MOLECULAR ANALYSIS OF HUMAN T- REGULATORY AND ACCESSORY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Ihab H. Younis, MS

* * * * *

The Ohio State University

2005

Dissertation Committee: Approved by Dr Patrick Green, Advisor

Dr Kathleen Boris-Lawrie ______Advisor Dr Michael Lairmore Graduate Program in Molecular, Cellular, Dr Larry Mathes and Developmental

ABSTRACT

The Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are

two pathogenic human that are closely related at the amino acid sequence

and organization, but highly distinct in their pathogenesis. In vitro, they both

have the capacity to transform human primary T cells. On the other hand, HTLV-1 is

the causative agent for adult T-cell leukemia/ and HTLV-1 associated

myelopathy, whereas HTLV-2 is less pathogenic and has been reported to be endemic

in intravenous drug users. In this dissertation, we report molecular studies regarding the

regulation of HTLV replication and its impact on viral persistence in vivo. In Chapter

2, we generate a novel HTLV-1 clone (H1IT) in which the two regulatory proteins, Tax and Rex, have been separated in an attempt to provide a better reagent to study mutants of these proteins in the context of the provirus and analyze their contribution to HTLV- mediated transformation. Transient of H1IT shows that it expresses both functional Tax and Rex and is able to produce p19Gag. In short and long-term coculture assays, data indicate that H1IT is replication competent and is capable of cellular transformation of primary human T-cells. On the other hand, H1IT was not able to persist in vivo, emphasizing the importance of temporal and quantitative regulation of specific RNA to viral replication in vitro and in vivo.

ii

In Chapter 3, we report that both HTLV-1 and HTLV-2 have evolved accessory

whose products are able to restrict viral replication at a post-transcriptional level.

The HTLV-1 p30II and the related HTLV-2 p28II proteins act as negative regulators of

both Tax and Rex by binding to and retaining tax/rex mRNA in the nucleus, thereby

inhibiting virion production. Reduction of viral replication in a cell carrying the

provirus may allow escape from immune recognition in an infected individual.

In Chapter 4, we follow up on data in Chapter 3 to show that p28II is recruited to

the viral in a Tax-dependent manner. After recruitment to the promoter, p28II

or p30II then travels with the transcription elongation machinery until its target mRNA

is synthesized. The result of experiments artificially directing these proteins to the

promoter indicated that p28II, unlike HTLV-1 p30II, displays no transcriptional activity.

Furthermore, the tethering of p28II directly to tax/rex mRNA resulted in repression of

Tax function.

Since data in Chapters 3 and 4 are consistent with a critical role of these

accessory proteins in viral persistence in vivo, in Chapter 5, we used an animal model of

HTLV-1 and HTLV-2 infection to study the specific contribution of p28II on HTLV-2

survival in rabbits. In this study, all wtHTLV-2 infected rabbits showed persistent infection, whereas those infected with HTLV-2∆p28 were able to eliminate the virus as early as 2 weeks, indicating that p28II is critical for early viral infectivity, spread and/or

persistence in infected rabbits. Collectively, data presented within this thesis support

the conclusion that the regulation of HTLV expression a complicated but a tightly

controlled process.

iii

Dedicated to my mother

iv

ACKNOWLEDGMENTS

First and foremost, I would like to thank my advisor Dr Patrick Green for his

valuable advice and continual support. It would not have been possible to reach this

stage without him teaching and allowing me to be independent. He was always there to

discuss new ideas and gave me the freedom to test them.

I would like to express a special thanks to Dr Kathleen Boris-Lawrie, Dr Michael

Lairmore, and Dr Larry Mathes for serving on my Ph.D. committee. I appreciate their

input, critical review of my dissertation, time and interest in my progress.

Special thanks to past and current members of the Green lab who created a unique

and productive atmosphere: Dr Murli Narayan, Dr Jianxin Ye, Dr Matt Anderson, Dr Li

Xie, Lyne Khair, Dr Brenda Yamamoto, Joshua Arnold, Min Li, Matt Kesic, and Mary

Forget. I also want to thank members of the Boris-Lawrie lab especially Tiffiney

Roberts and Alper Yilmaz who were always willing to help. Special thanks to members of the Lairmore lab especially Dr Andrew Phipps who provided invaluable help in the rabbit studies. Kate Hayes and Tim Vojt provided editorial and techniqual support that was important in completing these projects.

Scot Erbe has always been the friend who listens and never hesitated to provide valuable advice. He helped me go through my journey as a graduate student. I will always cherish the friendship of my first true American “buddy”.

v I would also like to extend thanks to all of my family for their unwavering support.

In particular, I would like to acknowledge my mother, who always believed in me, and through her dedication and hard work inspired me and gave me the motivation to succeed.

I would, of course, like to thank Nadine Bakkar. Among all the many things she is great at, she makes me a better human being.

vi

VITA

September 2, 1975 ...... Born – Beirut, Lebanon

1993 - 1997 ...... Bachelor of Sciences-Biology The American University of Beirut Beirut, Lebanon

1997 - 1999 ...... Masters of Sciences-Biology The American University of Beirut Beirut, Lebanon

1999 - 2000 ...... Research Associate-Biology Department The American University of Beirut Beirut, Lebanon

2000 - present...... Graduate Research Associate The Ohio State University Ohio, USA

PUBLICATIONS

Research Publications

1. Younis I, Green PL. 2005. The human T-cell leukemia virus Rex . Front Biosci. 10:431-45.

2. Younis I, Khair L, Dundr M, Lairmore MD, Franchini G, Green PL. 2004. Repression of human T-cell leukemia virus type 1 and type 2 replication by a viral mRNA-encoded posttranscriptional regulator. J Virol. 78(20):11077-83.

3. Narayan M, Younis I, D'Agostino DM, Green PL. 2003. Functional domain structure of human T-cell leukemia virus type 2 Rex. J Virol. 77(23):12829-40.

vii 4. Gali-Muhtasib HU, Younes IH, Karchesy JJ, el-Sabban ME. 2001. Plant tannins inhibit the induction of aberrant crypt foci and colonic tumors by 1,2- dimethylhydrazine in mice. Nutr 39(1):108-16.

5. Gali-Muhtasib HU, Haddadin MJ, Rahhal DN, Younes IH. 2001. Quinoxaline 1,4-dioxides as anticancer and hypoxia-selective drugs. Oncol Rep. 8(3):679-84.

FIELDS OF STUDY

Major Field: Molecular, Cellular, and Developmental Biology

viii

TABLE OF CONTENTS

Page Abstract...... ii

Dedication...... iv

Acknowledgments...... v

Vita...... vii

List of Tables ...... xii

List of Figures...... xiii

Chapters:

1. Literature review...... 1 HTLV discovery and epidemiology ...... 1 HTLV classification, genome organization and life cycle...... 3 HTLV pathogenesis and disease association ...... 5 HTLV-1 pathogenesis...... 6 ATLL ...... 6 HAM/TSP ...... 7 HTLV-2 pathogenesis ...... 8 HTLV structural and enzymatic proteins ...... 9 Gag ...... 9 Pro ...... 10 Pol ...... 10 ...... 11 HTLV accessory proteins ...... 11 HTLV-1 accessory proteins ...... 12 ORF-I p12 and p27 ...... 12 ORF-II p30 and p13 ...... 13 HTLV-2 accessory proteins ...... 15 ORF-I p10 ...... 16 ORF-II p28 ...... 17 ORF-V p11 ...... 17 ix HTLV regulatory proteins ...... 18 Tax ...... 18 Tax-mediated nuclear transcription activation through CREB/ATF ..... 19 Tax-mediated signaling through NFκB ...... 20 Role of Tax in cellular transformation ...... 20 Rex ...... 22 Role of Rex in viral RNA export ...... 22 Rex proteins: structure, subcellular localization, and functional domains ...... 25 The Rex responsive element ...... 28 Interaction of Rex with host cellular factors ...... 30 Mechanism of Rex function and the Rex transport cycle...... 32 Role of phosphorylation in the regulation of Rex...... 36 Role of Rex in cellular transformation, viral spread & tropism 38

2. Human T-cell leukemia virus type 1 expressing non-overlapping Tax and Rex genes replicates and immortalizes primary human T-lymphocytes but fails to replicate and persist in vivo ...... 44

Abstract...... 44 Introduction...... 45 Materials and Methods...... 47 Results...... 52 Discussion...... 58

3. Repression of human T-cell leukemia virus type 1 and type 2 replication by a viral mRNA-encoded posttranscriptional regulator...... 70

Abstract...... 70 Introduction...... 71 Materials and Methods...... 73 Results...... 77 Discussion...... 81

x 4. Human T-cell leukemia virus ORF II encodes a post-transcriptional repressor that is recruited at the level of transcription ...... 92

Abstract...... 92 Introduction...... 93 Materials and Methods...... 96 Results...... 100 Discussion...... 109

5. Human T-cell leukemia virus type 2 ORF II encoded p28 is required for viral persistence in vivo ...... 128

Abstract...... 128 Introduction...... 129 Materials and Methods...... 132 Results...... 135 Discussion...... 138

5. Synopsis and future studies...... 146

References...... 154

xi

LIST OF TABLES

Table Page

5.1 Viral load of HTLV-2 and HTLV-2∆p28 in PBLs of inoculated rabbits...... 144

5.2 Serum response to HTLV-2 and HTLV-2∆p28 from inoculated rabbits ...... 145

xii

LIST OF FIGURES

Figure Page

1.1 Genome organization of HTLV-1 and HTLV-2 and their unspliced (U), singly spliced (S) and doubly spliced (D) mRNAs ...... 40

1.2 Domain structure of the HTLV-1 and HTLV-2 Rex ...... 42

1.3 The Rex-dependent export of viral mRNA...... 43

2.1 Schematic diagram of the generation of the H1IT...... 62

2.2 Functional activities of Tax and Rex expressed from H1IT...... 63

2.3 Characterization of a 729H1IT stable producer cell line...... 64

2.4 H1IT immortalizes human PBMCs ...... 66

2.5 Representative Kaplan-Meir plot for T-lymphocyte proliferation in short- term microtiter ...... 67

2.6 Immortalized human PBMCs harbor integrated viral sequences in their genome...... 68

2.7 H1IT virus fails to persist in vivo...... 69

3.1 HTLV-1 p30II inhibits viral replication...... 85

3.2 HTLV-1 p30II inhibits Tax and Rex function when both are expressed from HTLV proviral clones ...... 86

3.3 HTLV-2 p28II inhibits viral replication and Tax function...... 88

3.4 p28II inhibits Rex function when Rex is expressed from an HTLV proviral clone ...... 89

3.5 p28II retains HTLV-2 tax/rex mRNA in the nucleus ...... 90 xiii

3.6 In vivo binding of p28II to tax/rex mRNA ...... 91

4.1 Schematic diagrams of Tax-expression plasmids used in this study ...... 114

4.2 HTLV-2 p28II inhibits Tax-2 and Rex-2 expression only when they are expressed from a full length proviral clone ...... 116

4.3 p28II is a post-transcriptional repressor...... 117

4.4 Intron sequences/splicing and 5’UTR are not required for p28II-mediated inhibition of Tax activity ...... 119

4.5 p28II-mediated inhibition directly correlates with Tax expression in the context of the native HTLV-2 promoter (LTR)...... 121

4.6 p28II associates with HTLV-2 promoter as well as downstream DNA sequences ...... 123

4.7 p28II associates with components of the transcription machinery...... 125

4.8 p28II overrides the TAP/p15 export pathway...... 126

4.9 A model for the mechanism of action of p28II...... 127

5.1 Schematic diagram of HTLV-2∆p28 and its gene products ...... 141

5.2 Functional activity of Tax and viral replication are not affected by p28II deletion in vitro...... 143

xiv

CHAPTER 1

INTRODUCTION

Human T cell Leukemia Virus Discovery and Epidemiology

The first pathogenic to be isolated and associated with a human was human T-cell leukemia virus type 1 (HTLV-1) 1,2. T-cell lines established from patients with coetaneous T-cell lymphoma 2 and leukemia 1 were shown to produce detectable retroviral particles as well as that are reactive against sera from adult T-cell leukemia/lymphoma (ATLL) patients. A plethora of epidemiological and molecular studies have since shown that HTLV-1 is the etiological agent for ATLL and a slowly progressive neurological disorder termed HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) 3.

Human T-cell leukemia virus type 2 (HTLV-2) was discovered a few years after the isolation of HTLV-1 in a patient with a rare variant of hairy cell leukemia 4 and in a second patient with CD8+ T-cell leukemia and coexistent B-cell hairy cell leukemia 5.

Although HTLV-2 appears to play a role in rare T-cell lymphoproliferative 6,7 as well as neurological 8-10 disorders, a definitive association between viral infection and the above diseases is yet to be established.

1 Despite the close similarity in sequence and genomic organization between HTLV-1

and HTLV-2, the current hypothesis for the origin of human T-cell leukemia

(HTLVs) and their evolutionary relationship is that they originated independently from distinct lineages of simian T-lymphotropic viruses (STLVs) type 1 and 2, respectively

11. Very recently, two novel isolates of HTLV have been reported. HTLV-3 is

phylogenetically related to STLV-3 12,13, whereas HTLV-4 belongs to a phylogenetic

lineage that is distinct from all known HTLVs and STLVs 13.

HTLV-1 and HTLV-2 have distinct distribution in different regions of the world.

Between 10-20 million individuals world-wide are estimated to be infected with HTLV-

1 14, which is endemic in Japan, the Caribbean basin, Central and West Africa,

Southeastern United States, Melanesia, parts of South America, and in certain

populations in the Middle East and India 15-22. After a long latency of 20 or more years

23,24, it is predicted that among HTLV-1 infected individuals only 2-5% will develop

ATLL 25, and 0.25-3% will develop HAM/TSP 26-28. Unlike, HTLV-1, the geographic

origin of HTLV-2 is unknown, and its distribution is somewhat limited. HTLV-2 is

endemic in intravenous drug users (IVDUs) and their sexual contacts in the United

States, Europe, South America, and Southeast Asia 29-32. Interestingly, certain

Amerindian populations with distinct cultural, ethnical, and geographical distribution

are also endemically infected with HTLV-2 33-37.

The low percentages of infected people that develop HTLV-associated diseases

indicate that HTLV induces a chronic, lifelong, but asymptomatic infection. Because

HTLV cell free virions are poorly infectious, the main route of transmission occurs via

cell-cell contact from breast feeding 38,39, sexual contact 40, blood transfusion 41, and by

2 needle sharing among IVDUs.

Mother-to-child transmission depends on ingestion of infected milk-borne lymphocytes, duration of breast feeding, and maternal against HTLV.

Although sexual transmission could be bidirectional, there is higher risk of male-to- female transmission. For example, there is 60% likelihood that a woman would be infected due to sexual contact with an HTLV positive male compared to 0.4% for a man. These numbers are also affected by other factors such as genital ulcers, high viral load, and anti-HTLV antibodies. The most efficient route of HTLV transmission that leads to rapid disease development is blood transfusion as long as it has cellular blood components as opposed to plasma. It is estimated that the seroconversion rate in this case is up to 50%.

Finally, despite a wide variety of human and nonhuman cells that can be infected by

HTLV in vitro 42,43, the in vivo tropism or cellular targets are preferentially CD4+ T cells for HTLV-1, and CD8+ T cells for HTLV-2 44-47.

HTLV Classification, Genome Organization, and Life Cycle

HTLVs are members of the Retroviridae family with C-type morphology. C-type virions assemble at the plasma membrane and possess a central, symmetrically placed, spherical inner core that contains a homodimer of linear, positive sense, single stranded

RNA genome. Based on recent taxonomy, HTLVs have been placed in the deltaretrovirus genus together with bovine leukemia virus (BLV) and STLV-1, -2, and -

3 48. Taxonomical classification of HTLVs further divides HTLV-1 into subtypes 1a,

1b, 1c and 1d, whereas HTLV-2 has three subtypes 2a, 2b and 2c. The subtype

3 grouping is based on nucleotide sequence divergence.

Another feature of HTLVs is that they are complex retroviruses due to their ability to encode regulatory and accessory gene products in addition to the typical structural and enzymatic proteins (Figure 1.1) 79. The complexity of HTLVs is due to at least

three mechanisms that they utilize to generate diverse proteins and transcripts from a

relatively small genome. First, frame shifting results in the generation of Gag, Pro and

Pol proteins from a single transcript with three reading frames. Second, the cleavage of

certain large protein precursors such as Gag generates smaller peptides with distinct

functions. Finally, alternative splicing leads to multiple transcripts that encode different

proteins.

The RNA genome organization as well as that of the DNA intermediate (provirus)

of all HTLVs is very similar. The RNA genome is capped at the 5’ end and contains a

poly(A) chain of around 200 nucleotides at the 3’ end. The proviral genome contains a

(LTR) at each end which encompasses sequence blocks that are

essential for viral replication and regulation of . Each LTR is composed

of a unique 3’ (U3) region present only at the 3’ end of the viral RNA genome, a

repeated (R) region present at both ends of the viral RNA genome, and a unique 5’ (U5)

region present only at the 5’ end of the viral RNA genome. The bulk of the HTLV

genome between the LTRs is the coding region for Gag, Pol and Env in addition to four

open reading frames (ORFs) in HTLV-1 and five ORFs in HTLV-2 present in what was

initially referred to as the X region (Figure 1.1).

The overall replication life cycle of HTLVs is similar to that of other retroviruses.

Briefly, the viral glycoprotein SU component of Env recognizes and binds to a cell

4 surface receptor(s) leading to the viral and host membrane fusion that is facilitated by

the TM component of Env. This is followed by the release of the uncoated viral core

into the cytoplasm of the host cell. Then, due to a unique enzyme produced by all

retroviruses (), including HTLVs, the RNA genome is reverse

transcribed to generate a double stranded linear DNA intermediate that is transported to

the nucleus where it gets stably integrated into the host genome due to the enzymatic

activity of the viral protein, . At this point, the integrated HTLV genome is

referred to as a provirus and becomes part of the host genome. Afterwards, the cellular

RNA polymerase II mediates transcription from the viral promoter present in the U3

region of the 5’ LTR, resulting in multiple viral transcripts that are spliced (with the

exception of the genomic mRNA), processed, exported to the cytoplasm, and translated

into viral proteins. The assembly of the virion and packaging of genomic RNA follows.

HTLV utilizes a sequence called the Psi element (ψ) for encapsidation 79. Since ψ is

spliced out in all other transcripts, only unspliced genomic RNA is packaged into the

virion that buds at the cytoplasmic membrane. The released virions are immature and

the activity of protease is needed for the proteolytic cleavage of structural proteins and

maturation of the virions 48,49.

HTLV Pathogenesis and Disease Association

HTLV-1 and HTLV-2 are oncogenic retroviruses that have the capacity to infect

and promote T-lymphocyte activation and proliferation both in vitro and in vivo 50-53. In vitro, primary human T-lymphocytes that are cocultivated with lethally irradiated

HTLV-producing cells get transformed based on their indefinite proliferation in an IL-

5 2-independent manner. The transforming ability of HTLV is dependent on the viral

protein, Tax, which makes HTLV unique among oncogenic viruses in that it has no

cellular counterpart or proto- 54. Interestingly, most of the HTLV-1

transformed T-lymphocytes are CD4+, whereas HTLV-2 preferentially transforms

CD8+ T-cells. Moreover, ex vivo derived HTLV-1-infected T-cells show spontaneous

proliferation up to 2 weeks independently of IL-2. Although both HTLV-1 and HTLV-

2 transform T-cell in vitro and in vivo, only a small fraction of infected individuals

develop ATLL or HAM/TSP in the case of HTLV-1 and even a smaller portion develop

HTLV-2-mediated . This suggests that the diseases associated with HTLV

especially leukemia are multistep processes that require the accumulation of several

genetic mutations over a long period of time. However, there is ample evidence to

suggest that HTLVs either initiate or potentiate these processes. Below are specific

examples of HTLV-associated diseases.

HTLV-1 Pathogenesis

Two major diseases have been linked to HTLV-1 infection: ATLL and HAM/TSP.

Furthermore, HTLV-1 has been implicated as an etiologic agent for polymyositis,

polyarthritis, uveitis, infectious dermatitis, and virulent strongyloidiasis 3,55-60.

ATLL

Due to the unusual clustering of ATLL patients in some areas of Japan, it had been suggested that a transmissible agent was involved as early as 1977 61. Later, it was

found that HTLV-1 is indeed the causative agent for ATLL. Five different stages

6 characterize this aggressive lymphoproliferative disease: asymptomatic, pre-leukemic,

chronic/smoldering, lymphoma, and acute. Although the majority of infected people

are asymptomatic, these individuals are still able to transmit the virus. Infected T-cells

typically have highly lobulated or “flower-shaped” nuclei with a CD2+, CD3+, CD4+,

CD8-, CD25+, and HLA-DR+ 61. While almost one-half of the pre-leukemic people undergo spontaneous regression, some progress to the next phase, smoldering

ATLL which is characterized with skin lesions and marrow involvement, or chronic

ATLL with elevated numbers of circulating leukemic cells. Finally, a proportion of patients develop acute ATLL which is clinically characterized by hypercalcemia, elevated levels of LDH, skin lesions, lymphadenopathy, lymphomatous meningitis, lytic bone lesions, spleen or liver involvement, and immunodeficiency 62. The median

survival time for acute ATLL patients is 6-10 months even with intense

63.

Although the site of HTLV integration seems to be random in infected individuals

64, using rearrangement of the T-cell receptor gene and Southern blotting, it was shown

that leukemic cells are mainly clonal 65,66. The clonality of infected cells indicates that

viral replication is mainly a consequence of mitotic division of infected cells rather than

the typical reverse transcriptase-mediated expansion and reinfection.

HAM/TSP

Like ATLL only a small fraction of infected people (0.2-5%) develop HAM/TSP 67, a chronic and progressive demyelinating disease that predominantly affects the spinal cord and is more prevalent in women than men 27,68. In contrast to ATLL, the onset of

7 HAM/TSP is faster and can be as little as 6 months after transfusion 69,70. Initial

symptoms of HAM/TSP include weakness and stiffness of the lower limbs. As the

disease progresses, more symptoms such as constipation, impotence and hyperreflexia

may present. Pathologic analyses have revealed perivascular and parenchymal infiltration of mononuclear lymphoid cells that correlate with myelin and axonal degeneration, ultimately resulting in severe degeneration of white matter.

Several lines of evidence indicate that virus-host immune system interaction plays a

critical role in the development of HAM/TSP. Briefly, high proviral load accompanied

with increased viral gene expression lead to the processing and presentation of HTLV-

1-specific peptides, especially those of Tax-1. This results in the activation and

expansion of -specific CD4+ and CD8+ T-cells. Localized infiltration of

HTLV-1-specific CD8+ cytotoxic T lymphocytes (CTL) in the central nervous system

71 implicate those cells in disease development 3.

HTLV-2 Pathogenesis

Unlike HTLV-1, the disease association for HTLV-2 is less clear and lacks solid epidemiological evidence. However, the isolation of HTLV-2 from patients with a rare

variant of hairy cell leukemia 4, CD8+ T-cell leukemia 5, mycosis fungoides 7, and large

granular lymphocytic leukemia 6 suggests some association between HTLV-2 and T-

cell lymphoproliferative disorders. Consistent with the in vitro tropism of HTLV-2,

several of the HTLV-2 associated leukemia cases show CD8+ T-cell lineage.

Furthermore, there are reports that associate HTLV-2 with spastic ataxia 8 and chronic neurodegenerative diseases 9. Interestingly, HTLV-2 infection has been reported in a

8 patient with a chronic progressive neurological disease that is clinically identical to

HAM/TSP 10. HTLV-2 is also associated with increased incidence of pneumonia and

bronchitis, inflammatory conditions such as arthritis, and perhaps with increased

mortality 72,73. Finally, although HTLV-2 and HIV coinfection has not been proven to

alter the course of HIV disease, patients with such coinfection may have altered levels

of CD4+ and CD8+ lymphocytes, and antiretroviral therapy may paradoxically increase

HTLV-2 proviral load 71.

HTLV Structural and Enzymatic Proteins

HTLVs encode for the following structural and enzymatic proteins: Gag, Pol, Pro,

and Env. The structural proteins are encoded by gag and env genes, whereas the

gene provides most of the enzymatic activity. A unique feature of HTLV-1 and HTLV-

2 is that although their structural proteins have the general characteristics of those of

other retroviruses, they do not appear to ensure proper and efficient transmission 74.

The reason for this low effieciency is not clear.

Gag

The main function of Gag is to promote the assembly and release of virus particles, even in the absence of any other viral protein. HTLV Gag is made as a polypeptide precursor. A critical processing step leads to the proteolytic cleavage and generation of

the matrix (MA, p19), capsid (CA, p24), and nucleocapsid (NC, p15) proteins 75.

Targeting of MA and its precursor p55 to the inner surface of the plasma membrane is made possible by myristylation at the N-terminal end of the protein 76,77. Like other

9 retroviruses, HTLV MA has an N-terminal cluster of basic amino acids that have been

shown to play multiple roles in virus replication such as infectivity, particle release,

precursor cleavage and ultimately cell-to-cell transmission 78. The other two Gag

products (CA and NC) of HTLV have not been studied in detail but their contribution to

the overall replication of the virus is likely similar to other retroviruses including

Moloney MLV and HIV-1.

Pro

A frameshift during the synthesis of Gag 79 is needed to produce protease (Pro)

whose reading frame overlaps the 3’ end of gag and the 5’ part of pol 80. The function

of Pro is the processing of the precursor Gag and Pol polypeptides to produce smaller functional units. Thus, Pro is essential for the maturation of HTLV viral particles 79.

Pol

Another ribosomal frameshift during the translation of Gag results in the synthesis of Gag-Pol. The C-terminal region of Gag-Pol is cleaved by Pro to release integrase, the protein needed for the stable integration of the provirus into the host genome. The remaining part of Pol has the reverse transcriptase activity at the N-terminus and the

RNase H activity at the C-terminus. These activities cooperate after HTLV infection to generate the double-stranded DNA intermediate from the RNA genome 81.

10 Env

A singly spliced mRNA in HTLV encodes for the Envelope (Env) protein which is

post-translationally modified, thus resulting in a 61-69 kDa glycoprotein 82,83. The Env

precursor is cleaved by a cellular protease to form the surface (SU, gp46) and

transmembrane (TM, gp21) subunits. Like other retroviral Env proteins that are organized as oligomers, HTLV Env is assembled at an early stage in the ER as a dimer

84. The SU glycoprotein is composed of three domains that harbor the receptor binding

determinants. The TM on the other hand possesses a fusion peptide at the N-terminus

and is directly responsible for the initiation of fusion pores. Mutational analysis of TM

also indicates that it functions in post-fusion events that are required for infection 78.

Since the discovery of HTLV, several molecules have been speculated as being

receptors or coreceptors for viral infection 85-89. However, it was not until recently that

convincing data have shown that a vertebrate glucose transporter, GLUT1, is an HTLV

receptor 90,91. This does not rule out that other cell surface molecules can be used by

HTLV for infection. Indeed, cellular partners of GLUT1 seem to be acting as

coreceptors 91.

HTLV Accessory Proteins

As complex retroviruses, HTLVs utilize alternative splicing and internal initiation

codons to generate several regulatory and accessory proteins. The term “accessory”

was initially given to the gene products that were believed to be dispensable for viral

replication 92,93. However, extensive work since then has been done to show that these proteins are critical for different aspects of HTLV replication ranging from regulation of

11 transcription to infectivity, maintenance of viral load, host cell activation, and modulation of immune response (reviewed in 94). Although the presence of the accessory proteins themselves have not been directly shown in vivo, there is ample indirect evidence for their expression from studies that detected serum antibodies and

CTL specific for ORF-I and -II gene products in asymptomatic carriers as well as patients 95-97. These data suggest that the accessory proteins are produced at some point during infection and to levels high enough to elicit a specific immune response.

HTLV-1 Accessory Proteins

In HTLV-1, the accessory proteins are produced from pX ORF-I (p12, p27), ORF-II

(p30, p13), and ORF-III (p21Rex) (see Figure 1.1). Below is a brief summary of what has been done to elucidate the function of ORF-I and ORF-II proteins.

ORF-I p12 and p27

HTLV-1 p12 is generated from a singly spliced mRNA and is a 99 amino acid (a.a.) long, highly hydrophobic protein with two putative transmembrane domains, four predicted SH3-binding motifs, a leucine zipper region, and a calcineurin-binding motif

94,98-100. p12 localizes to the ER and cis-Golgi apparatus where it interacts with two ER resident proteins that regulate calcium signaling, calreticulin and calnexin 101. It has also been reported that p12 interacts with the immature forms of interleukin-2 receptor

β (IL-2Rβ) and γ chains, leading to their reduced expression on the cell surface 102.

Further analysis of p12 in the context of the HTLV-1 provirus suggested that p12 is not responsible for IL-2-dependent proliferation response or JAK/STAT activation of

12 HTLV-1-immortalized cells 102. Since the expression of p12 may activate quiescent T

cells as well as provide growth advantage for primary human PBMCs in the presence of

suboptimal antigen stimulation, it is more likely to play a role in early HTLV-1

infection 103,104. Moreover, p12 binds to and directs the degradation of immature forms of the major histocompatibility complex class I (MHC-I) in transiently transfected and transduced cells but not immortalized T-lymphocytes. This observation suggests that

p12 may interfere with antigen presentation and help infected cells escape from the

immune surveillance early after infection 103,105,106. Finally, it has been shown that p12

plays a role in T-cell activation by inducing NFAT gene expression in a calcium-

dependent manner 107,108 and stimulating IL-2 production 109. Collectively, these

functions of p12 are consistent with its overall role in enhancing infectivity and

facilitating viral replication as observed in an in vivo rabbit model 107,110.

The 152 a.a. p27 is generated from a doubly-spliced mRNA and shares the first 20 a.a. with Rex and the last 99 a.a. with p12. The exact role of p27 in HTLV-1 replication is not known; however, a study established that CTLs against p27 protein are generated

during HTLV-I infection providing evidence for the in vivo chronic production of p27

96.

ORF-II p30 and p13

Two alternatively spliced mRNAs can produce proteins encoded by ORF-II 111. p30 is a 241 a.a. nuclear/nucleolar protein with domains that share homology with the DNA binding domain and homeodomain of the Oct-1 112, and a bipartite nuclear localization signal 113. Early biochemical analysis of p30 together with its

13 homology to known transcription factors suggested that it might play a role in

modulating transcription. Indeed, studies from Lairmore and colleagues provided

evidence that p30 is involved in modulating HTLV-1 as well as cellular gene expression

114. It was reported that low amounts of transfected p30 enhanced HTLV-1 LTR- mediated gene expression, whereas high levels of p30 show repressive effects on LTR- driven gene expression 114. Further analysis of p30 showed that it co-localizes and

interacts with the conserved KIX domain of the transcriptional coactivator p300.

Evidence that p30 acts as a transcriptional repressor comes from experiments showing

that p30 disrupts CREB-Tax-CBP/p300 complexes that are bound to the Tax-response

elements (TREs) present in the HTLV promoter 115. Using microarrays, the same group

showed that p30 has the capacity to alter the expression of multiple cellular gene

clusters that are involved in transcription and translation, T-cell activation, apoptosis,

, and cell adhesion 116. Another report demonstrated that numerous cellular

genes are transcriptionally modulated by p30 in a TIP60-dependent manner 116. Since

TIP60 is a partner of the transcription activator, Myc, the authors went on to show that p30 is a modulator of Myc transcriptional and transforming activities, which might significantly contribute to ATLL progression 117. Recently, a study by Nicot et al

demonstrated that p30 negatively regulate viral gene expression by specifically

associating with and blocking the export of tax/rex mRNA, resulting in reduced protein

production and lower viral replication 118.

The smaller protein expressed from ORF-II represents the last 87 a.a. of p30 (p13).

This ORF initiates at an internal methionine initiation codon and encodes a protein that

is targeted to the mitochondria 119,120. The role for p13 in the induction of apoptosis is

14 based on the observation that p13 disrupts the mitochondrial inner membrane potential

and eventually alters mitochondrial morphology 121. In the mitochondria, p13 interacts

with farnesyl pyrophosphate synthase that is involved in the synthesis of a substrate

required for the prenylation of Ras 122. Furthermore, p13 seems to sensitize cells to

apoptosis induced by pro-apoptotic agents such as ceramide 123. The role of p13 in

apoptosis and its interaction with the Ras signaling pathway will ultimately result in

reduction of tumor incidence and growth rate in oncogenic conversion assays 124.

Using rabbits as an animal model, the role of p13 and p30 in viral infectivity,

propagation, and persistence in vivo has been recently tested. The results from these studies demonstrate a critical role for both proteins combined 125 or separately 126 in maintaining viral load and persistence in vivo.

HTLV-2 Accessory Proteins

The accessory proteins in HTLV-2 are produced from pX ORF-I (p10), ORF-II

(p28), ORF-III (p22/p20), and ORF-V (p11). Limited detailed or comprehensive analyses have been done to elucidate the function of these proteins. However, the fact that the pX region from which they are expressed is conserved among deltaretroviruses indicates an important functional contribution of these proteins to the virus. More direct evidence for the functional importance of the accessory proteins comes from a study in which an HTLV-2 clone has been modified to eliminate the accessory genes of the pX region. Although this virus was able to replicate and infect human PBMCs in vitro, it was attenuated in terms of infectivity and persistence in an in vivo rabbit model 127.

Since all accessory genes were deleted simultaneously in that study, the individual

15 contribution of each gene was not elucidated. We dedicated a chapter in this

dissertation to study the in vivo role of the HTLV-2 accessory protein, p28. Using RT-

PCR on RNA isolated from HTLV-2-chronically infected cell line (MoT), Ciminale et al were able to generate and sequence cDNAs corresponding to HTLV-2 accessory gene products 128. p22 and p20 are truncated forms of Rex-2 that seem to have cytoplasmic

localization, but their function has not been elucidated. Below is a brief description of

the proteins generated from transient transfection of these cDNAs of ORF-1, ORF-II,

and ORF-V and their localization.

ORF-I p10

HTLV-2 p10 is expressed from a doubly spliced bicistronic mRNA that also

encodes ORF-V p11. p10 is an 83 a.a. protein that consists of the first 21 a.a. of Rex-2

linked to ORF-I-encoded sequences. The protein is generally hydrophobic and shows some homology to HTLV-1 p12. Similar to p12, p10 associates with MHC-I but does not seem to bind to IL2-Rβ or γ or 16K, suggesting that despite the homology between p12 and p10, the two proteins may be functionally distinct 105. Consistent with this concept, p12 localizes to the ER and cis-Golgi, whereas p10 accumulates in the periphery of nucleoli and in nuclear speckles. This unique localization of p10 could be attributed to the first 21 arginine-rich amino acids that are derived from the Rex-2 ORF and serve as nuclear/nucleolar localization signal 128.

16 ORF-II p28

HTLV-2 p28 can be translated from two singly spliced, bicistronic mRNAs. The

protein is 216 a.a. in size with a predicted molecular mass of 23.9 kDa, suggesting that

p28 is post-translationally modified. Interestingly, the first 49 N-terminal amino acids

of p28 reveal 77.5% identity with the C-terminal portion of HTLV-1 p30. Moreover,

there is 83% homology between a.a. 3-14 of p28 and a portion of HTLV-2 reverse

transcriptase. p28 localizes predominantly to the nucleus. Although p28 could

potentially be translated on tax/rex mRNA, it has been reported that when the AUGs for

Tax and Rex are functional, there is a barely detectable amount of p28 that is produced from this mRNA 128. We dedicated two chapters in this thesis to study the role of p28

in the regulation of viral replication and determine the molecular mechanism of action

of p28.

ORF-V p11

Using the AUG of Tax/Env that is linked to ORF-V sequences, p11 is produced but

seems to be larger than its predicted molecular weight of 8.4 kDa. A stretch of 10 a.a. in p11 shows high homology to part of the musculoaponeurotic fibrosarcoma (MAF)

nuclear transforming protein, but a functional significance of this homology has not been tested yet 128. Like p10, p11 associates with MHC-I but does not bind to 16K, or

IL2Rβ or γ 105. Finally, p11 localizes to the nucleus and, to a lesser extent, to the

cytoplasm 128.

17 HTLV Regulatory Proteins

A single bicistronic mRNA is translated into the two HTLV trans-acting regulatory

proteins Tax and Rex that are essential for viral replication both in vitro and in vivo.

Tax is a transcriptional regulator needed for efficient transcription from the viral

promoter that is present in the U3 region of the 5’ LTR. In addition, Tax modulates cellular gene expression either directly by regulating expression from their promoters or indirectly by interacting with cellular signal pathways such as NFκB and

SRF, resulting in regulation of their target genes 129. Rex, on the other hand, is a post-

transcriptional regulator that binds to and facilitates the cytoplasmic expression of viral

unspliced and incompletely spliced mRNAs 130. Although Rex has been extensively studied, a comprehensive review on Rex is lacking. Thus, the Rex section in this chapter is extensive and will cover almost all aspects of the protein.

Tax

HTLV Tax is a pleiotropic phosphoprotein that regulates several aspects of viral replication as well as HTLV-mediated transformation of T-lymphocytes. HTLV-1 Tax protein is predominantly nuclear but has been shown to shuttle between the nucleus and cytoplasm 131. Tax-2, on the other hand, was reported to be located predominantly in

the cytoplasm of the HTLV-2 immortalized or transformed infected T-cells 132. Tax-1 is 40 kDa and 353 a.a. in size, while Tax-2a is 37 kDa and 331 a.a. in size 80.

18 Tax-mediated nuclear transcription activation through CREB/ATF

Key elements that are needed for transactivation of the HTLV promoter reside in the

U3 region of the 5’LTR and consist of three 21-bp imperfect repeats called Tax-

response elements (TREs) 132. In the center of the TREs resides a cAMP response element (CRE) that is flanked by GC-rich sequences 133,134. Tax itself does not directly bind to the TREs, but forms a ternary complex with the transcription factor, CREB, as well as other members of the CREB family such as ATF-1 and the TRE 135. The

significance of the presence of Tax in this complex is that it overrides a necessary

phosphorylation step on serine 133 of CREB that is needed to activate the protein 136,137.

Thus, Tax mediates the assembly of active complexes under conditions in which CREB

would be inactive in the absence of Tax. After the formation of the proper ternary complex on the TRE, Tax recruits other transcriptional co-activators such as CBP, p300, P/CAF and ATF-4 138-143. Ultimately, proteins in this complex such as CBP/p300

contact the RNA polymerase II holoenzyme and help remodel chromatin resulting in

activation of transcription from the viral LTR.

Tax interaction with p300/CBP is not only important for the activation of

transcription, it has also been implicated in Tax-mediated repression of some cellular

genes such as cyclin A, cMyb and Lck 144-146. A body of evidence indicates that the

mechanism of this repression is due to Tax sequestering p300/CBP leading to its limited

availability for other transcription complexes. Another possible mechanism for Tax-

mediated trans-repression is through its recruitment of histone deacetylases such as

HDAC-1 to the promoter, implicating a role in chromatin remodeling that leads to repression of transcription, 147,148.

19 Tax-mediated signaling through NFκB

Upon HTLV infection, and specifically Tax expression, a variety of NFκB

responsive genes are upregulated suggesting that Tax interacts with this pathway 149. A simplistic view of the NFκB pathway is that the transcription-activator subunits (NFκB) are retained in the cytoplasm via their interaction with the inhibitory subunit IκB. Upon

IκB-Kinases (IKKs)-mediated phosphorylation and subsequent degradation of IκB, the

NFκB subunit translocates to the nucleus and regulates transcription of target genes.

The mechanism with which Tax activates the NFκB pathway is distinct from that of p300/CBP, and involves the dysregulation of the NFκB signaling pathway via at least two strategies. First, through its interaction with MEKK1, which normally phosphorylates IKKs in a regulated manner, Tax promotes constitutive IKK phosphorylation 150,151. On the other hand, Tax can induce the degradation of IκB by

directly interacting with IKKs and increasing their kinase activity or by recruiting the

proteasome to the cytoplasmic NFκB complexes 152-155.

Role of Tax in cellular transformation

Tax expression is not only necessary for immortalization and transformation, but in some settings it has been shown to be sufficient for the establishment of these processes

135. Although, the exact mechanism by which this oncogenic protein induces cellular

transformation is not fully understood, Tax-mediated modulation of several cellular

genes and/or proteins involved in T-cell activation, cell cycle regulation, DNA repair, genomic instability and aneuploidy, and apoptosis may play a crucial role in this

20 process. Since transformation is a multistep process, it is reasonable to speculate that

several of the distinct Tax functions may play important roles at different stages during

transformation. However, further changes in the cell that are independent of Tax

activity may be acquired over time in order to achieve full transformation.

Several Tax activities have been directly implicated in immortalization and

transformation. For example, HTLV-transformed cells show a clear deregulation of cell

cycle, and Tax has been reported to regulate different steps or checkpoints of the cell

cycle 156. More specifically, Tax has been shown to induce G1-S progression 157 via its functional crosstalk with p16INKa as well as p18INK4c 158-160. In addition, Tax modulates

the level or activity of several cyclins such as cyclin D 161,162, cyclin-dependent kinases

(CDK) such as CDK4 and CDK6 163,164, retinoblastoma protein (pRB), transforming

growth factor betta (TGF-β) 165-167, the tumor suppressor protein, p53 168-171, and the mitotic spindle checkpoint protein MAD-1 172.

Other features of transformation are genomic instability, aneuploidy, and impaired

DNA repair and apoptosis, all of which are affected or regulated by Tax 129,172-175.

Through its interaction or modulation of DNA polymerase β and PCNA, Tax has the potential to influence base excision repair 176,177. On the other hand, Tax disrupts nucleotide excision repair of DNA 178 which also correlates with the ability of Tax to induce PCNA 179. Finally, ample evidence point at an intimate link between Tax and

apoptosis despite the conflicting reports on whether Tax actually induces or protects

from apoptosis 180-186.

21 Rex

A requirement for successful replication of all retroviruses is their ability to override

the nuclear retention of intron-containing mRNAs, resulting in the efficient export of

the viral unspliced RNA to the cytoplasm. Like other complex retroviruses 14,187-189,

HTLV-1 and HTLV-2 have evolved to encode a protein that binds to and facilitates the nucleo-cytoplasmic export of viral unspliced and singly spliced mRNAs. The Rex protein of HTLV, first defined in 1988 190, was found to be a post-transcriptional

regulator that utilizes specific host machinery to actively export incompletely spliced

mRNA species from the nucleus 191-193. Since Rex has been shown to be essential for

HTLV replication 191,194, the full understanding of its function and regulation remains a

critical objective in HTLV research that could yield new strategies for therapeutic

intervention.

Role of Rex in viral RNA export

Since the accumulation of viral structural proteins is dependent on Rex, and Rex

itself is generated from completely spliced mRNA, the virus has a biphasic life cycle:

an early Rex-independent phase and a late Rex-dependent phase. Early during

infection, when insufficient Rex protein is being made, most of the viral mRNAs are

doubly spliced, due to default splicing by the host cellular machinery. Accumulation of

sufficient levels of Rex results in the expression of incompletely spliced mRNA in the

cytoplasm, leading to the production of structural and enzymatic gene products and

assembly of virus particles. Therefore, Rex is considered to be a positive regulator that

controls the switch between early, latent and late, productive infection.

22 The ability of Rex to function depends on several characteristics: (a) its ability to

recognize and bind a responsive element on specific mRNA species in the nucleus prior

to their splicing, (b) its ability to utilize defined cellular pathways to export the

incompletely spliced mRNA cargo, and (c) its potential to be regulated through specific

domains in order to ensure that the protein functions best when it is needed. Below, we

will discuss in detail each characteristic, its contribution to Rex function, and

significance to the virus life cycle. Mapping of functional domains of Rex and their

regulation may ultimately provide us with therapeutic tools to disrupt Rex function and

HTLV replication and pathogenesis.

First, it is important to note that the unspliced and incompletely spliced viral

mRNAs have essential features that render them Rex responsive. In addition to

containing a unique Rex response element (RxRE), these mRNAs have cis-acting

repressive sequences (CRS) that retain and stabilize the unspliced mRNA in the nucleus

to ensure the availability of sufficient amount of Rex substrate 195,196. The fact that

unspliced pre-mRNA in the nucleus is unstable and is targeted for processing (splicing

to completion followed by export) or degradation 197,198 gives the combination of CRS

and RxRE a unique role in providing suboptimal conditions for mRNA splicing

efficiency and hence is a prerequisite for the ability of Rex to activate cytoplasmic

transport. Drawing from the similarity between HIV-1 Rev and HTLV Rex, one can

assume that the absence of these elements corresponds with rapid splicing which

depletes the nuclear pool of Rex substrates. On the other hand, if these elements were to render splicing too inefficient, the mRNA would be recognized by the cell as intronless mRNA that would be constitutively exported to the cytoplasm in a Rex

23 independent manner 199-201. Thus, the working model for these elements is that the CRS

retains the unspliced mRNA in the nucleus until Rex binds to the RxRE, overrides the

repressive effects of the CRS, and exports the mRNA to the cytoplasm 195.

There are two suggested models for the mechanism by which Rex induces

cytoplasmic expression of unspliced mRNA. The first model proposes that Rex

actively transports the unspliced mRNA to the cytoplasm where it is translated. Here,

Rex would directly bind to the RxRE on the mRNA, override the nuclear retention

signals, and carry this mRNA cargo through the nuclear pore to the cytoplasm. The

second model suggests that Rex actively inhibits splicing of mRNA by stripping it of

splicing factors 195,202,203. Once the mRNA is free of splicing factors the cellular machinery would recognize it as a processed mRNA and export it efficiently to the

cytoplasm. Although there is some evidence for both models, the first is supported by a

greater amount of published work.

A less investigated model for Rex function is that Rex may also increase the

translational efficiency of Rex-responsive mRNA. Kusuhara et al have shown that Rex-

2 increases the levels of incompletely spliced mRNA in the cytoplasm by 7 to 9-fold,

while Gag protein production increases by 130-fold 193. These results are consistent

with HIV-1 Rev data 204,205, and suggest that Rex has an effect on translation efficiency.

Additional support for this hypothesis comes from studies that show an association

between Rex-1 and translational initiation factor 5A (eIF-5A) 206,207. Despite some

evidence for this mechanistic effect of Rex, the primary function of Rex remains the

nucleo-cytoplasmic export of unspliced and partially spliced viral mRNA.

24 Rex Proteins: Structure, subcellular localization, and functional domains

The 189 amino acid Rex-1 and 170 amino acid Rex-2 proteins share 60% homology

at the amino acid level. When analyzed by SDS-PAGE, Rex-1 has an apparent size of

27 kDa, and Rex-2 is detected as two major bands of 24 and 26 kDa 80. The two isoforms of Rex-2, p24rex and p26rex, have the same amino acid backbone and differ by

a post-translational modification, specifically a conformational change induced by

serine phosphorylation 208,209. HTLV-1 and HTLV-2 also produce truncated forms of

Rex from alternatively spliced mRNAs. These proteins, named p21rex-1 and p22/20rex-2, lack N-terminal sequences of Rex responsible for nuclear import and RNA binding, and interfere with Rex localization and function 210-212.

Both Rex-1 and Rex-2 are phosphoproteins that localize to nucleus, nucleoli, and nucleolar speckles in transiently transfected cells as well as HTLV infected cell lines

128,211,213-215. Studies using a recombinant baculovirus expressing Rex-2 in SF-9 cells, as well as cell fractionation analysis following transfection of human lymphoid cells, have shown that the p24rex isoform is predominantly cytoplasmic, while the p26rex isoform is

nuclear/nucleolar 216. Despite the nuclear and nucleolar localization of Rex at steady

state, studies have shown that Rex is a shuttling protein. Thus, Rex localization is in dynamic equilibrium between the nucleus and the cytoplasm. In fact, this property is essential for Rex to exert its function as a nucleo-cytoplasmic mRNA export facilitator.

There is no clearcut evidence for the role of the nucleolar localization of Rex in its function. Using in situ hybridization, it was shown that env mRNA can be detected in the nucleoli of cells cotransfected with Rex 217. In addition, several cellular mRNAs

such as c-myc, n-myc and myo-D can be detected in nucleoli 218. Recently, the

25 nucleolus has been considered to be a site for mRNA processing prior to export 219,220.

Thus, a likely hypothesis is that Rex interacts with its target mRNA at the site where the mRNA is to be processed. Studies of HIV-1 Rev suggest that the nucleolus might also serve as a storage compartment that ensures preservation of Rev and Rex in the cell 221.

Mutational analyses allowed the assignment of biochemical and functional

properties to discrete protein domains of Rex (Figure 1.2). Both Rex-1 and Rex-2 have

homologous nuclear localization signals (NLS), RNA binding domains (RBD),

multimerization domains, and an activation domain which encompasses the nuclear

export signal (NES) 209,222-226. In addition, a unique C-terminal domain has been

described for Rex-2 that is a target for serine phosphorylation and may also contribute

to efficient nucleocytoplasmic shuttling 215.

The amino terminal 19 residues of Rex contain an arginine rich cluster that serves as

both an NLS and RBD. Upon fusion to heterologous proteins such as β-galactosidase,

the arginine rich motif of Rex, is sufficient to impose nuclear import of the chimeric

protein, thus establishing it as an NLS 210,227,228. Hammes et al showed that substitution

of all 7 arginines in the NLS of Rex-1 with positively charged lysine residues does not

affect nuclear and/or nucleolar localization. This suggests that the mere presence of a

stretch of positive charges within this domain is sufficient for nuclear localization 226.

However, mutating certain arginine residues in the NLS alters the nucleolar but not the

nuclear localization of Rex. Several studies indicate that targeting of Rex to its correct

subcellular compartment is critical for its proper function 229,230.

The same stretch of arginine residues also serves as an RBD for the RxRE and is

similar to domains found in a diverse group of proteins including HIV Rev 231, HIV Tat

26 232, and RNAase P 233. The RBD binds specifically to a cis-acting regulatory element in

viral mRNAs called the Rex responsive element (RxRE) 234,235. The specific binding of

Rex to its RxRE is a critical step in its transactivation of mRNA export by allowing the

mRNA to overcome nuclear retention due to the inhibitory CRS 236. The CRS is located

in the 5’LTR in HTLV-1. In HTLV-2, it is located in the R/U5 region, within the 5’

RxRE and downstream of the splice donor site 195,236. Another CRS in HTLV-2

overlaps with the 3’RxRE.

Although the RxRE harbors a single high affinity binding site for Rex, it has been

suggested that multiple Rex proteins bind to a single mRNA molecule prior to export

via cooperative protein-protein and protein-RNA interactions 237,238. There are two

regions in both Rex-1 and Rex-2 that serve as multimerization domains 215,224,239. The multimerization domains map to amino acids 57-66 and 106-124 in Rex-1 and approximately residues 60-70 and 120-130 in Rex-2 215. Mutations in the

multimerization domains render Rex non-functional. In fact, multimerization mutants

behave as transdominants and hinder wild type Rex when cotransfected in transient

assays, suggesting that the ability of Rex to assemble into high order complexes on the

mRNA is critical for its function. However, there are some conflicting reports on the

role of multimerization in Rex function. First, Rex-1 has been shown to form stable

homo-oligomers in the absence of RxRE in vivo using a mammalian-two hybrid assay

207. Furthermore, studies by Heger et al indicated that despite the critical role for multimerization in Rex function, mutants defective in multimerization were still efficient in nucleo-cytoplasmic shuttling, suggesting that multimerization of Rex protein on its target mRNA does not play a direct role in export 240.

27 The activation domain (AD) of Rex was originally identified as the minimal region

that could functionally replace the HIV Rev activation domain in cis. It maps to

residues 79-99 in Rex-1 223,241 and residues 81 to 94 in Rex-2 215. Subsequent studies

showed that the sequence responsible for nuclear export of Rex, NES, is in fact its

minimal effector domain 242,243. The NES in Rex is a leucine rich motif involved in

protein-protein interactions that are critical for its function 231. First identified in HIV-1

Rev, this type of NES is found in many cellular and viral proteins with shuttling properties including visna virus Rev 244,245, adenovirus E4 34-kDa 246, RanBP1 247, TAP

248 249 , and IκBα . The consensus leucine-rich NES is LX2-3λX2-3LXL/I (with X being any amino acid and λ being an amino acid with a bulky hydrophobic side chain) with the core tetramer being LXLX. As described later, the most critical protein-protein interaction that occurs at the Rex AD/NES is with region maintenance interacting protein 1 (CRM1)/exportin1 250,251. Other proteins that interact with

AD/NES include eIF-5A 207, and human nucleoporin-like protein (hRIP/Rab) 252.

The Rex Responsive Element

In the mRNA, the Rex-1 responsive element (RxRE-1) is a stem loop structure of

205 nucleotides that is found in the 3’ LTR 253,254. The Rex-2 responsive element

(RxRE-2) is 226 nucleotides and is located in the 5’LTR and maps to the region in the

R/U5 that also contains the CRS 195,196,255,256. Mutational analyses indicate that the

actual Rex binding subdomains in the ~200 nucleotide RxRE are relatively short. A

stretch of 43 nucleotides constitutes the high affinity Rex-1 binding motif 234,235, whereas two short motifs (nucleotides 361-483 and 460-520) in RxRE-2 are sufficient

28 for maximal Rex-2 binding 216. Studies showing that only a short sequence or a single

stem loop structure is sufficient to mediate Rex function 235,257 raises the question of the

significance of the complex and relatively large stem loop structure formed by the

RxRE. It is likely that the free energy from a longer secondary structure ensures proper

folding into a stable secondary structure with a high affinity binding site. Another

explanation is that additional stem loops form secondary and less efficient binding sites

for Rex, or allow binding of cellular factors involved in Rex response. Also, the ability

of the RxRE to fold into stem loop structure brings the polyadenylation signal

(AAUAAA) into close proximity to the GU rich polyadenylation site 222,258, giving the

full RxRE a novel role in ensuring efficient polyadenylation of viral mRNA.

Substitution and deletion studies have demonstrated that proper folding of RxRE is

required to provide a docking site for Rex, and hence, is essential for its in vivo function

194,259.

Unlike HIV-1, all HTLV transcripts contain the RxRE. However, it is still unclear

how Rex preferentially regulates the unspliced and incompletely spliced versus the fully

spliced mRNAs. One likely explanation is that Rex binds to viral mRNA prior to the

commitment of splicing factors. On the other hand, Rex could actively inhibit the

splicing machinery prior to facilitating mRNA export 192,260. Indeed, Rex has been

shown to bind pre-mRNA splicing factors such as SF2/ASF and inhibit mRNA splicing

in vitro 203,261,262. In addition, studies by Seiki et al and King et al provide evidence that

the RxRE acts as a negative element in the absence of Rex 263,264.

Finally, more recent reports indicated that certain cellular mRNA-binding proteins could bind to the RxRE. One such factor is hnRNP A1, which binds to RxRE-1 and

29 interferes with Rex function 265. One study suggests that hnRNP A1 competes with

Rex-1 for binding to RxRE. The consequence of this competition might be impairment of Rex function in cells over-expressing hnRNP A1. Indeed, Rex-1 exhibits impaired function in Jurkat lymphoblastoid T-cells 266 due to interference from hnRNP A1,

whereas HTLV-1 infected cell lines such as MT-2 and HUT-102, where Rex is fully

functional, have low levels of this protein 265.

Interaction of Rex with host cellular factors

Several studies have identified cellular proteins that interact with Rex and either

augment its function or inhibit its activity. Of particular interest are proteins that

interact with the AD/NES, given the essential role of this domain in Rex function.

Among the proteins that interact with NES, CRM1 250,251, eIF-5A 207, and hRIP/Rab 252 have been studied in more detail. The most functionally significant interaction is with

CRM1, which bridges Rex to several cellular proteins at the nuclear pore complex as well as the Ran family of proteins. Using chemical crosslinking studies, it has been shown that while CRM1 and eIF-5A bind directly to the Rex NES, other proteins such as hRIP/Rab are brought to the NES via their association with CRM1 267.

CRM1, also called exportin 1, is a ~112 kDa nucleoprotein which is a member of

the importin-β family of transport receptors. The role of CRM1 in nuclear transport

was first identified using the antibiotic leptomycin B (LMB) 268, which covalently modifies CRM1 and subsequently inhibits the exit of Rev and Rex from the nucleus 269.

CRM1 localizes to the nucleoplasm, nucleolus, and NPC, and associates specifically

with CAN/Nup214; this association as well as NPC localization can be inhibited by the

30 FG-repeat domain of CAN/Nup214. 251,270,271. Since the importin-β family had been

shown to be involved in mRNA export 272, and CRM1 itself can shuttle between the

nucleus and cytoplasm 273, it was hypothesized that CRM1 could be a transport receptor for leucine rich NES-containing proteins. Indeed, CRM1 can directly interact with Rex-

1 and Rex-2 and mediate their function in vivo 274,275. At the molecular level, CRM1,

through its interaction with a wide range of proteins, can mediate the export of leucine-

rich NES-containing proteins. CRM1 can interact with GTP-bound Ran, but only in the

presence of the NES 271,273,276. Since Ran-GTP is predominantly a nuclear protein, it is

assumed that the formation of Rex NES/CRM1/Ran-GTP complex can only occur in the

nucleus. CRM1 is then thought to extensively interact with members of the NPC to facilitate the export of this assembled complex 277-280. Studies using LMB have

confirmed the essential role of CRM1 in the export of NES-containing proteins 271,281.

Interestingly, the overexpression of CRM1, partially rescues the ability of Rex mutants to multimerize indicating that CRM1 facilitates Rex multimerization 275.

The role for eIF-5A in mRNA export was first indicated in studies of HIV-1 Rev 282,

and was subsequently implicated in Rex-mediated mRNA transport 207. Studies using

Glutathione S-transferase (GST)-fusion proteins containing the NES of HTLV-1 Rex

showed that eIF-5A inhibitors selectively block the nucleo-cytoplasmic export of Rex

NES and indicated a role for eIF-5A upstream of CRM1 281. Although it was initially

thought that eIF-5A is only involved in translation initiation, this small protein (~19 kDa) is pleiotropic and functions in cell proliferation, gametogenesis, senescence and apoptosis 283-285.

31 Yeast two-hybrid screens revealed another protein that interacts with the NES of

Rev and Rex named Rev/Rex interacting protein or Rev/Rex activation domain binding protein (RIP/Rab) 252. This protein is classified as a nucleoporin because it contains

FG-repeat sequences and is homologous to the FG-nucleoporin Nup153p/CAN1. The

ability of Rex to interact with RIP/Rab was utilized to identify the leucine rich NES 241.

In yeast, RIP/Rab is essential for Rev nucleoplasmic as well as nuclear pore complex

(NPC) localization. However, in yeast strains that are deficient in RIP/Rab, Rev is still partially functional 201,286. A recent study of the role of RIP/Rab in Rev function reveals

that it promotes the release of Rev/RRE complexes from the perinuclear region 287.

Based on the similarity between Rex and Rev, it is reasonable to conclude that RIP/Rab plays a similar role in Rex function. Yeast two-hybrid screens have identified a number of FG-repeat containing proteins that interact with Rev and Rex 288. Interestingly, it

was later shown that the interaction between the NES and these FG-repeat containing

proteins is lost in yeast strains lacking the export receptor CRM1/exportin 1, indicating

that the latter is important for bridging Rex/Rev and the nucleoporins 267.

Mechanism of Rex function and the Rex transport cycle

The mechanism of Rex function involves several discrete steps that must occur in a

sequential order for proper RxRE-dependent export of intron-containing mRNA. As shown in Figure 1.3, the proposed model reflects the transport cycle of Rex. A

prerequisite for proper Rex activity is the localization of newly synthesized Rex to the nucleus/nucleolus. The transport cycle itself involves: (a) binding of Rex to the RxRE present in incompletely spliced mRNA, resulting in the protection of this mRNA from

32 splicing and/or degradation; (b) Rex multimerization; (c) formation of an

RNA/Rex/CRM1/Ran-GTP complex; (d) interaction of the complexes with NPC and exit from the nucleus; (e) hydrolysis of Ran-GTP to Ran-GDP, resulting in dissociation of the complex and subsequent release of the cargo mRNA; and (f) return of Rex to the nucleus to start another cycle.

As mentioned earlier, the nascent Rex protein is actively imported into the nucleus through the NPC. Passage of proteins through the NPC is strictly selective and involves bridging factors such as the import/transport receptors that bind to the FG-repeat containing nucleoporins resulting in the transport of their cargo through the NPC 289-291.

Rex is able to localize to the nucleus due to the arginine-rich NLS at its N-terminus.

Transport receptors that recognize and import NLS-containing proteins are termed importins 292. Importin-β heterodimerizes with importin-α and enters the nucleus with

cargo protein (NLS-containing protein) 293. Classically, importin-α is the subunit that

directly binds to the prototype NLS (lysine-rich or basic). In contrast, the Rex NLS is

arginine-rich and hence does not interact with importin-α. Indeed, the results of

digitonin-permeabilized nuclear import assays demonstrated that Rex does not require

importin-α for its import and that importin-β alone is sufficient for this purpose 294.

Due to the between the Rex NLS and the importin-α-binding

domain on importin-β, it is suggested that Rex NLS binds directly to importin-β without the adaptor protein, importin-α 231. The nuclear Ran-GTP binds to the N-terminus of

importin-β once it reaches the inner side of the nuclear membrane and possibly as early

as the nuclear pore basket, resulting in the release of Rex into the nucleus 295,296. The newly formed Ran-GTP/importin-β complex is then transported back into the 33 cytoplasm, where the GTP on Ran is hydrolyzed into GDP, leading to the dissociation

of the complex. This makes importin-β available for another round of import, whereas

Ran-GDP is translocated into the nucleus, where it is converted back to Ran-GTP 297.

Some earlier reports indicated that other molecules are also involved in Rex import into

the nucleus such as B23 298 and p32 or its murine homologue YL2 299. Using affinity column chromatography with Rex-1 NLS, it has been shown that the NLS specifically binds to B23 from the nuclear extract of a variety of cell lines (Jurkat, HUT 102, Molt-

4, Hela) 298. However, later studies failed to confirm co-localization of B23 and Rex

proteins 214. YL2 has been shown to interact with Rex NLS using a yeast two-hybrid

screen 299. YL2 also copurifies with SF2/ASF, a factor involved in alternative splicing

300. The exact role of these factors in Rex function or nuclear import is still not clearly

understood.

Once Rex is released from importin-β, the RNA-binding domain, which overlaps

with the NLS, becomes available for binding to the RxRE. As stated earlier, several

events could explain the mechanism of Rex function at the molecular level. First,

binding of Rex increases the stability of mRNA, which might in turn facilitate its

cytoplasmic transport 193. Alternatively, Rex could bind to the mRNA and inhibit the

cellular splicing machinery 192,203,260,299.

Rex likely binds with high affinity to its primary site in the RxRE, and then

additional molecules of Rex are bound to secondary sites due to cooperative protein- protein and protein-RNA interactions. The multimerization of Rex on its target mRNA has been shown by several groups to be essential for its function 215,224,239,275. It should

be noted that multimerization deficient mutants have been shown to be able to export

34 RxRE-containing mRNA with the same efficiency as wild type protein as long as there

is an intact NES 240,301. Thus, although multimerization of Rex is important for function, it is not essential for mRNA export. It is likely that multiple Rex molecules

are required to effectively counteract the nuclear retention signals on intron-containing

mRNA.

Rex-dependent mRNA export proceeds once a stable Rex/RxRE/CRM1 complex is

assembled. CRM1 interacts directly with Rex and its cargo mRNA and facilitates their

transport through the nuclear pore complex through a series of protein-protein

interactions with the FG-repeat-containing nucleoporins 250,251,271,275,302. The complex

formed between CRM1, Rex and the mRNA is then recognized and bound by Ran-GTP

276. This last association leads to the final steps needed to exit the nuclear pore 303.

Other proteins that play a role in the export of Rex have been identified. Ran binding protein (RanBP3) contains FxFG and FG repeats and is proposed to interact with NPC

277,278,304. Since RanBP3 is a non-shuttling protein 279, it is suggested that this protein is

essential for the steps leading to the migration of the transport complex to the nuclear

face of the NPC, but is released upon exit to the cytoplasm. As mentioned earlier, eIF-

5A and RIP/Rab may also play a role in Rex-mediated mRNA export. More recently,

another nuclear protein termed Src-associated protein in mitosis (Sam) 68 has been

reported to augment Rex/RxRE and Rev/RRE function 305,306. The synergistic effects of

Sam 68 on Rex/RxRE function appear to be LMB independent, suggesting that Sam 68 augments Rex function by utilizing a pathway that is distinct from that of CRM1 305.

The Rex transport cycle ends in the cytoplasm upon the release of cargo mRNA.

Cytoplasmic RanGP1 and RanBP bind to Ran-GTP and catalyze the hydrolysis of GTP

35 to GDP. This hydrolysis step likely causes the dissociation of the complex and the

subsequent release of the mRNA. Free Rex and CRM1 proteins shuttle back to the

nucleus to start another cycle, whereas the mRNA can be used for translation of

enzymatic and structural viral proteins or ultimately used for packaging into virions.

Role of phosphorylation in the regulation of Rex

Due to the critical role of Rex in the viral life cycle, it is unlikely that such a protein would lack regulation. Posttranslational modification by phosphorylation is one of the most common mechanisms of protein regulation. It has been well documented that a common consequence of phosphorylation is a charge-induced conformational change in the protein’s 3-dimensional structure, leading to different interactions with distinct partners and ultimately function. Examples of conformational changes causing a mobility shift that is detectable by SDS-PAGE include Fos 307, Myc 308, the polyomavirus large T-antigen 309, and adenovirus E1A protein 310. In most cases, the

shift results from phosphorylation of one or more residues. As mentioned earlier,

HTLV-2 Rex migrates as two isoforms in SDS-PAGE, p24 and p26 311. These forms

differ in their level of phosphorylation 208. There is no such distinction for

phosphorylated versus unphosphorylated Rex-1 312.

Early reports indicated that Rex-1 and Rex-2 are phosphoproteins and that phosphorylation is essential for their function. For example, treatment of HTLV-1 infected cells with the protein kinase C inhibitor H-7 results in a decreased level of Rex- mediated accumulation of viral unspliced mRNA 313, suggesting that phosphorylation is

important for Rex-1 function; a subsequent study showed that Rex-1 is phosphorylated

36 at serines 70 and 177 and threonine 174 312. In HTLV-2, the two forms of Rex-2 are

detected in virus-infected cells as well as cells transfected with Rex expression plasmids

190,208,216,311,314. Using immunofluorescence techniques, it was possible to show that p24

predominantly localizes to the cytoplasm whereas p26 has nuclear/nucleolar

localization 211,215, suggesting that the active form of Rex-2 is the phosphorylated one.

Indeed, it was shown that phosphorylation of Rex-2 enhances its ability to bind to

RxRE-containing mRNA 315. In addition, the phosphorylated form of Rex-2 has been implicated in the inhibition of mRNA splicing 203. A more detailed mutational analysis

of Rex-2 targeting all serines and threonines revealed that phosphorylation of two key serines in the C-terminus (S151, S153) is critical for Rex-2 function. Replacement of these serines with alanine relocalizes Rex-2 to the cytoplasm and renders it functionally inactive, whereas aspartic acid substitutions lock the protein in a constitutively active form that localizes mainly to the nucleolus 209. Several pieces of evidence suggest that

Rex-2 is also phosphorylated on other residues, leading to the hypothesis that additional

phosphorylation sites are required to stabilize the active form of Rex-2. A subsequent

study suggested that the C-terminus of Rex-2, including the phosphorylation sites S152

and S153, is involved in nuceocytoplasmic shuttling of the protein, thus providing a better molecular understanding of the influence of phosphorylation on Rex function 215.

The implication of phosphorylation and the critical role of this event on the function of

Rex is that HTLV gene expression is regulated at multiple levels by cellular factors.

Such regulation might be essential for the virus to better adapt by responding to regulatory signals of the cells it infects, providing HTLV with an additional level of replication control.

37 Role of Rex in cellular transformation, viral spread, and tropism

At the molecular level, the basic role of Rex is to regulate the cytoplasmic levels of

viral genomic mRNA and the expression of the structural and enzymatic gene products that are essential for production of viral progeny 80. Therefore, it is proposed that Rex

plays a critical role in the transition from the early, latent phase to the late, productive

phase of HTLV infection and hence is required for efficient viral spread. Despite the

critical role of Rex for efficient viral replication, it has been shown that low, but

detectable levels of HTLV p19Gag and p24Gag are produced from T-cells transfected

with HTLV-1 and HTLV-2 proviruses that are Rex-deficient 193,316. These bring into

question the absolute requirement of Rex for HTLV replication. It is thus important to understand the contribution of Rex to HTLV outside its function in replication.

The pleiotropic protein Tax is the main transcriptional activator of HTLV genes,

and its role in cellular immortalization/transformation has been very well established

53,317,318. The role of Rex in this process has been addressed in a study that utilized a

Rex-deficient HTLV-1 proviral clone and characterized the role of Rex in cellular

immortalization/transformation, viral replication, spread, and persistence in inoculated

rabbits 316. This report confirmed that Rex is not absolutely required for structural

protein expression, as the production of p19Gag was still detectable, albeit at much

lower levels compared to the wild type virus (by a factor of 116). Using an in vitro

system in which irradiated producer cells were cocultured with peripheral blood

mononuclear cells (PBMCs), it was shown that Rex-deficient viruses were able to

sustain IL-2-dependent, long-term growth of T-cells, suggesting that Rex is dispensable

for cell-mediated infection/spread of HTLV as well as immortalization of primary T

38 lymphocytes. However, when inoculated into immune competent rabbits, the Rex-

deficient provirus failed to infect, spread, persist or induce a detectable immune

response, indicating that Rex is important for the establishment of viral infection and

presistence in vivo 316.

Although HTLV-1 and HTLV-2 can infect a variety of cell types in vitro, they only

induce transformation and/or pathogenesis in T lymphocytes 319-322. However, the

tropism of these two viruses in T cells is distinct. While HTLV-1 has a preferential

tropism for CD4+ T cells in both ATLL patients and those with neurological disease 47, the in vivo tropism of HTLV-2 is less clear. Some reports indicate that it can be detected in both CD4+ and CD8+ T cells, with greater viral burden in CD8+ T-cells

46,323,324. In vitro, HTLV-2 has a preferential tropism for CD8+ T cells 44,45. Given the

critical role of the post-transcriptional regulator Rex in viral gene expression and

replication, it was proposed that Rex may be the genetic viral determinant responsible

for the transformation tropism of HTLV-1 and HTLV-2. A study in which Tax and Rex

ORFs were exchanged between the two viruses indicated that despite the altered Tax

and Rex transactivation activities in the new recombinant viruses, both recombinants

were able to replicate, infect and immortalize T lymphocytes 44. Therefore, neither Tax

nor Rex confers the distinct transformation tropism of HTLV-1 and HTLV-2 45. Further

gene exchange between HTLV-1 and HTLV-2 will be required to identify the viral

determinants responsible for their distinct tropism.

39

Figure 1.1. Genome organization of HTLV-1 and HTLV-2 and their unspliced (U), singly spliced (S) and doubly spliced (D) mRNAs. A. HTLV-1 expresses 8 major

mRNA species. The genomic unspliced mRNA encodes the Gag, Pol and Pro proteins.

Four singly spliced mRNA species are the result of splicing of exon 1 (nt 1-119) to

unique splice acceptors at positions 4641 (Env), 6383 (p12), 6950 (p21rex), and 6875

(p13). The three doubly spliced mRNAs include exon 1, exon 2 (4641-4831) and a

third exon that starts at 6950 (Tax/Rex), 6478 (p30), or 6383 (p27, a putative Rex-p12

hybrid). Exons (solid lines) are designated by their positions in the viral mRNA. B.

The similar HTLV-2 genome expresses at least 7 mRNAs. In addition to the unspliced

species, three singly spliced mRNAs contain exon 1 (nt 1-134) linked to splice acceptor

sites at 4729 (Env), 6629 (p28, p22/p20), or 6899 (p28, p22/p20). The major doubly

spliced mRNA encodes Tax/Rex and contains exon 1 and exon 2 (nt 4729-4868) linked

to a splice acceptor at position 6899. The other doubly spliced mRNAs contain exon 1

and 2 linked to a splice acceptor at 6629 or 6491; their protein products are less

characterized. In both panels, the positions of the RxRE and CRS are indicated by

horizontal bars; nucleotide numbering starts at the beginning of the R region. Open and

black arrow heads indicate the positions of splice donors and splice acceptors,

respectively.

40

41

Figure 1.2. Domain structure of the HTLV-1 and HTLV-2 Rex. The functional domains or regions of the 189 aa Rex-1 and 170 aa Rex-2 proteins are depicted in shaded boxes. The RNA binding domain (RBD) and nuclear localization signal (NLS) are located within the first 19 amino acids in the N-terminus. The Core activation domain (AD), which encompasses the nuclear export signal (NES), lies between residues 79 and 99 in Rex-1 and 81-94 in Rex-2, respectively. Two multimerization domains span amino acids 57-66 and 106-124 in Rex-1, whereas residues 57-71 and

124-132 constitute the multimerization domains in Rex-2. A unique C-terminal domain has been described for Rex-2 spanning residues 144 to 164 that is important for efficient function and includes key phosphorylation sites (Ser 151 and Ser 153). Mutations in this region are also impaired for efficient nucleo-cytoplasmic shuttling.

42

Figure 1.3. The Rex-dependent export of viral mRNA. Shortly after transcription the

RxRE-containing mRNA is recognized and bound by Rex through its RBD (a). Rex multimerization and CRM1 binding generates a complex (b) that is recognized by Ran-

GTP (c). The passage of RNA/Rex/CRM1/Ran-GTP complex through the nuclear pore depends on extensive protein-protein interactions between CRM1, the FG-repeat domains of nucleoporins, and other factors (d). Once in the cytoplasm, Ran-GTP is hydrolyzed into Ran-GDP, causing the dissociation of the complex (e). The free mRNA is now available for either translation or packaging into virions. The other components of the complex shuttle back into the nucleus (f) to start another cycle. Rex itself utilizes importin-β for nuclear import.

43

CHAPTER 2

HUMAN T-CELL LEUKEMIA VIRUS TYPE 1 EXPRESSING NON-

OVERLAPPING TAX AND REX GENES REPLICATES AND

IMMORTALIZES PRIMARY HUMAN T-LYMPHOCYTES BUT FAILS TO

REPLICATE AND PERSIST IN VIVO

ABSTRACT

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus associated primarily with adult T-cell leukemia and neurological disease. HTLV-1 encodes the positive trans-regulatory proteins Tax and Rex, both of which are essential for viral replication. Tax activates transcription initiation from the viral long terminal repeat and modulates the transcription or activity of a number of cellular genes. Rex regulates gene expression post-transcriptionally by facilitating the cytoplasmic expression of incompletely spliced viral mRNAs. Tax and Rex mutants have been identified that have defective activities or impaired biochemical properties associated with their function. To ultimately determine the contribution of specific protein activities on viral replication and cellular transformation of primary T-cells, mutants need to be characterized in the context of an infectious molecular clone. Since the tax and rex genes are in partial overlapping reading frames, mutation in one gene frequently disrupts the other, confounding interpretation of mutational analyses in the context of the virus. Here we generated and characterized a unique proviral clone (H1IT) in which

44 the tax and rex ORFs were separated by an internal ribosome entry site. We showed that H1IT expresses both functional Tax and Rex. In short- and long-term coculture assays, H1IT was competent to infect and immortalize primary human T-cells similar to wt HTLV-1. In contrast, H1IT failed to efficiently replicate and persist in inoculated rabbits, thus, emphasizing the importance of temporal and quantitative regulation of specific mRNA for viral survival in vivo.

INTRODUCTION

Human T-cell leukemia virus type-1 (HTLV-1) is a complex retrovirus that is associated with adult T-cell leukemia as well as a neurological disorder termed HTLV- associated myelopathy/tropical spastic paraparesis 325-328. The genome organization of

HTLV-1 resembles that of other complex retroviruses that contain overlapping regulatory and accessory genes in addition to structural and enzymatic genes 80,329,330.

Tax and Rex are two trans-acting regulatory gene products that are essential for viral

replication 190,329. Upon HTLV-1 infection of a susceptible host cell, the randomly

integrated provirus is expressed at low levels. Tax, being the major transcriptional

activator of the viral long terminal repeat (LTR), mediates high level viral gene

expression necessary for efficient viral protein production. Tax-mediated activation of

HTLV-1 LTR is dependent on three imperfect 21 repeats, termed the Tax

response elements (TRE) 133,138,331. Tax also has been shown to disrupt cellular gene

expression by recruiting or interacting with cellular transcription coactivators, by

indirectly activating the NFκB activation pathway, or by modulating the activity of

cellular proteins 151,153,332,333. Many of the cellular genes modulated by Tax are involved

in growth, differentiation, apoptosis, or cell cycle control 177,334-337. Thus, a strong body

45 of evidence suggests that the effects of Tax on cellular processes are required for the

transforming or oncogenic capacity of HTLV 50,53,317,338. Indeed, mutational analysis in

the context of a replicating virus directly demonstrated that Tax of both HTLV-1 and

HTLV-2 are essential for virus-mediated cellular transformation of primary human T-

cells and that Tax activation of NFκB and CREB/ATF signaling plays a key role in the

malignant process 53,317,318.

Rex is a post transcriptional regulator that is essential for efficient cytoplasmic

expression of incompletely spliced viral mRNA 130. The HTLV structural and

enzymatic proteins are expressed from intron-containing mRNAs that would be targeted

for splicing or degradation in the nucleus in the absence of Rex 193,194,260. Rex binds to

its viral RNA responsive element (RxRE), stabilizes the mRNA, and facilitates its export from the nucleus. Based on the critical role of Rex in the expression of structural

and enzymatic gene products, it is considered to positively regulate the switch between

the early, latent phase and the late, productive phase of HTLV life cycle 130. Although

Rex is not absolutely required for cellular transformation in vitro, it is essential for efficient viral infectivity, spread, and survival in vivo 316.

Extensive mutational analyses of both Tax and Rex have mapped specific activities and biochemical properties to distinct regions or domains of the protein 129,130.

However, the majority of these mutations, particularly those that maintain replication competence, have been studied in cDNA expression systems in non lymphoid cells and their overall contribution to the virus life cycle or HTLV-mediated transformation has yet to be tested. Since Tax and Rex are encoded on the same viral mRNA in partial overlapping reading frames, it has been difficult, if not impossible, to introduce

46 mutations in one gene while maintaining the integrity of the other. Therefore, studying

such mutants precludes a clear interpretation of structure and function analyses in the

context of the virus.

The goal of this study was to generate and characterize an infectious proviral clone

in which the tax and rex genes were separated by expressing Tax from an internal

ribosome entry site (IRES) 339. In this uniquely modified provirus, HTLV-1-IRES-Tax

(H1IT), Rex is expressed from the natural doubly spliced mRNA and Tax is initiated 3’

of Rex as a result of the inserted IRES. Since the IRES-Tax was introduced at the 3’

end of the provirus, Tax now has the potential to be expressed from all viral messages.

Transient transfection studies in 293T cells indicated that H1IT expresses both

functional Tax and Rex and is able to produce p19 Gag. Furthermore, we showed that

729-H1IT stable transfectants produced replication competent virus with the capacity to immortalize primary human T-cells. Interestingly, H1IT was not able to persist in a rabbit model of infection, consistent with the conclusion that temporal and quantitative regulation of specific mRNAs is key to viral survival in vivo. This new reagent will be particularly valuable for future studies designed to dissect the contribution of specific activities of either Tax or Rex on HTLV-1 pathobiology, such as viral transformation of primary T-lymphocytes.

MATERIAL AND METHODS

Cells. 293T cells and 729 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and Iscove’s modified Eagle’s medium (IMEM), respectively. The media were supplemented to contain 10% fetal bovine serum, 2 mM glutamine,

47 penicillin (100 U/ml), and streptomycin (100 µg/ml). Human peripheral blood mononuclear cells (PBMCs) were isolated from freshly drawn human blood as previously described 316. Briefly, blood was diluted 1:1 with phosphate buffered saline

(PBS) and layered over 12 ml of Ficoll-hypaque (Amersham, Piscataway, NJ), then centrifuged for 20 min. The Ficoll/plasma interphase (buffy coat layer) was gently removed, transferred to a new tube and washed once with RPMI. The cell pellets were resuspended in RPMI supplemented with 20% fetal bovine serum, antibiotics, and 10

U/ml of human interleukin-2 (IL-2) (Roche, Indianapolis, IN).

Plasmids. The plasmid containing the wild type (wt) HTLV-1 proviral clone, pACHneo, was used in this study 316,340. HTLV-1 F4Term is a Tax knockout proviral

clone. It was generated by site directed mutagenesis (QuickChange, Stratagene, La

Jolla, CA) of pACHneo introducing TC to AG point mutations (nt 7307 and 7308) that

resulted in a stop codon at amino acid 4 of Tax while maintaining the Rex reading

frame. HTLV-1 F4Term was used as a template to generate the H1IT proviral clone.

An EcoR1 restriction enzyme site was introduced in sequences just downstream of the

Rex stop codon and was used to replace the remaining 3’ HTLV-1 proviral sequences

with the encephalomyocarditis virus (EMC) internal ribosome entry site (IRES), the

complete tax gene, and the 3’ LTR (Fig. 2.1). The full length H1IT construct was confirmed by restriction enzyme diagnostics and sequencing. The LTR-1-Luc Tax

reporter plasmid has been described previously 341. The HIV-1 Tat expression vector,

pctat, contains HIV-1 tat cDNA cloned downstream of the CMV promoter. The pCgagRxRE-I reporter contains the HIV-1 LTR promoter and gag gene linked to a

48 fragment of HTLV-1, spanning the RxRE (R-U5 region of the LTR) 211. CMV-βgal or

CMV-Luc plasmids were used to control for transfection efficiency in each experiment.

Transfection, luciferase reporter assay, and Gag ELISA. To measure Tax

CREB/ATF activating function (viral LTR), 2x105 293T cells were transfected using

Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendation. The total amount of DNA was kept constant and was composed of

0.2 µg LTR-1-Luc, 0.1 µg CMV-βgal, and 1 µg wt HTLV-1 or H1IT proviral clone.

After 48 h of growth, cells were pelleted and 450 µl of cell supernatants were used for p19 Gag enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s recommendations (Zeptometrix, Buffalo, NY). The cell pellets were lysed in passive lysis buffer (PLB) (Promega, Madison, WI) and Tax activity was measured as luciferase light units. All experiments were performed independently three times in triplicate, and results were normalized for transfection efficiency using β-galactosidase. The Rex functional assay was performed as described 209. Briefly, 0.4 µg of control plasmid,

HTLV-1, or H1IT proviral clone was cotransfected with 0.1 µg CMV-Luc, 0.1 µg pctat, and 0.3 µg of pCgagRxRE-I reporter. Cell lysates were prepared in PLB 48 h post- transfection and luciferase activity was determined to control for transfection efficiency.

HIV-1 p24 Gag level in cell lysate was measured using p24 Gag ELISA (Beckman-

Coulter, Fullerton, CA).

Generation and characterization of a stable 729-H1IT cell line. To generate stable transfectants, proviral plasmid clones containing the Neor gene were introduced into 49 cells by as described previously 342,343. Stable transfectants were

isolated following incubation in 24-well culture plates in medium containing 1 mg/ml of

geneticin. After four-to-five weeks of selection, viable cells were single cell cloned,

expanded, and maintained in culture for further analysis. The clones were screened for p19 Gag expression in the cell supernatant using an ELISA. 729ACHneo, a previously generated and characterized stable wt HTLV-1 producer cell line 340, was used as a

positive control.

DNA preparation, PCR, and Western blotting. Genomic DNA was isolated from

permanently transfected cell clones or from immortalized PBMCs using the

PUREGENE DNA purification system (Gentra, Minneapolis, MN). A primer set that

amplifies a 270 bp fragment from both HTLV-1 and H1IT is 670: 7335CGG ATA CCC

AGT CTA CGT GT7354 and ACH3AS: 7605GGG TGG AAT GTT GGG GG7589, whereas

the primer set LTax 5’CGA TGA TAA TAT GGC CCA CTT CCC AGG GTTT G and

ACH3AS amplifies a 318 bp fragment from H1IT only. PCR products were run on an

agarose gel and visualized by ethidium bromide staining.

To detect viral proteins, 729 stable transfectants were lysed with modified RIPA

buffer (50mM Tris-Cl [pH8.0], 150mM NaCl, 1% Nonidet P-40, 0.5% desoxycholate,

0.1% sodium dodecyl sulfate (SDS), 2.0mM phenylmethanesulfonyl fluoride, 20µg/ml

aprotinin, 1.0 mM Na3VO4, and 1mM NaF) on ice for 30 min. After centrifugation, the cell lysates were subjected to 4-12% SDS-PAGE, and transferred to nitrocellulose

(Amersham, Piscataway, NJ). Rabbit anti-Tax-1 or anti-HTLV-1 patient serum was used to detect different viral proteins. Viral proteins were visualized using the ECL

50 Western blotting analysis system (Santa Cruz , Santa Cruz, CA).

Short-term coculture microtiter proliferation and long-term immortalization

assays. Short-term microtiter proliferation assays were performed as described

previously with slight modifications 344. Briefly, freshly isolated human PBMCs were

prestimulated with 2µg/ml phytohemagglutinin (PHA) and 10U/ml IL-2 for two days.

729 HTLV producer cells (200 cells) were gamma-irradiated with 10,000 rads and cocultured with 104 prestimulated PBMCs in the presence of IL-2 in 96-well round

bottom plates. Wells were enumerated for growth and split 1:4 at weekly intervals.

This procedure ensured that only dividing cells growing fast enough to show visible

pellets at the bottom of the wells were maintained, whereas in the wells with non-

dividing cells or limited proliferation, pellets became smaller each week and usually disappeared by 3-4 weeks. For the long-term immortalization assays, 106 irradiated

729 producer cells were cocultivated with 2 × 106 freshly isolated PBMCs with 10U/ml

IL-2 in 24-well culture plates 343. The presence of HTLV expression was confirmed by

detection of p19 Gag protein in the culture supernatant using an ELISA at weekly

intervals. Viable cells were counted weekly by trypan blue exclusion. Cells inoculated

with wt HTLV-1 or H1IT that continued to produce p19 Gag antigen and proliferate

eight weeks post-coculture in the presence of exogenous IL-2 were identified as HTLV

immortalized.

Rabbit inoculation procedures. Twelve-week-old specific pathogen-free New

Zealand White rabbits (Hazelton, Kalamazoo, MI) were inoculated via the lateral ear

51 vein with approximately 1 x 107 gamma-irradiated (7500 rad) 729-HTLV-1 (6 rabbits),

729-H1IT (2 rabbits), or 729 uninfected control cells (2 rabbits). Cell inoculums were

equilibrated based on their p19 Gag production. At weeks 0, 2, 4, 6, and 8 after

inoculation, 10 mL of blood was drawn from the central ear artery of each animal.

Serum reactivity to specific viral antigenic determinants was detected using a

commercial HTLV-1 Western blot assay (Zeptometrix, Buffalo, NY) adapted for rabbit plasma by use of avidin-conjugated goat anti-rabbit IgG (1:200 dilution) (Sigma, St

Louis, MO) 110. Serum showing reactivity to Gag (p24 or p19) and Env (gp21 or gp46)

antigens was classified as positive for HTLV-1 seroreactivity. To detect integrated

proviruses, genomic DNA was harvested using the PUREGENE DNA purification

system (Gentra Systems, Minneapolis, MN) and 0.5µg of DNA was subjected to 40-

cycle PCR using primer pair 670 and 671 to amplify a 159-bp fragment specific for the

HTLV-1 tax/rex region 193 and rabbit GAPDH was detected using primer pair

rGAPDH-S, 5’GAT GCT GGT GCC GAG TAC GTG G and rGAPDH-AS, 5’GTG

GTG CAG GAT GCG TTG CTG A. PCR products were run on an agarose gel and

visualized by ethidium bromide staining.

RESULTS

Construction and characterization of an HTLV-1 proviral clone with non-

overlapping Tax and Rex sequences. In order to separate Tax and Rex open reading

frames (ORFs) in an HTLV-1 proviral clone, we designed a strategy that utilizes an

internal ribosomal entry site (IRES) from the encephalomyocarditis virus. The insertion

of the IRES between 2 ORFs in a bicistronic mRNA allows the translation of the

52 downstream ORF in a cap-independent manner. The strategy for the generation of an

HTLV-1 proviral clone with non-overlapping Tax and Rex (H1IT) is highlighted in

Figure 2.1. Although the addition of the IRES would result in a severely truncated (173

amino acids) nonfunctional Tax 345, the original Tax ORF was truncated further by

mutating the fourth amino acid codon of Tax to introduce a termination codon. In this

construct (HTLV-1 F4Term) the proviral clone is completely deficient for Tax activity

while Rex is still functional (data not shown). It should be noted that the F4Term

mutation introduces a single amino acid substitution of proline to alanine in the p30/p13

reading frame, which did not disrupt the repressive function of p30 on tax/rex mRNA

118,341 (data not shown). Using an EcoR1 site that was introduced immediately downstream of the Rex stop codon, a cassette that contains IRES-Tax cDNA in addition to the rest of the 3’LTR was introduced. The new proviral clone termed H1IT is ~1 Kb larger than wt HTLV-1.

Since efficient replication and virion production are highly dependent on functional

Tax and Rex, we tested the activities of these two proteins expressed by H1IT as compared to the wt parental clone. Co-transfection of 293T cells with the HTLV-1 or

H1IT proviral clone and the Tax reporter LTR-1-Luc resulted in 20-30 fold increase in

Tax-dependent gene expression from both clones (Fig. 2.2A). Similarly, when these clones were co-transfected with a Rex reporter that expresses HIV-1 p24 Gag under the control of Rex-response element, there was approximately a six-fold induction in Rex functional activity (Fig 2.2B). Our data indicates that Tax or Rex activity expressed from H1IT was not significantly different from wt HTLV-1. We were surprised at this result since it has been reported that IRES-dependent expression is less efficient than

53 cap-dependent expression 346. We attribute the recovery of full Tax activity and,

indirectly, Rex activity in H1IT to the fact that in H1IT, the primary viral transcriptional regulator, Tax, is potentially expressed from all viral transcripts as compared to a single doubly spliced transcript in wt HTLV-1.

Generation and characterization of H1IT viral producer cell lines. To assess the ability of the H1IT proviral clone to produce viral proteins, direct viral replication, and induce cellular immortalization, stable H1IT producer cells were generated. 729 B-cells were electroporated with the H1ITneo plasmid, and three-to-four weeks after transfection, wells containing visible clumps of Geneticin-resistant cells were single cell cloned and further characterized. Several cell clones were screened using ELISA for the production of p19 Gag released into culture supernatants. At least two cell clones showed robust p19 Gag production (Fig. 2.3A) and the H1IT clone 11 was used for the rest of this study. The integration of intact proviral sequences was determined using diagnostic PCR analysis of genomic DNA isolated from the stable producer cells.

Using a primer that is specific for the IRES-Tax junction (see Fig. 2.1), it was possible to differentiate between wt HTLV-1 and H1IT proviruses. As shown in Figure 2.3B, only 729-H1IT cells showed a product when using the LTax/ACH3AS primer pair, whereas a PCR product was visible in both 729-H1IT and 729-HTLV-1 cells when using the 670/ACH3AS primer pair. Further characterization was conducted to confirm that the IRES was still functional in the stable producer cells. Since Tax translation in this context is tightly dependent on a functional IRES, we confirmed the expression of a functional Tax in 729-H1IT following transfection of the Tax reporter LTR-1-Luc (Fig.

54 2.3C).

We next monitored viral protein production in 729-H1IT as compared to 729-

HTLV-1. Total proteins from each cell line were extracted and separated by SDS-

PAGE. Using HTLV-1 specific antibodies and HTLV-1 patient antisera, we were able

to detect comparable levels of p24Gag, Tax, gp46Env and gp55 from 729-H1IT as well

as 729-HTLV-1, but not 729 control cells (Fig. 2.3D). Taken together, these results

indicate that viral gene expression in producer cells was not significantly disrupted

either by the separation of Tax and Rex or by IRES-dependent Tax expression.

H1IT immortalizes PBMCs. We next assessed the capacity of the H1IT viruses to immortalize human PBMCs. 729, 729-HTLV-1 and 729-H1IT cells were lethally irradiated and co-cultured with freshly isolated PBMCs in the presence of 10 U/ml of human IL-2 in 24-well plates. Cell number and viability were monitored at approximately weekly intervals to follow the immortalization process and the characteristic expansion of cells from the PBMC mixed cell population. It is important to note that the irradiated cells die off within three weeks of the coculture, hence detection of HTLV-1 p19 Gag beyond that point would be attributed to the infected and proliferating PBMCs. A growth curve of a representative assay indicated a progressive loss of viable cells over time in cocultures containing irradiated uninfected 729 and

PBMCs (Fig. 2.4A). In contrast, PBMCs cocultured with either 729-HTLV-1 or 729-

H1IT showed very similar progressive growth patterns consistent with immortalization.

Since viral p19 Gag production is a good indicator of viral replication, supernatants from the immortalized cells also were monitored at weekly intervals for the release of

55 p19 Gag (Fig. 2.4B). The first few weeks in coculture reflect residual p19 Gag from the producer cell lines, while starting at week four there was a consistent and continuous accumulation of p19 Gag in the culture supernatant indicating viral replication and virion production. Our data indicated that H1IT, like wt HTLV-1, had the capacity to productively infect PBMCs and induced sustained proliferation or immortalization of T- lymphocytes in vitro.

In order to get a more quantitative measure of the ability of the H1IT virus to infect and transform PBMCs, a short term limiting dilution infectivity assay was employed.

In this assay, a fixed number of PBMCs is cocultured with different dilutions of virus producing cells in a 96-well plate. Each week, the cells are resuspended and split at a ratio of 1:4. Since three-fourths of the cells in each well are discarded, only wells containing actively dividing cells will be able to replenish the cell number and appear as a white cell pellet at the bottom of the well. Within three weeks, irradiated producer cells as well as cell debris were not visible and cell pellets reflected growing cells (Fig.

2.5). Since this assay is very stringent, slowly growing or non-dividing cells are eliminated very quickly and the percentage of surviving wells is an accurate measure of the immortalization efficiency of viruses. A Kaplan-Meir plot of HTLV-1-induced T- cell proliferation indicated the percentage of wells containing proliferating lymphocytes was not significantly different between wt HTLV-1 and H1IT (Fig. 2.5). Our results are consistent with the conclusion that H1IT and wt HTLV-1 have equivalent immortalization efficiency.

Lastly, we confirmed by PCR that the immortalized PBMCs harbor viral sequences in their genome. Our data showed that PBMC-H1IT cells harbor the unique sequences

56 of IRES-Tax suggesting that viral transmission was responsible for the immortalization

of PBMCs (Fig. 2.6).

H1IT fails to persist in vivo. Our data shows that H1IT expresses similar levels of Tax

as compared to the wt HTLV-1 clone despite the fact that IRES-dependent expression is

less efficient than cap dependent expression. This likely can be attributed to

introducing the Tax cassette at the 3’end of the provirus where it becomes a component

of all viral transcripts. While this expression pattern of Tax did not affect in vitro

immortalization of primary T-lymphocytes, we next tested if this genome modification

had an overall affect on viral survival in vivo. It has been hypothesized that temporal

and quantitative expression of different transcripts in HTLV-1 is tightly balanced and

critical for virus survival and pathogenesis. To evaluate the importance of this tight

regulation in vivo, we compared the abilities of 729, 729-HTLV-1, 729-H1IT cell lines

to establish infection and persistence in our rabbit model. Rabbits were inoculated with

lethally irradiated cell lines and blood was drawn at weeks 0, 2, 4, 6, and 8 after

inoculation. Rabbit PBMCs were isolated from blood to determine viral DNA

integration by PCR, and rabbit serum was assessed for anti-HTLV-1 response

by Western blot. All rabbits inoculated with wt HTLV-1 were positive for HTLV-1 provirus at all time points post inoculation, indicating that the virus was able to infect and persist in rabbits (Fig. 2.7A). Conversely, the H1IT virus failed to productively

infect and persist in rabbit PBMCs. Only one of the rabbits inoculated with 729-H1IT

showed a transient signal by PCR, which was lost by the end of the eight week study.

We also used sera from these rabbits to test the immune response to the different

57 viruses. HTLV-1 induced a potent antibody response starting week two, whereas

response to H1IT was low and transient (Figure 2.7B). These data suggest that

unregulated Tax expression is not advantageous for the virus that needs to have strict

temporal control over its specific transcripts for persistence in vivo.

DISCUSSION

Tax and Rex are two regulatory proteins that are essential for HTLV replication 80.

Abundant information has been accumulated about the biochemical properties of both proteins. Mutational analyses have identified Tax or Rex mutants with defective activities or impaired biochemical properties that are associated with protein function.

For example, Tax mutants that specifically diminish the protein’s ability to activate viral transcription without affecting its ability to activate the NFκB pathway or vice versa have been identified 138,150,347-349. Conversely, certain Tax mutants are deficient

for interacting with cell cycle components but still are able to induce NFκB activity as

well as transcription from the LTR 156,163,334,350,351. Such mutants are of special

importance because they allow for the dissection of the several Tax activities and their

specific role in HTLV-1-mediated transformation of primary human T-lymphocytes. In

addition, several Rex mutants that either inhibit Rex activity or render the protein

constitutively active have been described 130,240,312,352. Thus, it is worthwhile to study

the effect of loss of Rex regulation on the viral replication cycle. Although some of

these mutants have been known for several years, our ability to study their effects

directly in the context of a full length replication competent provirus is hindered by the

fact that a significant portion of the tax and rex ORFs is overlapping. Mutations in one

58 gene would inevitably alter the other making interpretation of viral very

difficult.

In this study we developed a novel HTLV-1 molecular clone (H1IT) in which tax

and rex ORFs are separated. We replaced the original tax gene by introducing an IRES followed by the entire Tax cDNA immediately downstream of the Rex termination codon. In doing so, both Tax and Rex still are expressed from one bicistronic mRNA, but they are on consecutive rather than overlapping ORFs. Our results demonstrated that the rearrangement of tax and rex ORFs did not affect their expression or function in transient assays. Stable cell lines harboring the H1IT provirus had significant expression of matrix p19 Gag protein as measured by ELISA as well as Env glycoprotein, Tax, and capsid p24 Gag detected by Western blotting. Based on previous reports that suggest that an IRES is less efficient in expressing a downstream

ORF to the same level of cap-dependent expression 353,354, Mizuguchi, 2000 #3358, we did not

expect the expression or activity of Tax produced by H1IT to be indistinguishable from

that of wt HTLV-1. We explain the efficient expression of Tax from H1IT by

introducing the IRES-Tax cassette at the 3’ end of the provirus. Since all viral

transcripts share the same 3’ end, it is inevitable that the IRES-Tax cassette is part of all

these transcripts. Therefore, the low efficiency of IRES-mediated expression is

compensated for by multiple copies of the IRES-Tax cassette. A myriad of functions is

attributed to Tax, ranging from expression of viral genes, dysregulation of cell cycle

and interference with DNA repair to HTLV-induced transformation of T-cells. Thus, it

is critical that Tax be expressed at efficient levels to study these functions in vitro.

59 While our H1IT provirus was able to express adequate levels of Tax to induce

sufficient viral replication and more importantly, HTLV-induced immortalization of

primary human T-lymphocytes, we also had a unique opportunity to evaluate the

dysregulation of Tax expression on viral persistence in vivo. Although not studied in

detail, it has been suggested that there is strict temporal and quantitative control on

expression of specific viral transcripts during the course of viral infection, replication,

latency, and persistence in vivo. For example, tax/rex mRNA is one of the initial

transcripts to be made upon viral integration 80. The products of this transcript are

essential for regulating the expression of other mRNAs. Rex, for instance, is needed for

full expression of incompletely spliced gag, pol and env mRNA, leading to the

assumption that these mRNAs are late products of infection 130. On the other hand, in

late stage HTLV-1-associated , Tax is rarely detected, which suggests that

this protein is down-regulated and not required to maintain the tumorigenic phenotype

355-358. Collectively, these observations lead to the conclusion that Tax expression is temporally regulated during the life cycle of HTLV-1 infection and disease induction.

In our H1IT provirus, in addition to being expressed from its original tax/rex transcript,

Tax has the potential to be expressed from all viral mRNA species, which causes a disruption of its temporal regulation. For example, data from our lab and others showed that p30 of HTLV-1 and p28 of HTLV-2 play a role in specifically down-regulating tax/rex mRNA expression at a post-transcriptional level 114,115,118,341. This negative

regulation of Tax and Rex was proposed to be important for the virus to evade immune

recognition in vivo. In H1IT, p30 is still able to down-regulate tax/rex mRNA at a

certain point during infection but Tax would still be expressed from other transcripts

60 that are unaffected by p30, such as gag/pol mRNA. Our in vivo data are in agreement with the hypothesis that temporal regulation of Tax is important for viral persistence in an immune competent animal model. In summary, our novel H1IT provirus provides a valuable reagent to study the effects of specific Tax or Rex mutants on viral replication and viral-induced transformation in vitro. Furthermore, it also gives us insight into the paramount importance of strict regulation of viral transcript expression in a temporal and quantitative manner in vivo.

61

Figure 2.1. Schematic diagram of the generation of the H1IT proviral clone. wt

HTLV-1 was expressed from pACHneo. Only Tax and Rex ORFs are represented here in black and gray, respectively. HTLV-1 F4Term is a Tax deletion proviral clone that was generated from pACHneo. A stop codon at amino acid 4 of Tax (asterisk) was introduced without affecting the Rex reading frame. HTLV-1 F4Term was used as a template to generate the H1IT. An EcoR1 restriction enzyme site was introduced in sequences downstream of the Rex stop codon and subsequently was used to replace the remaining 3’ HTLV-1 proviral sequences with an IRES from encephalomyocarditis virus (white box), the complete tax gene, and the 3’ LTR. Arrows indicate primer pairs used for diagnostic PCR. Primer pair a and b amplifies sequences in both wt HTLV-1 and H1IT whereas c and b amplify sequences from H1IT only.

62

Figure 2.2. Functional activities of Tax and Rex expressed from H1IT. (A)

Transiently transfected 293T cells were assayed for Tax activity by cotransfection of wt

HTLV-1, H1IT, or an empty vector along with the Tax reporter LTR-1-Luciferase.

CMV-βgal was used to control for transfection efficiency. The data shown is

representative of three independent experiments. There was no significant difference in

Tax activity (20-30 fold induction over background) when it was expressed from either

proviral clone. (B) Transiently transfected 293T cells were assayed for Rex activity by

cotransfection of wt HTLV-1, H1IT, or an empty vector along with pcTat and pCgag-

RxRE reporter. CMV-Luc was used to control for transfection efficiency and data

shown is representative of three independent experiments. Rex activity measured as a

function of p24Gag production using ELISA was not significantly different between the two proviral clones. 63

Figure 2.3. Characterization of a 729H1IT stable producer cell line. (A) Supernatants

from individual cell clones were tested for the expression of p19 Gag by ELISA. At

least two cell clones showed robust p19 Gag production and the H1IT clone #11 was

used for the rest of this study. (B) The stable integration of proviral sequences into 729

B cells was determined using diagnostic PCR analysis of genomic DNA. 729H1IT cells showed a product unique to H1IT when using the LTax/ACH3AS primer pair (see c and

b Fig 1 for location), whereas a PCR product was visible in both 729H1IT and

729HTLV-1 cells when using 670/ACH3AS primer pair (see a and b, Fig. 1). (C)

Since Tax expression is hightly dependent on IRES in H1IT, Tax functional activity

was measured in order to confirm that the IRES is still functional in the stable producer

cells following transfection of the Tax reporter LTR-1-Luc. (D) Viral protein

production in 729H1IT as compared to 729HTLV-1 was determined using western blot

analysis. Total proteins from each cell line were extracted and separated by SDS-

PAGE. Using HTLV-1 specific antibodies and HTLV-1 patient antisera, comparable

levels of p24Gag, Tax, gp46Env and gp55 from 729-H1IT (lane 3) as well as 729-

HTLV-1 (lane 2), but not 729 control cells (lane 1), were detected.

64

65

Figure 2.4. H1IT immortalizes human PBMCs. 1 x 106 729, 729HTLV-1 and

729H1IT producer cells were lethally irradiated and cocultured with 2 x 106 freshly isolated PBMCs in 24 well plates. (A) To follow the immortalization process, growth curves were determined by monitoring cell number and viability by trypan blue exclusion at weekly intervals. Cells were fed once per week with RPMI 1640 supplemented with 20% FBS and IL-2. The mean and standard deviation of each time point were determined from three independent samples. (B) The presence of HTLV gene expression was confirmed by detection of structural Gag protein in the culture

supernatant by p19 Gag ELISA. The mean and standard deviation of each time point

were determined from three independent samples. 66

Figure 2.5. Representative Kaplan-Meir plot for T-lymphocyte proliferation in short- term microtiter assay. Prestimulated PBMCs (104) were cocultured with 200 irradiated

729 stable producer cells in 96 well plates. The percentages of proliferating wells were plotted as a function of time (wks). Kaplan-Meir plots for wtHTLV-1 and H1IT, and uninfected 729 control are shown. Results indicated that the percentage of wells containing proliferating lymphocytes was not significantly different between wt HTLV-

1 and H1IT.

67

Figure 2.6. Immortalized human PBMCs harbor integrated viral sequences in their genome. Diagnostic PCR was performed on genomic DNA extracted from immortalized cells 8 wks post coculture. Using primers that differentiate between wt

HTLV-1 (primers 670 and ACH3AS) and H1IT (primers LTax and ACH3AS) proviruses, it was confirmed that immortalized peripheral blood lymphocytes (PBL) contained the expected proviral sequences.

68

Figure 2.7. H1IT virus fails to persist in vivo. (A) DNA isolated from PBMCs of two rabbits from each group inoculated with lethally irradiated cell lines was tested for viral

DNA integration by PCR at weeks 0, 2, 4, 6, and 8 after inoculation. In contrast to wt

HTLV-1 whose sequences were detectable at all the time points post inoculation

(arrow), H1IT failed to productively infect and persist in rabbit PBMCs. Only one of the rabbits inoculated with 729-H1IT showed a transient signal. (B) Rabbit sera were assessed for anti-HTLV-1 antibody response by western blot. The representative seroconversion patterns from each of the inoculated groups are summarized. HTLV-1 induced a potent antibody response starting at week two, whereas H1IT response was low and transient. 69

CHAPTER 3

REPRESSION OF HUMAN T-CELL LEUKEMIA VIRUS TYPE 1 AND TYPE 2

REPLICATION BY A VIRAL-ENCODED POST-TRANSCRIPTIONAL

REGULATOR

ABSTRACT

Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are complex

retroviruses that persist in the host eventually causing leukemia and neurological disease in a small percentage of infected individuals. In addition to structural and

enzymatic proteins, HTLV encodes regulatory (Tax and Rex) and accessory (ORF I and

II) proteins. The viral Tax and Rex proteins positively regulate virus production. Tax

activates viral and cellular transcription to promote T-cell growth and, ultimately,

. Rex acts post-transcriptionally to facilitate cytoplasmic

expression of viral mRNAs that encode the structural and enzymatic gene products, thus positively controlling virion expression. Here, we report that both HTLV-1 and HTLV-

2 have evolved accessory genes to encode proteins that act as negative regulators of both Tax and Rex. HTLV-1 p30II and the related HTLV-2 p28II proteins inhibit virion

production by binding to and retaining tax/rex mRNA in the nucleus. Reduction of

70 viral replication in a cell carrying the provirus may allow escape from immune

recognition in an infected individual. These data are consistent with the critical role of

these proteins in viral persistence and pathogenesis in animal models of HTLV-1 and

HTLV-2 infection.

INTRODUCTION

Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are distinct

complex oncogenic retroviruses that persist in the infected individual despite a robust

virus-specific host immune response 80. HTLV-1 is the causative agent of adult T cell

leukemia (ATL), a malignancy of CD4+ T lymphocytes, and a chronic neurological

disorder termed HTLV-1 associated myelopathy/tropical spastic paraparesis

(HAM/TSP) 1,2,26,27. The association between HTLV-2 infection and disease is less

clear in that only a few cases of variant hairy cell leukemia (CD8+ T cell origin) and

several cases of neurological disease have been reported 5,9,359.

In addition to structural and enzymatic proteins, Gag, Pol, and Env, HTLV encodes

the Tax and Rex trans-regulatory gene products that are essential for efficient viral

replication and cellular transformation. Tax increases the rate of transcription from the

viral long terminal repeat (LTR) 360-362 and modulates the transcription or activity of

numerous cellular genes involved in cell growth and differentiation, cell cycle control,

and DNA repair 177,363-366. In addition, Tax is highly immunogenic in vivo 367,368. Rex acts post-transcriptionally by preferentially binding, stabilizing and selectively exporting the unspliced and incompletely spliced viral mRNAs from the nucleus to the

71 cytoplasm, thus controlling the expression of the structural and enzymatic proteins

193,209,259.

Proteins encoded by open reading frame (ORF) I and ORF II near the 3’ end of the

viral genome 99,111,369 promote viral persistence in vivo (see Figure 3.1A) 191,370,371.

These proteins are dispensable for replication and immortalization of primary T lymphocytes in vitro 92,93,343. However, ORF II has been shown to be important for viral

persistence in vivo using a rabbit model of infection 110,125-127. The HTLV-1 ORF II protein, p30II, localizes to the nucleolus and nucleus 112 and has the capacity to

modulate viral gene expression by interacting with the coactivator p300 and

destabilizing the Tax-CREB interaction 114,115. The HTLV-2 ORF II protein, p28II, also localizes to the nucleus and its N-terminal 49 amino acids share 77.5% identity with the

C-terminal portion of HTLV-1 p30II, suggesting that the two proteins might have a

similar function 128. The mechanism of action for these proteins in viral replication and

survival in vivo remains unclear.

Only a subset of HTLV infected cells actively expresses viral RNA in vivo 372 leading to the hypothesis that a negative regulator(s) of HTLV gene expression is required for the survival of the virus in the infected host. Indeed, the p30II protein of

HTLV-1 recently was shown to act as a negative regulator of viral gene expression 118.

Since HTLV-2 is genetically related to HTLV-1, we investigated whether the HTLV-1

p30II also may function reciprocally as a negative regulator of HTLV-2 expression. Our

data demonstrate not only that p30II blocks HTLV-1 and HTLV-2 replication but that

HTLV-2 encodes a functionally related protein, p28II, which inhibits HTLV-2 as well as

HTLV-1 replication. Both p30II and p28II inhibit Tax-1 and Tax-2 but only when Tax is

72 expressed from a full-length proviral clone. Similarly, p30II and p28II inhibit Rex-1 and

Rex-2. Since Tax and Rex are expressed from the same doubly spliced mRNA, we hypothesized that this inhibitory effect may occur at the RNA level. We show that p28II, like p30II, binds to and retains tax/rex RNA of HTLV-2 in the nucleus, thereby

reducing its level in the cytoplasm. By repressing Tax and Rex functions, both p30II and p28II down-modulate viral expression and, in turn, promote viral persistence. This

phenomenon provides an example of the evolutionary conservation of a common

regulatory pathway by two distinct retroviruses.

MATERIALS AND METHODS

Cells, plasmids and antibodies. 293T cells were maintained in Dulbecco’s modified

Eagles medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-

glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

The HTLV-1 proviral clone, ACH 373, and HTLV-2 proviral clone, pH6neo 374,

were used in this study. pME-p30II-HA (a kind gift from Dr. B. Michael, Ohio State

University) was generated from the ORF II of the ACH proviral clone, tagged with HA

at the C-terminus, and cloned into the expression vector pME-18S at the EcoRI and

NotI sites. The protein was detected by Western blot using anti-HA monoclonal antibody (Covance). Tax and Rex were expressed from a vector encoding the respective cDNA under the control of the cytomegalovirus (CMV) immediate early gene promoter and has been described previously 45. An HTLV-2 p28II expression

vector (p28-AU1) was generated from ORF II of the pH6neo proviral clone, tagged

with AU1 (DTYRYI) at the C-terminus and cloned into the CMV-based expression

73 vector BC12 at the HindIII and KpnI sites. The protein was detected by

immunoprecipitation using anti-AU1 monoclonal antibody (Covance). p28II-GFP (GFP

fused to the amino terminus) was constructed by inserting the HindIII-EcoRI p28II cDNA fragment into the EGFP-N3 vector (Promega). The LTR-luciferase Tax reporter plasmid 375, pcTat, and the Rex-1 (pCgag-RxRE-I) or Rex-2 (pCgag-RxRE-II) reporter

plasmids were previously described 211,316. TK-Renilla luciferase plasmid was used to

control for transfection efficiency.

Transfection, luciferase assay, p19 and p24 ELISA. To measure Tax function, 1.5 x

105 293T cells were transfected using Lipofectamine (Invitrogen) according to the manufacturer's recommendations. The total amount of DNA was kept constant and was composed of 0.1µg of LTR-luciferase reporter along with 0.4 µg of an empty plasmid,

Tax cDNA expression plasmid or HTLV proviral clone. Increasing concentrations (0.4-

1.6 µg) of p30II or p28II expression plasmids were co-transfected to test the effect of

p30II or p28II on Tax activity. After 48 h, cells were pelleted and the cell supernatants

were used for p19 enzyme-linked immunosorbent assay (ELISA) (Zeptometrix)

according to manufacturer’s recommendations. The cell pellets were lysed in passive

lysis buffer (PLB) (Promega) and Tax activity was measured in light units as described

211,316. The Rex functional assay was performed as described 211,316. Briefly, 0.4 µg of

an empty plasmid, Rex cDNA expression plasmid or HTLV proviral clone were co-

transfected with 0.1 µg pcTat and 0.3 µg of the Rex reporter plasmid pCgag-RxRE, which contains the HIV-1 LTR promoter and gag gene linked to the Rex response

element (RxRE). Cell lysates were prepared in PLB 48 hours post-transfection and 74 luciferase activity was determined to control for transfection efficiency. HIV-1 p24

Gag levels in the cell lysates were determined by ELISA (Beckman-Coulter). All

transfection experiments were performed in triplicate and normalized for transfection

efficiency using Renilla luciferase.

RNA preparation, radiolabeled RT-PCR and Real Time RT-PCR. Transfected

293T cells were lysed in hypotonic lysis buffer (10mM HEPES-KOH pH7.9, 1.5mM

MgCl2, 10mM KCl, and 0.5mM DTT) for 10 minutes on ice. The cytoplasmic and

nuclear fractions were separated by centrifugation at 700 xg for 8 minutes. The supernatant (cytoplasmic fraction) was further cleared by centrifugation at 3300 xg for 5

minutes. The pellet served as the nuclear fraction. The RNA was extracted using Tri-

reagent (Molecular Research Center) and samples were treated three times with RNase-

free DNase.

Semi-quantitative RT-PCR was performed as previously described 193 using primers

LA79 (5085CCG GTG GAT CCC GTG GCG AT5104) and LA78 (7234GTC CAA ATC

CTG GGA AAT GG7214) to detect tax-2/rex-2, and #20 (1314AGC CCC CAG TTC ATG

CAG ACC1334) and #21 (1412GAG GGA GGA GCA TAG GTA CTG1392) to detect gag-

2/pol-2. Briefly, the antisense primer from each set was end-labeled for 1 h with 32P-γ-

ATP using T4 polynucleotide kinase (New England Biolabs). The reverse transcription reaction was performed using the labeled antisense primer at 65°C for 10 minutes followed by 30 cycles of amplification. The radiolabeled products were separated on a

6% acrylamide gel and quantified using Image Quant NT (Molecular Dynamics).

75 For real-time PCR, first strand cDNA was generated using SuperScript II reverse

transcriptase (Invitrogen) and oligo-dT primers. Then 10% of the cDNA was mixed

with SybrGreen master mix (Stratagene) and 0.5µM of primers RT-tax2s (5143GAA

CTC GCC GAG CAC GCC5160) and RT-tax2as (7320GGA ACA TAG ACC ACC

TGA7303) to amplify tax-2/rex-2, or #20/#21 to amplify gag-2/pol-2. The real-time PCR

reaction was performed using the Roche LightCycler system (Roche). Calibration

curves were generated using serial dilutions of linearized plasmid DNA. The expected

size of the amplified fragments was confirmed by agarose gel electrophoresis.

In vivo RNA binding. Detection of RNA bound to p28II was performed as described

215 with some modifications. Briefly, transfected 293T cells were lysed in NP-40 lysis

buffer (50mM KCl, 10mM Tris pH 8.0, 5mM MgCl2, 0.65% NP-40, 2mM PMSF, and

100U RNasin) for 30 minutes on ice. Lysates were cleared by incubating with 50 µl

protein-A-sepharose beads (Amersham) for 2 h at 4°C. Then 10% of the cleared lysate

was used as input RNA, and the rest was immunoprecipitated with either anti-AU1

antibody to capture p28II or anti-HA (nonspecific antibody). The immune complexes

were washed three times with lysis buffer, and the RNA was extracted using Tri-reagent

and subjected to radiolabeled RT-PCR as described above.

Immunofluorescence. Hela cells were electroporated with 5µg of p28-GFP or p28-TA

(HA tagged). For p28-GFP detection, cells were plated and visualized using a Zeiss

LSM 510 microscope. For p28-TA detection, plated cells were fixed with 2%

paraformaldehyde, permeabilized with 0.2% Triton X-100, and incubated with anti-HA 76 monoclonal antibody (1:100). Cells then were washed and incubated with anti-mouse

IgG conjugated with Cy3 at 1:1000 (Jackson Laboratories) and visualized using a Zeiss

LSM 510 microscope.

RESULTS

HTLV-1 p30II represses HTLV-2 replication. Recently, it was demonstrated that the

HTLV-1 p30II protein encoded by ORF II suppresses HTLV-1 replication 118. Tax-1 and Tax-2 activate transcription, though at different levels, through the HTLV-1 and

HTLV-2 promoters. Similarly, the Rex-1 and Rex-2 proteins bind to RxRE-1 and

RxRE-2 and transport the unspliced and singly spliced viral mRNA from the nucleus to the cytoplasm, thereby positively regulating structural and enzymatic protein expression and virion production 316. To assess whether p30II also was able to inhibit HTLV-2

replication, we co-expressed increasing concentrations of p30II protein with the

replication-competent HTLV-1 proviral clone ACH, as well as the HTLV-2 proviral clone pH6neo. The addition of 30II resulted in significant reduction of p19 Gag

production in the supernatant of transfected cells, indicating that p30II represses HTLV-

1 expression as expected 118, but also reduced HTLV-2 expression, indicating

significant inhibition of virus replication (Fig. 3.1B).

p30II inhibits Tax-1, Tax-2, Rex-1 and Rex-2 at a post-transcriptional level. Since

Tax-1 and Tax-2 are the key transactivators of transcription from the viral promoter, and since p19 Gag and other viral gene expression are highly dependent on functional

Tax, we investigated whether the repressive effect of p30II could be due to inhibition of

77 Tax transcriptional activity. Co-transfection of either the HTLV-1 or HTLV-2 proviral

clone as the source for Tax-1 or Tax-2 respectively, and the LTR-Luc reporter with

increasing concentrations of p30II (0.4-1.6 µg) resulted in a dose-dependent inhibition

of both Tax-1 and Tax-2 function (Fig. 3.2A). We then ruled out the possibility that the

repressive effect of p30II is a direct result of the inhibition of Tax mediated transcription

from the LTR. Co-expression of p30II with LTR-Luc in the absence of Tax did not

result in any inhibition of LTR-mediated transcription at the doses used (data not shown and reference 118. Also, the p30II repressive effect was not observed if either Tax-1 or

Tax-2 were expressed from a cDNA expression vector (Fig. 3.2B), ruling out a more

downstream block involving the Tax protein and its function. Our data indicate that

p30II does not affect the basal level of transcription mediated by Tax-1 and Tax-2 or

directly disrupt the protein itself, thus, suggesting that p30II inhibits Tax-1 and Tax-2 by

a post-transcriptional mechanism.

Since Tax and Rex are expressed from the same viral RNA in both HTLV-1 and

HTLV-2 (Fig. 3.1A), we hypothesized that p30II also may inhibit Rex function,

confirming that the effect of p30II is at the RNA level. Rex-1 or Rex-2 was co-

transfected into 293T cells with either a cDNA plasmid or full-length proviral clone

(HTLV-1 or HTLV-2) with increasing concentrations of p30II (0.4-1.6 µg). Consistent

with the inhibition of p19 Gag production and Tax function, p30II expression resulted in

a dose-dependent inhibition of both Rex-1 and Rex-2 (Fig. 3.2C). As with Tax, p30II repression was not observed if either Rex-1 or Rex-2 were produced from a cDNA expression vector (data not shown). Western blot analysis confirmed that the amount of p30II protein expressed correlated directly with the amount of plasmid DNA transfected,

78 whereas a control cellular protein β-actin remained unchanged (Fig. 3.2D). It is

important to note for these experiments that although Tax and Rex activity expressed from 0.4 µg of transfected proviral clone can be quantitatively measured using a sensitive reporter assay, the level of protein expressed is below the limit of detection by

Western blot.

The functional homologue of p30II in HTLV-2 is p28II. Since the HTLV-1 p30II was able to inhibit the Tax and Rex functional activities of both HTLV-1 and

HTLV-2, we hypothesized that HTLV-2 must have evolved a similar function. The

3’ end of the HTLV-2 genome encodes a protein of 28 kDa (p28II) with unknown

function 374. Since the N-terminal 49 amino acids of p28II and the C-terminal region of p30II have 77.5% identity, we hypothesized p28II may be the functional

homologue of p30II. Indeed, when co-transfected with HTLV-1 and HTLV-2

molecular clones, p28II expression decreased p19 Gag production in a dose

dependent manner (Fig. 3.3A). Furthermore, like p30II, p28II repressed both Tax-1

and Tax-2 functions when Tax was expressed from HTLV-1 or HTLV-2 proviral clones (Fig. 3.3B). Next, we determined that the inhibitory effects of p28II were due

neither to inhibition of basal level (Fig. 3.3C), nor Tax-mediated (Fig. 3.3D)

transcription from the viral LTR when Tax-1 or Tax-2 was expressed from cDNA expression plasmids. Immunoprecipitation of p28II from transfected cells confirmed

an increase in p28II protein production as a function of increased plasmid DNA

transfected, whereas a control cellular protein β-actin remained unchanged (Fig.

3.3E).

79 We next evaluated the effect of p28II on Rex-2 function. Co-transfection of

increasing concentrations of p28II (0.4-1.6 µg) with the HTLV-2 proviral clone

(pH6neo) as the source for Rex-2, and the RxRE linked to HIV Gag reporter resulted in

a dose-dependent inhibition of Rex-2 function (Fig. 3.4A). Like p30II, p28II had no effect on Rex-2 when it was expressed from a cDNA expression plasmid (Fig. 3.4B).

This provides the first report of a functional activity for HTLV-2 p28II and supports the

overall conclusion that the p30II and p28II homologues exert their inhibitory effect at a

post-transcriptional level.

The nuclear p28II binds to and retains the doubly spliced tax/rex mRNA in the

nucleus. To investigate the mechanism of p28II suppression of HTLV-2 gene

expression, we assessed the cellular localization of p28II. Both p28-TA and p28-GFP

localized to the nucleus as expected, showing that the addition of either tag to the

protein does not affect its localization (Fig. 3.5A). Since we ruled out a transcriptional

effect of p28II, we investigated whether the p28II suppressive effects could be exerted at a post-transcriptional level. Therefore, we studied the distribution of select viral mRNA

species in HTLV-2 transfected 293T cells in the presence or absence of exogenous p28II. Semi-quantitative reverse-transcription PCR (RT-PCR) was conducted on

nuclear and cytoplasmic RNA fractions using a primer pair that spans exons 2 and 3 of

tax/rex mRNA, as well as a specific primer pair that detects the unspliced gag/pol

mRNA (see Fig. 3.1A). p28II resulted in an increase of tax/rex mRNA in the nucleus

and a consistent reduction of this mRNA in the cytoplasm (Fig. 3.5B). The nuclear

retention of tax/rex mRNA was specific because the distribution of gag/pol mRNA was

80 not affected by p28II (Fig. 3.5B). In order to get a better quantitative measure of the

nuclear retention of tax/rex mRNA by p28II, nuclear and cytoplasmic RNA fractions

were subjected to real-time RT-PCR. As shown in Figure 3.5C, expression of p28II lead to dose-dependent retention of tax/rex mRNA in the nuclear fraction with a concomitant

reduction of this mRNA species in the cytoplasm. Confirming the RT-PCR results,

gag/pol mRNA was not significantly affected by p28II (Fig. 3.5D).

We evaluated whether the p28II and tax/rex mRNA can associate with each other

using an in vivo RNA binding assay. We co-transfected 293T cells with an HTLV-2 proviral clone and p28II or an empty plasmid expression vector. Following immunoprecipitation of p28II, the RNA bound to the p28II immune complex was

extracted and subjected to RT-PCR. Our data indicate that p28II can specifically

associate with tax/rex mRNA but not gag/pol mRNA in vivo (Fig. 3.6). An antibody to

a HA-tagged epitope not contained in the p28II could not capture tax/rex mRNA,

confirming that the specificity of binding is dependent on the presence of p28II and its specific antibody. We conclude that p28II binds either directly or indirectly to the

tax/rex mRNA. Collectively our data support the conclusion that p28II and p30II accessory proteins decrease viral replication by forming a protein-RNA complex that is retained in the nucleus.

DISCUSSION

Efficient expression of the HTLV-1 and HTLV-2 structural and enzymatic proteins from a provirus is dependent on the regulatory proteins, Tax and Rex. Tax increases overall transcription, whereas the post-transcriptional regulator Rex is essential for

81 nuclear export of unspliced and partially spliced to the cytoplasm 193,194.

Inhibiting the activity of either regulatory protein has drastic effects on virus replication. On the other hand, an infected host cell that expresses high levels of foreign proteins could be eliminated by immune surveillance. Thus, in order for the virus to persist in the host, it would be advantageous to suppress, at least partially, the positive regulatory proteins, leading to a state referred to as viral latency. It is not fully understood whether in vivo HTLV-1 and HTLV-2 achieve complete latency at the molecular level. Some insight came from experiments in which the region encoding the accessory proteins was shown to be dispensable for viral replication and transformation of activated primary T-lymphocytes in vitro 92,343, but not in vivo, in rabbits with a

competent immune system 110,125,127. Based on these observations, we hypothesized

that the accessory proteins played a role in dampening the function of Tax and Rex and

overall viral expression and contributed to viral persistence in vivo. In the present

study, we showed that HTLV-1 p30II suppresses both HTLV-1 and -2 replication and

we uncovered the function of HTLV-2 p28II. Our data suggest that both proteins may

play a very important role in viral persistence by post-transcriptionally inhibiting Tax

and Rex gene expression and ultimately repressing viral replication.

Our data show that the co-expression of either HTLV-1 p30II or HTLV-2 p28II with

replication-competent HTLV-1 or HTLV-2 proviral clones results in a dose-dependent

inhibition of both Tax and Rex functions. This repression was not observed when Tax

and Rex were expressed from cDNA expression vectors, suggesting that p30II and p28II do not affect Tax and Rex at the protein level. In addition, neither p30II nor p28II inhibited basal level or Tax-1 and Tax-2 mediated transcription from the LTR.

82 Collectively, these data indicate that the inhibitory effects of p30II and p28II are post-

transcriptional. Thus, we examined the p28II effect on the distribution of tax/rex doubly

spliced mRNA expressed from a proviral clone. We provided evidence that p28II retains tax/rex mRNA in the nucleus with a concomitant reduction of this RNA in the cytoplasm. The implication is that this effect would lead to less protein production, which could allow the infected cell to have a lower profile and escape the immune system. Since Tax and Rex protein levels expressed from transfected proviral clones are below the limit of detection we cannot directly quantitate Tax and Rex protein levels by immunoprecipitation or Western blot. However, since HTLV Gag production is highly dependent on both functional Tax and Rex, we can indirectly correlate p19 Gag levels with Tax and Rex functional activities. Indeed, co-expression of p30II and p28II

with the full-length HTLV proviral clones caused significant reduction in the viral p19

Gag production. Finally, our in vivo RNA binding analysis revealed that p28II has the ability to specifically associate with the doubly spliced tax/rex mRNA but not the

unspliced gag/pol mRNA. Whether this interaction is direct or through another adaptor

protein remains to be tested.

The mechanism of action of p30II and p28II is a post-transcriptional regulation of

viral mRNA trafficking. In contrast to Rex that binds to and facilitates the nucleo-

cytoplasmic export of unspliced and singly spliced viral RNA, p28II and p30II specifically bind and retain the doubly spliced tax/rex mRNA in the nucleus. The exact mechanism of RNA retention still is unclear. One possibility consistent with the data is that p30II and p28II are binding to the exon-exon junction that is unique to tax/rex

mRNA, preventing the recruitment of factors required for efficient release of the mRNA

83 from the nuclear pore, essentially blocking mRNA export.

The fact that the inhibitory function of p28II and p30II is conserved in both HTLV-1 and HTLV-2 emphasizes its importance and suggests a common pathway for modulation of gene expression by these two distinct but related viruses. However, it is important to note some differences between these two proteins. Unlike p30II which

localizes to the nucleolus 118, we showed that p28II is primarily nuclear and excluded

from the nucleolus. This difference may reflect the previously reported ability of p30II to have general transcriptional effects 114,115. In our assays, p28II did not cause similar effects on the TRE-mediated transcription. Additional comparative experiments will be required to identify the protein domains responsible for these differences and may provide insight into other distinct functional activities leading to a better understanding of the pathological differences between HTLV-1 and HTLV-2. Understanding the exact mechanism of action of p30II and p28II ultimately could provide a means for

therapeutic targeting of these proteins to eradicate HTLV persistence in the host.

84

Figure 3.1. HTLV-1 p30II inhibits viral replication. (A) Schematic representation of an

HTLV genome. Proteins are indicated by horizontal rectangles. ORF-II depicted as an overlapping solid and dotted box shows the location of the HTLV-1 p30II and HTLV-2

p28II, respectively. The full-length gag/pol and doubly spliced tax/rex mRNAs are

depicted below the genome. Arrows denote the location of primers used to specifically

detect viral mRNAs by PCR. (B) Increasing amounts (in micrograms) of p30II

cotransfected with HTLV-1 or HTLV-2 proviral clones causes a dose-dependent

reduction of p19 Gag as measured by ELISA.

85

Figure 3.2. HTLV-1 p30II inhibits Tax and Rex function when both are expressed from

HTLV proviral clones. (A) p30II dose dependent inhibition of Tax expressed from

HTLV-1 or HTLV-2 proviral clones. Tax function was measured as firefly luciferase activity from LTR-Luc normalized to Renilla luciferase activity. (B) p30II does not

inhibit Tax activity, expressed as fold activation over basal level, if Tax-1 and Tax-2 are

expressed from cDNA expression vectors. (C) p30II dose-dependent inhibition of Rex

expressed from HTLV-1 or HTLV-2 proviral clones. Rex functional activity was

determined using the HIV-1 p24 Rex reporter assay as described in Materials and

Methods. (D) Western blot analysis to confirm increasing concentrations of p30II-HA used in panels A and B. β-actin levels were assessed as a loading control.

86

Figure 3.2.

87

Figure 3.3. HTLV-2 p28II inhibits viral replication and Tax function. (A) Increasing

concentration of p28II cotransfected with the HTLV-1 or HTLV-2 proviral clone causes dose-dependent reduction of p19 Gag. (B) p28II dose-dependent inhibition of Tax

expressed from HTLV-1 or HTLV-2 proviral clones. (C) Basal level transcription

from the viral LTR is not inhibited by p28II. (D) p28II does not inhibit Tax activity if

Tax-1 and Tax-2 are expressed from cDNA expression vectors. (E)

Immunoprecipitation of radiolabeled p28II-AU1 to confirm increasing concentrations of

p28II in panels A-D. β-actin levels were assessed as a loading control.

88

Figure 3.4. p28II inhibits Rex function when Rex is expressed from an HTLV proviral clone. (A) p28II dose-dependent inhibition of Rex expressed from HTLV-1 or HTLV-2

proviral clones. Rex activity is a measure of p24 Gag expression from the pcGagRxRE-

II reporter plasmid. (B) Same as panel A, but Rex-2 is expressed from a cDNA

expression plasmid.

89

Figure 3.5. p28II retains HTLV-2 tax/rex mRNA in the nucleus. (A) Nuclear

localization of p28II-TA and the GFP-p28 fusion protein. (B) p28II affects the

distribution of tax-2/rex-2 doubly spliced mRNA. Nuclear and cytoplasmic mRNAs

were extracted from 293T cells cotransfected with HTLV-2 proviral clone in the

presence or absence of exogenous p28II. RT-PCR was used to amplify viral specific

mRNAs using 32P-labeled primers. (C/D) Equal amounts of mRNA from panel B were

subjected to real-time RT-PCR to determine the ratio of nuclear to total and cytoplasmic

to total RNA using primers specific for tax/rex (C) or gag/pol mRNA (D).

90

Figure 3.6. In vivo binding of p28II to tax/rex mRNA. 293T cells cotransfected with

HTLV-2 and p28II were lysed in NP-40 buffer and lysates were immunoprecipitated using anti-AU1 or anti- HA monoclonal antibody. The specific mRNAs bound to the immune complex were identified by RT-PCR. Input bands represent 10% of the total

RNA before immunoprecipitation.

91

CHAPTER 4

HUMAN T-CELL LEUKEMIA VIRUS ORF II ENCODES A POST- TRANSCRIPTIONAL REPRESSOR THAT IS RECRUITED AT THE LEVEL OF TRANSCRIPTION

ABSTRACT

Human T-cell leukemia virus (HTLV) infection is a chronic, lifelong infection that is

associated with the development of leukemia and neurological disease after a long

latency period. The mechanism with which the virus is able to evade host immune

surveillance is elusive. Besides the structural and enzymatic proteins, HTLV encodes regulatory (Tax and Rex) and accessory (ORF-I and ORF-II) proteins. Tax activates viral and cellular transcription and promotes T-cell growth and malignant transformation. Rex acts post-transcriptionally to facilitate cytoplasmic expression of incompletely spliced viral mRNAs. Recently, we reported that the accessory gene products of HTLV-1 and HTLV-2 ORF II (p30II and p28II, respectively) are able to

restrict viral replication. These proteins act as negative regulators of both Tax and Rex

by binding to and retaining their mRNA in the nucleus leading to reduced protein

expression and virion production. Here, we show that p28II is recruited to the viral

promoter in a Tax-dependent manner. After recruitment to the promoter, p28II or p30II then travels with the transcription elongation machinery until its target mRNA is 92 synthesized. Experiments artificially directing these proteins to the promoter indicated

that p28II, unlike HTLV-1 p30II, displays no transcriptional activity. Furthermore, the tethering of p28II directly to tax/rex mRNA resulted in repression of Tax function which

could be attributed to the ability of p28II to block TAP/p15-mediated enhancement of

Tax expression. Since p28II-mediated reduction of viral replication in infected cells

may permit survival of the cells by allowing escape from immune recognition, our data

are consistent with the critical role of HTLV accessory proteins in viral persistence in

vivo.

INTRODUCTION

Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are distinct

complex oncogenic retroviruses that persist in the infected individual despite a robust

virus-specific host immune response 80. HTLV-1 and HTLV-2 share 65% homology at

the nucleotide sequence level, but remain distinct in their pathology. HTLV-1 is the

causative agent of adult T-cell leukemia (ATL), a fatal malignancy of CD4+ T-

lymphocytes, and a chronic neurological disorder termed HTLV-1-associated

myelopathy/tropical spastic paraparesis 1,2,26,27. HTLV-2 has a less clear disease

association in that only a few cases of variant hairy cell leukemia and neurological

disease have been reported 5,9,359. HTLV-1 and HTLV-2 have the capacity to promote

T-lymphocyte growth both in vitro and in vivo 50-53. The ultimate fate of infected T-

cells in vivo depends on their ability to balance proliferative, cell cycle, and anti-

apoptotic signals mediated by viral and cellular proteins, versus the ability to evade the

host immune response. Therefore, how the virus responds to environmental signals and

93 regulates viral and cellular gene expression is critical to its long-term survival and

persistence in the infected individual.

Upon viral infection, reverse transcription and random integration of the proviral

DNA into the host genome 64, the viral transactivator Tax directs HTLV gene

expression from a promoter that is located in the 5’ long terminal repeat (LTR) 361.

Three highly conserved 21-bp enhancer elements that are critical for Tax-mediated

transcription are contained in the U3 region of the LTR and are referred to as Tax-

response elements (TREs) or viral CREB-response elements (CREs) 331,376. The cellular

transcription factor CREB and/or other ATF/CREB family members bind to the TREs,

whereas Tax interacts with both CREB and the GC-rich DNA sequences that

immediately flank the CREB binding site 377-380. Tax-mediated protein-protein and

protein-DNA interactions on the HTLV promoter lead to the assembly of very stable

ternary complexes that are critical for the recruitment of the cellular co-activator

CBP/p300 139,141, which subsequently results in strong transcriptional activation of the

virus 381,382. This transcriptional activation results in the expression of all viral mRNAs

encoding structural and enzymatic proteins (Gag, Pol, and Env), trans-regulatory proteins (Tax and Rex), and accessory proteins (p12, p13, and p30 for HTLV-1 and p10, p11, p22, and p28 for HTLV-2) 80.

The HTLV-1 and HTLV-2 accessory proteins, encoded by open reading frames

(ORF) near the 3’ end of the viral genome 99,369 are the least conserved between the two related viruses and have been shown to be dispensable for in vitro replication and immortalization of activated primary T-lymphocytes 92,93,343. However, the accessory

proteins have been shown to be essential for viral infectivity and persistence in vivo

94 using a rabbit model of infection 110,125-127 and unpublished data. HTLV-1 p30II and

HTLV-2 p28II are nuclear proteins encoded by ORF II that share minimal homology at

the amino acid level. Recently, p30II and p28II have been shown to down-regulate

tax/rex mRNA expression post-transcriptionally by binding to and retaining this mRNA

in the nucleus 118,341. Interestingly, both p28II and p30II are able to inhibit tax/rex

mRNA only when the latter is expressed from a full-length proviral clone and not from

a cDNA.

There is a growing body of evidence that the different stages of gene expression,

from transcription, to pre-mRNA processing, to export to the cytoplasm and translation,

are functionally and physically coupled 383,384. For example, the mRNA export factors

Yra1p and Sub2p are co-transcriptionally recruited to the mRNA via their interaction

with Hpr1p, a component of the transcription elongation machinery 385. Similarly,

factors involved in splicing and polyadenylation associate with the C-terminal domain

(CTD) of RNA polymerase II, positioning them close to their mRNA substrate and enabling efficient processing 386-389. Collectively, these observations raise the question

of whether the discrimination between a tax/rex mRNA expressed from a proviral clone

or a cDNA by p28II or p30II is dependent on pre-mRNA processing and/or another co-

transcriptional process. A link between HTLV-1 p30II and the transcriptional

machinery is suggested by its ability to differentially modulate viral gene expression via

its interaction with CBP/p300 and destabilizing the Tax-CREB interaction 114,115.

To further characterize the role of p28II or p30II in post-transcriptional suppression

of tax/rex mRNA and define how and when it is recruited to the mRNA, we reconstructed several cDNA plasmids to mimic the mRNA that is expressed from a full-

95 length proviral clone. Our data shows that the 5’ untranslated region (UTR) of the

target RNA as well as the intron sequences play a minimal role in mediating the

inhibition. Conversely, we identified the component of the HTLV promoter, specifically Tax, as the factor required for efficient recruitment of p28II or p30II to the newly transcribed mRNA. Consistently, our chromatin immunoprecipitation (ChIP) experiments showed that these proteins are recruited co-transcriptionally and associate with the elongation machinery. Furthermore, we provide evidence that p28II itself does

not exhibit transcriptional activity but inhibits tax/rex mRNA transport by overriding

the TAP/p15 export pathway. These data reveal a complex interplay between the

transcriptional machinery and the post-transcriptional regulation of tax/rex mRNA,

thereby providing the first example of a retroviral protein that couples transcription with

post-transcriptional inhibition.

MATERIALS AND METHODS

Cells. 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM).

The media were supplemented to contain 10% fetal bovine serum, 2 mM glutamine,

penicillin (100 U/ml), and streptomycin (100 µg/ml).

Plasmids. The HTLV-2 proviral clone, pH6neo 374, was used in this study. pME-p30II-

HA 116, CMV-p28II-AU1 341 and the LTR-2-Luc Tax reporter plasmid 341 were

described previously. CMV-βgal was used to control for transfection efficiency in each

experiment.

96 The following are the sequences of the different cDNAs used to express HTLV-2

Tax, with numbers in parentheses based on proviral genome location; BC20.2 (Tax-2):

Partial exon 2 (bp 5091-5183), fused to exon 3 (bp 7214-8552). IY531: Exon 1 (bp

316-447), fused to full-length exon 2 (bp 4044-5183), fused to exon 3 (bp 7214-8552).

IY587: equivalent to pH6neo proviral clone but intron 1 is severely truncated (bp 1034-

3634 are deleted, which eliminates gag and pol gene expression). IY588: equivalent to

IY531 but the truncated intron 1 from IY587 is reinserted between exon 1 and exon 2.

IY594: equivalent to BC20.2 but the full-length intron 2 is reinserted between exon 2 and exon 3. IY595: equivalent to IY531 but the full-length intron 2 is reinserted between exon 2 and exon 3. BCHTLV-2 is equivalent to pH6neo but the U3 region of the 5’ LTR is replaced with CMV immediate early promoter. IY619 is equivalent to

Tax-2 but CMV promoter is replaced with U3 region of the HTLV-2 LTR. IY620 is equivalent to IY531 but the CMV promoter is replaced with U3 region of the HTLV-2

LTR. All the cDNAs, their promoters and corresponding RNAs are summarized in

Figure 4.1. TAP and p15 were transcribed from vectors under the control of a CMV promoter. For the Gal4 chimeras, the coding sequences of p28II or p30II were amplified

by PCR to generate a BamHI-SacI fragment. The PCR product was ligated in-frame

downstream of the first 147 codons of the Gal4 DNA binding domain contained within

the pSG424 plasmid (a kind gift from H. Bogerd, Duke University). For the MS2

fusion proteins, the coding sequences of p28II or GFP were amplified by PCR to

generate a Pac1-Not1 fragment. The PCR product was ligated in-frame downstream of

the MS2 protein. The Tax2-MS2(RE) plasmid is equivalent to Tax-2

with 6 MS2 RNA Elements (RE) inserted between the Tax stop codon and the poly A

97 signal.

Transfection, western blot, and Tax functional assays. To measure Tax CREB/ATF

activating function (viral LTR), 2x105 293T cells were transfected using Lipofectamine

(Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendation. The total

amount of DNA was kept constant and was composed of 0.1 µg LTR-2-Luc, 50ng

CMV-βgal, and 0.2-0.4 µg of Tax expression plasmid, HTLV proviral clone, or empty control plasmid. To test the effect of p28II on Tax activity, increasing amounts of p28II expression plasmid (2X and 4X the amount of Tax expression plasmid) was cotransfected. After 48 h of growth, cells were pelleted and lysed in passive lysis buffer

(PLB) (Promega, Madison, WI) and Tax activity was measured as luciferase light units.

All experiments were performed independently three times in triplicate, and results were

normalized for transfection efficiency using β-galactosidase.

To measure Tax protein levels in the presence or absence of p28II, equivalent amounts of cell lysates from the functional assay were separated on 4-12% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham, Piscataway, NJ). Rabbit polyclonal antibodies against Tax-2, Rex-2, and p28II were used to detect the viral

proteins. Rabbit polyclonal antibody to β-actin (Novus Biological, Littleton, CO) was

used as a loading control (LC). Proteins were visualized using the ECL western

blotting analysis system (Santa Cruz Biotechnology, Santa Cruz, CA).

Chromatin immunoprecipitation (ChIP) assay. 293T cells (2 x 105) were transfected

using Lipofectamine with 0.5 µg of pH6neo or 0.1 µg LTR-2-Luc with or without 0.5 98 µg p28II or p30II expression plasmid. After 48 h of growth, cells were crosslinked with

formaldehyde for 10 minutes at 37°C, washed twice with PBS, lysed in SDS lysis

buffer, and sonicated on ice to generate DNA fragments between 100-1000 bp. The

ChIP assay was performed as recommended by Upstate

(Charlottesville, VA). Briefly, cell lysates were pre-cleared with protein A-sepharose

beads and then incubated with anti-AU1 monoclonal antibody (p28II-AU1) or anti-HA

monoclonal antibody (p30II-HA) overnight at 4°C. The immune complexes were

captured on beads and washed extensively. The DNA-protein complexes were eluted

followed by treatment 65°C for 4 h to reverse crosslink. DNA was then extracted using

phenol/chloroform and ethanol precipitated.

PCR and primers. PCR on extracted DNA from the ChIP procedure was used to

detect regions in the LTR or within the HTLV genome (see Figure 4.6 for location).

The PCR primer pair for region “a” that amplifies bp 41-298 of U3 region was: TRE-

PH-S, 41GAG TCA TCG ACC CAA AAG G59; TRE-PH-AS, 298TGC GCT TTT ATA

GAC TCG GC279. Primer pair for region “b” (bp 1314-1412 of gag/pol coding region)

was: #19, 1314AGC CCC CAG TTC ATG CAG ACC1334; #20, 1412GAG GGA GGA

GCA TAG GTA CTG1392. Primer pair for region “c” (bp 5011-5204 of exon 2 of tax/rex) was: GP-S, 5011CAG CGG TGG AAA GGT CC5027; GP-AS, 5204AAA GTA

GGA AGA AAA CATT5186. Primers used to amplify region “d” (bp 7314-7434 of

exon 3 of tax/rex) were: R1p28, 7314ATG TTC CAC CCG CCT7328; TR2-AS, 7434GAG

GCG AGG GAT AAG GTA T7416.

99 Each primer set was optimized for the amount of input DNA (1% of total lysate

before immunoprecipitation) to give an amplified product in the linear range.

Conditions for PCR were as follows: 95°C for 5 minutes, followed by 25-30 cycles of

95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. A final extension

at 72°C for 4 minutes was also performed. The number of cycles and the concentration

of MgCl2 for PCR were optimized and were different for each primer set.

Co-immunoprecipitation assay. 293T cells transfected with 1 µg p28II, 1 µg Tax-2 or

both were grown for 48 h and cells were lysed in profound lysis buffer (Pierce,

Rockford, IL) on ice for 30 minutes. After centrifugation, ~500 µg of cleared lysates

were incubated overnight with 5 µl anti-AU1 antibody, 10 µl affinity ligand, and 20%

(w/v) capture resin on Catch and Release Spin Columns (Upstate Biotechnologies,

Charlottesville VA). The resin was washed five times with 1X wash buffer containing

1% NP-40 and 0.25% deoxycholic acid at pH 7.4. Immune complexes were eluted at

room temperature with 2X SDS-loading buffer, boiled and separated by SDS-PAGE.

Input samples represented 10% of the lysate prior to immunoprecipitation. Western

blots were performed as above using antibodies against p28II, Tax-2, and RNA Pol II

(Santa Cruz Biotechnologies, Santa Cruz, CA).

RESULTS

HTLV-2 p28II inhibits viral Tax activity and protein production. We recently showed that the HTLV-2 accessory gene product, p28II, is a nuclear protein that exhibits repressor activity on viral replication 341. We also showed that the inhibitory effects are 100 exerted at the RNA level. More specifically, p28II binds to and retains tax/rex spliced

mRNA in the nucleus. Using a full length provirus (referred to hereafter as HTLV-2)

and a cytomegalovirus (CMV) immediate early promoter driven Tax cDNA expression

plasmid (Tax-2) that are summarized in Figure 1B, we observed that p28II inhibited Tax

and Rex functional activity only when these proteins were expressed from the proviral

clone and not the cDNA expression plasmid (Fig. 4.2A). The inability of p28II to

inhibit Tax that is expressed from a cDNA could be attributed to over-expression in the case of the CMV expression plasmid as compared to the native promoter in the HTLV-2 provirus. To rule out that the saturation of Tax activity causes the unresponsiveness to p28II, we transfected 293T cells with 0.4 µg of an empty vector (negative control or

NC), 0.4 µg of HTLV-2 provirus, or 0.1 µg of Tax-2 cDNA plasmid which led to similar levels of Tax activity on an LTR-luciferase reporter in the absence of p28II (Fig

4.2A). Co-transfection of increasing concentrations of p28II expression plasmid

resulted in dose-dependent inhibition of Tax activity expressed from the HTLV-2

provirus but had no significant repression on Tax-2 activity expressed from the cDNA

expression plasmid (Fig. 4.2A). Similar results were obtained with the p30II of HTLV-1

(data not shown).

In order to validate that the inhibition of Tax activity was due to a reduction of Tax

protein expression and not to a disruption of Tax function per se, we performed western

blot analysis on lysates extracted from the same cells that were transfected with the

HTLV-2 provirus or Tax-2/Rex-2 cDNA in the presence or absence of exogenous p28II.

In addition, since both Tax-2 and Rex-2 are expressed from the same doubly spliced mRNA, we measured Rex-2 protein levels to confirm that p28II inhibited tax/rex mRNA

101 leading to a reduction in and therefore, functional suppression of, both proteins. As

shown in Figure 4.2B, both Tax-2 and Rex-2 proteins were significantly reduced in the

presence of p28II but only if expressed in the context of the HTLV-2 provirus. These

data corroborate the results of the functional activity of Tax and are consistent with the

overall conclusion that p28II specifically inhibits tax/rex mRNA expressed from a

provirus leading to repression of protein production.

Tethering of p28II to the promoter or tax/rex mRNA identifies its intrinsic post-

transcriptional repressor function. HTLV-1 p30II has been established as both a

transcriptional 114,115,117 as well as a post-transcriptional 118,341 repressor of HTLV-1

gene expression. Therefore, to address potential mechanistic differences between p28II and p30II, and to examine whether p28II has any transcriptional effects, p28II and p30II fusion proteins were generated to analyze their activity in tethering reporter assays.

Using the Gal4 DNA binding domain, we targeted either Gal4-p28II or Gal4-p30II fusion proteins to a promoter containing five Gal4 binding sites upstream of the luciferase gene. As previously reported 114, Gal4-p30II repressed the expression of

luciferase when tethered to the promoter. However, contrary to p30II, Gal4-p28II did not show any inhibitory effects on the same promoter dispensing the possibility that p28II has transcriptional repressor activity (Fig. 4.3A). The lack of p28II-mediated

transcriptional repression could not be attributed to inefficient expression of Gal4-p28II, which was expressed to similar levels of Gal4-p30II (Fig. 4.3B).

Our previous biochemical and functional studies indicated that p28II is a post-

transcriptional regulator that inhibits Tax and Rex expression at the mRNA level. To

102 couple the repression of p28II with its ability to associate with tax/rex mRNA 341, we tethered p28II to the mRNA using the previously established bacteriophage MS2 coat

protein RNA interaction approach. A chimeric protein in which MS2 coat protein fused

to the amino terminus of either p28II (MS2p28II) or GFP (MS2GFP) was generated (Fig.

4.3C). A reporter plasmid was generated containing MS2-binding sites at the 3’UTR of

Tax-2 cDNA (Tax-2MS2 depicted in Fig. 4.3C). To examine whether tethered p28II inhibited tax/rex mRNA, 293T cells were transfected with either Tax-2 or Tax-2MS2 cDNA together with the Tax reporter plasmid (LTR-Luc) and different p28II constructs.

As expected, the effects of p28II or the control chimeric protein MS2GFP on Tax

expressed from cDNA were negligible. Conversely, only Tax expressed from a cDNA

containing MS2 binding sites was inhibited by MS2p28II indicating that targeting p28II to the mRNA led to inhibition of Tax activity (Fig. 4.3D). To control for expression of the different fusion proteins, western blot analysis performed on lysates from the transfected cells indicated that all the proteins were expressed (Fig. 4.3E).

The 5’UTR and splicing of tax/rex mRNA do not contribute to p28II-mediated

inhibition. In an effort to understand the mechanism of the p28II-mediated inhibition

and its differential effects on expression from the provirus versus cDNA, we next

focused on the mRNA itself. As highlighted in the panel of constructs shown in Figure

4.1, there are three differences between the mRNA expressed from the HTLV-2

provirus versus the Tax cDNA expression plasmid. The tax/rex mRNA expressed from

the provirus contains a 5’ untranslated region (5’UTR) and is doubly spliced. Both

features are excluded from mRNA expressed from the cDNA expression vector.

103 Furthermore, expression of the tax/rex mRNA from the provirus is driven by the

HTLV-2 natural promoter that is present in the LTR, whereas expression of the Tax-2

cDNA is directed by the CMV immediate early promoter.

Since data indicates that sequences in the UTR of genes can regulate mRNA

processing at different steps ranging from overall stability 390,391 to translational

efficiency 392,393, we examined whether the 5’ UTR could play a role in recruiting p28II to tax/rex mRNA leading to its inhibition. A cDNA construct expressing full-length tax/rex mRNA that includes the 5’ UTR (Fig 4.1, IY531) was generated and tested for susceptibility to inhibition by p28II. 293T cells were transfected with either HTLV-2 or

IY531 in the presence or absence of p28II. Tax activity (Fig 4.4A) and protein levels

(Fig. 4.4B) were then measured. The results show that the addition of the 5’ UTR to the

Tax cDNA did not result in p28II-mediated inhibition of Tax activity.

Several studies demonstrate that pre-mRNA splicing imprints the mRNA in the

nucleus with a range of proteins needed for later processes such as export, translation

and stability 383,394-397. In light of our previous results showing that the p28II inhibitory

effect is dependent on its ability to bind to the spliced tax/rex mRNA and retain it in the

nucleus 341, we sought to determine if the splicing process and subsequently loading of

splicing factors and/or the exon junction complex (EJC), might recruit p28II to tax/rex

mRNA. We generated a panel of constructs that reinserted intron-1 or intron-2 into the

tax/rex cDNA plasmids with or without the 5’ UTR (see Fig. 4.1). To simplify our

analysis, we truncated intron-1 to eliminate the gag/pol open reading frame. This

truncated intron did not have any detrimental effect on Tax activity itself or the ability

of p28II to inhibit Tax activity (Fig. 4.4, IY587). Further analysis of the cDNA

104 plasmids containing introns indicated that splicing of neither intron-1 nor intron-2 is

necessary or sufficient for p28II-mediated inhibition since none of these constructs was significantly responsive to p28II (Fig 4.4A, IY588, IY594, IY595 as compared to

HTLV-2 or IY587). Western blot analysis results were consistent with the Tax

functional assay revealing that the p28II-mediated reduction in Tax protein that is

expressed only from HTLV-2 and IY587 (Fig. 4.4B). Taken together, we conclude that

recruitment of p28II to tax/rex mRNA and repression of Tax/Rex activity is not dependent on 5’UTR sequences, intron sequences or the loading of splicing factors and/or splicing itself.

Inhibition of tax/rex mRNA by p28II is coupled to the HTLV-2 promoter. To

determine whether the promoter from which tax/rex mRNA is expressed is directly

correlated to p28II-mediated inhibition, we replaced the native HTLV-2 core promoter

with a CMV immediate early promoter in the context of the provirus (Fig 4.1,

BCHTLV-2). Tax activity expressed from BCHTLV-2 was not repressed by p28II as

compared to the wild type provirus (Fig. 4.5A). Since expression from a CMV

promoter is more efficient than that from HTLV-2, two concentrations of BCHTLV-2

were tested to rule out the possibility that resistance to p28II inhibition was due high

expression levels and saturation. Furthermore, to examine the possible role of the

HTLV-2 promoter on the repressive activity of p28II, we replaced the CMV promoter in

BC20.2 and IY531 with the HTLV-2 promoter generating IY619 and IY620,

respectively (Fig 4.1). As shown in Figure 4.5B, p28II had the capacity to inhibit Tax

activity when Tax was expressed from constructs containing the native HTLV-2

105 promoter (HTLV-2, IY619, and IY620), but not the CMV promoter. Western blot

analysis results were consistent with the Tax functional assay revealing a p28II-

mediated reduction in Tax expressed from only HTLV-2, IY619, and IY620 (Fig.

4.5C). Taken together, these results indicated that the recruitment of p28II to tax/rex

mRNA and repression of Tax/Rex activity was dependent on the HTLV-2 promoter,

specifically the HTLV-2 U3 region.

HTLV-2 p28II and HTLV-1 p30II are recruited to the viral promoter and associate

with transcription elongation. To investigate whether the correlation between the

inhibition of Tax/Rex activity by p28II and the HTLV-2 promoter is due to a direct association, we used the chromatin immunoprecipitation (ChIP) approach on cells transfected with a construct expressing HTLV-2 LTR-Luciferase in the presence or absence of p28II. Since efficient transcription from the viral LTR requires Tax, we also transfected Tax to determine whether an association, if any, is dependent on a transcriptionally active promoter. Cross-linked and sonicated extracts prepared from transfected cells were immunoprecipitated using an antibody specific for the p28II-AU1.

Input DNA that was not subjected to immunoprecipitation as well as co-precipitated

DNA fragments were amplified by PCR using a primer pair specific for the viral promoter region. More specifically the primers span all three TREs that are present in the U3 region of LTR-2. Figure 4.6A shows that p28II is clearly associated with the

HTLV-2 viral promoter in a Tax-dependent manner, suggesting that the recruitment of

p28II occurs during transcription.

106 To define the role of this co-transcriptional recruitment in sequestering p28II to the newly transcribed mRNA, we examined the association of p28II with different regions

of the 9 kb HTLV-2 genome by ChIP analysis. Primer pair “a” detects the promoter

region, pair “b” detects gag sequences, pair “c” amplifies exon 2 of tax/rex sequences,

and pair “d” detects sequences in tax/rex exon 3 (see diagram in Fig. 4.6B). To validate

our experimental conditions, we also analyzed two other proteins whose distribution on

the HTLV-2 genome is known or can be anticipated. RNA polymerase II (Pol II) is

known to be evenly distributed over the promoter region, coding region and 3’UTR of

actively transcribed genes 385,398. HTLV-1 p30II, the functional homologue for HTLV-2

p28II, has a proposed role in both transcriptional 114,115,117 and post-transcriptional 118,341 regulation and would be expected to be found at the promoter.

As previously shown 385, Pol II association was detected at all regions of the viral

genome (Fig. 4.6B). The distribution of p28II and p30II was analyzed using the

monoclonal antibodies AU1 (for p28II) and HA (for p30II). Interestingly, these proteins

showed very similar distribution patterns over the HTLV-2 genome with positive signal

for regions “a”, “b” and “c”, whereas no clear association was detected for region “d”

(Fig. 4.6B). These profiles are consistent with the hypothesis that p28II and p30II are

recruited to the tax/rex mRNA at the transcription level and the lack of binding or cross-

linking at the 3’ coding region may reflect the redistribution of p28II and p30II from the

transcriptional machinery to the newly transcribed and/or processed mRNA.

p28II interacts with Tax-2 and components of the transcriptional machinery. The

data above indicated that p28II associates with a transcriptionally active HTLV-2

107 promoter, which leads to its recruitment to the tax/rex mRNA. To further verify the

interaction between p28II and components of the transcriptional machinery in intact

cells, 293T cells were transfected with plasmids expressing p28II, Tax-2 or both

followed by immunoprecipitation of p28II complexes. Proteins associated with p28II were analyzed by SDS-PAGE and individual proteins were identified by western blot analysis. Results from representative western blots in Figure 4.7 confirmed that the monoclonal AU1 antibody efficiently immunoprecipitated p28II-AU1, which was

detected using a rabbit polyclonal antibody to p28II. Importantly, in cells that co-

expressed both p28II and Tax-2, we were able to co-immunoprecipitate Tax-2 along with p28II. Nonspecific binding of Tax-2 to the beads or the antibody could not account for this result because Tax was not immunoprecipitated in cells that did not express p28II. Likewise, we were able to immunoprecipitate endogenous RNA Pol II with p28II.

These results demonstrated that p28II was able to associate with components of the

transcriptional machinery in mammalian cells.

Inhibition of tax/rex mRNA by p28II may involve the TAP/p15 pathway. Thus far,

our data along with our previous findings suggested two non-exclusive possibilities for

the mechanism through which p28II inhibits tax/rex mRNA: p28II may actively retain

the mRNA in the nucleus, or p28II may inhibit export of the mRNA by interfering with

its export pathway. In order to define the pathway used by tax/rex mRNA for

nucleocytoplasmic transport, we examined the contribution of two major pathways on

Tax expression. The CRM1 pathway is utilized by several viral mRNAs, including

HTLV unspliced and incompletely spliced gag/pol and env mRNA for transport to the

108 cytoplasm 130. Conversely, the majority of mammalian spliced mRNAs utilize the

TAP/p15 export pathway 399-401. The specific CRM1 inhibitor, Leptomycin B (LMB), was used to test whether CRM1 is involved in tax/rex mRNA export. LMB had no

inhibitory effects on Tax activity when expressed from a provirus or a cDNA

expression vector (Fig. 4.8A). In fact, there was a slight enhancement of Tax activity when expressed from a provirus. We attributed that enhancement to blockage of

gag/pol and env mRNA export and redistribution of the viral mRNA pools to favor

splicing. We next tested the effects of exogenous TAP/p15 on Tax activity in

transfected 293T cells. Consistent with the hypothesis that tax/rex mRNA utilizes

TAP/p15 for transport to the cytoplasm, all three concentrations of TAP/p15 enhanced

Tax activity (Fig. 4.8B). Lastly, we examined whether p28II was able to repress Tax expression in the presence of exogenous TAP/p15. Consistent with our previous data, p28II caused 70-80% inhibition of Tax activity when expressed from a provirus (Fig.

4.8C). While over-expression of TAP/p15 significantly enhanced Tax activity, p28II resulted in a 79% inhibition of that activity at the dose used (Fig. 4.8C). These data indicated that p28II can override or block the activity of TAP/p15.

DISCUSSION

The HTLV-2 p28II accessory protein is a potent repressor of viral gene expression by specifically interacting with and retaining tax/rex mRNA in the nucleus 341 leading to

a decrease in protein production. Tax and Rex are critical proteins that play essential

roles in HTLV replication. Tax is the main transcriptional regulator of the HTLV

promoter, which is present in the 5’LTR and drives expression of all viral genes 361.

109 Rex is a post-transcriptional regulator that facilitates the cytoplasmic expression of

unspliced and incompletely spliced viral mRNAs 130. Thus, by lowering Tax and Rex

expression, p28II can suppress viral replication 341 leading to a state of viral latency that

is suggested to be critical for viral persistence in vivo. Consistent with this principle, an

infectious molecular clone of HTLV-2 that is deficient for p28II is unable to spread and

persist in a rabbit model of HTLV infection (Younis et al, unpublished data).

In this report, we have identified the mechanism by which p28II is selectively

recruited to tax/rex mRNA. We previously showed that the inhibitory effects of p28II were restricted to tax/rex mRNA expressed from a full length provirus and not a cDNA expression plasmid (Fig. 4.2) and 341. Putative candidates that may aid in recruiting

p28II to the former mRNA but not the latter include 5’UTR, splicing or the promoter

context. Thus, we generated a panel of Tax expression plasmids to test for the

contribution of these variables separately or in combination. Our data indicated that

neither the 5’UTR nor splicing of the pre-mRNA was sufficient to make tax/rex mRNA

a substrate for p28II repression (Fig. 4.4). Conversely, we showed a positive correlation between expression from the native HTLV promoter (LTR) and p28II-mediated

inhibition of Tax activity (Fig. 4.5). Replacing the LTR in a proviral clone with a CMV

promoter was sufficient to block the repressive effects of p28II. Moreover, switching

the CMV promoter with the LTR in a previously resistant Tax cDNA expression

plasmid rendered tax mRNA completely susceptible to inhibition by p28II.

In the last few years, considerable evidence has accumulated to support the concept

that transcription directed by RNA Pol II and downstream mRNA processing events

such as splicing, export, and polyadenylation are functionally coupled 388. In this study,

110 we confirmed that p28II is not a transcriptional regulator (Fig. 4.3), but is recruited co-

transcriptionally to the promoter in a Tax-dependent manner. Chromatin

immunoprecipitation was used to show the association profiles of p28II, its HTLV-1

homologue p30II, and Pol II with a transcriptionally active HTLV-2 genome. Our results showed an association between p28II and p30II with the promoter region as well

as the 5’ and middle of the coding region of HTLV-2. This observation, together with

the loss of association at the 3’ end of the coding region, supports the conclusion that

these proteins not only are recruited to the promoter but also are components of the

transcription-elongation complex until their response element on the newly transcribed

tax/rex mRNA is generated (Fig. 4.6). Interestingly, the similar association profiles of

p28II and p30II suggest that these two proteins utilize a similar mechanism for their

recruitment to their target mRNAs. It is worth noting, however, that although p28II has negligible transcriptional activity, p30II has the capacity to modulate transcription of

HTLV-1 differentially due to its interaction with the transcriptional co-activator p300

114. Moreover, p30II has recently been shown to interact with and stabilize the

Myc/TIP60 transcription complexes that assemble on Myc responsive promoters such

as cyclin D2 leading to enhanced Myc-transforming potential 117. Conversely, p28II does not interact with p300 (data not shown), but physically associates with Tax-2 and

Pol II, providing more evidence that it is recruited at the level of transcription and becomes a component of the elongation complex.

Mammalian mRNAs utilize at least two nuclear export receptors to achieve export to the cytoplasm including CRM1 and TAP/p15. Retroviruses, including HTLV, depend on these pathways for the export of their own mRNA 401. For example, both

111 HIV and HTLV require the export and cytoplasmic expression of unspliced and

incompletely spliced mRNAs that encode for structural and enzymatic proteins. HIV

Rev and HTLV Rex are adaptor proteins that provide access to the CRM1 pathway to

export such mRNAs 130,231. MPMV, on the other hand, recruits TAP/p15 to a specific

sequence on its unspliced mRNA (CTE) to allow transport to the cytoplasm 402,403.

Since our previous study indicated that p28II interferes with the nucleocytoplasmic

export of tax/rex mRNA, we examined the contribution of CRM1 and TAP/p15 to this

process. Our data suggested that while CRM1 did not contribute to Tax expression and

activity, exogenous TAP/p15 significantly enhanced Tax activity in a dose-dependent

manner (Fig. 4.8). When p28II was transfected into cells along with TAP/p15, we

observed that the suppression of Tax activity was sustained. This possibly could be

attributed to p28II overriding or blocking the TAP/p15 pathway. The fact that in the

presence of TAP/p15, p28II did not show complete inhibition of Tax activity could be

due to some TAP-dependent rescue of tax/rex mRNA export. In either case, there

appeared to be cross-talk between p28II and TAP/p15 that needs to be analyzed further.

Another possible explanation for the ability of p28II to inhibit Tax expression in the

presence of exogenous TAP/p15 is based on previous reports indicating that in 293T

cells, Tap/p15 complexes are likely to play an important role in translational regulation

beyond their previously proposed function as RNA export receptors. More specifically,

Tap/p15 augments protein expression from mRNAs that have already been exported by

enhancing polyribosome association and translation of these mRNAs 404,405. Thus, if

TAP/p15-mediated enhancement of Tax expression in 293T cells is due to their

translational effects, p28II would still be able to override this enhancement by simply

112 retaining the RNA in the nucleus making it unavailable for TAP/p15.

Collectively, our findings suggest that Tax-mediated transcription of HTLV LTR is

a process that serves not only to drive the expression of viral genes, but also to recruit a

negative factor that suppresses the expression of two of the major positive regulators of

HTLV expression, leading to a complicated but tightly regulated feedback loop (see

Figure 4.9 for model). Such regulation might hold the key for latency and evasion of

the immune response in vivo. In addition, it is of significance that p28II and p30II, two

suppressors of gene expression, are recruited to transcriptionally active genes, which suggests that the functional coupling between transcription and mRNA export could be utilized to promote as well as suppress gene expression. A better understanding of the role of the accessory proteins is critical not only to expand our knowledge of their contribution to basic cellular biology and , but also for viral-associated disease progression and therapeutic development.

113

Figure 4.1. (A) Schematic diagrams of HTLV-2 genome and the different mRNAs

(horizontal lines) as well as proteins (Gray boxes). Dashed lines represent introns removed. (B) Tax-expression plasmids used in this study. wtHTLV-2 and BCHTLV-2 are full length proviral clones that express doubly spliced tax/rex mRNA. IY619 and

Tax-2 express the entire tax/rex coding sequence as a cDNA, but lack the 5’UTR.

IY620 and IY531 express full length tax/rex cDNA which includes the 5’UTR. IY587

is a proviral clone with truncated intron-1 (gag/pol sequences) that still expresses

doubly spliced tax/rex mRNA. IY594 expresses tax/rex mRNA lacking 5’UTR but

contains intron-2. Both IY588 and IY595 express full length tax/rex mRNA that

contains a truncated intron-1 (gag/pol sequences) or intron-2 (env sequences),

respectively. For more detailed description of the plasmids see Materials and Methods.

Plasmids wtHTLV-2, IY619, IY620, and IY587 express tax/rex mRNA from HTLV-2

promoter (LTR), whereas plasmids BCHTLV-2, Tax-2, IY531, IY588, IY594 and

IY595 utilize CMV immediate early promoter. Asterisks represent Rex and Tax start

codons.

114

Figure 4.1

115

Figure 4.2. HTLV-2 p28II inhibits Tax-2 and Rex-2 expression only when they are

expressed from a full length proviral clone. (A) 0.4 µg of wtHTLV-2 plasmid or 0.1 µg of Tax-2 cDNA plasmid were transfected into 293T cells along with the LTR-luciferase reporter in the absence or presence of 2X or 4X molar ratio of p28II expression plasmid.

After 48 h of culture, cells were lysed and luciferase activity was measured.

Experiments were done in triplicate and CMV-gal was used to adjust for transfection

efficiency. Negative control (NC) represents cells that are transfected with an empty vector. (B) The same lysates from panel A were separated on 4-12% SDS PAGE and transferred onto nitrocellulose membranes followed by western blot assay using antibodies specific for Tax-2, Rex-2, p28II, or actin (loading control, LC). Rex-2

migrates as two bands, p24 (hypophosphorylated) and p26 (phosphorylated).

116

Figure 4.3. p28II is a post-transcriptional repressor. (A) Gal4 DNA binding domain

fused with either p28II or p30II were transfected into 293T cells together with the 5G-

Luc reporter that contains a minimal promoter with 5 Gal4 binding sites upstream of a

luciferase reporter. Experiments were done in triplicates and error bars reflect standard

deviations. (B) Gal4-p30II and Gal4-p28II were detected by immunoprecipitation using

a Gal4 specific antibody. (C) Schematic diagram of tax-2MS2 mRNA with 6 MS2

response elements (MS2-RE) inserted after the Tax-2 termination codon and before the

polyadenylation signal. The MS2-p28II fusion protein was targeted to the tax-2MS2

mRNA via the interaction between the MS2 protein and the MS2-RE. (D) 293T cells

were transfected with LTR-luciferase reporter together with a control, p28II, MS2-p28II, or MS2-GFP expression vector. Tax-2 was expressed from a Tax-2 or Tax-2MS2 cDNA expression plasmid. were performed in triplicates and error bars reflect standard deviations. Tax activity was measured as luciferase units and averaged.

(E). Expression of p28II, MS2-p28II, GFP and MS2-GFP proteins were confirmed by

western blot analysis.

117

Figure 4.3

118

Figure 4.4. Intron sequences/splicing and 5’UTR are not required for p28II-mediated

inhibition of Tax activity. (A) 0.4 µg of wtHTLV-2 and IY587 plasmids or 0.1 µg of the indicated Tax-2 cDNA plasmids were transfected into 293T cells along with the

LTR-luciferase reporter in the absence (-) or presence of 4X molar ratio of p28II expression plasmid. After 48 h of culture, cells were lysed and luciferase activity was measured. Experiments were done in triplicates and error bars reflect standard deviations. CMV-gal was used to adjust for transfection efficiency. p28II inhibited

Tax-2 activity that was expressed from both wtHTLV-2 and IY587 indicating that truncating intron-1 does not influence p28II-mediated inhibition or Tax-2 expression.

All cDNA expression plasmids showed minimal or no inhibition by p28II. (B) The

same lysates from panel A were separated on 4-12% SDS PAGE and transferred onto

nitrocellulose membrane followed by western blot using antibodies specific for Tax-2,

p28II, or actin (loading control, LC). Reduction of Tax protein correlates with loss of

Tax activity in panel A.

119

Figure 4.4

120

Figure 4.5. p28II-mediated inhibition directly correlates with Tax expression in the

context of the native HTLV-2 promoter (LTR). (A) 0.4 µg of wtHTLV-2 or 0.4 or 0.2

µg of BCHTLV-2 were transfected into 293T cells along with LTR-luciferase in the

absence or presence of 4X molar ratio of p28II expression plasmid. After 48 h of culture, cells were lysed and luciferase activity was measured. Experiments were done in triplicates and error bars reflect standard deviations. CMV-gal was used to adjust for

transfection efficiency. p28II consistently inhibits Tax-2 activity when expressed from

wtHTLV-2 but not BCHTLV-2. (B) Expression of Tax-2 from cDNA with (IY620) or

without (IY619) 5’UTR but under the control of the HTLV-2 promoter reverses the

resistance to inhibition by p28II that is observed in the Tax-2 CMV-expression plasmid.

(C) Lysates from panels A and B were separated on 4-12% SDS PAGE and transferred

into nitrocellulose membrane followed by western blot using antibodies specific for

Tax-2, p28II, or actin (LC). Reduction of Tax protein correlates with loss of Tax

activity in panels A and B.

121

Figure 4.5

122

Figure 4.6. p28II associates with HTLV-2 promoter as well as downstream DNA

sequences. (A) Chromatin immunoprecipitation (ChIP) analysis reveals that p28II associates with HTLV-2 promoter (LTR-2) but only in the presence of the transcription activator Tax-2, indicating that p28II associates only with a transcriptionally active

promoter. Input represents 10% of DNA prior to IP. (B) Schematic diagram of the

HTLV-2 provirus with primer pairs used in the PCR of the following ChIP are indicated. For exact location of primers refer to Materials and Methods. TRE indicates the three 21bp repeats representing Tax response elements in the viral promoter. The

ChIP assay was performed as in panel A with antibodies specific for p28II, HTLV-1

p30II, or RNA polymerase II. PCR conditions for each primer pair were optimized to amplify a product in the linear range. Lanes 1 and 4 are lysates from mock transfected cells. Lanes 2, 5, 7, and 9 are lysates from cells expressing wtHTLV-2. Lanes 3, 6 and

10 are lysates from cells expressing wtHTLV-2 and p28II. Lane 8 is lysate from cells

expressing wtHTLV-2 and p30II.

123

Figure 4.6

124

Figure 4.7. p28II associates with components of the transcription machinery.

Immunoprecipitation using AU1 antibody was performed on 293T cells transfected with

p28II-AU1, Tax-2 or both. Immune complexes were resolved on SDS-PAGE followed by western blot analysis using Tax-2, pol II or p28II specific antibodies. Prior to

immunoprecipitation, 10% of lysates were saved (Input) and blotted as above.

125

Figure 4.8. p28II overrides the TAP/p15 export pathway. (A) Expression of Tax-2

from full length provirus (HTLV-2) or cDNA plasmid (Tax-2) was tested in the absence

or presence of the CRM1 specific inhibitor LMB. (B) Expression of Tax-2 from

HTLV-2 or Tax-2 plasmid was tested in the absence or presence of increasing

concentrations of TAP and p15 expression plasmids. In panels A and B, Tax-2

expression was indirectly measured using the LTR-luciferase reporter. (C) p28II-

mediated inhibition of Tax-2 that is expressed from HTLV-2 was tested in the absence

or presence of 10ng of TAP and p15. Percent inhibition is indicated in each case. All

experiments were done in triplicates and error bars reflect standard deviations. CMV-

gal was used to adjust for transfection efficiency.

126

Figure 4.9. A model for the mechanism of action of p28II. After recruitment to the

HTLV-2 promoter in a Tax-dependent manner, p28II associates with RNA Pol II and moves with the transcription elongation machinery. As the RNA is being synthesized, capped and spliced, a novel mRNA element is generated (possibly exon-2/exon-3 junction) and recognized by p28II which translocates to the mRNA and retains it in the

nucleus.

127

CHAPTER 5

HUMAN T-CELL LEUKEMIA VIRUS TYPE 2 OPEN READING FRAME II

ENCODED P28 IS REQUIRED FOR VIRAL PERSISTANCE IN VIVO

ABSTRACT

Human T-cell leukemia virus (HTLV) persists and causes leukemia and neurological

disease in a small percentage of infected people. HTLV encodes for structural and

enzymatic proteins, regulatory (Tax and Rex) and accessory (ORF-I and ORF-II)

proteins. It has been reported that the accessory gene products of HTLV are dispensable for in vitro viral replication and viral-induced cellular transformation.

However, an HTLV-2 mutant that was deleted for the region encoding the accessory genes was unable to persist in an in vivo rabbit model of infection. We have previously reported that the HTLV-2 ORF II gene product, p28II, is a nuclear protein that is able to restrict viral replication. p28II is recruited to the viral promoter in a Tax-dependent

manner without leading to transcriptional effects but rather leads to a downstream post- transcriptional inhibition. More specifically, p28II binds to and retains tax/rex mRNA in

the nucleus leading to reduced protein expression and virion production. These data are

consistent with a critical role of p28II in viral persistence in vivo since a reduction of

viral replication in a cell carrying the provirus may allow escape from immune

128 recognition. Since the previous in vivo study deleted all accessory proteins collectively,

we aimed at selectively deleting p28II in the context of the provirus to determine its

contribution. In this study, an HTLV-2 virus that is deleted for p28II expression

(HTLV-2∆p28) showed no significant difference from wtHTLV-2 in vitro. However,

when inoculated into immune-competent rabbits, all wtHTLV-2 infected animals

showed persistent infection, whereas rabbits infected with HTLV-2∆p28 were able to eliminate the virus as early as 2 weeks, indicating that p28II is critical for early viral

infectivity, spread and/or persistence in infected rabbits.

INTRODUCTION

Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are distinct

complex oncogenic retroviruses that persist in the infected individual despite a robust

virus-specific host immune response 80. Although HTLV-2 shares 65% homology at

the nucleotide sequence level with HTLV-1, it seems to be less pathogenic where only a

few cases of variant hairy cell leukemia and several cases of neurological disease have

been reported to be associated with HTLV-2 infection 5,9,359. Both HTLV-1 and HTLV-

2, however, have the capacity to promote T-lymphocyte growth in vitro and in vivo 50-53.

The ultimate fate of infected T-cells in vivo depends on their ability to balance proliferative, cell cycle, and anti-apoptotic signals mediated by viral and cellular proteins and to evade the host immune response. Therefore, the ability of the virus to respond to environmental signals and regulate viral and cellular gene expression is critical to its long-term survival and persistence in the infected individual.

129 The HTLV gene products include structural and enzymatic proteins (Gag, Pol, and

Env), trans-regulatory proteins (Tax and Rex), and accessory proteins (p12, p13, p30 for

HTLV-1 and p10, p11, p22, p28 for HTLV-2) 80. Tax drives transcription from three

21-bp enhancer elements referred to as Tax-response elements (TREs) 331,376 that are

located in the U3 region of the 5’ long terminal repeat (LTR) 361. Tax-mediated protein-

protein and protein-DNA interactions on the HTLV promoter are critical for the

assembly of complexes that contain CREB/ATF and CBP/p300 139,141,377-380, and

subsequently lead to strong transcriptional activation of the virus 381,382. Rex acts post-

transcriptionally to facilitate cytoplasmic expression of incompletely spliced viral

mRNAs. Based on the critical role of Rex in the expression of structural and enzymatic

gene products, it is considered to positively regulate the switch between the early, latent

phase and late, productive phase of HTLV life cycle 130.

The HTLV-1 and HTLV-2 accessory proteins, encoded by open reading frame

(ORF) I and ORF II near the 3’ end of the viral genome 99,369 are the least conserved

between the two related viruses. Little is known about the role of these proteins in the

replication cycle and pathogenesis of the virus, especially in the case of HTLV-2.

Furthermore, they have been shown to be dispensable for in vitro replication and

immortalization of activated primary T lymphocytes 92,93,343. However, using a rabbit

model of infection, all of the accessory proteins tested thus far have been shown to be

essential for viral infectivity and persistence in vivo 110,125-127.

HTLV-1 p30II and HTLV-2 p28II are nuclear proteins encoded by ORF II and share

minimal homology at the amino acid level. We have recently shown that p30II and p28II down regulate tax/rex mRNA expression post-transcriptionally by binding to and

130 retaining this mRNA in the nucleus 118,341. Furthermore, we identified the component of

the HTLV promoter, specifically Tax, as the factor required for efficient recruitment of

p28II to the newly transcribed mRNA. We also provided evidence that p28II itself does

not exhibit transcriptional activity but inhibits tax/rex mRNA transport possibly due to

an early block of the TAP/p15 export pathway (Younis et al submitted). Our data

revealed a complex interplay between the transcription machinery and the post-

transcriptional regulation of tax/rex mRNA that is consistent with a critical role for

p30II and p28II in regulating viral replication in vivo. Indeed, Silverman et al have

recently reported that p30II is required for HTLV-1 persistence in a rabbit model of

infectivity. Their data further showed that in vivo the virus was pressured to revert its

mutation to wild type in order to be maintained or survive 126. The only in vivo study

targeting the accessory proteins of HTLV-2 was designed to eliminate all accessory

proteins, and showed that collectively these proteins are also critical for in vivo survival

of HTLV-2 343.

In light of our previous findings that despite the minimal homology between p28II and p30II, they both share similar functions that are important for regulating viral

replication, we aimed at specifically deleting p28II in order to determine its contribution

in vivo. Here we report that, an HTLV-2 virus that is specifically mutated to delete p28II (HTLV-2∆p28), behaves similarly to wild type HTLV-2 (wtHTLV-2) in in vitro

immortalization assays, but fails to replicate and/or persist in infected rabbits. The

block on HTLV-2∆p28 survival seems to be early in the infection process since at week

2 we were unable to detect any viral sequences or antibody response to the virus in

131 animals infected with HTLV-2∆p28 as opposed to wtHTLV-2. We conclude that p28II is essential for viral persistence in vivo.

MATERIALS AND METHODS

Cells and plasmids. 293T and 729 cells were maintained in Dulbecco’s modified

Eagle’s medium (DMEM) and Iscove’s modified Eagle’s medium (IMEM), respectively. The media were supplemented to contain 10% fetal bovine serum, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml). Human peripheral blood mononuclear cells (PBMCs) were isolated from freshly drawn human blood as previously described 316. Briefly, blood was diluted 1:1 with phosphate buffered saline

(PBS) and layered over 12 ml of Ficoll-hypaque (Amersham-Pharmacia), then

centrifuged for 20 minutes at 2100 rpm. The Ficoll/plasma interphase (buffy coat layer)

was gently removed into a new tube and washed once with RPMI. The pelleted cells

were resuspended in RPMI supplemented with 20% fetal bovine serum and antibiotics.

10 U/ml of human IL-2 was added as indicated.

The HTLV-2 proviral clone, pH6neo 374, was used in this study. To generate an

HTLV-2 proviral clone that is deficient for p28 expression (HTLV-2∆p28), a single

nucleotide substitution was introduced into HTLV-2 at position 7333 that changed the

seventh serine of p28 into a termination codon without affecting Tax or Rex sequences.

LTR-2-Luc Tax reporter plasmid 341 has been previously described. CMV-βgal was

used to control for transfection efficiency.

132 Transfection, luciferase assay, and p19 ELISA. For Tax functional assay, 1.5 x 105

293T cells were transfected using Lipofectamine (Invitrogen, Carlsbad, CA) according

to manufacturer recommendations. The total amount of DNA was kept constant and

contained 0.2 µg HTLV-2 LTR-luciferase reporter, 0.1 µg CMV-βgal, and 1 µg HTLV-

2 or HTLV-2∆p28 proviral clones. After 48 h of growth, cells were pelleted and lysed

in passive lysis buffer (PLB) (Promega, Madison, WI) and Tax activity was measured

as luciferase light units after adjusting for β-galactosidase. Each experiment was done

in triplicates, averaged and standard deviations were indicated as error bars. For the

detection of viral p19Gag matrix antigen, 450 µl of cell supernatants were used in a

commercially available enzyme-linked immunosorbent assay (ELISA) (Zeptometrix

Corporation, Buffalo, NY). Samples were diluted accordingly in order to be in the

linear range of the standard curve.

Generation of a stable 729-HTLV-2∆p28 cell line. Stable viral producer cells were

generated as follows. Five million 729 cells were washed once with RPMI, then

resuspended in 250 µl of electroporation buffer (60% 2X RPMI, 20% FBS, 20%

deionized distilled water). The cell suspension was mixed with 15 µg of H1IT plasmid

on ice and transferred to a 0.5 mm gap electroporation cuvette and electroporated in a

BioRad GenePulser at 250 V and 975 µF. After incubation on ice for 10 min, the cells were diluted into 5 ml of IMEM supplemented with 10% FBS and 1% antibiotics. One day later, the cells were washed with PBS, resuspended in 25 ml of complete IMEM containing 1 mg/ml of active geneticin (Invitrogen, Carlsbad, CA), and divided into 24- well plate. Selection for integrated plasmid was carried for 4 weeks, after which wells 133 containing visible cell clumps and depleted medium were screened for p19Gag

production using ELISA. Single cell clones were generated by resuspending cells from

positive wells at 1-2 cells/ml in IMEM + geneticin and expanded for additional 4

weeks. A 729-based stable HTLV-2 producer cell line is previously described 45 and was used as positive control throughout this study.

Detection of proviral sequences. For detection of provirus in cell lines and rabbit

PBMC, genomic DNA was harvested by salt purification (Gentra, Minneapolis, MN)

and examined for the presence of HTLV-2 sequences following PCR amplification.

Five hundred nanograms of DNA was amplified using a primer pair specific for the

HTLV-2 pX region (670: 5’CGG ATA CCC AGT CTA CGT GT and 671: 5’GAG

CCG ATA ACG CGT CCA TCG), which yielded a 159-bp product. The amplified

products were separated on a 2% agarose gel and visualized by ethidium bromide

staining.

Rabbit study. To measure viral infectivity, replication and persistence in vivo, female

specific-pathogen-free New Zealand White rabbits (Harlan, Indianapolis IN) were inoculated via the lateral ear vein with approximately 107 729, 729-HTLV-2 or 729-

HTLV-2∆p28 cells. All cells were gamma-irradiated (7500 Rad) prior to injection to prevent outgrowth of the cellular inoculum in vivo but allow virus transmission.

Inoculated cell were equilibrated based on their p19Gag production. Prior to inoculation, 13ml of whole blood as well as sera were withdrawn from each rabbit and

used as week 0 control. At week 2, 4, 6 and 8 additional blood and sera were collected

134 followed by isolation of PBMCs. For the detection of viral sequences, genomic DNA

was isolated as described above and PCR was performed using the following primer

sets. 670 and 671 for detection of viral sequences, whereas rabbit GAPDH was

detected using primers: rGAPDH-S: 5’GAT GCT GGT GCC GAG TAC GTG G,

rGAPDH-AS: 5’GTG GTG CAG GAT GCG TTG CTG A.

The rabbit antibody response to specific viral antigenic determinants was detected

by a commercial western blot assay (GeneLabs Diagnostics, Singapore) that was

adapted for rabbit plasma by use of alkaline phosphatase-conjugated goat anti-rabbit

immunoglobulin G (1:1000 dilution; BioMerieux, Inc. Durham, N.C.). Plasma showing

reactivity to Gag (p24 or p19) and Env (p21 or gp46) antigens was classified as positive

for HTLV-2 seroreactivity. Rabbits were regularly evaluated for any overt signs of

clinical disease. At the end of week 8, rabbits were euthanized for necropsy.

RESULTS

Generation and characterization of an HTLV-2 proviral clone that is deficient for

p28II expression. In order to generate an HTLV-2 proviral clone with deleted p28II, we

introduced using site-directed mutagenesis a single point mutation at position 7333 of the wtHTLV-2 provirus. This change introduced a nonsense mutation at serine 7 of p28II without affecting Tax-2 or Rex-2 sequences (See Figure 5.1). The introduced

mutation was confirmed by sequencing. In order to evaluate Tax and Rex functions, the

wtHTLV-2 clone or HTLV-2∆p28 were transfected into 293T cells along with the Tax

reporter, LTR-2-luciferase. As shown in Figure 5.2A, Tax-2 that is expressed from

either clone is fully functional. Interestingly, in some experiments, Tax-2 expressed

135 from HTLV-2∆p28 showed better activity which could be attributed to the loss of p28II.

We have previously reported that p28II is a negative post-transcriptional regulator of

Tax expression (Younis 2004 JV), but we did not anticipate detecting any major effects

in a short term transient assay. Indeed, at best, we were able to measure 1.5 fold

enhancement of Tax-2 activity in HTLV-2∆p28.

Next, we measured p19Gag production and secretion into the medium by ELISA.

Since p19Gag production is dependent on functional Tax and Rex, we used it as an

indirect measure of Rex activity as well as a measure of viral replication. We show that

p19Gag release is not different between wtHTLV-2 and HTLV-2∆p28 (Figure 5.2B),

indicating that the mutation introduced in HTLV-2∆p28 did not have any overt effects

on viral protein production or function in vitro. Consistent with these observations, we

have previously reported that an HTLV-2∆p28 provirus that is stably integrated into

729 cells maintained its ability to infect and immortalize primary human PBMCs 343.

When compared to wtHTLV-2, the mutated clone induced similar levels of syncycia.

Moreover, PBMCs infected with wtHTLV-2 or HTLV-2∆p28 showed very similar

growth curves and eventually were immortalized at similar efficiencies 343.

Since cell free transmission of HTLV is very inefficient, in order to asses the ability

of the HTLV-2∆p28 proviral clone to make viral proteins and replicate in vivo, stable expression of HTLV-2∆p28 is needed. 729 B-cells were electroporated with HTLV-

2∆p28neo plasmid, and 3-4 weeks after transfection, wells containing visible clumps of

Geneticin-resistant cells were further characterized. Several wells were screened for the production of p19Gag. The integration of intact proviral sequences was determined using diagnostic PCR analysis of genomic DNA isolated from the stable producer cells 136 (data not shown). Supernatants from individual wells were also tested for the presence

of p19Gag by ELISA. Some clones showed robust p19Gag production and the clone

shown in Figure 5.2C was used for the rest of this study.

In vivo replication of wtHTLV-2 and HTLV-2∆p28.

To evaluate the function of HTLV-2 p28II in vivo, we compared the abilities of

wtHTLV-2 and HTLV-2∆p28 cell lines to establish and maintain infection in the well

established rabbit model (refs from lairmore and ratner). To ensure comparable

infection potential, inocula were equilibrated by HTLV-1 p19Gag production on a per

cell basis (Fig. 5.2C). Prior to inoculation, the fidelity of the ORF II mutation was

confirmed by sequencing. Five female rabbits were inoculated with wtHTLV-2, six

with HTLV-2∆p28, and 2 with either 729 negative cells or media control. Prior to

inoculation, blood and sera were collected from all rabbits and designated as the week 0 control. At weeks 2, 4, 6 and 8, PBMCs were isolated from freshly drawn blood in addition to sera and plasma from each individual rabbit.

In order to detect the presence of HTLV-2 provirus in rabbits, we used PCR to amplify HTLV-2-specific pX region proviral sequences from rabbit PBMC DNA using primer pair that is indicated in Figure 5.1. Provirus was detected in wtHTLV-2- inoculated rabbits by 2 weeks postinoculation and was persistently present till the termination of the study; however, results in the HTLV-2∆p28-inoculated rabbits were variable. In four of the six rabbit, proviral sequences were not detected throughout the study (Table 5.1; R6-R9). On the other hand, two of the rabbits showed a weak signal only at week 8, but not at earlier time points. The control rabbits (R12 and R13) were 137 HTLV-2-negative by PCR throughout the duration of the study (Table 5.1).

Collectively, these data indicate that HTLV-2∆p28 was eliminated very early after

infection, and the two rabbits that showed much delayed signal could reflect escape

mutants.

Serological responses were assayed at 2, 4, 6, and 8 weeks post-inoculation. All

wtHTLV-2-inoculated rabbits developed reactivity to specific HTLV-2 antigens at 2-

week intervals throughout the study by western blot analysis (Table 5.2). wtHTLV-2-

inoculated rabbits ranged from mild to strong seropositive for HTLV-2 (positive

reactivity was considered after clear visual band to both Gag and Env antigens).

Rabbits inoculated with HTLV-2∆p28 showed reactivity that correlated with the PCR

data. Four of the rabbits were seronegative (no reactivity to either Gag or Env

antigens), whereas two rabbits were weakly seropositive (weak reactivity to both Gag

and Env antigens) at weeks 4 and 6, respectively. Both rabbits, however, were

seronegative at week 8. Control rabbits failed to seroconvert to any HTLV-2 specific

antigens (Table 5.2).

DISCUSSION

The HTLV-2 p28II accessory protein and its HTLV-1 counterpart p30II are putative repressors of viral gene expression that specifically interact with and retain tax/rex

mRNA in the nucleus 341 leading to less protein production. Tax and Rex are critical

proteins that play essential roles in HTLV replication. Tax is the main transcriptional

regulator of the HTLV promoter present in the 5’LTR that drives expression of all viral

genes 361. Rex is a post-transcriptional regulator that facilitates the cytoplasmic

138 expression of unspliced and incompletely spliced viral mRNAs 130. Thus, by lowering

Tax and Rex expression, p28II and p30II induces repression of viral replication 341 leading to a state of viral latency that is suggested to be critical for viral persistence in vivo.

Lairmore and colleagues have demonstrated that selective mutations of an HTLV-1 clone designed to eliminate p30II expression do not affect in vitro viral infectivity of

HTLV-1 in human PBMC or influence Tax function in transfected cell lines 94,406.

However, this clone was unable to replicate or persist in vivo 126. On the other hand, the accessory proteins of HTLV-2 collectively have also been shown to be dispensable for in vitro replication and immortalization of primary T lymphocytes, but are required for

in vivo replication 343. Moreover, mRNA, serum antibodies and cytotoxic CD8+ T cells

specific for the accessory proteins have been demonstrated in HTLV-infected

individuals 95,97,111,369, indicating that they play a significant role the survival of the

virus in vivo. Given the functional similarity between p28II and p30II, and our recent

data concerning their modular role in viral replication, we aimed at determining the

contribution of p28II to viral replication in vivo. Overall, our data provide the first

direct evidence that p28II-dependent modulation of viral gene expression and replication is critical for infection, spread, and persistence in vivo. Our in vitro transient transfection assays revealed that, as expected, deletion of p28II did not have overt effects on viral Tax activity or p19Gag production. This data corroborate a previous report showing that an HTLV-2∆p28 proviral clone behaved similarly to wtHTLV-2 with respect to infectivity and viral-induced immortalization of human PBMCs in vitro

343.

139 Here we used our reproducible rabbit model that has been successfully used to study

HTLV-1 and HTLV-2 infection, transmission, and persistence, and is established as an appropriate model of the persistent asymptomatic infection in humans 110,407,408. We inoculated rabbits with lethally irradiated wtHTLV-2 and HTLV-2∆p28 cell lines.

Inocula were equilibrated by p19 production. As expected, viral persistence and an antiviral immune response were easily detectable in wtHTLV-2-inoculated rabbits.

Antibodies against major specific viral antigens were detected in these rabbits beginning at 2 weeks post-inoculation which correlated with detectable HTLV-2 sequences in rabbit PBMCs by PCR. However, the response to the HTLV-2∆p28 cell line varied from weak and transient in two rabbits to no response in four rabbits. In contrast to the wtHTLV-2-inoculated rabbits, in HTLV-2∆p28-inoculated rabbits we could amplify a weak HTLV-2-specific signal in two rabbits, but only at week 8.

Regarding the antibody response in these two rabbits, we detected faint bands by western blot at weeks 4 and 6, respectively. However, we were unable to detect HTLV-

2-specific antibody response at week 8 in any of the six HTLV-2∆p28-inocluated rabbits. The transient antibody response and the much delayed detection of viral DNA sequences in rabbit PBMCs suggest that the HTLV-2∆p28 virus was unable to establish infection in an immune competent rabbit. The delayed signal however could be attributed to an escape mutant that either reverted to wild type or accumulated some other mutations which enabled it to persist after a long incubation time.

The implication of this data is that HTLV-2 ORF II, like that of HTLV-1 is absolutely required for successful HTLV-2 survival in vivo. Previous work demonstrated that simultaneous ablation of all accessory proteins of HTLV-2 resulted in 140 reduced proviral loads in our rabbit model 343. That study did not attempt to separate

the in vivo effects of eliminating p28II versus other accessory gene products. This study

provide a direct evidence that p28II does indeed contribute to viral replication in vivo without ruling out that other accessory proteins are also required. The exact mechanism or role of p28II in vivo is still elusive; however, we have recently shown that p28II is a negative post-transcriptional regulator of Tax and Rex gene expression. Thus, it remains to be determined if the in vivo requirement of p28II is dependent on this specific

function or whether p28II, like other retroviral proteins, is pleiotropic and has another

direct role in maintaining viral load and persistence in vivo such as regulating the innate

immune response. A more detailed understanding of the in vivo role of the accessory

protein could open the door for the development of vaccines or a better therapeutic

strategy.

141

Figure 5.1. Schematic diagram of HTLV-2∆p28 and its gene products. The wtHTLV-

2 provirus was used as a template to generate the HTLV-2∆p28 using site directed mutagenesis that introduced a point mutation at position 7333 (asterisk). The mutation changed amino acid #7 (serine) of p28II into a termination codon. The overlapping tax and rex ORFs are not affected by this change. Arrows indicate the position of the primers used for diagnostic PCR.

142

Figure 5.2. Functional activity of Tax and viral replication are not affected by p28II deletion in vitro. (A) Transiently transfected 293T cells were assayed for Tax activity by cotransfection of wtHTLV-2, HTLV-2∆p28, or an empty vector along with the Tax reporter LTR-2-Luciferase. CMV-βgal was used to control for transfection efficiency.

The data shown is representative of three independent experiments and error bars indicate standard deviations. (B) HTLV-2 p19Gag release into the medium was assayed in transiently transfected 293T cells using an ELISA. Data shown is representative of three independent experiments. (C) Supernatants from individual cell clones of 729, 729HTLV-2, and 729HTLV-2∆p28 stable producer cell lines were tested for the expression of p19Gag by ELISA. The cell clones showed here were used for the rest of this study.

143

Table 5.1. Integrated HTLV-2 sequences in rabbit PBLs were detected by PCR using primers 670 and 671. Positive signals were either weak (+) or strong (++). The two negative controls (R12, R13) as well as four of the HTLV-2∆p28-inoculated rabbits

(R6-R9) did not show any signal (-) throughout the study.

144

Table 5.2. HTLV-2-specific antibody responses in the serum of inoculated rabbits were detected by western blot analysis. Positive reactivity to Gag (p24 or p19) and Env (p21 or gp46) antigens was either weak (+) or strong (++) in all HTLV-2-inoculated rabbits.

Two of the HTLV-2Dp28-inoculated rabbits showed transient and very weak (-/+) response to either Gag (p24 or p19) or Env (p21 or gp46) antigens. The negative controls (R12, R13) did not show any response (-) throughout the study.

145

CHAPTER 6

SYNOPSIS AND PERSPECTIVE STUDIES

The human T-cell leukemia virus Type 1 (HTLV-1) and Type 2 (HTLV-2) are pathogenic human retroviruses that have the capacity to transform primary human T lymphocytes in vitro and in vivo. However, the exact mechanisms with which HTLV induces cellular transformation remain elusive. The specific viral determinants in this process are still not clear despite several studies indicating that Tax plays a critical role in this process. More specifically, in vitro coculture assays confirmed that the pleitropic protein Tax is a key player in HTLV-mediated transformation but more studies are needed to dissect the specific activities of Tax that drive this process. In this dissertation, we sought to broaden our knowledge of HTLV both at the molecular level and in vivo with a focus on the pX region. Incorporated as part of Chapter 1, we provide a comprehensive review of the HTLV Rex protein summarizing more than two decades of reports and suggest some future directions that would help better understand the contribution of this protein not only to viral replication, but also to disease progression and the development of novel therapies.

Tax and Rex are the two major trans-regulatory proteins that are essential for HTLV replication 80,190,329. A plethora of data and mutational analyses have identified Tax or

146 Rex domains or regions that are associated with a specific activity among the several

functions of these proteins. For example, mutants that specifically diminish the ability

of Tax to activate transcription without affecting its ability to activate the NFκB

pathway or vise versa have been identified 138,150,156,163,334,347-351. Although some of

these mutants have been identified for several years, our ability to directly study their

effects in the context of a full length replication competent provirus was hindered by the

fact that a significant portion of the tax and rex ORFs is overlapping, making the

analysis of the phenotypes very difficult. In Chapter 2, we describe our novel HTLV-1

molecular clone (H1IT) in which tax and rex ORFs are separated by introducing an

IRES after the Rex termination codon followed by tax cDNA. Our data indicate that

H1IT provirus is able to express enough Tax to induce proper viral replication and more importantly, HTLV-induced transformation of primary human T-lymphocytes. These

properties make H1IT a valuable and unique reagent to study several Tax or Rex

mutants in the context of the full length provirus. Moreover, we took advantage of the

fact that in H1IT, Tax mRNA is deregulated and studied the effect of this phenomenon

on viral persistence in vivo. Our data are in agreement with the hypothesis that

temporal regulation of Tax is important for viral persistence in an immune competent

animal model.

In the future, some of the interesting Tax mutants that would be studied using H1IT

include those that disrupt the ability of Tax to interact with components of the cell cycle

such as p53, CDK4, CDK6, p21WAF1/CIP1, and p16INK4. These mutants are of specific interest since the disruption of the cell cycle may lead to the accumulation of mutations and other forms of DNA damage that can ultimately result in the

147 transformation of cells. The Tax mutants Tax∆7-16 and Tax∆3-6 are of particular

importance because they are unable to interact with p16INK4 and CDK4, respectively.

However, in the context of the virus, they introduce a deletion in the Rex coding region.

Thus, the only way to analyze these mutants is to study them the context of H1IT.

In the natural settings of HTLV infection, Tax and Rex function in the context of the virus which expresses several other proteins that might affect each other. Thus more detailed analysis of the role of other HTLV proteins is critical for our comprehensive understanding of HTLV-mediated pathogenesis. We dedicated three chapters in this dissertation to dissect and analyze the molecular mechanism of action of HTLV-2 p28II and HTLV-1 p30II with respect to their contribution of viral replication in vitro and

survival in vivo. The HTLV-1 and HTLV-2 accessory proteins, encoded by ORF I and

II near the 3’ end of the viral genome, have been shown to be dispensable for in vitro

replication and immortalization of primary T lymphocytes. However, using a rabbit

model of infection, ORF II of HTLV-1 has been shown to be important for viral

persistence in vivo. In Chapter 3, we describe for the first time a detailed molecular

analysis of the HTLV-2 ORF II protein, p28II, which localizes to the nucleus and has

minimal homology to HTLV-1 p30II. Our data show that p28II inhibits HTLV-2 as well

as HTLV-1 replication. More specifically, we show that p28II, like p30II, binds to and

retains tax/rex RNA of HTLV-2 in the nucleus, thereby reducing its level in the

cytoplasm. Given the critical roles of Tax and Rex, dampening their expression by

p28II can suppress viral replication 341 which may lead to a state of viral latency that is

suggested to be critical for viral persistence in vivo.

148 Upon further characterization of p28II in Chapter 4, we identified the mechanism by which p28II is selectively recruited to tax/rex mRNA. Our data indicated a strong

positive correlation between expression from the native HTLV promoter (LTR) and

p28II-mediated inhibition of Tax activity. Replacing the LTR in a proviral clone with a

CMV promoter was sufficient to override the repressive effects of p28II. We also

confirmed that p28II is not a transcriptional regulator, but is recruited co-

transcriptionally to the promoter in a Tax-dependent manner. Using chromatin

immunoprecipitation and coimmunoprecipitation assays, we showed that p28II as well as its HTLV-1 functional homologue p30II, not only associate with the promoter, but

move with the transcription elongation machinery until their response element on the newly transcribed mRNA is generated. Finally, we provided the first evidence that

tax/rex mRNA utilizes the TAP/p15 export pathway. Interestingly, the robust

enhancement of Tax expression in the presence of exogenous TAP/p15 was unable to

override the block on tax/rex mRNA by p28II. Collectively, our findings suggest that

Tax-mediated transcription of HTLV LTR is a process that serves not only to drive the

expression of viral genes, but also to recruit a negative factor that suppresses the

expression of two of the major positive regulators of HTLV expression, leading to a

complicated but tightly regulated feedback loop. Such regulation might hold the key for

latency and evasion of the immune response in vivo.

Some open questions remain regarding p28II and its regulation of viral replication.

First, more studies are needed to decipher the exact mechanism with which p28II

inhibits tax/rex mRNA export. Based on our data in Chapter 4, we suggest an early

block of the export machinery prior to recruitment of TAP/p15. A better understanding

149 of p28II cellular partners will help in shedding some light on this process. Thus, one of

the future directions for this study is to isolate and analyze the composition of

complexes associated with p28II using Mass Spectrometry. Proteins identified in these

complexes under different conditions will not only increase our knowledge of how p28II regulates tax/rex mRNA expression, but may also provide new insights into different functions of p28II. Based on our knowledge of all studied retroviral proteins, we predict

that p28II is pleiotropic. It is also empirical to biochemically map the different domains

on the p28II protein. Lastly, due to the difference between the predicted and the actual

molecular weight of p28II, we suggest that the protein is post-translationally modified.

This begs the question of whether p28II itself is regulated by phosphorylation or other

modifications, and if this regulation is critical for its function. If this is proven to be the

case, then one could hypothesize that p28II acts as a sensor of environmental and

cellular cues. That is, the viral gene expression would be inhibited by p28II only when the later picks up a cellular signal in the form of specific post-translational modification.

Such control could be of paramount importance for viral survival in the host.

One of our future goals with regard to p28II is to expand our preliminary data that

suggest a role of p28II in regulating T-cell survival under different conditions (data not

shown). We generated a lentiviral vector that expressed p28II and used it to infect

Jurkat T-cells. After selection of clones that stably expressed p28II, these cells were

placed under different stress conditions. Interestingly, cells that expressed p28II showed

preferential growth and survival under low serum conditions, a phenomenon that was

minimally observed under normal serum conditions or when Jurkat cells were

stimulated with human IL-2. These data indicate that p28II provide some advantage to

150 infected cells under stress, and more importantly may counteract the apoptotic effects of

Tax. More detailed analysis is needed to confirm this hypothesis.

Another intriguing observation of p28II is its ability to shuttle between the nucleus

and the cytoplasm. Indeed, upon examination of the amino acid sequence of the

protein, we identified a putative leucine-rich nuclear export signal (NES) in the C-

terminal region of p28II. Given role of p28II in binding to and retaining tax/rex mRNA

in the nucleus, it is very interesting to determine whether the NES is required for this

particular activity of p28II. We plan on using site-directed mutagenesis to eliminate the

NES in order to determine its contribution on the established function of p28II. Another

critical experiment is to examine whether p28II is transported to the cytoplasm with or

without its mRNA substrate. Finally, the presence of p28II in the cytoplasm begs the

question of whether it regulates some cytoplasmic processes that might relate to the

virus per se such as downregulating tax/rex mRNA translation, or the host cell such as

blocking apoptosis. Using polysomal loading assays, we will check for the presence of p28II on active polysomes and whether this association, if any, enhances or represses

translation of p28II target RNAs. Moreover, we will directly test the effects of p28II on apoptosis in Jurkat T-cells using Annexin V and flow cytometry. As mentioned above, identification of cellular proteins that interact with p28II will help us identify other

pathways that crosstalk with p28II in the cytoplasm.

Although the accessory proteins of HTLV-2 have been shown to be collectively

dispensable for cellular transformation, specific contribution of select proteins has not

been tested 343. In Chapter 5, we provide evidence that p28II is also dispensable for in

vitro transformation but is absolutely required for viral survival in vivo. It has been

151 reported that selective mutations of an HTLV-1 clone designed to eliminate p30II expression do not affect in vitro viral infectivity of HTLV-1 in human PBMC or influence Tax function in transfected cell lines 94,406. However, this clone was unable to

replicate or persist in vivo 126. Another indication that the accessory proteins play a

significant role in the survival of the virus in vivo is implied from the successful

isolation of their mRNAs, as well as serum antibodies and cytotoxic CD8+ T cells

specific for the accessory proteins from infected individuals 95,97,111,369. In the light of

the above observations we tested whether p28II-dependent modulation of viral gene

expression and replication is critical for infection, spread, and/or persistence in vivo. In

transient transfection assays, deletion of p28II did not have overt effects on viral Tax

activity or p19Gag production which corroborated previous reports showing that an

HTLV-2∆p28 proviral clone behaved similarly to wtHTLV-2 with respect to infectivity

and viral-induced immortalization of human PBMCs in vitro 127,343. We then used our

reproducible rabbit model that has been successfully used to study HTLV-1 and -2

infection, transmission, and persistence and is established as an appropriate model of

the persistent asymptomatic infection in humans 110,127,343,408. As expected, viral

persistence and an antiviral immune response were easily detectable in wtHTLV-2-

inoculated rabbits. Antibodies against major specific viral antigens were detected in

these rabbits beginning at 2 weeks post-inoculation which correlated with detectable

HTLV-2 sequences in rabbit PBMCs by PCR. Interestingly, the response to the HTLV-

2∆p28 cell line varied from weak and transient in two rabbits to no response in four

rabbits. The transient antibody response and the much delayed detection of viral DNA

sequences in rabbit PBMCs suggest that the HTLV-2∆p28 virus was unable to establish

152 infection in an immune competent rabbit. The delayed signal however could be

attributed to an escape mutant that either reverted to wild type or accumulated some

second site mutation(s) that enabled it to persist after a long incubation time. We have recently shown that p28II is a negative post-transcriptional regulator of Tax and Rex gene expression. Thus, it remains to be determined if the in vivo requirement of p28II is dependent on this specific function or whether p28II, like other retroviral proteins, is

pleiotropic and has another direct role in maintaining viral load and persistence in vivo such as regulating the innate immune response. A more detailed understanding of the in vivo role of p28II especially early after infection is needed. Rabbit PBMCs infected

with wtHTLV-2 or HTLV-2∆p28 could be checked for proviral load, and viral transcripts could be detected using real-time RT-PCR to determine if the absence of p28II disrupted the balance of the different viral mRNAs.

Thorough investigations of these future directions have implications on several

fronts. First and foremost, they will expand our understanding of basic retroviral

biology which, thus far, has proven to be critical in understanding the biology of the

host cell. Second, HTLV-associated malignancies remain to be a significant problem in

endemic regions. Thus, the need to develop a vaccine is urgent. Understanding the

exact role of the accessory proteins, how they regulate viral replication, and how they

themselves are regulated might prove to be critical for the development of vaccines or

novel therapeutic strategies.

153

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