. The idea that p30 plays a role in transformation by enhancing Myc-responsive genes was supported by the data showing that p30 cooperated with Myc in focus-forming transformation assays (Awasthi et al., 2005). More evidence for regulation of cellular transcription by p30 comes from a study showing that p30 can interact with the PU.1 transcription factor through its DNA binding domain resulting in the suppression of PU.1 DNA binding. Subsequently, the expression of p30 decreased cell surface expression of TLR4 as the expression of TLR4 is directly under the influence of PU.1 transcription activation. Concurrently, reduced TLR4 expression by p30 resulted in reduced pro-inflammatory cytokines TNF-α, MCP-1 and IL-8 whereas the expression of anti-inflammatory cytokine IL-10 was increased. Additionally it was found that p30 increased phosphorylation of GSK3-β leading to an increase in IL-10 production (Datta et al., 2006). These data indicate that p30 can influence innate and adaptive immunity to potentially facilitate viral spread by influencing cellular transcription. Similar experiments to investigate the ability of p28 to regulate transcription of cellular genes demonstrated that unlike p30, p28 has no transcription function, although chromatin immunoprecipitation assays (ChIP) show that p28 is recruited to the LTR along with the transcriptional machinery (Younis et al., 2006).

In order to further characterize the role of p30 in cellular transcription, various groups have performed microarray analysis. The initial microarray analysis showed that p30 altered expression of cellular genes involved in apoptosis, cell cycle regulation, cell adhesion, transcription, and T-cell signaling and activation. It was found that p30 selectively activates genes involved in T cell signaling/activation and also enhances transcription mediated by NFAT, NF-κB, and AP-1 when stimulated (Michael et al., 2004). The ability of p30 to transcriptionally activate numerous cellular genes was also reported, where p30 binds and stabilizes the Myc/TIP60 transcriptional complex (Awasthi et al., 2005). Recently, similar microarray analysis also showed that p30 was capable of altering transcription of cellular genes involved in apoptosis and transcription (Taylor et al., 2009). Overall, these data indicate that p30 regulates transcription of viral genes as well as cellular genes to create an environment that promotes viral gene expression and survival of infected cells.

#### **POST-TRANSCRIPTIONAL REGULATION**

HTLV-1 p30 controls viral gene expression at the posttranscriptional level by interacting with and retaining *tax/rex* mRNA in the nucleus. The expression of p30 results in decreased levels of *tax/rex* mRNA specifically: this effect was not observed with other viral mRNAs. p30 binds to the second splice junction of *tax/rex* mRNA, which then is retained in the nucleus to repress tax expression and, in turn, viral gene expression (Nicot et al., 2004). HTLV-2 p28 mediates nuclear retention of the tax/rex mRNA via a similar post-transcriptional mechanism (Younis et al., 2004). Younis et al. (2006) went on to demonstrate that both p28 and p30 are recruited by Tax to the viral promoter, which then bind to their respective *de novo* transcribed response elements leading to the retention of *tax/rex* mRNA in the nucleus.

Rex and p30 have antagonistic functions where the Rex protein facilitates the nuclear-cytoplasmic export of unspliced and singly spliced mRNA while p30 retains*tax/rex* mRNA in the nucleus. The Rex and p30 interaction is favored when p30 is bound to *tax/rex* mRNA. The interaction of p30 with Rex has no effect on the ability of Rex to export mRNA from the nucleus to the cytoplasm whereas Rex moderately rescues the nuclear retention of *tax/rex* mRNA in the nucleus by p30. This interplay between p30 and Rex may be critical to establishing HTLV-1 latency (Sinha-Datta et al., 2007). Immunofluorescence confocal microscopy showed that p30 and Rex localized with CRM1, suggesting an interaction between Rex and p30 in HTLV-1 post-transcriptional control (Baydoun et al., 2007). On the other hand, p28 inhibited *tax/rex* mRNA export through the TAP/p15 pathway and not CRM1, where exogenous expression of TAP/p15 rescued p28 post-transcriptional repression (Younis et al., 2006).

Recently, microarray studies demonstrated that p30 can regulate cellular gene expression at the post-transcriptional level as well. Cytoplasmic expression of genes involved in cell signaling, transcription, translation, replication, cytoskeleton, DNA, and metabolism was decreased while those involved in repair, apoptosis, cell adhesion, and signaling were increased (Taylor et al., 2009). Studies to evaluate the effect of p28 post-transcriptional regulation on cellular genes have not been performed and are worth investigating.

#### **CELL CYCLE REGULATION**

Cell cycle control is an intricate, highly regulated pathway that guarantees proper cellular turnover and involves various cyclin dependent kinases (CDK) and Cyclins that phosphorylate key molecules allowing cell cycle progression. Different studies have shown that p30 also influences cell cycle progression. The G2/M checkpoint is regulated by a tight balance of phosphorylation and dephosphorylation events, where Cdc25c plays a crucial role in the onset of mitosis (Draetta and Eckstein, 1997). p30 activates the G2/M checkpoint by increasing the phosphorylation of Chk1 and subsequently increasing phosphorylation of Cdc25c. Another mechanism of p30-induced G2/M cell cycle arrest would be via reduction of PLK1 levels and phosphorylation resulting in decreased phosphorylation of Cdc25. In support of this mechanism, cells immortalized with an HTLV-1 molecular clone lacking p30 were more susceptible to apoptosis when treated with camptothecin and etoposide (Datta et al., 2007).

In addition to the effects of p30 on the G2M checkpoint, p30 activates Myc-driven genes leading to increased S phase progression and polyploidy (Awasthi et al., 2005). However, a recent study reported that expression of p30 delays S phase entry (Baydoun et al., 2010). The CDK2–Cyclin E complex is known to phosphorylate Rb, which in turn releases the transcriptional factor E2F, thereby controlling the expression of several genes required for S phase entry. It was shown that p30 delays S phase entry by interacting with both Cyclin E and CDK2 affecting their complex formation and resulting in reduced phosphorylation of Rb (Vermeulen et al., 2003). Hence, p30 expression causes a decrease in E2F and Cyclin E levels, and an increase in p21Waf expression. In contrast to p30, the expression of p28 had no effect on S phase entry and p28 was incapable of binding to Cyclin E (Baydoun et al., 2010).

#### **DNA DAMAGE AND REPAIR**

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