HUMAN T LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1) ACCESSORY
PROTEIN P30(II) MODULATES CELLULAR AND VIRAL GENE EXPRESSION
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
by
BINDHU MICHAEL, B.V.Sc. & A.H., M. Sc., M.S.
* * * * *
The Ohio State University
2004
Dissertation Committee: Approved by Professor Michael Lairmore, advisor
Professor Patrick Green
Professor Michael Oglesbee ______Adviser Professor Stefan Niewiesk Veterinary Biosciences Graduate Program ABSTRACT
Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes
adult T-cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated disorders. In addition to structural and enzymatic proteins, the HTLV-1 provirus encodes
various regulatory and accessory genes in four pX open reading frames. Our laboratory
has elucidated the functional characteristics of the HTLV-1 accessory proteins, and it has
become clear that these proteins are, in fact, essential for the virus life cycle and may
determine disease outcome associated with HTLV-1 infection. We have identified the
functional properties of the pX ORF-II encoded p30II, but the role of this viral protein in
virus replication or pathogenesis remain incompletely defined.
Studies from our laboratory have demonstrated that pX ORF-II mutations
diminish the ability of the virus to maintain high viral loads in vivo in our rabbit model of
HTLV-1 infection. The ORF-II encoded p30II, a nuclear-localizing protein that interacts
with the KIX domain of CREB-binding protein (CBP)/p300, disrupts CREB-Tax-
CBP/p300 complexes bound to viral Tax-Responsive Element (TRE) repeats and
differentially modulates CREB and TRE-mediated transcription. Herein, we have further
characterized the role of p30II in regulation of cellular gene expression, using a stable
p30II expression system employing lentiviral vectors to test cellular gene
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expression with Affymetrix U133A arrays, representing ~33,000 human genes. Our data reveals alterations of interrelated pathways of cell proliferation, T-cell signaling,
apoptosis and cell cycle in p30II expressing Jurkat T-lymphocytes. Moreover, we have
verified our findings by reporter assays and RT-PCR in Jurkat and primary CD4+ T-
lymphocytes. We are the first to test the overall effect of an HTLV-1 accessory protein,
on cellular gene expression and demonstrate that p30II activates many key transcription
factors involved in T-cell signaling/activation, such as nuclear factor of activated T-cells,
nuclear factor-kappa B and activator protein-1. Collectively, these data indicate that this
complex retrovirus, associated with lymphoproliferative diseases, rely upon accessory
gene products to modify cellular environment to enhance clonal expansion of the virus
genome and thus maintain proviral loads in vivo.
Next, we further characterized the role of p30II in regulation of viral gene
expression, by identifying motifs critical in binding p300 and regulating TRE-mediated
transcription in the absence or presence of provirus. Analysis of the amino acid domain
of p30II (100-179) critical for repressing LTR mediated transcription, was used to identify
a lysine residue at amino acid 106 (K3) of p30II, that is critical for repressing TRE- mediated transcription. Additionally, we have found that p300 reverses the p30II- dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, in a dose-responsive manner. Our data confirms the role of p30II as a regulator
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of viral gene transcription, in association with p300. However, in contrast to wildtype
p300, p300 histone acetyl transferase (HAT) mutants only partially rescue p30II-mediated
LTR repression. In addition, we also show that p30II is acetylated and that deacetylation enhances p30II-mediated HTLV-1 LTR repression. Inhibition of deacetylation decreased
p30II-mediated LTR repression. We are the first to demonstrate that the HAT activity of
p300 is crucial in modulating viral gene expression from HTLV-1 LTR by p30II. Overall, our data indicates that HTLV-1, a complex retrovirus associated with lymphoproliferative disorders, uses accessory genes to modulate viral gene expression in conjunction with host cell proteins in part, through the acetylation of p30II. Collectively, our data suggests
that HTLV-1 enhances clonal expansion of the virus genome by regulating viral and
cellular gene expression, avoids immune elimination by the host and thereby maintains proviral loads in vivo.
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Dedicated to my wonderful parents, siblings and husband who gave me the freedom to be
who I want to be and were there for me always, no matter what, and
Vavlo who reminds me of the little joys in everyday life
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ACKNOWLEDGMENTS
First and foremost, I would like to express my sincere gratitude and appreciation to my thesis advisor, Dr. Michael Lairmore for his valuable guidance, motivation, support and leadership. As stated in Dr. Lairmore’s recent University Distinguished Scholar
Award biography, he is truly a mentor who invests a great deal of himself in his students and believes it is through their success that he is best reflected. With his exceptional
mentoring skills, he always keeps his students’ best interest in his mind and trains us to
be independent investigators. Moreover, he was gracious enough to support me through my Pathology Training. In spite of his busy schedule, he is committed to meeting with every student frequently and providing constant support and guidance. I am fortunate to have the opportunity to learn from Dr. Lairmore, who successfully handles every aspect of his teaching, research, pathology services and administrative responsibilities.
I would like to thank my committee members Dr. Patrick Green, Dr. Michael
Oglesbee and Dr. Stefan Niewiesk for their helpful ideas and suggestions. I am also thankful to Dr. Larry Mathes and Dr. Kathleen Boris-Lawrie for their helpful discussions
in the Retrovirus Group lab meetings. I would also like to acknowledge Dr. Louis
Mansky who was a member of my committee during my candidacy examination and Dr.
Weisbrode who has been a great teacher during my Pathology residency training.
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I was fortunate to be a part of Dr. Lairmore’s research group and would like to
thank all my colleagues, both past and present. I extend my sincere appreciation to Antara
Datta, Hajime Hiraragi and Wei Ding for their help me with some of the experiments discussed in this dissertation, valuable scientific discussions and for their friendship. I also thank Chris Premandandan, Seungjae Kim, Rashade Haynes, Andy Montgomery,
Lee Silverman, Andrew Phipps, John Nisbet, Jessica Alcorn and Laurie Millward for their helpful ideas, productive scientific discussions and great times shared together. I appreciate the effort made by Cynthia Sagel, Jennifer Huck, Stacey Dietrich, Susie Vogel and Allison Blanton in the maintenance of the laboratory, equipment and reagents. I must also thank members of the Boris-Lawrie, Green and Mathes laboratories for their discussions, reagents, and friendship.
I am previlaged to have had many mentors who taught me that the sky is the limit and to not be afraid to pursue my dreams and ambitions: right from Ashan who taught me the alphabets, Sr. Rosia and other teachers who invested a great deal of themselves for me, Dr. Francis Xavier and Dr. Nanu E who supported me immensely while pursuing my ambitions. I have also been lucky to have some great friends like Sangeetha and Sajan who believed in me. Special thanks to Desi friends, David Joseph, Tanya, Stan, Jordan,
Rachelmol, Thomas and George who made my stay in Columbus enjoyable.
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I am blessed with a wonderful and loving family. My parents Michael and
Kuttiyamma have always been a source of motivation, inspiration and support throughout
my life. In a small town where women’s higher education particularly in a foreign
country, was not supported much, what my parents have done needs special appreciation.
Moreover, they have always been there even when I did not listen to them and was faced
with difficult circumstances in life. My sister Rani and brother Bibin have always
believed in me and have been very supportive, sincere and loving. I also thank my
cousins Baiju, Asha, Jose and Bitto who have been loving and supportive to my family. I
could not have completed my PhD and training, without the unconditional love and support of my husband, friend and colleague Amrithraj, who have always been very
encouraging and understanding. Last but not least, special thanks to Vavlo, who reminds
me of the little joys and fun of everyday life.
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VITA
Birthday...... Born – 12th August, 1974
1989-91 ...... Pre-Degree- Biology, Chemistry, Physics Mahathma Gandhi University, Kottayam, Kerala, India
1991-97 ...... BVSc and AH (DVM equivalent) Kerala Agricultural University, India
1997-1998 ...... Research Associate Department of Veterinary Public Health Kerala Agricultural University, India
1998-99…………………………………….. MSc (Wild animal Health) University of London, UK
1999-2001………………………………….. Graduate Teaching Assistant Department of Microbiology The Ohio State University
2001-present………………………………...Graduate Research Associate, Department of Veterinary Biosciences The Ohio State University
PUBLICATIONS
Research Publications
1. Ding, W., Kim, S., Nair, A., Michael, B., Boris-lawrie, K., Tripp, A., Feuer G and Lairmore, M. Human T-Lymphotropic Virus Type-1 p12I Enhances Interleukin-2 Production during T Cell Activation. Journal of Virology. 77: 11027-11039, 2003
2. Michael, B., Smith, J. N., Swift, S., Heffron, F. and Ahmer, B. M. SdiA of Salmonella enterica is a LuxR Homolog that Detects Mixed Microbial Communities. Journal of Bacteriology.183: 5733-42, 2001.
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3. Francis Xavier, Gigi K. Joseph and Bindhu Michael. A Coprological Survey of Parasites of Seven Mammal Groups at Silent Valley National Park, Kerala, Zoo’s Print. 15, 2000
4. Amrithraj, M., Bindhu Michael and Madhavan Pillai, K. Artyfechinostomum malayanum (Leiper, 1911) Mendheim, 1943 from a Small Indian Civet (Viverricula indica). Zoo's Print. 14: 6, 1999
5. Bindhu Michael, Gigi. K. Joseph and Francis Xavier. Comparison of Morphometric Indices of Large and Small Flying Squirrels. Zoo’s Print. 13: 46-47, 1998.
6. Vijayan, R., Devada, K., Balakrishnan, V.S., Bindhu Michael and Aleyas, N. M. Ehrlichiosis in Dog - a Case Report. Journal of Veterinary and Animal Sciences. 28: 101-103, 1997.
7. Bindhu Michael, M. Amrithraj and K. Madhavan Pillai. A Note on Isospora Infection in a Southern Red Whiskered Bulbul (Pycnonotus jocosus fascicaudatus). Zoo’s Print. 12: 5, 1997.
8. Bindhu Michael, Francis Xavier and Leo Joseph. Anomalous Animal Behaviour Preceding Earthquake. Zoo’s Print. 12: 30-31, 1997.
9. Bindhu Michael, Amrithraj M., Sajan George and P. J. Rajkamal. Case Study of a Specialised Buffalo Farmer in the Foot-hills of Western Ghats. Dairy Guide. 18: 13- 14, 1997.
10. Bindhu Michael, Gigi. K. Joseph and Francis Xavier. Morphometry and Feeding Habit of Small Travancore Flying Squirrel. Zoo’s Print. 11: 4-5, 1996.
Review Publications
1. Bindhu Michael, Amrithraj M. Nair and Michael D. Lairmore. Role of Accessory Proteins of HTLV-1 in Viral Replication, T Cell Activation, and Cellular Gene Expression. Frontiers in Bioscience. 9: 2556-2576, 2004.
2. M. Lairmore, Michael, B., Silverman. L and Nair. A. Human T-Lymphotropic Virus-Associated Neurological Disorders: Pathogenesis, Immunology and Clinical Management. Accepted as Chapter in textbook “Inflammatory disorders of the nervous system”
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Previous Thesis / Dissertations
Detection of Heterologous Bacterial Pheromones by SdiA, a LuxR Homolog in Salmonella enterica serovar typhimurium: Master’s Dissertation, The Ohio State University, Columbus, Ohio, 2001 Identification of Type C Retrovirus and Simian T-Lymphotropic Virus from a Brown Headed Spider Monkey (Ateles fusciceps robustus). Master’s Dissertation, The Royal Veterinary College, University of London, London, UK, 1999
FIELDS OF STUDY
Major Field: Veterinary Biosciences.
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TABLE OF CONTENTS
Page Abstract...... ii
Dedication ...... v
Acknowledgments...... vi
Vita...... ix
List of Tables ...... xv
List of Figures...... xvi
Chapters:
1. Literature Review: Molecular mechanisms of HTLV-1 infection, LTR mediated gene expression and role of accessory proteins in HTLV-1 pathogenesis.
1.1 First Recognition of Human T-lymphotropic virus (HTLV-1) as Etiologic Agent of Human Disease ...... 1 1.2 Geographical Distribution of HTLV-1 ...... 2 1.3 Transmission of HTLV-1...... 3 1.4 HTLV-1 Associated Diseases...... 4
1.4.1 Adult T cell Leukemia/Lymphoma...... 4 1.4.2 Tropical spastic paraparesis/HTLV-1-associated myelopathy ..... 8 1.4.3 Other diseases associated with HTLV-1-infection ...... 10
1.5 Genomic Organization and Replication of HTLV...... 12 1.6 Structural Proteins of HTLV-1 ...... 16 1.7 Regulatory Proteins of HTLV-1: Tax and Rex...... 18
1.7.1 Tax and Transactivation of the HTLV-1 LTR ...... 18 1.7.2 Tax and Regulation of Cellular Gene Expression...... 19 1.7.3 HTLV-1 Rex: Post-transcriptional Regulator...... 21
1.8 HTLV-I accessory proteins...... 24 xii
1.8.1 pX ORF I p12I...... 26
1.8.1.1 Biochemical Characteristics of p12I: Features of a Signaling Molecule ...... 26 1.8.1.2 Subcellular Localization of p12I and Interactions with Cellular Proteins...... 27 1.8.1.3 Role of p12I in Regulation of Viral Infectivity In vitro and In vivo...... 30 1.8.1.4 Role of p12I in Calcium-Mediated T Cell Activation..... 31 1.8.1.5 Putative Role of p12I Variants in HAM/TSP...... 36
1.8.2 pX ORF II p13II ...... 37
1.8.2.1 Biochemical Characteristics of p13II: Mitochondrial Targeting...... 37 1.8.2.2 p13II Alteration of Mitochondrial Morphology and Inner Membrane Potential...... 37 1.8.2.3 p13II Cellular Protein Interactions ...... 39
1.8.3 pX ORF II p30II ...... 40
1.8.3.1 Biochemical Characteristics of p30II: Features of a Transcription modulator...... 41 1.8.3.2 Role of p30II in Modulating Viral and Cellular Gene Expression...... 42 1.8.3.3 HTLV-1 Mediated T Cell Activation /Transformation and the Role of p30II ...... 45
1.8.4 References...... 48
2. Human T Lymphotropic Virus Type-1 P30II Influences Cellular Gene Expression and Enhances Transcription Activation of T Lymphocytes
2.1 Introduction...... 87 2.2 Materials and Methods...... 90 2.3 Results...... 96 2.4 Discussion...... 101 2.5 References...... 107
3. Motifs of Human T Lymphotropic Virus Type 1 P30II critical for its Interaction with p300 and Regulation of LTR Transcriptional Activity. xiii
3.1 Introduction...... 121 3.2 Materials and Methods...... 125 3.3 Results...... 129 3.4 Discussion...... 137 3.5 References...... 144
4. Histone Acetyltransferase (HAT) Activity of p300 Modulates Human T Lymphotropic Virus Type 1 P30II-mediated Repression of LTR Transcriptional Activity.
4.1 Introduction...... 158 4.2 Materials and Methods...... 162 4.3 Results...... 165 4.4 Discussion...... 171 4.5 References...... 178
5. Synopsis and future studies
5.1 Role of p30II in T cell signaling / activation and transformation. 191 5.2 Identify the mechanism by which p30II activates NF-κB, NFAT and AP-1 mediated transcription ...... 192 5.3 Determine if p30II has an effect on IL-2 production and lymphocyte proliferation...... 194 5.4 Does p30II affect cell cycle progression in T lymphocytes?...... 195 5.5 Does p30II affect cell-to-cell adhesion between T lymphocytes? 196 5.6 Determine the significance of critical functional domains and specific amino acids within p30II, in vivo ...... 197 5.7 Does HTLV-1 p30II form complexes at the HTLV-1 promoter, to modulate LTR mediated transcription? ...... 198 5.8 Identify the functional domains and specific amino acids within p30II, which are critical in nuclear retention of tax/rex mRNA and protein-protein interactions...... 199 5.9 Temporal expression pattern and interaction between HTLV-1 regulatory and accessory proteins during various stages of the HTLV-1 infection ...... 200 5.10 References...... 202
Bibliography ...... 207
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LIST OF TABLES
Table Page
2.1 Genes modulated by HTLV-1 p30II ...... 113
3.1 Lysine residues in p30II are conserved among various HTLV isolates...... 149
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LIST OF FIGURES
Figure Page
1.1 Diagram of HTLV-1 genome and alternatively spliced mRNA and protein species from pX ORFs...... 85
1.2 Diagram of p12I, p30II, and p13II with predicted functional motifs…...... 86
2.1 Schematic illustration of lentiviral vectors expressing both p30IIHA and GFP (sample vector) as bicistronic messages and GFP alone (control vector) from elongation factor 1 alpha promoter...... 117
2.2 Triplicate p30II samples express GFP and p30II while triplicate controls express only GFP ...... 118
2.3 Semiquantitative RT-PCR of CHP, JUN and NFATc in controls and p30II expressing Jurkat T lymphocytes and primary CD4+ T lymphocyte samples...... 119
2.4 p30II activates NFAT, AP-1 and NF-κB transcriptional activity in Jurkat T lymphocytes ...... 120
3.1 p30II localizes to the nucleus and colocalizes with p300 in the nucleus of transiently transfected HeLa-Tat cells ...... 150
3.2 Amino acids 1-132 of HTLV-1 p30II are critical for its physical interaction with p300 ...... 151
3.3 Subcellular localization characteristics of various p30II serial deletion mutants p30II in transiently transfected HeLa-Tat cells...... 152
3.4 HTLV-1 p30II differentially modulates LTR-mediated transcription in the absence or the presence of the provirus...... 153
3.5 p300 expression reverses the p30II-dependent repression of LTR driven transcription, in the absence or the presence of the provirus...... 154
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3.6 HTLV-1 p30II and Tax appear to compete with each other in modulating the LTR mediated transcription...... 155
3.7 Amino acids 100-179 of p30II is critical for its repression of LTR driven transcription, in the absence or the presence of the provirus ...... 156
3.8 Lysine 106 (K3) of p30II is critical for its repression of LTR driven transcription in the absence or the presence of the provirus...... 157
4.1 p300 HAT mutants only partially rescue p30II-dependent repression of LTR driven transcription ...... 183
4.2 Addition of HDAC-1 enhances p30II-dependent repression of HTLV-1 LTR driven transcription ...... 184
4.3 Addition of 100 nM Trichostatin A (TSA) causes a decrease in the enhancement of p30II-dependent repression of HTLV-1 LTR driven transcription by HDAC-1...... 185
4.4 Addition of Trichostatin A causes a decrease in the enhancement of p30II- dependent repression of HTLV-1 LTR driven transcription by HDAC-1 ...... 186
4.5 Expression of P/CAF does not rescue p30II-dependent repression of HTLV-1 LTR driven transcription...... 187
4.6 HTLV-1 p30II appears to be acetylated by the transcriptional co-activator p300 at lysine 106 (K3)...... 188
4.7 Schematic model diagram of the potential mechanism by which HTLV-1 p30II modulates LTR-driven transcription...... 189
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CHAPTER 1
LITERATURE REVIEW: MOLECULAR MECHANISMS OF HTLV-1
INFECTION, LTR-MEDIATED GENE EXPRESSION AND ROLE OF
ACCESSORY PROTEINS IN HTLV-1 PATHOGENESIS
1.1 First Recognition of Human T-Lymphotropic Virus Type 1 (HTLV-1) as
Etiologic Agent of Human Disease
Takatsuki et al 334reported a unique and aggressive CD4+ T cell malignancy called
adult T cell leukemia (ATL) in a small geographically clustered group of patients with
lymphoid neoplasms in Japan In 1977 245,334,343. Human T-lymphotropic virus type 1 was
first detected and isolated as a type C retrovirus particle from two T cell lymphoblastoid cell lines, HUT 102 and CTCL-3 and fresh peripheral blood lymphocytes of a patient in the United States with cutaneous T cell lymphoma (mycosis fungoides) by Poisez, Gallo and their colleagues in 1980 293. This patient was diagnosed with cutaneous T cell
lymphoma; however he had widespread systemic involvement typical of ATL and is now
thought to have been possibly misdiagnosed and was in fact suffering from ATL. In
1981, Hinuma et al 123demonstrated the presence of type C retroviral particles termed 1 adult T cell leukemia virus (ATLV) from a cell line termed MT-1, which was established
from a patient diagnosed with ATL 123,378. About the same time, Miyoshi et al
234,236,237established another T cell line, MT-2, from an ATL patient which was later
found to have type C retrovirus particles 377. Further sero-epidemiologic, immunologic,
genetic and molecular studies showed that the viruses in these two cell lines were the
same and helped to establish the close linkage between ATL and HTLV-1. Thus HTLV-
1 was established as the first retrovirus associated with a human disease122,377,378.
1.2 Geographical Distribution of HTLV-1
Although it is estimated that HTLV-1 infects 15 to 25 five million people worldwide, it is not uniform in its distribution103, it is in fact found in geographical
clusters208. HTLV-1 infection is endemic in southern Japan 122, the Caribbean basin 29,
Central and South America 229,298, among some Melanesian Islands in the Pacific basin 13 and in regions of central Africa 308. It is also a serious public health problem among at-
risk groups in Europe and North America and therefore a target of intervention policies
including blood donor screening57,80,84,250,252,253,310. HTLV-1 infection is also prevalent in
regions such as southern India, northern Iran and northern Australia103. The seroprevalence of HTLV-1 varies widely within endemic regions, between 0.1% and 30
% of the population, depending on age and gender103.
Recent studies indicate the presence of HTLV-1 sequences in a 1,500-year-old
Andean mummy, indicating the ancient nature of HTLV-1 infections200. Primate T
lymphotropic viruses are thought to have originated in Eastern Africa and spread 2 worldwide346. It is hypothesized that an early African lineage of the simian T
lymphotropic virus (STLV) evolved into STLV-1 in Asia and subsequently infected
humans prior to their migration to Melanesia/Australia, resulting in the origin of
"Melanesian" clad or subtype HTLV-1c, one of the 4 major widely-recognized clads of
HTLV-1 127,171,204,322. The Asian STLV-1 strains possibly re-entered Africa at a much
later stage, and multiple interspecies transmissions resulted in the establishment of the
second clad of HTLV-1, the African clad, which can be further subdivided into five sub-
clads (HTLV-1a, -b, -d, -e and –f) 347. The third clad, referred to as the "Cosmopolitan"
clad is considered to have originated from HTLV-1a, which spread throughout the world
due to the increased human host mobility, particularly during the slave trade 102,204.
Recent reports indicate the presence of subtype D in Central Africa, mainly in
Pygmies212. In addition, two other African HTLV-1 variants from Zaire (Efe1) and Gabon
(Lib2) have been discovered and may be yet considered as the sole human prototypes of
new subtypes (E and F) 344.
1.3 Transmission of HTLV-1
HTLV-1 is a highly cell-associated virus and free HTLV-1 particles have low
infectivity. Cell-cell contact through virus-infected T cells is required for efficient
transmission 373. The most efficient route of HTLV-1 transmission is by exposure to
infected blood or whole cell blood products, but not plasma (e.g., transfusion or through
145 the use of contaminated needles), with a seroconversion rate of approximately 50 %
and an estimated median time for seroconversion of 51 days 221. In fact, sharing of
3 needles among intravenous drug users constitutes one of the most common modes of blood-to-blood transmission among endemic population in the United States 153,154.
Transmission by infected blood products has also been a major problem in endemic areas, particularly in Japan 221,268. Nonetheless, most cases of HTLV-1 infections are due to
transmission from mother to child through prolonged breastfeeding for more than 6
months (through infected milk-borne lymphocytes) or by sexual contact later in life
120,121. The probability of mother-to-infant transmission is estimated to be 18–30% and
the risk factors involved are maternal factors, including higher HTLV-1 antibody titer,
prolonged ruptured membranes during delivery, and low socioeconomic status 358. The sexual route of transmission is less efficient than transmission involving blood, but has the potential to introduce infection into previously unexposed groups. Transmission from male to female through sexual contact is about four times as frequent as female to male transmission, at a rate of 4.9 per 100 person-years among females married to an infected male compared with 1.2 among males married to an infected female323. Transmission
risk to females is believed to be greater if the partner has high antibody titers or antibody
to Tax proteins while the risk of female-to-male transmission is associated with penile sores/ulcers and diagnosis of syphilis among males 251.
1.4 HTLV-1-Associated Diseases
1.4.1 Adult T cell Leukemia/Lymphoma
Typically, 1-5 % of the HTLV-1 infected individuals develop ATL after a latent
period of 20-30 years 50,371,372. ATL is an aggressive T cell malignancy with a leukemic
phase characterized by circulating, activated CD4+/CD25+ T cells343. Infection early in 4 life is associated with the development of ATL and the estimated lifetime risk is about 5
% in individuals infected before the age of 20 years 50,359. The incidence rate is 2–4 per
100,000 person-years and males have higher risk than females 50,170. The average age of
onset is 40 years in Jamaica, Trinidad, and Brazil and 60 years in Japan 50,295.
The clinical features of ATL are similar to non-Hodgkin’s lymphoma: malaise, fever, lymphoadenopathy, hepatosplenomegaly, jaundice, drowsiness, weight loss, and opportunistic infections. Typically widespread or localized skin involvement (in 40% of cases, there are large nodules, plaques, ulcers, and papular rash on the limbs, trunk, or face) and lytic bone lesions are noticed in these patients. Immunosuppression with bacterial and opportunistic infections such as Pneumocystis carinii pneumonia, fungal
infections, and strongyloidiasis has been reported in these patients, contributing to poor
prognosis 357. Unique clinical chemistry characteristics of this condition include
hypercalcemia, high serum concentrations of lactate dehydrogenase and elevated serum
levels of soluble interleukin-2 (IL-2) receptor α chain. Circulating ATL lymphocytes are
morphologically unique abnormal T cells with characteristic convoluted multi-lobulated
nuclei, which are defined as “flower cells” 317. Typical criteria to establish a diagnosis of
ATL in patients include seropositivity for HTLV-1, marked leukocytosis, characteristic
morphologic appearance of ATL cells, histology or cytology of malignant cells with a T
cell immunophenotype, usually CD4+/CD3+, hypercalcemia and increased circulating
levels of the IL-2 receptor α-chain (IL-2Rα/CD25) and elevated serum lactate
dehydrogenase (LDL) levels317. Diagnostic tools for asymptomatic carriers include an immunoassay to detect HTLV-1 antibodies and polymerase chain reaction (PCR)-based
5 amplification of lymphocyte genomic DNA to detect provirus 353. Based on restriction fragment length polymorphism (RFLP) analysis, the proviral integration into host cells is believed to be polyclonal, but the RFLP analysis of leukemic cells isolated from ATL patients show a monoclonal proviral integration pattern 353,378, suggesting that a sub- population of infected cells are clonally selected to proliferate while rest of the infected lymphocytes either die or get eliminated.
Based on clinical and laboratory criteria, ATL is classified into four subtypes: acute, lymphoma, chronic or smoldering 151,317,370. Distribution of these subtypes vary depending on the geographic location 317, 115 and the median survival for ATL patients is
4-6 months depending on the subtype of ATL220. While the acute subtype has the highest incidence and is characterized by increased ATL cells, skin lesions, systemic lymphoadenopathy, hepatosplenomegaly and poor prognosis, particularly with high level of serum LDH, calcium and bilirubin 369,369,371, the chronic subtype ATL is characterized by increased white blood cells counts, cough and skin lesions. Smouldering subtype
ATL has the best prognosis among the four subtypes and is reported to have few ATL cells in the peripheral blood over a long time period.
The development of ATL has been the focus of many investigations, but the exact mechanism is not completely understood. After infecting CD4+CD25+ T cells, the
HTLV-1 provirus is randomly integrated into the host genome, where it persists for years.
Although HTLV-1 replicates via reverse transcription in vivo, HTLV-1 replication is thought to occur via mitosis rather than via reverse transcription242. In ATL, tumor cell
6 clones were identified against a general background of oligo to polyclonal expansion of
infected but non-transformed cells40. Typically there is a progression from polyclonal to
oligoclonal and then to monoclonal proliferation in vivo, which is achieved while the
cells become IL-2 independent 85,125,378. Based on the long clinical latency and the low
percentage of individuals who develop ATL, T cell transformation is believed to occur
after a series of cellular alterations and/or mutations. Transformation of infected
lymphocytes is believed to be initiated through induction of cellular genes and alterations in critical cellular activation and death pathways by the viral transactivator Tax 97.
Although the mechanism of cellular transformation by Tax is not fully understood, it is thought to involve dysregulation of cell cycle and suppression of DNA repair pathways, leading to the gradual accumulation of mutations over time and development of ATL.
However, transformed cells often contain latent or defective proviruses 87,88,175, indicating
the possibility that viral protein expression may not be essential to maintain the
transformed phenotype.
Intriguingly, despite a strong immune response mounted against the HTLV-1, the
virus is able to persist in the host. Several possible mechanisms have been suggested to
explain this observation: Often the genome is partly deleted, resulting in defective virus
which may provide a mechanism for escape from immune surveillance 18-20. Recent
reports 14 suggest that HTLV-1 may not be latent and the virus might be far more dynamic than previously thought. Based on in vitro studies on the half-life of Tax-
expressing cells, it is hypothesized that infected cells start expressing viral proteins as
early as within one day after infection and might have a turn over rate of ~ 109 to 1011
7 infected cells per day. Cytotoxic T lymphocytes (CTLs) are thought to be highly efficient in significantly reducing the viral load in vitro and possibly in vivo, resulting in equilibrium between the rate of production and the rate of clearance of cells expressing viral proteins. Based on this proposed mechanism, cell-free virus, viral mRNA and viral protein may be often below detection limits and infectious transmission is limited, explaining the relative lack of sequence diversity 14.
1.4.2 Tropical Spastic Paraparesis / HTLV-1-Associated Myelopathy (TSP/HAM)
In the mid-1980's HTLV-1 began to be associated through epidemiologic studies as a contributing factor in chronic neurodegenerative disorders. After detecting anti-
HTLV antibodies in the serum and cerebrospinal fluid (CSF) of infected patients, HTLV-
1 was associated with the development of a progressive neurological disease called
tropical spastic paraparesis (TSP) in Martinique (French West Indies) in 1985. In 1986,
Osame et al reported similar clinical findings in HTLV-1-positive individuals in Japan
and they named the syndrome HTLV-1-associated myelopathy (HAM) 101,278.
Subsequently, in 1988, both syndromes were understood to be identical in clinical presentation and the World Health Organization (WHO) declared HAM and TSP as the same disease, now known collectively as HAM/TSP.
HAM/TSP is characterized as a progressive chronic myelopathy, with preferential damage of the thoracic spinal cord 2,95,149,167,253. In affected patients the myelopathy
presents as paraparesis with spasticity in the lower extremities, hypereflexia, muscle
8 weakness, sphincter disorders including dysfunction of the urinary bladder and intestines.
Less frequently it may precede, or give rise to, a cerebellar syndrome with ataxia and intention tremor 39. Some HAM/TSP patients exhibit a predominance of sympathetic
nervous system dysfunction 6. Magnetic resonance imaging (MRI) of HAM/TSP patients
reveals the presence of multiple white matter lesions in both the spinal cord and the brain
involving perivascular demyelination and axonal degeneration 93,104,162,163.
Within the spinal cord, regions mainly affected are the so-called ‘watershed’
zones, suggesting a nonrandom distribution 132. Lesions include degeneration of lateral
corticospinal tract and spinocerebellar or spinothalamic tract of the lateral column as well
as perivascular and parenchymal cellular infiltration, consisting of mainly T cells (both
CD4+ and CD8+ T cells), macrophages, astrocytes, and glial cells 276. Typically, cellular
destruction and inflammation is a feature of HAM/TSP. CD4+ T cells, CD8+ T cells,
and activated macrophages are present within white matter lesions of HAM/ TSP patients133. Peripheral blood and the CSF of HAM/TSP patients have high levels of
proinflammatory cytokines, such as IFN-γ, TNF-α, IL-1 and IL-6, suggesting the
importance of these proinflammatory cytokines in HAM/TSP15,150,179,227. Additionally,
CSF of these patients contains activated lymphocytes, suggesting that activated
lymphocytes and macrophages also play a role in the development of HAM/TSP133.
The lifetime risk of developing HAM/TSP among carriers is estimated to be
0.23%, based on multiple risk factors 148. The progression to HAM/TSP is influenced by
multiple risk factors such as host genetic factors254, host immune response, high HTLV-1
9 proviral load 19,20,166,264,374, route of infection (exposure via blood transfusion) 277 and perhaps by specific viral characteristics such as variations in HTLV-1 tax19,65,82,106,212,306.
HAM/TSP patients develop a vigorous expansion of CD8+ T cells 307 and their
CSF contain high levels of anti-HTLV-1 antibodies 100 and HTLV-1 Tax specific
CTLs176. These CTLs are implicated in the cellular destruction and inflammation within
the central nervous system (CNS) 176, probably by directly killing of HTLV-1 infected
cells expressing Tax, CNS cells expressing a cross-reactive cellular determinant and
neighboring uninfected neuronal cells by apoptosis mediated via the toxic inflammatory
cytokines 21. Although the pathogenesis of HAM/TSP is not completely resolved,
immune mechanism is widely accepted due to the increased cellular and humoral immune
responses in HAM/TSP patients. Autoimmune mechanism has also been proposed due to
the presence of a unique T cell receptor CDR3 motif in infiltrating lymphocytes in the
spinal cord lesions of HAM/TSP patients, as in brain lesions of MS and experimental
autoimmune encephalomyelitis patients116. Moreover, antibodies to hnRNP-A1 may
cross-react with HTLV-1-Tax, during the immune response associated with HAM/TSP,
leading to a recent theory that molecular mimicry between HTLV-1 and autoantigens in
CNS play a role in the pathogenesis of HAM/TSP 195,196.
1.4.3 Other Diseases Associated with HTLV-1-Infection
HTLV-1 infection has been linked to many other immune-mediated diseases,
although some of these remain controversial. A high incidence of idiopathic uvetis with a
high rate of anti-HTLV-1 antibodies in HTLV-1 endemic areas of Kyushu, Japan led to
the speculation that HTLV-1 might be the etiological agent 165,239. Proviral DNA was 10 detected in cells from the patients’ aqueous humor. Moreover, peripheral blood
leukocytes of these patients had higher proviral loads than HAM/TSP patients 269.
Interestingly, proviral loads showed significant positive correlations with the severity of disease among uveitis patients with a previous history of Grave's disease 269.
Immunological and virological studies further confirmed these findings 270,270,305,305.
Moreover, all patients with HTLV-1-associated uveitis had hyperthyroidism preceding the onset of uveitis, implying a potential role for hyperthyroidism in the onset of HTLV-
1-associated uveitis 238,280.
In 1990, HTLV-1 was associated with infective dermatitis, characterized by
watery nasal discharge with crusting of the anterior nares and severe exudative dermatitis
in scalp, neck, ears, axillae and groin183. Later, these infective dermatitis patients were
confirmed to be seropositive for HTLV-1, however the pathogenesis is not yet
understood 182,183,185. Interestingly, the presence of infective dermatitis in children may
predispose them to subsequent development of ATL or TSP/ HAM 184.
HTLV-1 has also been associated with cutaneous T cell lymphoma including
mycosis fungoides and Sezary syndrome, supported by the detection of HTLV-1 DNA in a minority of cutaneous T cell lymphoma patients155,213. Although cutaneous T cell lymphoma is clinically and histologically similar to ATL, its association with HTLV-1 is debated, due to the failure to detect HTLV-1 proviral DNA in patients199,362,363, HTLV-1
seronegative nature in most of these patients114,213,281 and uncertainty on whether HTLV-
1 specific sequences in these patients are intact, full-length proviral genomes or non-
functional deleted proviruses199,362,363.
11 HTLV-1 has been linked to Sjögren’s syndrome based on epidemiological evidence in southwest Japan, a high endemic region of HTLV-1 infection. Sjögren's syndrome is a chronic inflammatory disorder characterized by lacrimal and salivary gland insufficiency, due to high HTLV-1 337. These patients also had high serum titers of
HTLV-1 antibodies 223, but low viral load and only tax sequences were detected in their labial salivary glands222 222,325. The pathogenesis and the causative role of HTLV-1 in
Sjögren's syndrome remains a topic of debate.
HTLV-1 has also been linked to HTLV-1 associated arthropathy 263. However, whether HTLV-1 associated arthropathy represents a separate clinical entity remains controversial. HTLV-1 infection is also associated with a variety of additional autoimmune disorders such as polymyositis 194, T lymphocyte alveolitis324, lymphadenitis
267, chronic respiratory disease159, acute myeloid leukemia 367 and conditions such as systemic sarcoidosis as well as Strongyloides stercoralis infection 368. Since the evidence linking these diseases to HTLV-1 infection are not as strong as those for ATL and
HAM/TSP, the etiological role of HTLV-1 in these diseases remains controversial.
Nonetheless, the association of HTLV-1 to an array of clinical disorders indicates the huge public-health impact of this human pathogen.
1.5 Genetic Organization and Replication of HTLV-1
Based on its morphologic appearance, HTLV-1 is a C-type retroviral particle, which is typically assembled directly at the cell membrane and does not mature until after budding from the infected cell 51. It is a complex retrovirus and a member of the 12 deltaretrovirus group, which includes bovine leukemia virus (BLV) and STLV. Unlike
simple retroviruses, complex retroviruses carry additional genes to encode for several
regulatory and accessory proteins. Mature HTLV-1 particles are spherical and have a
diameter about 110 to 140 nm with an outer envelope composed of both viral and cellular
proteins and lipids 51. The core of the virion is composed of the ribonucleoprotein
complex consisting of two copies of an identical ~9 kb RNA genome that contains the m7Gppp capping group, the primer tRNAPro of host cell origin, coding regions and 3’
poly(A) sequences51 with structural and enzymatic proteins, including nucleocapsid,
capsid, matrix, reverse transcriptase, integrase and protease.
The HTLV-1 life cycle starts when viral particles attach to the cell surface
through an interaction between envelope and HTLV-1 receptors, (which will be discussed
in later section) which are not yet fully characterized. After the outer membrane of the
viral particles fuse with cell membrane, the contents of virions enter the cytoplasm and
the viral genomic RNA is reverse transcribed to generate double-stranded DNA (dsDNA)
using the virally encoded reverse transcriptase (RNA-dependent/ DNA-dependent
polymerase). Viral dsDNA traffics to the nucleus and randomly integrates into the host
genome using the viral encoded integrase (the genome is now referred to as the
provirus)108. Host cellular machinery drives transcription of the proviral DNA using host cell RNA polymerase II to produce viral mRNA. The unspliced full-length (~9 kb) viral genomic RNA is used to generate progeny virus particles (virions) and to generate Gag
(internal group specific antigen), Pol (polymerase, reverse transcriptase and integrase
enzymes needed for viral replication and maturation) and Pro (protease enzyme required
13 for maturation of structural and enzymatic proteins) proteins while the singly spliced
subgenomic ~4.3 kb mRNA is used to generate Env protein (envelope glycoprotein) and doubly-spliced (two introns removed) RNA (~ 2.1 kb) is used to translate the regulatory proteins Tax (open reading frame (ORF) IV) and Rex (ORF III). Immature virus particles are assembled at the plasma membrane and become mature only after budding
52. Additionally, by alternatively splicing, several accessory proteins are also produced
from pX ORF I and II, which will be discussed further in the following sections 24.
HTLV-1 proviral genome is 9032 nucleotide long, 312 has 5’ and 3’ long terminal repeats (LTR). The LTR is 755 nucleotides long and contain cis-acting regulatory sequences necessary for viral gene expression and replication, including sequences essential in transcription start and termination, splicing, mRNA polyadenylation, strand transfer during reverse transcription and RNA transport 24,47,173. LTR is divided into three
regions, termed U3, R and U5 110. The U3 region contains the viral transcriptional
enhancer and promoter elements 316, including three imperfect 21-base-pair repeats,
known as Tax-responsive elements-1 (TRE-1). The TRE-1 in 5’ LTR of HTLV-1
proviral genome at positions –251 to –231 (promoter distal repeat, pd), -203 to –183
(promoter central repeat, pc) and –103 to –83 (promoter proximal repeat, pp) relative to
the transcription start site 107,339,355,356, and is necessary for
transactivation34,107,164,339,355,356. The TRE-1 regions contain a conserved TGACG
sequence, with consensus to the TGACG cyclic adenosine monophosphate (cAMP)
response element (CRE), present in the promoters of several cellular genes. Thus, TRE-1
elements are in fact defective/ imperfect CREs, which bind multiple transcription factors
14 such as cyclic AMP response element binding protein (CREB)326,382, cAMP response
element modulator (CREM) 326, activating transcription factors (ATFs) 111 and other
cellular proteins that facilitate transcription including TRE binding proteins (TREB-1 335,
TREB-5 379, TREB-7 379, c-Jun 135, c-Fos 91, activator protein-2 (AP-2) 244,266, HTLV enhancer binding protein (HEB)-1p67 and HEB-1p94 205. Between the pc and pp repeats
is another second enhancer element, namely, Tax-responsive element 2 (TRE-2), also
critical in viral transcription 224,336. In addition, many cellular proteins such as Ets family proteins (Ets-1, Ets-2, Elf-1 and TIF-1) and c-Myb bind to TRE-232,33,49,53,340. Based on sequence analysis, NF-κB, AP-2 and PU.1 are also thought to potentially interact with
TRE-2 69. Additionally, inducible cAMP early repressor protein (ICER) binds TRE-1 and/or TRE-2 and decreases transcription from the LTR. Newbound et al257 reported that
Tax-mediated transcription can be repressed by ICER protein in peripheral blood
mononuclear cells, with possible significance in decreasing viral gene expression, leading
to immune evasion and subsequent viral persistence. Tax mediated transactivation
through TRE is discussed further in the following sections. In addition, many cellular
proteins such as Sp-1 266 and nuclear factor-1 (NF-1)266 bind the LTR at regions outside
TRE and enhance transcription. Moreover, LTR also contains the Rex-responsive
element (RxRE). RxRE secondary structure directly interacts with Rex and is essential
for proper polyadenylation of HTLV-1 transcripts and Rex-mediated cytoplasmic
expression of incompletely-spliced RNAs. R and U5 provide the 1-119 leader sequence
for HTLV-1 transcripts 3,4.
15 1.6 Structural Proteins of HTLV-1
HTLV-1 uses ribosomal frame shifting and alternative splicing mechanisms to
produce an array of viral proteins from the same coding region 51. Ribosomal frame
shifting is the predominant mechanism employed to generate the structural and enzymatic
proteins and precursor proteins. First, the gag gene encoding the viral structural proteins is translated from an unspliced, full-length mRNA as a single precursor polyprotein p55(Gag) 275, which is myristoylated at its N-terminus, targeted to inner plasma
membrane 271 and later gets proteolytically cleaved by viral protease into three final
products of 19 kDa (matrix), 24 kDa (capsid) and 15 kDa (nucleocapsid) proteins 119,275.
The p19 matrix protein remains myristoylated and is localized to the inner surface of the viral envelope 256. HTLV-1 Gag protein alone has been found to be adequate for the
assembly and release of virus particles 188. The p19 matrix protein (MA) contains eleven
basic amino acids, critical in the release of HTLV-1 virus particles and cell-to cell
transmission 189.
The pol gene message is translated into the Gag/Pol precursor from the same
genomic mRNA as the Gag precursor, but in a different reading frame through a
ribosomal frame shift 255. This precursor is available in lower levels than the Gag
precursor and is subsequently cleaved by the protease to produce reverse transcriptase
(RT), integrase (IN) and the remaining structural Gag proteins 256. Pol also has RNase H
activity that is required during the process of reverse transcription 52.
16 Protease (PR) is encoded by a reading frame extending between 3' part of gag and
5' part of pol and is made by another frame shift256. Due to the absence of a methionine
codon at the 5’ end of the pro ORF, an mRNA processing event or translational frame
shifting is thought to occur prior to the synthesis of protease 256. In addition, protease is
synthesized in an immature form that subsequently undergoes self-cleavage to generate
the active form 110.
The viral envelope glycoprotein (Env) is synthesized from a ~4.3 kb singly-
spliced mRNA transcript as a gp61 envelope precursor in the endoplasmic reticulum
(ER), gets heavily glycosylated and cleaved into the mature surface protein (SU) gp46
and a transmembrane (TM) gp21 protein 289. SU and TM proteins form a heterodimer necessary for receptor binding and the fusion events involving the virus and host cell109.
The SU protein contains two functionally well-defined regions71 while the TM protein contains a leucine-zipper like region required for fusion169,304 and the region between C
terminus of TM ectodomain to the end of the cytoplasmic domain critical for viral
transmission, likely for post-fusion steps in viral infection72,139. In addition, TM protein
is recognized by neutralizing antibodies75. Although Env has been extensively
investigated, the identity of HTLV-1 receptor that facilitates its spread has remained
elusive until recently. Transferrin receptor, an inducible receptor which plays a crucial
role in the regulation of iron uptake and cell growth, is overexpressed in ATL cells285 and
a neutralizing monoclonal antibody directed against transferrin receptor induced
apoptosis of ATL cells in patients243. More importantly, Manel et al recently identified
GLUT-1, a ubiquitous glucose transport protein, as a receptor for HTLV-1215. It is not
17 clear how interactions of the HTLV-1 envelope with GLUT-1 affect cell-to-cell spread of
the virus, however if GLUT-1 is essential for the spread of both cell-free and cell-
associated HTLV-1, further characterization of this receptor may be valuable in
understanding of the complexities of HTLV-1 pathogenesis and in designing strategies to
prevent HTLV-1 transmission279.
1.7 Regulatory Proteins of HTLV-1: Tax and Rex
As a complex retrovirus, HTLV-1 contains regulatory genes in pX region, located
between env and 3’ LTR. By means of alternative splicing, utilizing sequences within the
5’ LTR, pol and pX region and internal initiation codons, virus produces regulatory and
accessory proteins from four open reading frames in the pX region. ORF IV and ORF III
encode regulatory proteins Tax and Rex respectively 168,313 while ORF I and ORF II encode four accessory proteins, p12I, p27I, p30II and p13II 24,47,94,173,41(Fig. 1.1).
1.7.1 Tax and Transactivation of the HTLV-1 LTR
Tax, (Transcriptional Activator of pX region) is a 40 kd nuclear protein, produced
from a doubly spliced ~2.1 kb mRNA, and is known as the viral transactivator, which
regulates viral gene expression from the HTLV-1 LTR 37,83,92,320,321. As discussed in a
previous section, HTLV-1 gene expression is dependent on three 21-bp imperfectly
repeated elements, termed TRE-1 34,107,164,339,355,356. Among the 3 repeats within TRE-1, at least two have been demonstrated to be necessary for Tax transactivation of LTR. Tax
18 does not directly bind to TRE-1 or TRE-2 sequences, but by protein-protein interactions,
Tax increases the DNA-binding activity of CREB/ATF proteins to the TRE-1 and several
basic leucine zipper (bZIP) proteins to the TRE-2 282. Interaction of Tax with CREB and
serum response factor (SRF) enhances the stability of the transcription enhancers - DNA complexes. However, Tax transactivation of the LTR requires direct binding of Tax to
GC-rich sequences flanking the TRE-1, at a region within the CREB binding protein
(CBP/p300) binding domain 160,161,193,207. Once cells are stimulated, protein kinase A
(PKA) phosphorylate CREB, which then binds CBP/p300 45,105,180. Tax recruits
CBP/p300 to the DNA bound CREB, allowing CBP/p300 to further associate with DNA
and facilitate transcription. By binding both CREB and CBP, Tax acts as a bridge
between these two proteins without the need for CREB phosphorylation, enabling the
virus to maintain constitutive activation (without CREB phosphorylation) through TRE-1 in HTLV-1 infected cells 25,333. However, CBP/p300 recruitment by Tax is not sufficient for transactivation 118 and requires CBP/p300 associated factor (P/CAF), which associates with the C-terminal activation domain of Tax 117,137.
1.7.2 Tax and Regulation of Cellular Gene Expression
Tax regulates the gene expression of numerous cellular genes as well, mostly by induction of the transcription factors NF-κB and SRF, independent of CREB activation.
An increasing list of cellular genes activated by Tax include cytokines such as IL-2 226,
IL-3 361, IL-4202, IL-6 249, IL-8 240, IL-2Rα 226, IL-1 241, GM-CSF 233, TNFα 58 and TNFβ
203, transcription factors such as c-myc 79, c-fos 89, c-sis 297, erg-1 90, c-rel 198, as well as
19 261,342 genes involve in apoptosis Bcl-xL and DNA repair (proliferating cell nuclear antigen or PCNA) 300. Tax has also been shown to repress expression of β-polymerase
(an enzyme involved in DNA repair), lck,and bax 35,136,192.
ATL cells have characteristic IL-2Rα overexpression 338, which is believed to be
induced by Tax-mediated NF-κB activation 128. Tax directly binds multiple NF-κB
family proteins such as p50, p65, c-Rel 327,329 and lyt10 248. Since NF-κB proteins and
Tax both bind CBP/p300 99,283,383, it is possible that Tax links NF-κB and CBP/p300 to
enhance the stability of the DNA-protein complex 25. In addition, Tax localizes in the
cytoplasm and associates with IκBα, the inhibitor of NF-κB, destabilizes IκBα, thereby
allowing NF-κB to translocate to the nucleus210,328. Moreover, Tax interacts with and
induces the activation of cytokine-inducible IκB kinase (IKK), which contains IKKα,
IKKβ and IKKγ (NEMO) subunit, resulting in dephosphorylation and degradation of
IκBα and subsequent NF-κB activation 201. Additionally, Tax activates cellular gene promoters controlled by the serum response element (SRE), such as c-fos. Similar to activation of CREB, Tax induces dimerization of the serum response factor (SRF) protein p67SRE in a complex with the SRE 10,90.
Tax has a significant effect on cell cycle regulation, by means of trans-repression
of multiple cellular genes including p18INK4c, p53 and Bax 35. By sequestering
CBP/p300, Tax downregulates the promoter activity of p18INK4c, an inhibitor of CDK4
that can arrest the cells at G1 phase of the cell cycle 181,333. Although similar mechanism
has been implicated in Tax-mediated repression of p53 12,345, it is not consistent with the
20 elevated p53 levels detected in HTLV-1 transformed 96,299,375 and Tax immortalized cell
lines 5. This led to the hypothesis that Tax does not impair the expression of p53, instead
impairs its function, supported by the detection of hyperphosphorylated and inactive p53
in HTLV-1 infected cells 247,292. In addition, Mulloy et al 247found CREB binding
domain of Tax to cause Tax-mediated p53 repression while Pise-Masison et al 291 suggested an NF-κB dependent pathway for p53 inactivation by Tax. In addition, Tax associates with other cell cycle regulators, such as p16INK4a 330 and p15INK4b 144,331, negatively influences their inhibitive functions on cyclin dependent kinase 4/6 (CDK4/6), leading to Rb phosphorylation and induction of G1/S phase transition. Tax also binds to
HsMAD1, a component of mitotic checkpoint that prevents anaphase until chromosomal alignment is completed and may abrogate the function of HsMAD1 to facilitate cell division 138. Furthermore, Tax expression leads to accumulation of DNA damage
expression 232,286, by inhibition of topoisomerase I 332 and DNA polymerase β 136.
Overall, Tax suppresses DNA repair, enhances DNA replication and cell-cycle progression, thereby increase cellular mutation frequency and contribute to HTLV-1- mediated cellular transformation 97.
1.7.3 HTLV-1 Rex: Post-transcriptional Regulator
Rex, a nucleolar localizing 27KDa protein encoded by pX ORF III, is responsible for viral post-transcriptional regulation. Rex facilitates transport of unspliced RNA
(gag/pol) and singly spliced RNA (env) from nucleus to cytoplasm and inhibits the
splicing, transport of doubly spliced RNAs that encode the regulatory proteins in the pX region, which include Rex itself. Overall, Rex is necessary for viral replication 129,130,168,
21 by regulating the balance between messages encoding for viral structural proteins and
messages encoding regulatory / accessory proteins. Rex functions by binding to the Rex response element (RxRE), a highly stable RNA stem-loop structure in the U3/R region of the HTLV-1 3’LTR 17,22,26,27,156,314, critical in polyadenylation of viral RNAs 3,73. RxRE
is present in doubly spliced, singly spliced and unspliced messages, and thus other cis-
acting sequences possibly determine the selectivity of Rex on various messages. In fact, cis-acting repressive sequence (CRS) in the U5 region of the viral LTR negatively regulate Rex 28. Additionally, Rex may inhibit mRNA splicing by altering early
splicosome assembly and stabilizing the mRNA of cellular gene, IL-2Rα chain by acting
in trans on its coding sequence 147.
Rex contains both an N-terminal located NLS responsible for nuclear localization
and RxRE binding 265,318, as well as a nuclear export sequence (NES) located in the
middle, with similarity to human immunodeficiency virus (HIV) Rev, influenza virus
NS2, herpes simplex virus type 1 (HSV-1) ICP27 protein 61 and cellular protein IκBα 140.
The molecular mechanism of Rex-mediated RNA export is not completely elucidated, but
current understanding involves the CRM1/exportin1 pathway 31,112, which is also
employed by HIV Rev to facilitate viral RNA transport 61,294. Interestingly, Rex can
functionally substitute for HIV Rev 131,301. Rex and Rev directly bind both the viral
mRNA and hCRM1 in the presence of RanGTP via their RNA binding domains and NES,
respectively, acting as adaptor proteins that bridge the target mRNA and the export
receptor. Interestingly, multimerization of Rex/Rev along the viral mRNA is necessary
for RNA export67,209,354, possibly because multimerization allows a number of Rex/Rev
22 proteins to shield RNA, preventing the attachment of factors involved in splicing and nuclear retention of RNA and thus increasing the number of CRM1 molecules that associate with the complex. The association of multiple CRM1 molecules may be important in overcoming the factors that retain RNA in the nucleus and allow RNA to pass efficiently through NPCs. Human CRM1 (hCRM1) functions in the Rex-mediated mRNA export of HTLV-1 as an export receptor and as an inducing factor for Rex multimerization on its cognate RNA113. Recently, Ye et al376 reported that Rex is critical for efficient infection of cells and persistence in vivo, but its function to modulate viral gene expression and virion production is not required for in vitro immortalization by
HTLV-1.
Interestingly, a variety of HTLV-1-infected cell lines including MT-1, MT-2, TL-
Su, H582, HUT-102 contain mRNA and protein for a truncated form of Rex (p21Rex)
41,94,272, which can be generated from both the doubly-spliced message encoding Tax and
Rex, as well as the message singly spliced from nt 119 directly to the splice acceptor at nt
6875 and only encoding p21Rex. The second type of message is highly expressed in primary cells isolated from asyptomatic HTLV-1 carriers, ATL and HAM/TSP patients
94,274. This protein contains only the NES sequence and not the NLS sequence and when coexpressed with the full-length Rex, it repressed the shuttle function of the full-length protein and increased the nuclear accumulation of full-length protein 177. HTLV-2 and bovine leukemia virus (BLV) have p21Rex homolog mRNAs 273, however the exact function of p21Rex in viral pathogenesis remains to be elucidated.
23 1.8 HTLV-1 Accessory Proteins
The HTLV-1 pX genome region includes ORF I and II that produce alternatively
spliced forms of mRNA, which encode four accessory proteins, p12I, p27I, p13II, and p30II (Fig. 1.1)24,46,172,173. pX ORF I mRNA is produced by alternative splicing events
that combine the second exon of Tax with additional downstream sequences and encodes
p27I (152 amino acids long) and p12I (99 amino acid long). p12I can be translated from a
singly spliced message produced by direct splicing of nucleotide 119 to the splice
acceptor at position 6383 or by initiation at an internal methionine codon in the p27I ORF
I47. p12I is thought to be preferentially expressed from the p27I mRNA since transfection
of expression plasmids containing HA1-tagged versions of either the full-length p27I
cDNA or the cDNA for the singly spliced p12I produced only the smaller p12I protein173.
However, using in vitro transcription-translation systems, Ciminale et al 47 produced p27I
from the doubly spliced mRNA. Therefore, removal of the internal p12I AUG start codon
could yield detectable levels of p27I. Interestingly, Pique et al 290 demonstrated
production of cytotoxic T cells in HTLV-1-infected subjects that were reactive against
peptides representing all putative pX accessory proteins, including p27I.
The accessory proteins encoded by pX ORF II of HTLV-1 are produced from two
alternatively spliced mRNAs. The larger protein, p30II, is encoded by a doubly spliced
message including the first and second exon of Tax spliced to the splice acceptor site at position 6478 24. p13II, the smaller protein contains the C-terminal 87 amino acids of
p30II and is produced from a singly spliced message by splicing of the first Tax exon
24 directly to the splice acceptor at position 6875 or translated from an internal methionine
codon within p30II24,173. HTLV-1 accessory proteins were originally thought to be
dispensable for viral replication 74. However, recent investigations performed by our laboratory and others have shed light into the role of the HTLV-1 accessory proteins in viral infectivity, maintenance of high viral loads, host cell activation, and regulation of gene transcription 7,8,23,48,55,64,70,76,141,157,186,341,380,381.There is evidence that pX ORFs I and
II mRNAs and proteins are expressed in infected cell lines and freshly isolated cells from
asymptomatic carriers, ATL and HAM/TSP patients 173,41. In addition, HTLV-1 infected
patients and asymptomatic carriers have antibodies 43,70 and cytotoxic T cells290 against recombinant proteins or peptides of the pX ORF I and II proteins. However, to date, there are no conclusive reports on the temporal expression of Tax, Rex and the four accessory proteins messages in terms of their specific quantities or relative levels.
Chronically infected cell lines were found to have pX-tax/rex mRNA at 500-fold to 2500- fold higher levels than pX tax-ORF II mRNA and 1000-fold higher levels than pX-rex-
ORF I mRNA296.
Interestingly, analogous gene regions encoding the accessory proteins, especially
the pX ORF I-encoded p12I, are highly conserved in the closely related virus HTLV-2
and the nonhuman primate counterpart, simian T cell lymphotropic virus type 1 (STLV-
1)9. Further illustration of the conserved nature of these gene regions comes from studies
of another member of the deltaretroviruses, bovine leukemia virus (BLV). BLV, like
HTLV-1, contains an X region between the env sequences and the 3’ long terminal
repeat. Two proteins are expressed from this region of BLV: the protein R3, which
25 shares a common NLS with the Rex protein of HTLV-1, and G4, an arginine rich protein
that may exist as two isoforms following protease processing 152,191. Similar to HTLV-1,
deletion of homologous sequences from BLV infectious molecular clones encoding these
accessory proteins, R4 and G3, results in decreased viral loads in the experimental sheep
model 152,190,360. Collectively these studies illustrate that these retroviruses, which are all
associated with lymphoproliferative disorders, during the course of their evolution have
retained conserved gene regions that apparently serve analogous functional roles.
1.8.1 pX ORF I p12I
1.8.1.1 Biochemical Characteristics of p12I: Features of a Signaling Molecule
HTLV-1 p12I is a highly hydrophobic protein, which contains a significant
percentage of leucine (32%) and proline (17%) residues173, a minimal number of soluble
regions and two putative transmembrane domains (aa 12 to 30 and aa 48 to 67)77,341, which overlap with two predicted leucine zipper motifs that form alpha-helices. These distinct structural features (Fig. 1.2) could contribute to membrane localization and dimerization341. Additionally, p12I contains four predicted SH3-binding motifs (Fig.
1.2), which are typically proline rich cellular signaling proteins with a minimal core of
PXXP 38. Interestingly, aa 8 to 11 and 70 to 74, encoding the first and third PXXP motifs
of p12I are highly conserved among viral strains, suggesting a possible role for these domains in the function of p12I. In addition, there is a conserved sequence (PSLP(I/L)T)
extending from aa 48 to 99 in p12I, highly homologous to the PXIXIT calcineurin-binding
motif of nuclear factor of activated T cells (NFAT) and found to be critical for the
26 interaction between p12I and calcineurin157. In addition, p12I contains a dileucine motif
(DXXXLL) at amino acid positions 26 to 31, with no functional role ascertained yet (Fig.
1.2). However, dileucine motifs have been described in viral proteins such as HIV Nef and are commonly involved in directing protein trafficking through endosomal compartments by mediating association of the protein with adapter protein 1 (AP-1) to
AP-360. Sequence analysis of p12I suggests possible post-translational modifications of
the protein due to the presence of potential phosphorylation sites, glycosylation sites and
a ubiquitynation motif surrounding the lysine at position 88, which might significantly
enhance the half-life of the protein341are also present in p12I. However, p12I was
determined to be not a glycoprotein or a phosphoprotein 77.
1.8.1.2 Subcellular Localization of p12I and Interactions with Cellular Proteins
HTLV-1 p12I was originally reported to localize in cellular endomembranes,
which was suggested by a spider-like staining of cells expressing the viral protein 172.
Subsequent immunofluorescent confocal microscopy, electron microscopy and subcellular fractionation studies confirmed that p12I accumulates in the ER and cis-Golgi
apparatus. In addition, p12I directly binds calreticulin and calnexin, two resident ER-
proteins which regulate calcium storage and calcium-mediated cell signaling, suggesting a role in the establishment of HTLV-1 infection by activation of host cells 77.
HTLV-1 p12I shares sequence homology with the bovine papilloma virus (BPV)
E5 protein and Epstein-Barr virus (EBV) LMP-1 protein 86. The region of highest
27 homology starts after the first transmembrane domain and extends into the second. Like
E5, p12I also binds to the 16 kD subunit of the vacuolar H+-ATPase (16K). Although this
association appears to be required for the E5-mediated transformation of epithelial
cells,86,174 additional studies are necessary to determine its functional significance.
Interestingly, Nef, a key accessory protein of simian immunodeficiency virus (SIV) and
HIV, binds the catalytic subunit NBP-1 of the ATPase206. NBP-1 association of Nef
mediated by the Nef C-terminal flexible loop is critical for Nef-dependent internalization
of CD4 and viral infectivity 214.
Analogous to E5, p12I also associates with immature form of IL-2 receptor β and
γ chain when transiently co-expressed, resulting in reduced surface expression of the
receptor chains246. The p12I-binding site on the IL-2R chain overlaps with the binding
site for Janus kinases, JAK 1 and 3 and the adapter protein Shc. p12I does not influence
JAK3 kinase activity directly, however it is considered to be accountable for the modest
increase in STAT5 DNA binding activity262. As a consequence, p12I-expressing cells
displayed a decreased requirement for IL-2 to induce proliferation during suboptimal
stimulation with anti-CD3 and anti-CD28 antibodies 262. However, peripheral blood
derived lymphocyte cell lines immortalized by a HTLV-1 proviral clone ablated for pX
ORF I expression, ACH.p12I, have intact IL-2 receptor signaling pathways 54. A possible
explanation for these conflicting observations is that p12I may modestly activate IL-2 receptor pathways during the early stages of HTLV-1 infection before immortalization.
Nevertheless, p12I does not appear to be necessary for the activation of the IL-2R-
associated JAK1 and JAK3, or their downstream effectors STAT3 and STAT5, after
28 immortalization. Collectively, these studies indicate that p12I may induce STAT activity
to confer a growth advantage to infected cells during the early stages of infection, before
immortalization. Future studies are necessary to elucidate the JAK3-independent
pathway p12I uses to induce STAT5 activation.
HTLV-1 p12I was reported to associate with immature forms of the major
histocompatibility complex class-I (MHC-I), interfere with the interaction of MHC-I with
β2-microglobulin, decrease the surface expression of MHC-I and direct its degradation in the proteasome 143,143, suggesting that p12I might help the virus escape immune surveillance. However, levels of MHC-I and II were similar between T-lymphocytes
immortalized with the wild type and p12I-mutant HTLV-1 molecular clones (ACH and
ACH.p12 respectively), indicating that p12I-mediated modulation of MHC-I surface
expression likely occurs only during the early stages of infection54. Intriguingly, the
accessory proteins p10I and p11V of HTLV-2 also associate with MHC-I, however these
do not bind to either 16K or IL-2Rβ or γ142. Additionally, HIV-1 Nef also binds to and
down regulates the cell surface expression of MHC-I and is believed to contribute to
immune evasion by HIV-1287. However, down regulation of MHC-I of virus-infected
cells does not explain the early loss of infectivity of a molecular clone of HTLV-1 that
lacks ORF I expression, as virus infection is blocked as early as 1 week post-inoculation, before a possible active immune response occurs55. Future studies of early virus
replication immediately after inoculation of virus infected cells in animal models might
provide evidence to whether p12I indeed down regulates MHC-I expression on infected peripheral blood mononuclear cells (PBMC) in vivo and actively contributes to viral spread or persistence. 29 In addition, p12I associates with two ER-resident calcium-binding proteins, calreticulin and calnexin77 and the calcium/calmodulin-dependent serine/threonine
phosphatase, calcineurin 157. Calreticulin, a highly conserved and ubiquitous protein,
serves as one of the major calcium-binding proteins in the ER, participates in calcium
signaling, and has been linked to activation of the transcription factor NFAT230. Through
protein-protein interactions, calcineurin regulates transcriptional activation of NFAT by
triggering the dephosphorylation and subsequent nuclear translocation of NFAT, which
results in transactivation of NFAT-inducible cytokine genes including IL-2178,228,385. It would be advantageous for a virus to target such conserved proteins to dysregulate calcium signaling pathways to activate and modulate NFAT in infected T lymphocytes.
1.8.1.3 Role of p12I in Regulation of Viral Infectivity In vitro and In vivo
Earlier reports showed that deletion of pX ORF I from HTLV-1 infectious molecular clones did not affect the viral infectivity and primary lymphocyte transformation mediated by HTLV-1 infection in vitro74,302. In contrast, our research
group demonstrated that selective elimination of pX ORF I from the molecular clone
ACH resulted in dramatically reduced viral infectivity in vivo 55. Rabbits inoculated with
ACH.p12, harboring selective mutations that abolish the expression of pX ORF I mRNA,
failed to establish persistent infection as indicated by reduced anti-HTLV-1 antibody
responses, failure to demonstrate viral p19 antigen production in PBMC cultures, and
only transient detection of provirus by PCR55. A major difference between these in vitro
and in vivo studies is that standard in vitro co-culture techniques used to transmit virus to
30 naive PBMC utilize target cells stimulated by IL-2 and mitogen. However, in vivo the
majority of circulating and tissue-associated lymphocytes are non-dividing.
In support of these findings, using co-culture assays that would allow
transmission of the virus to resting primary lymphocytes, our group demonstrated that pX
ORF I mRNA is critical for viral infectivity in non-activated/quiescent PBMCs in vitro7.
More importantly, viral infectivity was restored upon addition of IL-2 and mitogen to the co-cultured PBMCs7. These data provided the first evidence that HTLV-1 p12I is
required for optimal viral infectivity in nondividing primary lymphocytes and suggested a
role of p12I in T-lymphocyte activation and in the early stage of viral infection.
Analogously, studies of HIV-1 Nef indicate that the accessory protein is dispensable for
transmission of the virus to activated target cells in vitro but is required for viral
infectivity in nondividing lymphocytes219,284,364.
1.8.1.4 Role of p12I in Calcium-Mediated T Cell Activation
Reports from our laboratory illustrated that p12I expression in Jurkat cells results
in ~ 20-fold activation of NFAT dependent gene expression, while AP-1 or NF-κB-
mediated transcription remained unchanged8. p12I specifically activates NFAT in
synergy with Ras/MAP kinase activation stimulated by the phorbol ester, PMA. By inhibition of proximal signaling molecules, such as phospholipase C- γ (PLC-γ) and LAT,
as well as distal signals, including calcineurin and NFAT, the function of p12I was
mapped to be between PLC-γ and calcineurin8. p12I mediated NFAT activation was dependent on cytosolic calcium since this function was abolished by BAPTA-AM
31 treatment. Importantly, p12I functionally substituted for thapsigargin, which specifically
depletes the ER calcium store by blocking the ER calcium ATPase. Therefore, HTLV-1
p12I was found to activate NFAT-mediated transcription in lymphoid cells in a calcium- dependent manner. Indeed, p12I expression increases the base-line cytoplasmic calcium concentration, possibly secondary to reduced ER stores calcium content and subsequently
76 higher extracellular calcium entry . Both the calcium channel on ER membranes, IP3R,
and the calcium channels on plasma membranes, calcium release activated calcium
channels (CRAC), contribute to the p12I mediated NFAT activation, strongly indicating
the modulation role of p12I on calcium homeostasis. Induction of calcium release from the ER by p12I to activate NFAT 76, would be an advantage for the virus during the early stages of HTLV-1 infection. Interestingly, this is similar to the function of a cellular
protein CAML (Ca2+-modulating cyclophilin ligand), which also contain two putative
transmembrane domains like p12I, colocalizes with calreticulin in the ER, induces
calcium release from the ER and leads to NFAT activation 351. In addition, p12I mediated increase in NFAT activity could cause complete activation of cellular stimuli that would normally induce only partial activation of T cells (e.g., through AP-1). These stimuli could be triggered by cytokines or chemokines released from infected neighboring cells
or by direct contact between viral envelope proteins and certain cell surface receptors on
newly targeted lymphocytes prior to viral entry 16. Although localization of p12I to the
ER appears to be essential for NFAT activation, 78, direct binding between calreticulin
and p12I does not correlate with the NFAT activation76 and future studies are necessary to
identify the biological significance of p12I - calreticulin interaction in HTLV-1 infection
and pathogenesis.
32 Expression of NFAT induces a highly permissive state to overcome the blockade
at reverse transcription and permitted HIV replication in primary CD4+ T cells, therefore it is possible that p12I causes T cells to be hypersensitive to T cell receptor and CD28
stimulation and thus highly permissive for subsequent viral infection. Interestingly, susceptibility of these cells to HIV infection could be restored by mitogen treatment,
likely due to the phytohemagglutinin-induced upregulation of NFAT activity. This is
similar to earlier reports from our laboratory that addition of mitogens can rescue the
infectivity of a p12I mutant viral clone in resting PBMC7, likely by overriding the
requirement for p12I-induced activation of NFAT.
Other retroviruses also encode proteins regulating Ca2+- related signals by
analogous or different mechanisms in T lymphocytes or other cell types. HIV accessory
protein, Nef, also activates NFAT in synergy with Ras/MAPK pathway in a calcium dependent fashion217-219 and is dispensable for viral infection of activated target cells in vitro, but required for viral infectivity in quiescent lymphocytes216,352,364,384. Importantly
p12I appears to enhance the production of a downstream gene of NFAT activation, IL-2 in Jurkat T cells and primary lymphocytes, which could be abrogated by calcium chelator
BAPTA-AM and calcineurin inhibitor, cyclosporin A, suggesting the effect is calcium pathway-dependent78. This increase in IL-2 could account for the decrease in
requirement for the cytokine in proliferation of human primary lymphocytes in the
presence of p12I262. Overall, p12I expression promotes T cell activation and likely
facilitates the viral replication and productive infection, which correlates with our
previous finding that p12I is necessary for viral infectivity in a rabbit model of
infection55. 33 Recently, p12I was found to contain two positive (aa 33-47, aa 87-99) and two
negative (aa 1-14, aa 70-86) regions containing individual PXXP motif, that regulate the
NFAT activation78. In a recent report from our laboratory, proline residues in these
motifs were mutated into alanine residues to test the role of PXXP motifs in p12I- mediated NFAT activation. Interestingly, the third SH3 binding domain (aa 70-73) was responsible for the negative effect of region aa 70-86 on NFAT activation 78,157. Besides, mutations in the first two PXXP motifs (aa 8-11 and 35-38) caused only minimal changes in NFAT activation, while mutations in the last PXXP motif (aa 90-93) in the second positive region (aa 86-99), enhanced the NFAT activation. In addition, p12I competes
with NFAT for calcineurin binding. More strikingly, alanine substitution mutations in
PSLP(I/L) caused more NFAT nuclear translocation and increased NFAT transcriptional activity (~2-fold) than wild type p12I 157. Interestingly, PSLP(I/L)T calcineurin binding
site is within the aa 70–86 negative region and third PXXP motif which was responsible
for the negative effect of region aa 70-86 on NFAT activation. Additionally, NFAT-
inhibitory function of the PSLP(I/L)T motif in p12I was verified to be not from the
inhibition of calcineurin phosphatase activity. PSLP(I/L)T is homologous to the
conserved functionally critical calcineurin-binding motif (PXIXIT) in the N-terminal
regulatory domain of NFAT, which binds both inactivated and activated calcineurin 11.
Many calcineurin-binding proteins such as the anti-apoptotic protein Bcl-2, calcineurin B homologous protein, a kinase anchoring protein AKAP79, and myocyte-enriched calcineurin-interacting protein 1 inhibit either calcineurin phosphatase activity or its substrate NFAT transcriptional activity59,197,350. However, PXIXIT motif mediated binding itself does not inhibit the catalytic activity of calcineurin, because NFAT
34 activation requires enzymatic activity of calcineurin as well as binding via this motif.
Not surprisingly, p12I binding to calcineurin via a motif similar to PXIXIT did not inhibit calcineurin catalytic activity but instead influenced NFAT and calcineurin interaction by competing for binding with NFAT similar to artificial peptides representing this motif 11.
Due to the existence of a calcineurin-binding motif in p12I, it may have at least
two regulatory actions to modulate NFAT activation: (1) positive modulation by
increasing cytosolic calcium concentration from ER stores and (2) negative modulation
by calcineurin binding. It is unclear why p12I has two regulatory functions for NFAT
transcriptional activity. It is notable that Bcl-2 has these similar properties like p12I, however the functional relationship between calcium release from the ER and calcineurin binding of Bcl-2 is also still unresolved. Bcl-2 maintains calcium homeostasis and prevents apoptosis by localizing not only at the mitochondrial membrane but also in the
ER membranes 1,288. p12I may function like Bcl-2 at the ER membrane, acting like an ion
channel protein to increase ER calcium permeability. Like Bcl-2, p12I may affect
apoptosis in HTLV-1-infected T cells. Another ER membrane protein, CAML that
activates NFAT by increasing calcium flux binds with calcineurin indirectly, through its
association with cyclophilin351. Overall, p12I interacts with calcineurin, an important
regulator of NFAT signaling, via a highly conserved PSLP(I/L)T motif, to further T cell
activation, an important antecedent to effective viral infection, via a
calcium/calcineurin/NFAT pathway.
35 1.8.1.5 Putative Role of p12I Variants in HAM/TSP
Proteasome destabilization of viral proteins is thought to be an intracellular
defense mechanism against viral infection 68,309. Lysine is a known target for covalent binding of ubiquitin 56,68,309and the metabolic instability p12I is mediated in part by
ubiquitylation at a single lysine residue at position 88 and subsequent proteasomal degradation, as well as by destabilizing residues at its amino terminus341. Interestingly,
lysine carrying allele was found only in some TSP-HAM cases irrespective of
geographical locations, suggesting that a selective pressure over p12I might occur in the
host 85,341. It is hypothesized that the reduced stability of p12I in HAM/TSP patients due to this sequence variation may facilitate generation of a viral-specific CTL response, since degradation of p12I would alleviate the reduction of MHC-I molecules at the cell
surface21. Interestingly, in a study conducted at the same geographic location, lysine
residue at position 88 in p12I was not found in all HAM/TSP patients, but found in an
asymptomatic HTLV-1 carrier225. However, since all individuals in this study were born
in the same geographic region, it might simply represent a particular HTLV-1 carrier
population in which the selective pressure on the p12I sequence would not be occurring.
Overall, the significance of natural p12I alleles is unclear, and it is possible that the lysine
at position 88 of p12I might have a significant effect on the biological effects of the
protein in the host, including giving a possible selective advantage in individuals with a
certain MHC-I. Future studies including screening of HTLV-1-infected individuals in
other populations may elucidate this further.
36 1.8.2 pX ORF II p13II
1.8.2.1 Biochemical Characteristics of p13II: Mitochondrial Targeting
Less is known about the function of the smaller protein, p13II, encoded by the 87
carboxy-terminal amino acids of the 241-residue Tof or p30II protein. Initial studies
demonstrated p13II localization to the nucleus173, but more-recent reports show
mitochondrial localization of the protein48,62, specifically in the inner mitochondrial
membrane and cristae62. This localization is mediated by an atypical mitochondrial targeting sequence (MTS) in the N terminus of p13II between amino acids 22-31 48(Fig
1.2). In addition, p13II MTS is, at least in part, was able to override the potent NLS of
Rev66. While the p13II MTS is also present in p30II, p30II is not localized to the
mitochondria, suggesting that the signal has to be near the amino-terminus to direct mitochondrial targeting63. Analysis of the amino acid sequence of p13II reveals no
characteristic DNA-binding motifs and previous data show neither DNA binding nor
transcriptional activity303.
1.8.2.2 p13II Alteration of Mitochondrial Morphology and Inner Membrane Potential
Functionally, expression of p13II disrupts the mitochondrial inner membrane
potential and alters mitochondrial morphology and architecture, leading to apparent
mitochondrial swelling and fragmentation of their normal interconnected string-like network, suggesting a role for p13II in induction of apoptosis48. Additionally, it is
thought that p13II might possibly cause changes in mitochondrial permeability and/or
alter processes like calcium signaling that relies on the mitochondrial network and the
ER. Since p13II does not cause substantial cytochrome c release48 or show any 37 involvement of the permeability transition pore (PTP) in driven cation fluxes62, the biological significance of p13II mitochondrial localization and disruption of membrane
potentials remains unclear. D’Agostino et al64 consider that p13II might initially induce
selective permeability changes causing swelling and subsequent de-energization and
irreversible swelling, and suggested that p13II triggers apoptosis. However, thus far,
increased levels of apoptosis have never been demonstrated in p13II-expressing cells,
leaving open the possibility for other mitochondrion-based functions of this viral protein.
Such functions could simply include an increased respiratory activity of mitochondria,
which is often accompanied by swelling or Ca2+ signaling and Ca2+ dependent regulation
of the oxidative phosphorylation machinery. Thus, p13II may facilitate later stages of
HTLV-1 infection such as assembly and release.
D’Agostino et al 62 demonstrated that amino acids 9–41 of p13II which includes
the MTS, folds into an α helix in the context of a membrane-like environment and has
specific effects on the permeability of isolated mitochondria to small cations.
Furthermore, the presence of the four arginines in the MTS is essential for the increase in mitochondrial ion conductance and in situ effects on mitochondrial morphology but not
for mitochondrial targeting. However, the observation that the p13II MTS is not cleaved
upon import into the mitochondria and that it does not require positively charged residues
distinguishes it from most MTS, and suggests that the protein might utilize a
mitochondrial import pathway distinct from that described for most MTS, which involves
binding to a series of acidic receptors followed by cleavage of the signal.
38 1.8.2.3 p13II Cellular Protein Interactions
Hou et al.,126 reported that p13II associates with two novel cellular proteins
designated C44 and C254. C254 appears to be rabbit actin binding protein 280
(ABP280) present in the cytoskeleton of many different cell types and functions in the
modulation of cell shape and polarity and is essential for migration in melanocytes and
other cultured cells. More importantly, ABP-280 is important in the insertion of adhesion
molecules into the cell membrane. While the region of ABP-280 that interacts with p13II is also involved in interactions with integrin B1, tissue factor, and presenilin, other regions of ABP-280 binds to glycoprotein and the cytoplasmic domain of furin126. C44 shares homology with archeal adenylate kinases, the eukaryotic homologues of which localize to mitochondria and are involved in energy metabolism. Interestingly, the human homologue of C44 is expressed in the Jurkat T cell line and proliferating, but not resting, PBMC 126. These studies were performed using two molecular clones of HTLV-
1, K30p and K34p, which have amino acid differences in rex, p13II, and p30II.
Interestingly, only p13II K34, but not p13II K30 allow the interaction with C44. The
amino acid sequence of the p13II variant used by Ciminale et al 48 was most similar to
p13II K30, as it had the 25- amino-acid carboxyl tail and that it would not bind C44. This
might provide clues to the role of specific amino acids within p13II, which are critical in binding C44 and in causing apoptosis. Additionally, p13II binds to farnesyl
pyrophosphate synthetase (FPPS), an enzyme involved in the mevalonate/squalene
pathway, and in the synthesis of farnesyl pyrophosphate, a substrate required for
prenylation of Ras190. Interestingly, G4, a mitochondrial targeting accessory protein of
39 BLV also binds to FPPS. Future studies on the functional significance of the interaction between p13II and these cellular proteins will possibly elucidate the role of p13II in altering mitochondrial physiology and thus in HTLV-1 replication and pathogenesis.
Furthermore, Mahana et al., 211 reported an increase in Vav phosphorylation in
rabbit cells transfected with an HTLV-1 molecular clone that contains two mutations in
pX ORF II, resulting in expression of truncated p13II and p30II. Vav is a
hematopoietically restricted guanine nucleotide exchange factor for the Rac/Rho family
of GTPases and is necessary for T cell activation146. These findings suggest that p13II may play a role in controlling the activation state of Vav, which may relate to viral infectivity and leukemogenesis.
1.8.3 pX ORF II p30II
Earlier studies suggested that ORF II was dispensable for expression of HTLV-1
proteins Tax, Rex, or Env, viral replication and immortalization of primary lymphocytes
in vitro74,302. In addition, the isolation of a viral clone containing a premature stop codon in pX ORF II, from leukemic cells led to the conclusion that pX ORF II was not necessary for the outgrowth of leukemic clones in vivo 44. However, possible functional
role of pX ORF II during early infection was not ruled out by these initial studies. To
specifically test the functional role of pX ORF II in viral replication in vivo, our
laboratory group inoculated rabbits with lethally irradiated cell lines expressing the wild-
type molecular clone of HTLV-1 (ACH) and a clone containing selected mutations in pX
40 ORF II (ACH.p30/13)23. While all ACH-inoculated rabbits became infected as early as 2 weeks postinoculation, ACH.p30/13-inoculated animals failed to become infected or maintained low proviral copy numbers in their blood leukocytes. These animals also had weak and transient ex vivo p19 antigen production from their PBMC cultures and anti-
HTLV-1 antibody titers declined towards the end of the study. Most strikingly, using quantitative competitive PCR, our laboratory group demonstrated a dramatically reduced
(up to 100-fold) viral load in the ACH.p30/13-infected animals 23. Taken together, these
data suggested that pX ORF II is necessary for maintenance of high viral loads in vivo.
1.8.3.1 Biochemical Characteristics of p30II: Features of a Transcription modulator
Several lines of evidence indicate that p30II acts as a transcription factor.
Importantly, the protein localizes to the nucleus, specifically the nucleolus of cells
transiently transfected with a p30II expression vector172,381. p30II contains a highly
conserved bipartite NLS between amino acids 71 to 98, which can be functionally
substituted for the NLS of Rex66. In addition, p30II contains serine and threonine-rich
regions that share distant homology to the activation domain of cellular transcription factors, such as Oct-1/2, Pit-1, and POU-1 47 (Fig. 1.2). Taken together, these characteristics suggest that p30II has a role in transcription of viral and cellular gene expression.
41 1.8.3.2 Role of p30II in Modulating Viral and Cellular Gene Expression
Our laboratory group has reported that, when provided in limiting concentrations,
p30II expression stimulates HTLV-1 LTR-driven reporter gene activity, even in the presence of Tax, whereas higher concentrations represses LTR and CRE-driven reporter gene activity381. These activities are analogous to herpes simplex virus type 1 (HSV-1)
regulation of its immediate-early (IE) gene promoter. VP16, a potent transcription factor
from HSV-1, binds the host cell protein HCF, which facilitates the stable complex
formation of the viral protein with Oct-1187,366. The IE gene promoter contains an Oct-1- like motif (TAATGARAT) that is important for IE gene expression, with both positive and negative effects, depending on the context of these cellular transcription factors and
VP16366.
Our laboratory group has also demonstrated that p30II co-localizes with p300 in
the nucleus and physically interacts with CBP/p300, at the highly conserved KIX domain,
the domain HTLV-1 Tax also interacts with. In addition, p30II is able to disrupt CREB-
Tax-CBP/p300 complexes bound to the viral 21-bp TRE repeats380. Since p30II and Tax
interacts with CBP/p300 through the same KIX domain, it is possible that the competitive
CBP/p300 binding between p30II and Tax might be the mechanism by which p30II attenuate the formation of these multiprotein complexes and thereby repress transcription on CREB-responsive promoters. Similarly, Tax expression has been demonstrated to interfere with the transcriptional activity of c-Myb and the binding of Tax and c-Myb to
KIX domain of CBP was found to be mutually exclusive36,53,259,260.
42 The coactivators CBP and p300 mediate transcriptional control of various cellular
and viral DNA binding transcription factors. Although these coactivators have divergent
functions, they are similar in nucleotide sequence, are evolutionarily conserved, and are
commonly referred to as CBP/p30030,134. CBP and p300 are highly related and share many functional properties, however there is evidence that these factors are not interchangeable. Several cellular and viral proteins that interact with either CBP or p300
have been identified, including steroid and retinoid hormone receptors, CREB, c-Jun, c-
Myb, Sap-1a, c-Fos, MyoD, p53, Stat-1/2, NF-κB, pp90rsk, TATA-binding protein,
TFIIB, HTLV-1 Tax, adenovirus E1A, Kaposi’s sarcoma-associated herpes virus viral
interferon regulatory factor protein, and simian virus 40 large T antigen30,134,349.
CBP/p300 protein is available only at limiting concentrations within the cell nucleus, causing an environment for competition between coactivators and transcription factors, thus providing an additional layer of regulated gene expression98,315,319. There is evidence of a functional antagonistic relationship between transcription factors, as a result of competition for binding to common regions of CBP/p30081,98,134. Under such a
condition of tight competition, relative concentrations of Tax/ p30II at various stages of
disease might be a critical factor in determining the levels of viral transcription. It is
possible that, at higher concentrations, p30II may support viral persistence by reducing
viral expression, and thus reducing immune elimination of HTLV-1 infected cells.
Additionally, p30II might also repress cellular genes necessary to maintain viral
persistence. However, at low concentrations, p30II enhances TRE/viral over
CRE/cellular mediated transcription. p30II has the potential to play a role in promoting viral transcription, cell proliferation, competitively repressing CBP/p300-dependent
43 cellular gene transcription (e.g., p53-dependent p21WAF1/CIP1 gene activity), and for
promoting the spread of the virus in vivo. This is also consistent with previous finding
from our laboratory that an infectious HTLV-1 molecular clone failed to maintain viral
loads in vivo when p30II and p13II expression was abolished 23.
Recently, it was reported that p30II modulates LTR mediated transcription, in the context of the entire provirus, by a post-transcriptional mechanism 258. However, data
presented in this thesis further confirm the role of CBP/p300 in the modulation of LTR
mediated transcription by p30II, in the context of the provirus. In the presence of
increasing concentrations of p300, we were able to rescue the p30II-mediated repression on LTR driven gene transcription, in a dose-dependent manner, irrespective of the presence or absence of the provirus. CBP and p300 bridge transcription factors to relevant promoters, has intrinsic histone acetyltransferase (HAT) activity, and form
complexes with proteins such as CBP/p300 binding protein-associated factor, which also
exhibits HAT activity 134. Recently, there is increasing knowledge of the mechanism and functional significance of the interactions between many viral proteins and CBP/p300. In the case of adenovirus oncoprotein E1A, interaction with CBP/p300 is critical for regulation of transcription, suppression of differentiation, and immortalization of cells in culture 30,134,348. The T antigen of SV40 regulates the expression of a group of cellular genes by modifying the HAT activity of CBP/p300 or by bridging the gap between DNA binding transcription factors and components of the general transcription machinery30,134.
Identifying the molecular mechanism and functional significance of the interaction between p30II and p300 is very crucial in understanding of the role of p30II in the
44 pathogenesis and replication of this important human pathogen. Therefore to further
understand the molecular mechanism and functional significance of the interaction
between p30II and p300, using N terminal and C terminal deletion mutants of p30II, we have identified the motifs within p30II that are critical in binding CBP/p300 and in
regulating LTR mediated transcription, in the presence/ absence of the provirus. Herein,
we confirm the role of p30II as a regulator viral gene transcription, in association with p300 and identified the amino acid sequence 1-132 to be critical in binding CBP/p300 and amino acids 100-179 of p30II to be critical for its function as a repressor of LTR-
mediated transcription, irrespective of the presence or absence of the HTLV-1 provirus.
Amino acid domain 100-179 contains important features such as the DNA binding
domain, serine and threonine rich residues with homology to Oct-1 and POU family of
transcription factors and five lysine residues, four of which represent potential acetylation
sites for CBP/p300. Data presented in this thesis define the roles of these four lysine
residues in p30II-mediated LTR repression. While CBP/p300-mediated acetylation has
become a common theme for regulation of protein function, herein we demonstrate that
the intrinsic histone acetyltransferase activity of CBP/p300 is critical in regulation of
p30II-mediated LTR repression.
1.8.3.3 HTLV-1 Mediated T Cell Activation /Transformation and the Role of p30II.
Mechanisms of HTLV-1 mediated T cell activation and transformation are not
completely understood. Unlike other oncogenic viruses, HTLV-1 does not contain a viral
oncogene or integrate at specific sites in the host cell genome, causing activation of
45 cellular proto-oncogenes 311. However, HTLV-1 transforms human primary CD4+CD8-
and immortalizes CD 8+CD4-, double positive, as well as double negative T cells 54 and a multistep oncogenic process is believed to occur during HTLV-1 infection, leading to cell proliferation and subsequent cell transformation. First, TCR engagement results in intracellular calcium increase and Ras/MAPK pathway activation, leading to IL-2 production and IL-2Rα accumulation at cell surface, causing induction of T cells to transit from G0 to G1 phase. Second, IL-2 and IL-2R complex interaction leads to activation of IL-2R/Jak/Stat and Shc/Ras/MAPK pathways, triggering T cell progression from G1 to S phase and eventually inducing cell proliferation. The association between these T cell activation pathways and T cell growth is not fully understood. Based on the studies on the initial stages of HTLV-1-induced T cell activation in ex vivo cultured infected T cell clones from HAM/TSP patients with HAM/TSP, infected T cell clones spontaneously proliferate in vitro up to 2 weeks after stimulation in the absence of exogenous IL-2 (involving autocrine IL-2/IL-2R stimulation), while uninfected T cell clones revert to G0 stage125. Tax is thought to increase the expression of IL-2Rα, thereby
induce this initial activation 128. Peripheral blood T cells can be immortalized and subsequently transformed by coculturing with HTLV-1-producing T cells124,125,235,293. In
vitro immortalization is thought to occur in multiple stages: At ~4 weeks after the
infection, T cell cultures reach a crisis stage with dramatic decrease in cell number,
followed by IL-2 dependent proliferation of cells surviving the crisis characterized by
upregulated expression of CD25 as well as MHC-II, polyclonal proviral integration,
scarse expression of tax/rex mRNA and transient expression of IL-2. Later, at ~14 weeks
after infection, oligoclonal proviral integration, abundant expression of tax/rex mRNA
46 and increased CD25 expression, but not IL-2, are detected158. Since transformed cells are resistant to rapamycin treatment, 365. HTLV-1 infection is believed to circumvent the rapamycin sensitive IL-2/IL-2R pathway. Interestingly, HTLV-1 transformed T cells
(but not immortalized T cells) show constitutive activation of IL-2R-associated Jak1/3 and Stat3/5 54,231. HTLV-1 Tax and p12I proteins have been linked to HTLV-1-induced
immortalization and transformation. Tax is known to upregulate p21WAF expression,
inhibit p53 42, inhibit p16INK and thus indirectly activate cyclin D/CDK4/6330, functional
disruption of mitotic checkpoint protein MAD1138 while p12 is reported to increase Stat5 activation and modestly enhance proliferation 262.
Data presented in this thesis establishes the role of p30II as a regulator of cellular
gene expression. More importantly, herein we illustrate that p30II activated many key transcription factors involved in T cell signaling/ activation, such as NFAT, NF-κB and
AP-1, and deregulated the expression of multiple genes involved in T cell activation or cell signaling and in regulation of transcription, translation, apoptosis, cell cycle, cell adhesion as well as immune response. The mechanism of function of p30II is not elucidated; however it is likely involved in T cell activation and subsequent T cell proliferation.
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84
Figure 1.1. Diagram of HTLV-1 genome and alternatively spliced mRNA and protein species from pX ORFs. Numbers correspond to nucleotide positions of exon splice acceptor and donor sites with respect to the full length HTLV-1 genome. Dotted lines indicate the mRNA while the boxes below the dotted lines indicate coding regions of each protein. Open reading frame from which each protein is produced, is indicated as superscript, on the right of each protein.
85
Figure 1.2. Diagram of p12I, p30II, and p13II with predicted functional motifs. Abbreviations: TM, transmembrane region; LZip, leucine zipper motif; DxxxLL, dileucine motif; PxxP, SH3 binding motif; K/R, lysine-to-arginine variant at position 88 (arginine at this position increases stability of protein). Boxes below p30II indicate regions sharing sequence homology with the DNA binding domain and homeodomain of Oct-1. Numbers below bars indicate amino acid position numbers.
86
CHAPTER 2
HUMAN T LYMPHOTROPIC VIRUS TYPE-1 P30II INFLUENCES CELLULAR GENE EXPRESSION AND ENHANCES TRANSCRIPTION ACTIVATION OF T LYMPHOCYTES
INTRODUCTION
Human T-lymphotropic virus type 1 (HTLV-1), the first characterized human
retrovirus, causes adult T cell leukemia/lymphoma (ATL) and is associated with several
lymphocyte-mediated disorders such as HTLV-1-associated myelopathy/tropical spastic
paraparesis (HAM/TSP)7. Mature CD4+ T lymphocytes are the primary targets of HTLV-
1 infection25. Although the mechanism by which the virus causes oncogenic
transformation of host T lymphocytes is incompletely understood, altered gene
expression has been associated with the initiation or progression of ATL 47. This complex
retrovirus encodes structural and enzymatic gene products, as well as regulatory and
accessory proteins from open reading frames (ORF) in the pX region between env and the
3’ long terminal repeat (LTR) of the provirus19. The well characterized Rex and Tax
proteins are encoded in the ORF III and IV respectively. Rex is a nucleolus-localizing
87 phosphoprotein, involved in nuclear export of unspliced or singly spliced viral RNA 24.
Tax is a nuclear localizing phosphoprotein that interacts with cellular transcription factors and activates transcription from the viral promoter, Tax-responsive element (TRE) and enhancer elements of various cellular genes associated with host cell proliferation 22.
Emerging evidence has documented the role of pX ORF I and II gene products in the replication of HTLV-13. There are four proteins expressed from these ORFs- p12I, p27I, p13II, and p30II. pX ORFs I and II mRNAs are present in infected cell lines and freshly isolated cells from HTLV-1-infected subjects36 as well as in ATL and HAM/TSP patients12. Antibodies13,16 and cytotoxic T cells46 that recognize recombinant proteins or
peptides of the pX ORF I and II proteins are present in HTLV-1 infected patients and
asymptomatic carriers.
Using molecular clones of HTLV-1 with selective mutations of ORF I and II, we have tested the requirement of p12I and p13II/p30II in the establishment of infection and
maintenance of viral loads in a rabbit model of infection 1 9. ORF II protein p30II contains a highly conserved bipartite nuclear localization signal (NLS) and localizes within the nucleus of cells 35,58. In addition, p30II contains serine- and threonine-rich regions with
distant homology to transcription factors Oct-1 and -2, Pit-1, and POU-M114. p30II also co-localizes with p300 in the nucleus and physically interacts with CREB binding protein
(CBP)/p300 and differentially modulates cAMP responsive element (CRE) and TRE mediated transcription 57 58. Based on these reports, we hypothesized that p30II functions as a regulator of cellular and viral gene expression to promote HTLV-1 replication.
88 Gene arrays have primarily been employed to study the changes in gene
expression profile of HTLV-1-immortalized and transformed cell lines or in cells from
ATL patients and attempts to test the influence of individual HTLV-1 viral proteins on
cellular gene expression have been limited to Tax 15,23,32,33,33,44,47. Herein we used the
Affymterix U133A human gene chip to confirm the role of p30II as a regulator of gene
expression and identified several novel and important alterations in gene expression
profiles, unique to cell cycle regulation, apoptosis and T cell signaling/ activation. In
addition, using semi-quantitative RT-PCR, we have confirmed the expression of multiple
genes modulated by p30II in primary CD4+ T lymphocytes. Moreover, we have validated
the function of p30II in T cell signaling/ activation, using reporter assays. This is the first
report that demonstrates the role of p30II as an activator of many key transcription factors
involved in T cell signaling/ activation, such as Nuclear Factor of Activated T cells
(NFAT), Nuclear Factor-Kappa B (NF-κB) and Activator Protein-1 (AP-1). Together, our data indicates that HTLV-1, a complex retrovirus associated with lymphoproliferative disorders, uses accessory genes to promote lymphocyte activation to enhance clonal expansion of infected cells and maintain proviral loads in vivo.
89 MATERIALS AND METHODS
Lentiviral vectors and other plasmids
The plasmid pWPT-IRES-GFP was generated by cloning the internal ribosome entry site (IRES) sequence from pHR’CMV/Tax1/eGFP 54 (Gerald Feuer, SUNY,
Syracuse) into pWPT-GFP plasmid (Didier Trono, University of Geneva). Subsequently, the plasmid pWPT-p30IIHA-IRES-GFP was created by cloning p30II sequence from
ACH30 with the downstream influenza hemagglutinin (HA1) tag (Fig. 2.1). Sanger sequencing confirmed both the plasmids to have the correct sequence and were in frame.
GFP and p30IIHA expression were confirmed by fluorescence activated cell sorting
(FACS) analysis (Beckman Coulter, Miami, FL) and western blot respectively. GFP expression from each of the plasmids was confirmed by flow cytometry (Beckman
Coulter) and the p30IIHA expression from pWPT-p30IIHA-IRES-GFP plasmid was confirmed by western blot using mouse monoclonal anti- hemagglutinin antibody
(1:1000) (Covance, Princeton, NJ) as described previously57,58. The plasmid pME- p30IIHA was created by cloning p30II sequence from ACH with HA1 tag, into pME-18S
(G. Franchini, NIH). Other plasmids used include previously reported pRSV-βGal 58and
AP-1, NF-κB and NFAT-luciferase reporter plasmids 2.
Recombinant lentivirus production and infection of Jurkat T lymphocytes and primary CD4+ T lymphocytes
Recombinant lentiviruses was produced by transfecting pHCMV-G, pCMV∆R8.2 and pWPT-p30IIHA-IRES-GFP (sample) or pWPT-IRES-GFP (control) as described
90 previously18. Briefly, 293T cells (5 × 106) were seeded in a 10-cm dish and transfected the following day with 2 µg of pHCMV-G, 10 µg of pCMV∆R8.2 and 10 µg of pWPT- p30IIHA-IRES-GFP or pWPT-IRES-GFP using the calcium phosphate method.
Supernatant from 10 to 20 dishes was collected at 24, 48 and 72 h post transfection, cleared of cellular debris by centrifugation at 1000 rpm for 10 min at room temp and then filtered through a 0.2 µm filter. The resulted supernatant was then centrifuged at 6,500 g for 16 h at 4° C. The viral pellet was suspended in cDMEM (DMEM containing 10%
FBS and 10% streptomycin and penicillin) overnight at 4° C and the concentrated virus was aliquoted and stored at –80° C. To determine the virus titer, serial dilutions of the virus stock were used to spin infect 293T cells and 48 h post infection, eGFP expression and p30II expression was measured by flow cytometry and RT-PCT respectively. Briefly,
on the day before infection, 293T cells (1 × 105) were seeded in a 6 well plate. The medium was removed the following day and the cells were then incubated with the
diluted virus containing 8 µg/ml polybrene (Sigma, St. Louis, MO). Cells were then spin-
infected by centrifugation at 2700 rpm for 1 h at 30o C, supplied with fresh medium and
cultured for 48 h. Then cells were treated with trypsin (Invitrogen, Carlsbad, CA),
pelleted and resuspended in D-PBS (Invitrogen) for fluorescence activity cell sorting
(FACS) analysis on an ELITE ESP flow cytometer (Beckman Coulter). 1 X 106 cells were used to perform western blot to detect the expression of p30 II HA. Jurkat T
lymphocytes (clone E6.1, American Type Culture Collection) were transduced with
recombinant virus at multiplicity of infection of 4 in the presence of 8 µg/ml polybrene
(Sigma) and spin-infected at 2700 rpm for 1 h at 25o C. Primary CD4+ T cells were
extracted using dynabead CD4 positive isolation kit (Dynal Biotech, Lake Success, NY) 91 according to manufacturer’s instructions. Primary CD4+ T cells were stimulated with
Phytohemagglutinin (PHA) for 48 hours, transduced with recombinant virus at
multiplicity of infection of 20 in the presence of 8 µg/ml polybrene (Sigma) and spin- infected at 2700 rpm for 1 h at 25o C. At 10 days post-transduction, GFP expression of
controls and samples were verified to be above 90% by FACS analysis and the presence of p30II mRNA expression in samples (and absence in controls) was verified by RT-PCR
(Fig. 2.2).
Probe preparation and microarray analysis
According to the instructions of manufacturers, total cellular RNA was isolated
from transduced Jurkat T lymphocytes using RNAqueous (Ambion, Austin, TX). To test
the concentration and purity of the RNA samples, absorbance at 260 nm and 280 nm
were measured and the 260/280 ratio was calculated using a spectrophotometer
(Genequant, Amersham Pharmacia, Piscataway, NJ). The 260/280 ratio of all the RNA
samples were between the range of 1.9-2.1. The probe preparation for GeneChip was
performed according to the Affymetrix GeneChip Expression Analysis Technical Manual
(Affymetrix, Santa Clara, CA). Briefly, cDNA was synthesized using genechip T7-Oligo
(dT) promoter primer kit (Affymetrix) and superscript double stranded cDNA synthesis
kit (Invitrogen), according to the manufacturers instructions. cDNA cleanup was done
using Genechip Sample Cleanup module (Affymetrix). In vitro transcription was
performed on the cDNA to produce biotin-labeled cRNA with ENZO RNA Transcript
labeling kit (Affymetrix), according to the manufacturers instructions. cRNA cleanup was
92 done using Genechip Sample Cleanup module (Affymetrix). The quality of total RNA and biotin-labeled cRNA of all the samples and controls were checked by calculating the
ratio of absorbance at 260 nm and 280 nm (between 1.9 to 2.1) using a spectrophotometer
(Genequant) and agarose gel electrophoresis. The labeled cRNA was fragmented to 50–
200 nucleotides, and hybridized to U133A arrays (Affymetrix) using GeneChip®
Hybridization Oven (Affymetrix). Arrays were washed and stained using GeneChip®
Fluidics Station 400 (Affymetrix) and scanned by GeneArray Scanner (Affymetrix).
Quality control criteria evaluations done as part of the basic analysis include (1)
The ratios of 3’ signal to 5’ signal of two housekeeping genes, beta-actin and GAPDH
were between 0 and 3. (2) The hybridization controls BioB, BioC, BioD, and Cre were all
present and in a linear relationship of intensity. (3) The scale factors between arrays did
not vary by 3 fold. (4) The background intensity was not significantly higher than
expected. (5) The percent of gene present was monitored and found to be not less than
the standard 30%. To determine the quantitative RNA level, the average differences representing the perfectly matched minus the mismatched for each gene-specific probe set
was calculated. Differential gene expression and comparative analysis was done using
Data Mining Tool (Microarray suite 5) to identify probes with at least 1.5 fold
difference in expression between control and p30II and verified for cluster formation by
dCHIP software39. The biological and molecular functional grouping of these probes was done using Gene Ontology Mining Tool (Affymetrix)6. The selected probe lists are presented in the supplemental data.
93 RT-PCR
One µg of RNA was converted to cDNA (Reverse Transcription system,
Promega, Madison, WI) as described by the manufacturer. cDNA from 100 ng of total
RNA was amplified with AmpliTaq DNA polymerase (Perkin Elmer, Boston, MA), PCR
products were separated by agarose gel electrophoresis, normalized to GAPDH and
quantified using alpha imager spot densitometry (Alpha Innotech, San Leandro, CA).
DNA contamination was tested by performing a control with no reverse transcriptase.
The PCR primers for p30II were as follows: TAG CAA ACC GTC AAG CAC AG
(forward) and CGA ACA TAG TCC CCC AGA GA (reverse). The PCR primers for CHP
were as follows: CCC ACA GTC AAA TCA CTC GCC (forward) and ATG GTC CTG
TCT GCG ATG CTG (reverse). The PCR primers for JUN were as follows: CTC TCA
GTG CTT CTT ACT ATT AAG CAG (forward) and TTA TCT AGG AAT TGT CAA
AGA GAA GATT (reverse). The PCR primers for NFATc were as follows: TTG GGA
GAG ACA TGT CCC AGA TT (forward) and TCA TTT CCC CAA AGC TCA AAC A
(reverse). The results were expressed as a graph. Statistical analysis was performed using
Student’s t test, P < 0.05.
Transient transfection and reporter gene assay
Analysis of AP-1, NF-κB, and NFAT transcriptional activity in pME- and pME- p30II-transfected Jurkat T lymphocytes was performed as described previously (25).
Briefly, transient transfection of Jurkat T lymphocytes was done by electroporating 107
94 cells in cRPMI (RPMI 1640 containing 10% fetal bovine serum (FBS) and 10%
streptomycin and penicillin) at 350V and 975 µF using Bio-Rad Gene Pulser II (Bio-
Rad,Laboratories, Hercules, CA) with 30 µg of pME-p30 or pME empty plasmid, 10 µg
of reporter plasmid (NFAT-Luc, AP-1 Luc or NF-κB Luc), and 1 µg of pRSV-Gal
plasmid or 1 ug pWPT-IRES-GFP plasmid. The transfected cells were seeded in six-well
plates at a density of 5 X 105/ml and were either left untreated or stimulated with 20
ng/ml of phorbol myristate acetate (PMA) (Sigma) or with 2 µM ionomycin (Sigma), or both at 6 h post-transfection, followed by incubation for 18 h prior to lysis for analysis of luciferase activity. Stimulations with anti-CD3 and/or anti-CD28 antibodies (each at 3
µg/ml) (BD Pharmingen, Heidelberg, Germany) were carried out 18 h post-transfection.
Following 8 h of stimulation, to measure luciferase activity, the cells were lysed with Cell
Culture Lysis Reagent (Promega), and the cell lysates were tested for luciferase activity according to the manufacturer’s protocol. Transfection efficiency was normalized by staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) (Sigma) and counting β-Gal expressing cells. Transfection efficiency was also normalized by counting
GFP positive cells under the fluorescence microscope. Results were expressed as mean of optimized luciferase activity (luciferase activity / percentage cells stained positive for β-
Gal expression) in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test, P <
0.05.
95 RESULTS
At 10 days post-transduction, GFP expression of Jurkat T lymphocytes transduced
with recombinant lentivirus expressing GFP alone (controls) or p30II and GFP (samples)
was confirmed to be > 95%. Presence of expression of p30II mRNA in the sample cells
and absence of p30II mRNA expression in control cells were confirmed by RT-PCR.
Differential gene expression and comparative analysis was done to identify probes with at least 1.5 fold difference in expression between control and p30II and verified for cluster
formation39. We categorized genes deregulated by HTLV-1 p30II into those upregulated
or downregulated in expression. We further grouped genes deregulated by p30II based on
their functions, such as apoptosis, cell cycle, cell adhesion, transcription / translation
factors and T cell activation or cell signaling. In all the categories, p30II appeared to be an
overall repressor of cellular gene expression, while selectively increasing the expression
of certain key regulatory genes (Table 2.1).
Apoptosis
Based on changes in gene expression in p30II expressing cells (Table 2.1), p30II would be predicted to modulate apoptosis. These include Bcl-2 related/interacting genes such as pro-apoptotic Bcl-2-related protein A1, anti-apoptotic MCL1, cell-death regulator
Harakiri, apoptotic protector BNIP1 (downregulated) and pro-apoptotic BIK
(upregulated). In addition, p30II expression correlated with downregulation of genes
associated with Fas mediated apoptosis pathway such as tumor suppressing
subtransferable candidate 3 and TNF receptor superfamily member 25. p30II expression
96 was also associated with decrease in caspases (2 and 4) and increase in genes associated
with the DNA fragmentation pathway (CIDE-B and CIDE-3). In addition, p30II expression correlated with decreased expression of many other apoptosis related genes including CD28, Lck, cyclin B1, Cullin 5, Adenosine A2a receptor, TAF4B and NCK- associated protein 1.
Cell cycle
p30II expression altered the expression of multiple genes involved in regulation of
different stages of cell cycle. These include checkpoint suppressor 1, cytosolic branched- chain amino acid transaminase 1, histone deacetylase 6, cyclin B1, WEE1 kinase,
CDC14A, Lck, JAK2, GAS7, BZAP45, Cullin, Rab6 GTPase activating protein
(downregulated) and TERF1, AKAP8, DDX11, MSH2 and JUN (upregulated). Another gene downregulated by p30II expression was MDM2, which, interestingly is
overexpressed in certain leukemias34 and capable of enhancing the tumorigenic potential
of cells by inhibiting p300 / PCAF mediated p53 acetylation 40.
Cell adhesion
p30II expression was associated with altered expression of several genes involved
in cell adhesion. These include decrease in integrin (integrin β8) immunoglobulin
(MADCAM1), a counter-receptor for P-selectin (SELPLG), cadherin (desmocollin 3),
protocadherin (PC-LKC) liprin (PPF1BP1), CD84 / Ly-9, CD58, CD43 / sialophorin and
glycosyl-phosphatidyl-inositol phospholipase D1. Expression of p30II correlated with increase in integrin receptor α1 subunit and KIT ligand.
97 Transcription and translation
Expression of p30II was associated with decrease in expression of transcriptional
control genes such as TATA-binding protein associated factor 4 (TAF4), two co-
repressors (Enolase-1 and Chromosome 19 ORF2 protein), a novel specific coactivator
for mammalian TEFs, namely TONDU 53, homeo box genes (mesenchyme homeo box 1,
homeobox A1), T-box genes (T-box 21) and proteins containing helix-loop-helix domain,
which are known to be critical in cell growth/differentiation and tumorigenesis (neuronal
PAS domain protein 2, Myc-associated factor protein, inhibitor of DNA binding-3).
Additionally, p30II expression correlated with downregulation of zinc finger proteins
(zinc finger protein 36), a group of transcription regulators proposed to be candidates in
malignant disorders 51 and coiled coil proteins (JEM-1). p30II was also associated with downregulation of many genes with positive transcriptional effects (including SEC14- like 2, Nurr 1, CITED2 / MRG1, LXR alpha and SMARCA2). Downregulation of
HDAC6, a histone deacetylase and nuclear receptor coactivator 3 (CBP interacting protein) with histone acetyltransferase and pCAF/CBP recruiting abilities 50 are
particularly interesting, since p30II contains multiple highly conserved lysines, which
could play a role in acetylation 57,58. Expression of p30II was associated with decrease in
GAS 7, which has sequence homology to Oct and POU family of transcription factors27.
Genes upregulated by p30II also included HTLV enhancer factor, c-Jun, TAF1C,
Kruppel-type zinc finger, PQBP1, AF4 and SOX4. Based on this, p30II appears to
regulate transcription through various mechanisms and different levels, some of which
could be attributed to the interaction between p30II and CBP/p30057. Additionally, p30II
98 expression was associated with decrease in translation initiation factor 2 (IF2) and
eukaryotic translation elongation factor 1δ (EEF1D) and increase in eukaryotic
translation elongation factor 1α (EEF1A2), a putative oncogene 4
p30II alters the expression of genes involved in T cell signaling / activation
Expression of p30II was associated with decrease in mRNA levels of CD28, a co- stimulatory molecule with a distinct role in T lymphocyte activation 10. Cells expressing
p30II had upregulated levels of Vav-2 and CD72 and downregulated levels of CD46 and
Lck tyrosine kinase, a member of the Src family protein tyrosine kinases that participates
in signal transduction pathways initiated by T cell surface receptors such as TCR/CD3,
CD2, CD4, CD8, and CD28 43. Additionally, p30II expression correlated with decrease in
the level of CHP, an endogenous calcineurin inhibitor, indicating a likely increase in
calcineurin activity, which would be predicted to correlate with upregulation of NFAT
expression by p30II. Moreover, p30II expression was associated with increased expression
of c-Jun and c-Fos, suggesting activation of AP-1 mediated transcription. p30II appeared
to downregulate the expression of protein kinase D (PKD), which negatively modulates
JNK signaling pathway 26, mediates cross-talk between different signaling systems, and is
critical in processes as diverse as cell proliferation, apoptosis, immune cell regulation and
tumor cell invasion 52. Interestingly, p30II expression was also associated with decrease in
epidermal growth factor (EGF), which stimulates calcium influx 56 and activates ETS
domain transcription factor Elk-1 48. In Jurkat T lymphocytes expressing p30II, there were
no detectable levels of IKKγ, which is important for NF-κB signaling in response to both
99 T cell activation signals and Tax 49. However p30II expression increased HPK-1
(Hematopoetic Progenitor Kinase-1), a known NF-κB activator5. p30II expression was
associated with decreased Ras GRP2, a guanyl nucleotide exchange factor that increases
Ras-GTP, suggesting a decrease in the level of activated Ras (Ras-GTP).
We performed semiquantitative RT-PCR analysis in Jurkat T lymphocytes and
primary CD4+ T lymphocytes to confirm the altered expression of genes involved in T cell activation / signaling pathway (Fig. 2.3A, 2.3B, 2.3C and 2.3D). Overall, HTLV-1 p30II downregulated the expression of multiple upstream regulators of T cell activation / signaling pathway to enhance NFAT, NF-κB and AP-1 driven transcription, key factors in T cell proliferation and leukemogenesis.
HTLV-1 p30II enhances transcription mediated by NFAT, NF-κB and AP-1 in
Jurkat T lymphocytes.
Using luciferase reporter assays, we directly tested the ability of p30II to enhance
NFAT, NF-κB and AP-1 driven transcription. NF-κB, AP-1, and NFAT luciferase
reporter plasmids and p30II expression plasmid were transiently transfected into Jurkat T
lymphocytes, and stimulated with PMA or ionomycin or both, CD3 or CD28 or both.
p30II increased the NFAT driven luciferase reporter gene activity from 2.2 to 10.7 fold
depending on co-stimulatory treatment e.g., PMA, ionomycin, CD3, CD28 etc. (Fig.
2.4A), indicating that p30II effectively enhanced NFAT driven transcription, when
stimulated with ionomycin or CD3. HTLV-1 p30II increased NF-κB driven luciferase
reporter gene activity from 3.1 to 11.4 fold, depending on co-stimulation (Fig. 2.4B),
100 indicating that p30II is able to effectively enhanced NF-κB driven transcription,
significantly even under unstimulated conditions. HTLV-1 p30II modestly increased the
AP-1 driven luciferase reporter gene activity from 1.2 to 5.2 fold in the presence of co-
stimulator treatments (Fig. 2.4C), indicating that p30II enhanced AP-1 driven
transcription, significantly when stimulated with PMA.
DISCUSSION
Our study is the most comprehensive analysis of changes in gene expression patterns caused by an individual retrovirus accessory protein. Our approach included methods to strengthen the reliability of our data by (a) use of triplicate samples and appropriate controls (b) use of multiple software for data analysis (c) minimization of
nonspecific hybridization and background signals by using Affymetrix chip 42 (d) use of a
well-characterized T lymphocyte system (Jurkat) and (e) verification of microarray data
by semiquantitative RT-PCR in Jurkat T lymphocytes and primary CD4+ T lymphocytes
(f) validation of microarray data by reporter assays, all of which were consistent with our
micro array findings. Some of these findings are consistent with previous studies using
gene arrays to test HTLV-1-transformed cell lines. For example, HTLV-1 infected cell
lines contain low levels of caspase-4 and high levels of JUN 47 and cyclin B1 levels are
low in HTLV-1 leukemic T cells 28. Overall, this study not only confirmed that p30II is a
regulator of cellular genes, but also identified several potential new functional roles for
p30II, in T cell activation or cell signaling and in regulation of apoptosis and cell cycle.
101 Others have used gene array approaches to study HTLV-1-related changes in gene
expression. Harhaj et al 23 studied the gene expression in HTLV-1 mediated oncogenesis
using human cDNA array analysis of normal and HTLV-1 immortalized T cells and
found that the expression of a large number of genes involved in apoptosis were deregulated in HTLV-1 immortalized T cells. Subsequently, the same type of cDNA arrays were employed by De La Fuente et al 15 to study upregulation of a number of
transcription factors in HTLV-1-infected cells, including zinc fingers, paired domains,
and basic helix-loop-helix (bHLH) proteins. Gene expression profiles of fresh peripheral
blood mononuclear cells (PBMC) from acute and chronic ATL patients were used to
identify the genes associated with progression of ATL including a T cell differentiated
antigen (MAL), a lymphoid specific member of the G-protein-coupled receptor family
(EBI-1/CCR7) and a novel human homolog to a subunit (MNLL) of the bovine
ubiquinone oxidoreductase complex31. Using NIH OncoChip cDNA arrays containing
2304 cancer related cDNA elements, Ng et al, 2001 44 compared normal and Tax-
expressing Jurkat T lymphocytes and identified Tax induced changes in gene expression,
associated with apoptosis, cell cycle, DNA repair, signaling factors, immune modulators,
cytokines, growth factors, and adhesion molecules. Recently, Affymetrix, GeneChip
microarrays containing oligonucleotide hybridization probes representative of ~7000
genes were used to compare the expression profiles of normal activated peripheral blood
lymphocytes to HTLV-I-immortalized and transformed cell lines 47. In this study, by
employing a gene chip representing ~33000 genes, we tested the role of p30II on cellular
gene expression profile of a larger number of genes.
102 Expression from the IL-2 promoter requires binding of several transcription factors, including NFAT, AP-1 and NF-κB. NFAT is vital to proliferation of peripheral lymphocytes for HTLV-1 infection 55 while AP-1 is linked to the dysregulated
phenotypes of HTLV-1 infected T cells 20 and malignant transformation 41. Activation of
AP-1 occurs through Tax-dependent and independent mechanisms in HTLV-1-infected T cells in vitro and in leukemia cells in vivo 20. NF-κB is highly activated in many
hematopoietic malignancies, HTLV-1 infected T cell lines and in primary ATL cells, even when Tax expression levels are low 41 and due to its anti-apoptotic activity, it is
considered to be a key survival factor for several types of cancer. Ours is the first report
demonstrating the ability of a retroviral accessory protein to have broad modulating
activities on the transcriptional activity of NF-κB, NFAT and AP-1.
Mechanisms employed by p30II to enhance NFAT, AP-1 and NF-κB mediated
transcription will require additional research. We have previously reported that another
HTLV-1 accessory protein p12I stimulates NFAT mediated transcription, when
stimulated with PMA, indicating that p12I acts synergistically with Ras/ MAPK pathway
to promote NFAT activation and thus may facilitate host cell activation and establishment
of persistent HTLV-1 infection 2 However p30II enhances NFAT driven transcription
significantly when stimulated with ionomycin or CD3, and therefore likely uses a
different mechanism than p12I. To modulate NFAT driven transcription and subsequent T
cell activation / signaling, it is possible that these two accessory proteins act
synergistically. AP-1 is able to interact with transcriptional coactivator CBP/ p300 8 as well as viral CREs and mediate HTLV-I gene expression (50). Intriguingly, we have 103 previously reported that p30II interacts with CBP/p300 at KIX domain of CBP 11, influences CRE and TRE mediated transcription 58 and disrupts CREB-Tax-p300
complexes on TRE probes 57. NF-κB (52) and NFAT 21 are also known to interact with
transcriptional coactivator CBP/p300. Therefore it is possible that p30II regulates the transcriptional activity of NFAT, NF-κB and AP-1, at least in part, by its interaction with
CBP/p300.
HTLV-1 mediated interference with normal T-cell apoptosis is thought to be a mechanism of tumorigenicity 25, but specific mechanisms by which HTLV-1 infection or
any particular HTLV-1 gene products influence on T-cell survival are not fully
understood 25. Similar to the effect of HTLV-1 Tax on apoptosis related genes23,44, we
found that p30II also deregulates multiple genes resulting in possible pro-apoptotic and
anti-apoptotic effects. Since apoptosis is a well-known mechanism of cellular defense
against viral infection, a possible role of p30II in lymphocyte apoptosis might correlate with the requirement of p30II in maintaining proviral loads in vivo 9. Previous studies
indicate that several members of the cell cycle machinery have altered expression in
HTLV-1 infected cells 47. Several studies examined the aberrations in cell cycle caused
by HTLV-1 Tax 25; however, not much is known about the role of other HTLV-1 proteins
in causing abnormalities in cell cycle.
p30II appears to regulate viral gene expression and modulate immune response.
We have previously reported that, p30II activated HTLV-1 LTR at lower concentrations
and repressed at higher concentrations. 58. Interestingly, p30II expression was associated
104 with downregulation of lck (p56), which suppresses the HTLV-1 promoter 45 and upregulate HTLV enhancer factor, which is known to bind to LTR at a region involved in regulation of gene expression by the ets family of transcription factors38. Additionally, p30II expression was associated with altered expression of cellular genes involved in
immune modulation such as CD46, CD43, CD58, IFNγ and CD72.
Overall, this study supports our earlier reports 2,9,57,58 and sheds light on the
mechanisms by which p30II functions in HTLV-1 pathogenesis and in leukemogenesis.
Many of the effects of p30II are similar to that of other HTLV-1 proteins like Tax or p12I.
It is possible that these proteins act coordinately or synergistically. We postulate that, by modulating the expression of various HTLV-1 proteins, the virus employs selective use of different viral proteins during different stages of the infection. However, since information on the expression profile of HTLV-1 proteins during different stages of the infection is limited, additional studies are required to explore this possibility. Further studies will also be required to verify our data in primary T lymphocytes. Such future studies might provide new directions in the development of therapeutic interventions against HTLV-1 disorders, which are associated with immune mediated mechanisms.
HTLV-1 accessory proteins are functionally homologous to the accessory proteins of bovine leukemia virus (BLV), a deltaretrovirus with conserved organizational structure similar to HTLV-1 that is associated with a naturally occurring lymphoma in cattle 17,29,37.
BLV-wild type and mutant proviruses that contained deletions in the G4 or R3 genes infected B lymphocytes and permitted the infected cell to resist apoptotic signals 17. G4
105 protein interacts with farnesyl pyrophosphate synthetase (FPPS), an enzyme in the
mevalonate/squalene pathway that is critical for synthesis of FPP, a substrate required for
prenylation of Ras 37. Infectious molecular clones of BLV with mutations in gene regions
encoding G4 and R3 were limited in their ability to maintain proviral loads and producing
lymphosarcomas in infected sheep29. These reports taken together with our data indicates
that complex retroviruses associated with lymphoproliferative diseases rely upon accessory gene products to modify their cellular environment to enhance clonal expansion of the virus genome and thus maintain proviral loads in vivo.
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112
Genbank ID Description / Title Apoptosis NM021960 myeloid cell leukemia sequence 1 AF017061 cullin 5 NM000675 adenosine A2a receptor NM004049 BCL2-related protein A1 NM003806 Harakiri NM001197 BCL2/adeno E1B interacting protein 1 BC005034 TSSC 3 AF314174 caspase 2 AL050391 caspase 4 Y09321 TAF4b RNA polymerase II AK001291 NCK-associated protein 1 NM003790 TNF receptor superfamily, member 25 U70056 seven in absentia homolog 1(Drosophila) AF222341 CD 28 antigen U07236 lymphocyte specific PTK (lck) AJ276888 Mdm2 *NM001197 BCL2-interacting killer, BIK *NM014430 cell death-inducing DFFA-like effector b *NM022094 cell death activator CIDE-3 Cell Adhesion BC002630 integrin, beta 8 AI335208 integrin, alpha 6 NM007164 MADCAM1 U02297 selectin P ligand AI797281 desmocollin 3 NM003622 PPFIBP1 (liprin beta 1) AF244129 CD84 (lymphocyte antigen 9) D28586 CD58 antigen X60502 CD43 (sialophorin) *NM000899 KIT ligand
Continued
Table 2.1. Genes modulated by HTLV-1 p30II *Genes upregulated by p30II, indicating that the signal intensity was either increased by a minimum of 1.5 fold or turned on (not expressed in controls) in at least 2 of the 3 p30II expressing samples. Unmarked genes are downregulated by p30II, indicating that the signal intensity was either decreased by a minimum of 1.5 fold or completely shut down in at least 2 of the 3 p30II expressing samples.
113 Table 2.1 continued
Apoptosis NM021960 myeloid cell leukemia sequence 1 AF017061 cullin 5 NM000675 adenosine A2a receptor NM004049 BCL2-related protein A1 NM003806 Harakiri NM001197 BCL2/adeno E1B interacting protein 1 BC005034 TSSC 3 AF314174 caspase 2 AL050391 caspase 4 Y09321 TAF4b RNA polymerase II AK001291 NCK-associated protein 1 NM003790 TNF receptor superfamily, member 25 U70056 seven in absentia homolog 1(Drosophila) AF222341 CD 28 antigen U07236 lymphocyte specific PTK (lck) AJ276888 Mdm2 *NM001197 BCL2-interacting killer, BIK *NM014430 cell death-inducing DFFA-like effector b *NM022094 cell death activator CIDE-3 Cell Adhesion BC002630 integrin, beta 8 AI335208 integrin, alpha 6 NM007164 MADCAM1 U02297 selectin P ligand AI797281 desmocollin 3 NM003622 PPFIBP1 (liprin beta 1) AF244129 CD84 (lymphocyte antigen 9) D28586 CD58 antigen X60502 CD43 (sialophorin) *NM000899 KIT ligand Cell cycle AL545982 chaperonin containing TCP1, subunit 2 β AA860806 checkpoint suppressor 1 NM003672 CDC14 cell division cycle 14 homolog A AF001362 Janus kinase 2 NM003644 growth arrest-specific 7 AI652662 cytosolic BCAT-1 AK022408 rab6 GTPase activating protein
Continued 114 Table 2.1 continued
AJ277546 WEE1 homolog BC005872 histone deacetylase 6 AL518328 basic leucine zipper & W2 domains 1 BE407516 cyclin B1 *AI347136 telomeric repeat binding factor 1 *AW341501 A kinase (PRKA) anchor protein 8 *NM004399 DEAD/H box polypeptide11 *U04045 mutS homolog 2 *BG491844 JUN Transcription NM000281 TCF1 (PCBD, PCD, DCOH) NM003666 basic leucine zipper nuclear factor 1 NM005693 nuclear receptor subfamily1-H, member 3 NM012429 SEC14-like 2 (S. cerevisiae) NM006186 nuclear receptor subfamily4-A member 2 NM000965 retinoic acid receptor, beta NM002518 neuronal PAS domain protein 2 NM004527 mesenchyme homeo box 1 NM014724 zinc finger protein 305 NM003070 SMARCA2 NM003438 zinc finger protein 137 NM002167 ID3 (HEIR-1) NM006079 CITED2 (MRG1) M64240 MAX protein U80737 nuclear receptor coactivator 3 AB006572 chromosome 19 ORF2 X79067 zinc finger protein 36 L1 S79910 homeo box A1 BC004145 trinucleotide repeat containing 4 BE542323 TONDU Y09321 TAF4b RNA polymerase II X59740 zinc finger protein, X-linked U88968 enolase 1, (alpha) NM024567 hypothetical protein FLJ21616 NM013351 T-box 21 AF016005 arginine-glutamic acid dipeptide repeats *NM003489 nuclear receptor interacting protein 1 *AW015313 TBP associated factor I C *NM005341 GLI-Kruppel family member HKR3
Continued 115 Table 2.1 continued
*NM004956 ets variant gene 1 *NM002158 HTLV enhancer factor *AB041834 polyglutamine binding protein 1 *BC000052 peroxisome proliferative activated receptor α *L22179 MLLT2 (AF-4) *AI989477 SRY box 4 *NM021020 leucine zipper, putative tumor suppressor 1 Translation NM004564 PET112-like (yeast) BE138647 translation initiation factor IF2 AI335509 eukaryotic translation elongation factor 1 δ *NM001958 eukaryotic translation elongation factor 1 α 2 T cell activation / signaling NM002389 CD46 NM001963 epidermal growth factor NM005825 RAS guanyl releasing protein 2 AF309082 Protein Kinase D AF074382 IKKγ NM007236 calcium binding protein P22 (CHP) *NM003371 vav 2 oncogene *AF283777 CD72 Other *M29383 IFNγ
116
Figure 2.1. Schematic illustration of lentiviral vectors expressing both p30HA and GFP (sample vector) as bicistronic messages and GFP alone (control vector) from elongation factor 1 alpha promoter. Abbreviations: LTR - Long Terminal Repeats; RRE - Rev Response Element; EF1 α - Elongation Factor 1 alpha promoter; IRES – Internal Ribosome Entry Site; WPRE – Woodchuck Hepatitis Post-transcriptional Regulatory Element.
117
Figure 2.2. Triplicate p30II samples express GFP and p30II while triplicate controls express only GFP. (A) Flow cytometric analysis illustrating the expression of GFP in Jurkat T cells 10 days post spin-infection with lentiviral vectors. Both sample (expressing p30II and GFP) and control (GFP alone) group contains relatively high and similar levels of GFP. (B) RT-PCR demonstrating the expression of p30II in Jurkat T cells 10 days post spin-infection with lentiviral vectors. Jurkat T cells spin-infected with sample vector express p30II while the control vector spin-infected cells do not express p30II. RT-PCR was performed with triplicate samples and controls. GAPDH was used as a control for the integrity of the message.
118
Figure 2.3. Semiquantitative RT-PCR of CHP, JUN and NFATc in controls and p30II expressing Jurkat T lymphocytes (A and B) and primary CD4+ T lymphocyte (C and D) samples. PCR products were separated by electrophoresis (A and C), normalized to GAPDH and quantified by densitometry (B and D). Black bars indicate control and grey bars indicate p30II expressing cells. Data points are mean of triplicates. CHP was downregulated while JUN and NFATc was upregulated by p30II. Fold decrease / increase in activity in the presence of p30II are indicated above each bar.
119
Figure 2.4. p30II activates NFAT, AP-1 and NF-κB transcriptional activity in Jurkat T lymphocytes. Black bars indicate control and grey bars indicate p30II. Data points are mean of triplicate experiments. Fold increase in activity in the presence of p30II is indicated above each bar. p30II increased the NFAT-luc activity from 2.2 to 10.7 fold depending on co-stimulatory treatment e.g., PMA, ionomycin, CD3, CD28 etc. (A), p30II increased NF-κB-luc activity from 3.1 to 11.4 fold (B) and modestly increased the AP-1 driven luciferase reporter gene activity from 1to 5 fold in the presence of co-stimulator treatments (C).
120
CHAPTER 3
MOTIFS OF HUMAN T LYMPHOTROPIC VIRUS TYPE 1 P30II CRITICAL
FOR ITS INTERACTION WITH P300 AND REGULATION OF LTR
TRANSCRIPTIONAL ACTIVITY.
INTRODUCTION
Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult
T cell leukemia/lymphoma (ATL), and HTLV-1-associated myelopathy/ tropical spastic
paraparesis (HAM/TSP) and many immune-mediated disorders48,26. Molecular
mechanisms used by the virus to circumvent immune elimination by the host and to
facilitate lymphocyte proliferation are not fully understood. The genome of HTLV-1
encodes Gag, Pol, and Env, the common structural and enzymatic proteins characteristic
of all retroviruses. Using alternative splicing mechanism and internal initiator codons,
this complex retrovirus makes several regulatory and accessory proteins encoded by four
open reading frames (ORFs) of the pX region (pX ORF I to IV) between the env gene
and the 3’ long terminal repeat16. ORF III and IV encode the well characterized Rex and
Tax proteins respectively. Rex is a nucleolar-localizing phosphoprotein, involved in
121 nuclear export of unspliced or singly spliced viral RNA21 while Tax is a nuclear-
localizing phosphoprotein, which interacts with cellular transcription factors and activates
transcription from the viral promoter containing repeats of TRE and enhancer elements of
various cellular genes associated with host cell proliferation18,34,37,38,47. pX ORFs I and II
also produce alternatively spliced forms of mRNA, which encode four accessory
proteins, p12I, p27I, p13II, and p30II7,12,17,28. Less is known about the role of pX ORF I
and ORF II in the replication or pathogenesis of HTLV-1. However, pX ORFs I and II
mRNAs are present in infected cell lines and freshly isolated cells from HTLV-1-infected
subjects 28 and in ATL and HAM/TSP patients10. Antibodies11,15 and cytotoxic T cells36 against recombinant proteins or peptides of the pX ORF I and II proteins are present in
HTLV-1 infected patients, and asymptomatic carriers.
We have reported that mutations in the ACH.p30II viral clone that insert an
artificial termination codon in the p30II mRNA prevent the virus from obtaining typical
proviral levels in the rabbit model of infection41. p30II contains a highly conserved bipartite nuclear localization signal14 as well as serine-and-threonine-rich regions with
distant homology to transcription factors Oct-1 and -2, Pit-1, and POU-M112. Taken
together, these characteristics suggest that p30II has a role in transcription of viral and
cellular gene expression. In addition, a recent report demonstrated that p30II modulates
LTR mediated transcription, in the context of the entire provirus, at the post-
transcriptional level, by retaining Tax/Rex mRNA in the nucleus33. We have previously
reported that, when provided in limiting concentrations, p30II expression stimulated
HTLV-1 LTR-driven reporter gene activity, even in the presence of Tax, whereas higher
concentrations repressed LTR and CRE-driven reporter gene activity50. 122 We have shown that p30II is co-localized with p300 in the nucleus and physically
interacts with CBP/p300, at the highly conserved KIX domain, the domain HTLV-1 Tax
also interacts with. CBP/p300 protein is available only at limiting concentrations within
the cell nucleus, causing an environment for competition between coactivators and
transcription factors, thus providing an additional layer of regulated gene expression35,43.
There is evidence of a functional antagonistic relationship between transcription factors, as a result of competition for binding to common regions of CBP/p3005,13,24. Under such
a condition of tight competition, relative concentrations of Tax/ p30II at various stages of
disease might be a critical factor in determining the levels of viral transcription. We have
previously demonstrated that p30II disrupts CREB-Tax-CBP/p300 complexes bound to
the TRE repeats49. At higher concentrations, p30II may support viral persistence by
reducing viral expression, thus reducing immune elimination of HTLV-1 infected cells
and at low concentrations, enhance TRE/viral over CRE/cellular mediated transcription,
thus promote viral transcription and spread of the virus in vivo. This is also consistent
with our previous results showing that an infectious HTLV-1 molecular clone failed to
maintain viral loads in vivo when p30II expression was abolished41.
Identifying the molecular mechanism and functional significance of the
interaction between p30II and p300 is crucial in understanding the role of p30II in the
pathogenesis and replication of HTLV-1. In this study, we have further characterized the
nature of this interaction, by identifying the motifs within p30II that are critical in binding
CBP/p300 and in regulating LTR mediated transcription. Using N terminal and C terminal deletion mutants of p30II, we have localized the binding site of p300 on p30II.
123 These serial deletion mutants were employed to identify the domain of p30II, which is
important in regulating LTR mediated transcription. Using our serial deletion mutants of
p30II, we identified the domain of p30II that is involved in regulating LTR mediated
transcription, in the context of the provirus. By dissecting this amino acid domain of p30II
(100-179) critical for repressing LTR mediated transcription further, we identified that lysine residue at amino acid position 106 (K3) of HTLV-1 p30II is critical for its
repression of TRE-mediated transcription. More importantly, by adding p300, we were
able to rescue the p30II-mediated repression on LTR driven gene transcription, irrespective of the presence or absence of the provirus. Our data confirms the role of p30II as a regulator viral gene transcription, in association with p300. This is the first report that dissects p30II and identifies the functional domains important for binding CBP/p300
and in regulating LTR mediated transcription. Furthermore, we found that HTLV-1 p30II represses Tax-mediated LTR transactivation, while HTLV-1 Tax rescues p30II-mediated repression of LTR driven transcription, indicating the ability of these proteins to compete with each other in modulating HTLV-1 LTR transcriptional activity. Taken together, our data indicates that HTLV-1, a complex retrovirus associated with lymphoproliferative disorders, uses accessory genes, in conjunction with regulatory proteins, to promote cell- to-cell transmission of the virus, clonal expansion of infected cells and maintenance of proviral loads in vivo.
124 MATERIALS AND METHODS
Cell lines.
293T cells were obtained from G. Franchini (National Cancer Institute, NIH,
Bethesda) and Hela-Tat cells were obtained from AIDS Research and Reference Reagent
Program, National Institute of Health. 293T and Hela-Tat cells were grown in modified
Dulbecco’s eagle medium containing 10% fetal bovine serum and 1% streptomycin and
penicillin at 37°C. Cells were split and cultured in six-well plates, 10 cm dishes or
chamber slides to 50-60% confluence 16-18 h before transfection.
Plasmids.
The pTRE-Luc plasmid and pRSV-B-Gal have been described previously49,50. pME-p30IIHA wildtype and serial deletion mutant plasmids were created by cloning the
p30II sequence from HTLV-1 molecular clone, ACH with downstream influenza
hemagglutinin (HA1) tag, into pME-18S plasmid (G. Franchini, National Cancer
Institute) between 5’ EcoRI and 3’ NotI sites. Site directed mutants were made by
substituting lysines in pMEp30IIHA with arginine. Fidelity of the plasmids were
confirmed by Sanger sequencing and p30II HA protein expression was confirmed by
western blot using monoclonal anti-HA antibody (Covance, Berkeley, CA). ACH.30
plasmid has a 24 bp insertion that causes an artificial termination codon in p30II40. pCMV-Tax expresses the HTLV-1 Tax protein and has been described previously32. pCMV-p300 expresses the full-length p300 protein from a CMV I/E promoter (Upstate
USA, Inc., Charlottesville, VA).
125 Cell transfection and reporter gene assay.
For each transfection, 0.3 µg of pTRE-Luc reporter plasmid was cotransfected with pME-p30IIHA wildtype or serial deletion / arginine substitution mutant plasmids and
0.4 µg ACH.30 plasmid when specified, using Lipofectamine Plus (Invitrogen,
Carlsbad, CA). When pCMV-p300 plasmid was used, it was also co-transfected. As an
internal control for transfection efficiency, 0.1 µg of pRSV-βGal (Invitrogen) was also
used in each transfection. pME 18S was used as carrier DNA to equalize DNA
concentrations for each transfection. Transfected cells were lysed with 400 µl lysis buffer
(Promega, Madison, WI) per well for 25 min. Twenty microliters of each lysate was used
to test luciferase reporter gene activity using an Enhanced Luciferase Assay kit
(Promega). To normalize transfection efficiency, cells were washed with PBS, stained
with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) (Sigma, St. Louis,
MO) and counted βGal expressing cells using 20X objective of light microscope. Co-
transfection of pME-p30IIHA had no effect on the X-Gal staining (data not shown).
Results were expressed as mean of optimized luciferase activity (luciferase activity /
percentage cells stained positive for β-Gal expression) in arbitrary light units (ALU) with
standard error (SE) from a minimum of triplicate experiments. Statistical analysis was
done using Student’s t test, P < 0.05.
Western immunoblot assay.
Transiently transfected cells were lysed in RIPA buffer containing PBS, 1%
Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Cell
126 lysates were prepared by centrifugation at 14,000 rpm (Beckman, Fullerton, CA) for 20
min at 4° C. Equal amounts of proteins were mixed with Laemmli buffer (62.5 mM Tris;
pH 6.8), 2 % SDS, 10 % glycerol, 0.2 % bromophenol blue, 100 mM dithiothreitol
(DTT). After boiling for 5 min, samples were electrophoresed through 6 or 10%
polyacrylamide gels. The fractionated proteins were transferred to nitrocellulose
membranes (Amersham Pharmacia Biotechnology, Piscataway, NJ) at 100 V for 1 h at 4°
C. Membranes were then blocked in Tris-buffered saline containing 5 % nonfat milk and
0.1 % Tween 20. Proteins were detected with primary monoclonal anti- hemagglutinin
(HA) antibody (Covance) followed by an anti-mouse (Cell Signaling Technology,
Beverly, MA) immunoglobulin G (IgG)-horseradish peroxidase-conjugated goat
antibody. Blots were developed using an enhanced chemiluminescence detection system
(Cell Signaling Technology).
Coimmunoprecipitation of p30II with p300.
Sixty percent confluent 293T cells were cotransfected by pME-p30IIHA, pME-
p30IIHA serial deletion or arginine substitution mutants and pCMV-p300 using
Superfect (Gibco BRL, Gaithersburg MD). After 48 h, the transfected cells were washed with PBS and resuspended in 400 µl of lysis buffer containing 50 mM Tris-HCl
(pH 8.0), 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 µg of leupeptin per ml, and 1 µg of aprotinin per ml. Cell suspensions were incubated on ice for
20 min and then lysed by homogenization. The lysates were centrifuged at 14,000 rpm for 20 min at 4° C and incubated overnight at 4° C with 100 ng of a polyclonal HA antibody (Covance). After adding 40 µl of 100 % of protein A-agarose slurry, the mixture
127 was incubated at 4° C for 1 h. The immunoprecipitated complexes were washed twice
with 10 volumes of lysis buffer and three times with PBS buffer. The components of the
complexes were resolved on 6% SDS-polyacrylamide gels and detected by western
immunoblot assay using anti-p300 antibody (Santa cruz biotechnology, Santa cruz, CA).
The density of each band was measured and compared to wildtype p30II using a commercial software package (Gel-pro Analyzer software; Media Cybernatic Inc., San
Diego, CA). Normal rabbit IgG was used as a negative control in immunoprecipitation assays and equal amounts of input cell extracts were processed to determine the expression of p30II by western immunoblot assay.
Localization of p30II and co-localization with CBP/p300 by confocal microscopy.
To detect cellular colocalization of p30II and p300 by immunofluorescence, Hela
Tat cells were seeded in two chamber slides (Fisher Scientific) at approximately 50 %
confluence 18 h prior to transfection. Transfection with 4 µg of pME-p30II-HA wildtype
or pME-p30IIHA serial deletion mutants alone or with 2 µg of pCMV-p300 was performed using Superfect (Gibco BRL). At 48 h posttransfection, media were removed and cells were fixed for 15 min using 4 % paraformaldehyde. Cells were then incubated with FITC (Fluorescin isothyocyanate) conjugated anti-HA antibody alone (diluted
1:1000; Santa Cruz) or with TRITC (Texas red isothyocyanate) conjugated anti-p300
antibody (diluted 1:1000; Molecular probes) in antibody dilution buffer containing 0.01
M sodium phosphate, 0.5 M NaCl, 0.5 % Triton-X 100 and 2 % bovine serum albumin overnight at 4° C. The expression of p30II-HA and p300 was evaluated using Zeiss LSM
510 confocal microscope.
128 RESULTS
HTLV-1 p30II localizes to the nucleus and colocalizes with p300 in the nucleus.
HTLV-1 p30II contains a nuclear localization signal, characteristic of most
proteins that function as a transcription factor. Using imunofluorescence and immunoblot
methods, previous reports have shown that HTLV-1 p30II was localized in the nucleus of
transfected cells27,50. To confirm the subcellular localization of p30II, we performed
confocal microscopy on HeLa-tat cells transiently transfected with pMEp30IIHA. Anti-
Phalloidin was used to stain the cytokeratin of the cells (Fig 3.1A). Consistent with previous reports, p30II was detected predominantly in the nucleus of our transiently
transfected HeLa-tat cells by confocal microscopy (Fig. 3.1B). Based on nuclear
localization, as typical of proteins that play a role in transcription, and the sequence homology of p30II with the POU family of transcription factors, we hypothesized that
p30II has a role in transcriptional regulation. To test whether p30II colocalizes with p300
in the nucleus, we performed confocal microscopy of Hela-Tat cells transiently
cotransfected with pME p30IIHA and pCMV-p300, and verified the subcellular
localization of p30II (Fig. 3.1B) and p300 (Fig. 3.1C). Hoechst was used as a nuclear
marker (Fig. 3.1D) and we found that p30II-HA colocalized with p300 in the nucleus of
cells that expressed both proteins (Fig. 3.1E).
129 HTLV-1 p30II physically interacts with p300
HTLV-1 p30II contains a bipartite nuclear localization sequence and regions with
sequence homology to the DNA binding domain and homeodomain of transcription
factors such as Oct-1 (Fig. 3.2A). To test if p30II physically interacts with the cellular
transcriptional co-activator p300, we transiently transfected 293T cells with pCMV-p300
and pME p30IIHA plasmid encoding full-length p30II, prepared whole-cell extracts and
performed co-immunoprecipitation assays. Our data indicated that p300 was
immunoprecipitated as a complex with p30II (Fig. 3.2B). In contrast, p300 could not be
detected in the complexes precipitated using preimmune serum while p30IIHA was detected from the cellular lysate (data not shown).
Amino acids 1-132 of HTLV-1 p30II are critical for its physical interaction with p300
To identify the domain of p30II that is required in its interaction with p300, we
performed transient co-transfection of 293T cells with pCMV-p300 and various amino-
terminal and carboxyl-terminal serial deletion mutants of p30II, and whole-cell extracts
were used for coimmunoprecipitation assays. These mutants of p30II contained serial
deletions of both the amino-terminal and carboxyl-terminal regions of p30II (Fig. 3.2A).
Each of the p30II mutant proteins was expressed at the expected molecular weight, as indicated by immunoblot analysis (Fig. 3.2B). The subcellular localization characteristics
of these mutants were tested by confocal microscopy. Mutants containing amino acids 1-
220, 1-179, 1-132 or 71-220 localized to the nucleus while mutants containing amino acids 1-71, 100-179 and 179-241 localized to the cytoplasm (Fig. 3.3). As seen in Fig. 130 3.2B, we have confirmed that amino acids 1-132 of p30II are critical for binding p300.
The p30II mutants 1-132, 1-220 and 1-179 (in the decreasing order) appeared to interact
with p300 more intensely compared to the wildtype p30II.
HTLV-1 p30II modulates LTR driven transcription.
To test the effect of p30II on LTR mediated transcription, we transiently transfected increasing concentrations of pME p30IIHA plasmid with constant amounts of
our LTR-luc reporter plasmid and tested for luciferase reporter activity. We found that
lower concentrations of the p30II plasmid (below 0.6 µg) consistently activated the
HTLV-1 LTR reporter gene activity, but increased amounts (above 0.6 µg) of the plasmid repressed LTR reporter gene activity (Fig. 3.4A). Consistent with our previous report, this data confirms that at low concentrations, p30II has the potential to enhance LTR-
mediated transcription while at higher doses, p30II represses the LTR-mediated
transcription 50.
We tested the ability of p30II to regulate LTR mediated transcription in the presence of the HTLV-1 provirus. By transiently transfecting pME-p30IIHA at various
doses with ACH.30, provirus deleted for p30II expression, we have found that p30II represses LTR mediated transcription, in a dose responsive fashion (Fig. 3.4B).
Interestingly, at concentrations that activated LTR mediated transcription in the absence of ACH.30 (below 0.6 µg), we found that p30II indeed repress LTR mediated
transcription if the provirus is present. This difference could be due to the presence of
other HTLV-1 proteins in the provirus.
131 p300 expression reverses the p30II-dependent repression of LTR driven
transcription, in the presence or absence of provirus, in a dose-responsive manner.
HTLV-1 Tax is known to activate the LTR. This transactivation function of
HTLV-1 Tax is dependent on the co-adaptors CBP/p300. Tax is reported to bind p300 at
the KIX domain, the same domain that p30II binds to. Our previous reports suggested that
p30II and Tax serve different roles in the regulation of transcription. Based on the
hypothesis that p30II represses LTR-driven reporter gene activities as a consequence of
competition for limited basal quantities of CBP/p300, we tested if overexpressing p300
rescues p30II-mediated repression of LTR-driven reporter gene expression. By transiently
transfecting increasing concentrations of pCMV-p300 with constant concentration of
pME-p30IIHA plasmid, herein, we demonstrated that p300 expression reverses the p30II- dependent repression of LTR-luciferase reporter gene activity, in the absence of the provirus (Fig. 3.5A). This data confirms that the transcriptional regulatory effect of p30II is p300 dependent.
To test if the p30II mediated repression of the LTR-mediated transcription is p300
dependent in the context of the provirus, we transiently transfected increasing amounts of
pCMV-p300 plasmid with constant amounts of pME-p30IIHA plasmid and ACH.30, the provirus deleted for p30II expression. Interestingly, even in the presence of the provirus,
increasing doses of p300 reversed the ability of p30II to repress the LTR mediated
transcription, in a dose responsive fashion (Fig. 3.5B). Taken together, this data further
confirm that the transcriptional regulatory function of p30II on the LTR is p300
dependent, in the presence of the provirus as well.
132 HTLV-1 p30II represses Tax-mediated LTR transactivation and HTLV-1 Tax
rescues p30II-mediated repression of LTR driven transcription
Since HTLV-1 p30II and Tax bind CBP/p300 at the KIX domain and since the
transcriptional regulatory functions of p30II and Tax on the LTR are p300 dependent, we
hypothesized that HTLV-1 Tax and p30II might competitively influence the
transcriptional regulatory function of each other on the HTLV-1 LTR. To test the effect
of p30II on Tax-mediated LTR transactivation, we transiently co-transfected increasing
concentrations of pME p30IIHA plasmid with constant concentration of LTR-luc reporter
plasmid, as well as pCMV-Tax plasmid and tested the luc reporter activity. We found that p30II consistently repressed the Tax transactivation of the HTLV-1 LTR reporter gene
activity, in a dose dependent manner (Fig. 3.6A). Conversely, to test the effect of HTLV-
1 Tax on p30II-mediated repression of the LTR transcriptional activity, we transiently co-
transfected increasing concentrations of pCMV-Tax plasmid along with constant
concentration of LTR-luc reporter plasmid as well as pME p30IIHA plasmid and tested
the luc reporter activity. We found that Tax consistently rescued the p30II-mediated repression of the LTR reporter gene activity, in a dose dependent manner (Fig. 3.6B).
HTLV-1 p30II contains multiple domains critical for its repression of LTR driven
transcription
To identify the structural domain of p30II that is critical for repression of LTR
driven transcription, 293T cells were transiently co-transfected with a series of carboxyl-
terminal and amino-terminal truncation mutants of p30II and LTR-luciferase reporter 133 plasmid, and was tested for their ability to reduce the LTR-luciferase activity. The
luciferase activity elicited by LTR-luciferase construct, in the presence of the full-length
p30II was compared to luciferase activity in the presence of each of the serial deletion mutants (Fig. 3.7A). Luciferase activity elicited by the serially deleted mutants 1-220, 1-
179, 1-132 and 1-71 were higher than that by the full-length p30II (in the decreasing order
of luciferase activity), while in the presence of mutants 179-241, 100-179 and 71-220 (in
the decreasing order of luciferase activity), the luciferase activity was less than that in the
presence of the wildtype p30II. This data shows that the region between 71-220 is able to
repress the LTR-luc reporter, even more than the wildtype p30II, indicating that these
amino acid sequences are critical for the repression of LTR driven transcription by p30II.
However, mutant containing amino acid sequences 1-220 appeared to elicit the highest
LTR driven reporter activity, which suggests the existence of a domain that inhibits the transcriptional repressive function of p30II in this mutant, which is likely amino acids 1-
71. This is also consistent with the higher luciferase activity elicited by the mutant
containing only amino acids 1-71, compared to the wildtype p30II. However, it appears
that amino acids 1-71 do not inhibit the repressive function of the wildtype p30II. A
comparison of 1-220 mutant p30II and the wildtype p30II raises the possibility of another
critical domain at amino acids 220-241, which helps the wildtype overcome the
inhibitory role by amino acids 1-71. This is also consistent with the increased ability of
the mutant containing 179-241 to repress LTR mediated transcription, compared to the
wildtype. However, we cannot rule out the possibility of a change in the physical
conformation or other properties of the mutant proteins, causing the observed differences
in their transcriptional activity. These data indicate that the amino acid sequence from 71-
134 220 of p30II is important for its function as a repressor of LTR-mediated transcription.
The mutant containing amino acid sequence 100-179 was also able to repress LTR
mediated transcription at levels similar to that of 71-220. The differences in activity
between these mutant was not statistically significant (p < 0.15), and therefore, the amino
acids 100-179 appears to be the structural motif within p30II, most critical for its
repression of LTR mediated reporter activity.
To determine if 100-179 itself is the structural domain that is critical for
repressing LTR mediated transcription, in the presence of the provirus, we performed
transient transfection of 293T cells with ACH.30, LTR-luc and the carboxyl- terminal
and amino-terminal truncation mutants of p30II described above and measured the luciferase activity. The luciferase activity elicited by LTR-luciferase construct, in the presence of the full length / wildtype p30II, in the context of the provirus was then
compared to luciferase activity in the presence of each of the serial deletion mutants (Fig.
3.7B). Similar to the findings in the absence of the provirus, luciferase activity elicited by
the serially deleted mutants 1-179, 1-220, 1-71 and 1-132 were higher than that by the
full-length p30II (in the decreasing order of luciferase activity), and in the presence of
mutants 100-179, the luciferase activity was less than that in the presence of the wildtype
p30II. Taken together, these data indicate that the amino acid sequence 100-179 of p30II is
critical in repression of LTR-mediated transcription in the presence or absence of the
HTLV-1 provirus.
135 Lysine 106 (K3) of HTLV-1 p30II is critical for its repression of LTR driven
transcription.
HTLV-1 p30II contains 6 lysine residues (K1-K6) (Table 3.1). Interestingly, 5 of
these are within the critical domain comprised of amino acids 100-179. Importantly, four
(K2-K5) of the lysine residues within this domain are part of a consensus acetylation
sequence (G/SK motif),6 representing potential acetylation sites for CBP/p300. To
determine the role of these lysine residues in p30II-mediated repression of LTR driven transcription, 293T cells were transiently co-transfected with the arginine substitution
mutants for each lysine residue of p30II and LTR-luciferase reporter plasmid, and was
tested for their ability to reduce the LTR-luciferase activity. The luciferase activity
elicited by LTR-luciferase construct, in the presence of the full-length p30II was
compared to luciferase activity in the presence of each of the arginine substitution
mutants (Fig. 3.8A). Luciferase activity elicited by the arginine substitution mutant K3
(amino acid 106) was higher than that by the full-length p30II, pME empty plasmid,
arginine substitution mutant K2 (amino acid 103), arginine substitution mutant K4
(amino acid 123) and arginine substitution mutant K5 (amino acid 143) (in the decreasing
order of luciferase activity), indicating a loss of repression function with substitution of
lysine at amino acid position 106. This data shows that the lysine residue at amino acid
position 106 is critical for repressing the LTR-luc reporter.
To determine the role of these lysine residues in p30II-mediated repression of LTR
driven transcription, in the presence of the provirus, we performed transient transfection
of 293T cells with ACH.30, LTR-luc and the arginine substitution mutants of p30II 136 described above and measured the luciferase activity. The luciferase activity elicited by
LTR-luciferase construct, in the presence of the full length / wildtype p30II, in the context
of the provirus was then compared to luciferase activity in the presence of each of the
arginine substitution mutants (Fig. 3.8B). Similar to the findings in the absence of the
provirus, luciferase activity elicited by the arginine substitution mutant K3 (amino acid
106) was higher than that by the full-length p30II, pME empty plasmid, arginine
substitution mutant K2 (amino acid 103), arginine substitution mutant K4 (amino acid
123) and arginine substitution mutant K5 (amino acid 143) (in the decreasing order of
luciferase activity), indicating a loss of repression function with substitution of lysine at
amino acid position 106. This data confirms that the lysine residue at amino acid position
106 is critical for repressing the LTR-luc reporter in the presence of the provirus as well.
Taken together, these data indicate that the lysine residue at amino acid position 106
within p30II is important for its function as a repressor of LTR-mediated transcription in the presence or absence of the HTLV-1 provirus.
DISCUSSION
Our data is the first to dissect p30II and demonstrate the functional motifs of p30II that are critical in binding p300 and in repressing LTR mediated transcription.
Identification of these motifs is important for defining the molecular mechanism of p30II- mediated repression of LTR-driven transcription. This information is vital in gaining more insight into HTLV-1 gene expression and pathogenesis. We found that the motif
137 critical for binding p300 is amino acid sequence 1-132 while the motif that plays a major
role in repressing LTR-mediated transcription is amino acid sequence 100-179, which
contains the entire DNA binding region, part of the homeodomain with homology to Oct-
1 and POU family of transcription factors as well as 5 lysine residues, four of which are
part of a G/SK consensus acetylation sequence6 representing potential acetylation sites
for CBP/p300.
The coactivators CREB binding protein (CBP) and p300 mediate transcriptional
control of various cellular and viral DNA binding transcription factors. Although these coactivators have divergent functions, they are similar in nucleotide sequence, are evolutionarily conserved, and are commonly referred to as CBP/p3009,19. CBP and p300
are highly related and share many functional properties, however there is evidence that
these factors are not interchangeable45. Several cellular and viral proteins that interact
with either CBP or p300 have been identified, including steroid and retinoid hormone
receptors, CREB, c-Jun, c-Myb, Sap-1a, c-Fos, MyoD, p53, Stat-1/2, NF-κB, pp90rsk,
TATA-binding protein, TFIIB4,19,22,23, HTLV-1 Tax, adenovirus E1A, Kaposi’s sarcoma-
associated herpes virus viral interferon regulatory factor protein, and simian virus 40
large T antigen1-3,13,25,29,30,43.
CBP and p300 bridge transcription factors to relevant promoters, has intrinsic
histone acetyltransferase (HAT) activity, and form complexes with proteins such as
CBP/p300 binding protein-associated factor, which also exhibits HAT activity20.
Recently, there is increasing knowledge of the mechanism and functional significance of
138 the interactions between many viral proteins and CBP/p300. In the case of adenovirus
oncoprotein E1A, interaction with CBP/p300 is critical for regulation of transcription,
suppression of differentiation, and immortalization of cells in culture1,3,31. Simian virus T
antigen regulates the expression of a group of cellular genes by modifying the HAT activity of CBP/p300 or by bridging the gap between DNA binding transcription factors
and components of the general transcription machinery4,19. Identifying the molecular
mechanism and functional significance of the interaction between p30II and p300 is very
crucial in understanding of the role of p30II in the pathogenesis and replication of this important human pathogen.
Previously, using deletion mutants of CBP/p300 in GST pull-down assays, we localized the binding site of CBP/p300 for p30II to a highly conserved KIX region.
Interestingly, KIX domain is the binding site of CBP/p300 for HTLV-1 Tax protein as well. In addition, p30II was able to disrupt CREB-Tax-CBP/p300 complexes bound to the
viral 21-bp TRE repeats49. These results indicate that p30II might act in contrast to the role of HTLV-1 Tax. The role of CBP and p300 coactivators in HTLV-1 gene expression has been the focus of many previous reports. HTLV-1 Tax, a transactivator of LTR mediated transcription, is critical in the activation of the HTLV-1 viral genes through its interaction with the p300 and CBP coactivators8.
Although CBP and p300 mediate the activities of various transcription factors,
many earlier reports prove that it is present in the cell only at limiting concentrations.
Even small reductions in the concentrations of these coactivator are damaging in many
139 instances, like the human Rubinstein-Taybi syndrome where loss of a single CBP allele
causes developmental defects35. In experiments where transcription factors are
overexpressed, it is possible that the capacity of the endogenous CBP/p300 is overridden
and the effects due to competition may be more obvious. Many such studies have
demonstrated the competition between different molecules for CBP/p300. Even though
some of these involve two mutually antagonistic transcription factors with the same
shared binding site on CBP/p300, competition does not necessarily require having the
same binding site. In circumstances where CBP/p300 levels are limiting, there could be
selective preference of one over the other, causing an exclusion of one of the proteins. In
our study, we found that HTLV-1 p30II and Tax appear to compete with each other in
modulating the transcriptional activity from the LTR. Since HTLV-1 p30II and Tax interacts with CBP/p300 through the same KIX domain, we believe that this is through competitive CBP/p300 binding between p30II and Tax. Similar mechanism involving
Tax and CBP/p300 has been previously reported. The binding of Tax and c-Myb to KIX
domain of CBP was found to be mutually exclusive and Tax expression was shown to
interfere with transcriptional activity of c-Myb13.
An environment of coactivator competition between transcription factors provides
an additional layer of regulated gene expression. The relative amounts of Tax and p30II may be critical in determining which protein binds to CBP/p300 and the consequent regulation of LTR mediated transcription of viral genes from the HTLV-1 viral promoter.
When its levels are low, p30II also enhance transcription from the viral promoter. Under
such circumstances, p30II and Tax might act synergistically. However, at higher
140 concentrations, p30II antagonizes the Tax transactivation of the HTLV-1 promoter. There
are similar reports on positive and negative effects by the same protein in other viruses,
such as herpes simplex virus type 1 (HSV-1), in which the interaction between cellular
and viral transcription factors play a critical role in the regulation of the immediate-early
(IE) gene promoter. VP16, a potent transcription factor from HSV-1, binds the host cell
protein HCF, which facilitates the stable complex formation of the viral protein with Oct-
146. The IE gene promoter contains an Oct-1-like motif (TAATGARAT) that is important
for IE gene expression, with both positive and negative effects, depending on the context
of these cellular transcription factors and VP1644.
CBP and p300 bridge transcription factors to relevant promoters and through its
intrinsic histone acetyl transferase (HAT) activity, acetylates lysine residues at the amino
terminus of histones, which results in modification of chromatin structure to allow access
of the transcriptional machinery. CBP/p300 is known to form complexes with proteins
such as CBP/p300 binding protein-associated factor, which also exhibits HAT activity20. p300 also directly acetylate transcription factors such as p53, GATA-1 and c-myb and thus augment their sequence-specific DNA-binding activity42. Simian virus T antigen
regulates the expression of a group of cellular genes by modifying the HAT activity of
CBP/p300 or by bridging the gap between DNA binding transcription factors and
components of the general transcription machinery4,19. c-myb oncoprotein of the avian
retrovirus group is known to be acetylated by CBP/p300 at its multiple lysine residues13.
This is considered to be the mechanism by which CBP/p300 regulates the functions of c- myb. p30II contains six highly conserved lysine residues, 5 of which are within the amino
141 acid region 100-179, four of which are part of the consensus acetylation sequence (G/SK
motif)6 and thus represent potential acetylation sites for CBP/p300 (Table. 3.1). We
found that the lysine residue at amino acid position 106 (K3) of HTLV-1 p30II is critical
for its repression of LTR driven transcription in the presence or absence of the provirus.
Based on this, it is possible that the intrinsic HAT activity of CBP/p300 is utilized to
acetylate and potentially modulate the transcriptional regulatory function of p30II.
Recently, it was reported that p30II modulates LTR mediated transcription, in the context of the entire provirus, by a post-transcriptional mechanism 33. Previous studies
from our laboratory demonstrated that HTLV-1 p30II directly interacts with CBP/p300 to
modulate gene transcription49,50. Data presented in this report further confirms the role of
CBP/p300 in the modulation of LTR mediated transcription by p30II, in the context of the
provirus. In the presence of increasing concentrations of p300, there was a dose-
dependent rescue of LTR driven gene transcription, irrespective of the presence or absence of the provirus. Although, we cannot rule out the possibility of a post- transcriptional mechanism by HTLV-1 p30II, this report presents convincing evidence for
a p300 dependent transcriptional regulatory function. Therefore, HTLV-1 p30II appears to
be a multi-functional protein with transcriptional and post-transcriptional role in regulating gene expression.
As of yet, there is not much information regarding the relative levels of various
HTLV-1 proteins during various stages of the infection. It is possible that differences in
expression levels of various viral proteins, leading to differences in transcriptional
142 regulation as described above might be the mechanism by which HTLV-1 infection/ disease progresses though various stages39. This is likely to be in synergy with differential regulation of cellular gene regulation by Tax and/or p30II and thus changing the immune responses in accordance with different stages of progression of HTLV-1 infection and disease. Our current data can be supported by recent findings from our laboratory where an infectious HTLV-1 molecular clone failed to maintain viral loads in vivo when p30II expression was abolished41. Taken together, our data indicates that
HTLV-1, a complex retrovirus associated with lymphoproliferative disorders, uses accessory genes to promote cell-to-cell transmission of the virus, clonal expansion of infected cells and maintenance of proviral loads in vivo.
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37. Pise-Masison, C. A., R. Mahieux, M. Radonovich, H. Jiang, and J. N. Brady. 2001. Human T-lymphotropic virus type I Tax protein utilizes distinct pathways for p53 inhibition that are cell type-dependent. J. Biol. Chem. 276:200-205.
38. Pise-Masison, C. A., R. Mahieux, M. Radonovich, H. Jiang, J. Duvall, C. Guillerm, and J. N. Brady. 2000. Insights into the molecular mechanism of p53 inhibition by HTLV type 1 Tax. AIDS Res. Hum. Retroviruses 16:1669-1675.
39. Princler, G. L., J. G. Julias, S. H. Hughes, and D. Derse. 2003. Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs. Virology 317:136-145.
40. Robek, M. D., F. H. Wong, and L. Ratner. 1998. Human T-Cell leukemia virus type 1 pX-I and pX-II open reading frames are dispensable for the immortalization of primary lymphocytes. J. Virol. 72:4458-4462.
41. Silverman, L. R., A. J. Phipps, A. Montgomery, L. Ratner, and M. D. Lairmore. 2004. Human T-cell lymphotropic virus type 1 open reading frame II- encoded p30II is required for in vivo replication: evidence of in vivo reversion. J Virol 78:3837-3845.
42. Sterner, D. E. and S. L. Berger. 2000. Acetylation of histones and transcription- related factors. Microbiol. Mol. Biol. Rev. 64:435-459.
43. Suzuki, T., M. Uchidatoita, and M. Yoshida. 1999. Tax protein of HTLV-1 inhibits CBP/p300-mediated transcription by interfering with recruitment of CBP/p300 onto DNA element of E-box or p53 binding site. Oncogene 18:4137- 4143.
44. Thomas, S., R. Coffin, P. Watts, G. Gough, and D. S. Latchman. 1998. The TAATGARAT motif in the herpes simplex virus immediate-early gene promoter can confer both positive and negative responses to cellular octomer-binding proteins when it is located within the viral genome. J. Virol. 72:3495-3500. 147 45. Vo, N. and R. H. Goodman. 2001. CREB-binding protein and p300 in transcriptional regulation. J. Biol. Chem. 276:13505-13508.
46. Wilson, A. C., R. W. Freeman, H. Goto, T. Nishimoto, and W. Herr. 1997. VP16 targets an amino-terminal domain of HCF involved in cell cycle progression. Mol. Cell. Biol. 17:6139-6146.
47. Yin, M. J. and R. B. Gaynor. 1996. Complex formation between CREB and tax enhances the binding affinity of CREB for the human T-cell leukemia virus type 1 21- base-pair repeats. Mol. Cell Biol. 16:3156-3168.
48. Yoshida, M. 1996. Molecular biology of HTLV-I: recent progress. J. Acquir. Immune. Defic. Syndr. Hum. Retrovirol. 13 Suppl 1:S63-8:S63-S68.
49. Zhang, W., J. W. Nisbet, B. Albrecht, W. Ding, F. Kashanchi, J. T. Bartoe, and M. D. Lairmore. 2001. Human T-lymphotropic virus type 1 p30(II) regulates gene transcription by binding CREB binding protein/p300. J. Virol. 75:9885-9895.
50. Zhang, W., J. W. Nisbet, J. T. Bartoe, W. Ding, and M. D. Lairmore. 2000. Human T-lymphotropic virus type 1 p30(II) functions as a transcription factor and differentially modulates CREB-responsive promoters. J. Virol. 74:11270-11277.
148
Lysine residue and amino acid position within p30II Percentage conservation
K1 (50) 87.5
K2 (103) 66.7
K3 (106) 87.5
K4 (123) 87.5
K5 (143) 91.7
K6 (152) 91.7
Table 3.1. Lysine residues in p30II are conserved among various HTLV isolates. Amino acid position of each lysine is indicated in parenthesis.
149
Figure 3.1. p30II localizes to the nucleus and colocalizes with p300 in the nucleus of transiently transfected HeLa-Tat cells. Hela Tat cells were seeded in two chamber slides (Fisher Scientific) at approximately 50 % confluence 18 h prior to transfection, and co- transfected with 4 µg of pME-p30II-HA and 2 µg of pCMV-p300 using Superfect (Gibco BRL). At 48 h post-transfection, media were removed and cells were fixed for 15 min using 4 % paraformaldehyde. Cells were then incubated with FITC conjugated anti-HA antibody (diluted 1:1000; Santa Cruz) and TRITC conjugated anti-p300 antibody (diluted 1:1000; Molecular probes) overnight at 4° C. Panel A represents the cytokeratin staining of the cells using anti-Phalloidin while Panel D represents the nuclear staining of the cell using Hoechst. The expression of p30II-HA (Panel B) and p300 (Panel C) were evaluated using Zeiss LSM 510 confocal microscope (40X) and the images were overlayed using Adobe photoshop (Panel E). These results are representative of a minimum of triplicate experiments.
150
Figure 3.2. Amino acids 1-132 of HTLV-1 p30II are critical for its physical interaction with p300. (A) Schematic diagram of the known/ putative functional domains of p30II. Schematic diagram of the carboxyl terminal and amino terminal deletion mutants of p30II is indicated. Bipartite NLS indicates the nuclear localization sequence. Regions with sequence homology to the DNA binding domain and homeodomain of Oct-1 are indicated. The numbers on both sides of p30II indicate length of the peptide as number of amino acids. Letters within the boxes indicate amino acids in p30II and Oct-1. (B) Top panel represents the immunoprecipitation of p300 with p30II and serial deletion mutants of p30II. Physical binding between various deletion mutants of p30II and p300 was tested by expressing pCMV-p300 and pME-p30IIHA wildtype or serial deletion mutant plasmids in 293T cells, p300 pull-down from the cellular lysates using polyclonal anti- HA antibody, followed western immunoblot assay with anti-p300 antibody. Below each line, the binding is indicated, as density of each band measured by Gel-pro Analyzer software (middle panel). The results above are representative of three experiments. The protein expression of the carboxyl terminal and amino terminal deletion mutants of p30II was tested using monoclonal anti-HA antibody (lower panel).
151
Figure 3.3. Subcellular localization characteristics of various p30II serial deletion mutants p30II in transiently transfected HeLa-Tat cells. Hela Tat cells were seeded in two chamber slides (Fisher Scientific) at approximately 50 % confluence 18 h prior to transfection, and co-transfected with 4 µg of each pME-p30II-HA serial deletion mutant using Superfect (Gibco BRL). At 48 h post-transfection, media were removed and cells were fixed for 15 min using 4 % paraformaldehyde. Cells were then incubated with FITC conjugated anti- HA antibody (diluted 1:1000; Santa Cruz) overnight at 4° C. Cytokeratin staining of the cells was performed using Anti-Phalloidin. Amino acid domains present in each mutant is indicated. The expression of p30II-HA was evaluated using Zeiss LSM 510 confocal microscope (40X). These results represent a minimum of triplicate experiments.
152
Figure 3.4. HTLV-1 p30II differentially modulates LTR-mediated transcription in the absence or the presence of the provirus. (A) In the absence of the provirus, p30II enhances LTR mediated transcription at low concentrations (upto 0.6 µg) and represses LTR mediated transcription at higher doses (above 0.6 µg) (B) In the presence of full-length provirus, p30II represses LTR mediated transcription in a dose responsive fashion. 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, increasing amounts of pME-p30II HA, +/- 0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test. * indicates P < 0.05.
153
Figure 3.5 p300 expression reverses the p30II-dependent repression of LTR driven transcription, in the absence or the presence of the provirus. (A) In the absence of the provirus, p300 expression reverses the p30II-dependent repression of LTR driven transcription, in a dose-responsive manner. (B) p300 reverses the p30II-dependent repression of LTR driven transcription, in the presence of the provirus, in a dose- responsive manner. 293T cells were transiently cotransfected with 0.3 µg of pLTR- luciferase reporter plasmid, 1.2 µg pME-p30II-HA, increasing quantities of pCMV-p300, +/- 0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test. * indicates P < 0.05.
154
Figure 3.6 HTLV-1 p30II and Tax appear to compete with each other in modulating the LTR mediated transcription. (A) p30II represses Tax-mediated LTR transactivation in a dose-dependent manner. (B) Tax rescues p30II-mediated repression of LTR driven transcription in a dose-dependent fashion. 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid along with either 0.4 µg of pCMV-Tax plasmid and increasing quantities of pME-p30II HA (Fig. 6A) or 1.2 µg of pME-p30II HA plasmid and increasing quantities of pCMV-Tax (Fig. 6B). pME 18S (Fig. 6A) or a plasmid containing CMV promoter (Fig. 6B) was used as carrier DNA to equalize DNA concentrations for each transfection. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test. * indicates P < 0.05.
155
Figure 3.7. Amino acids 100-179 of p30II is critical for its repression of LTR driven transcription, in the absence or the presence of the provirus. (A) In the absence of the provirus, amino acids 100-179 of p30II are critical for its repression of LTR driven transcription. (B) In the presence of the provirus, amino acids 100-179 of p30II are critical for its repression of LTR driven transcription. 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II-HA wildtype or serial deletion mutants, +/- 0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test. * indicates P < 0.05.
156
Figure 3.8. Lysine 106 (K3) of p30II is critical for its repression of LTR driven transcription in the absence or the presence of the provirus. (A) In the absence of the provirus, lysine 106 (K3) of p30II is critical for its repression of LTR driven transcription. (B) In the presence of the provirus, lysine 106 (K3) of p30II is critical for its repression of LTR driven transcription. 293T cells were transiently cotransfected with 0.3 µg of pLTR- luciferase reporter plasmid, 1.2 µg pME-p30II-HA wildtype or site directed mutants, +/- 0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test. * indicates P < 0.05.
157
CHAPTER 4
HISTONE ACETYLTRANSFERASE (HAT) ACTIVITY OF P300 MODULATES
HUMAN T LYMPHOTROPIC VIRUS TYPE 1 P30II-MEDIATED REPRESSION
OF LTR TRANSCRIPTIONAL ACTIVITY
INTRODUCTION
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of an aggressive and often fatal CD4+ T cell malignancy, known as adult T-cell leukemia
(ATL), a chronic neurodegenerative disease, called HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP) and is also implicated in various immune- mediated disorders50,25. The virus infection persists in 15-20 million people worldwide, but is a particular problem in endemic regions of the Caribbean, Japan, Africa, South
America, and among at-risk populations within the United States. HTLV-1 is a complex retrovirus, which encodes the typical gag, pol and env gene products, and unique regulatory and accessory genes encoded in four open reading frames (pX ORF I-IV) between the env gene and the 3’ long terminal repeat (LTR)13. Four accessory proteins designated p12I, p27I, p13II and p30II are expressed from these highly conserved ORFs.
158 Using molecular clones of HTLV-1 with selective mutations of pX ORFs I and II, our
laboratory was the first to identify functional roles of p12I, p13II/p30II for establishment
of infection in the rabbit model and for infection of resting T-cells7.
p30II, also known as Tof, is a 241 amino acid protein that localizes in the
nucleolus and nucleus of transiently transfected cells. The viral protein has a bipartite
nuclear localization signal (NLS) located between amino acids 73-98, which contains two
arginine-rich stretches separated by a 12 amino acid spacer and serine/threonine-rich
regions. This region shares distant homology to the activation domain of the POU family
of transcription factors. We have previously demonstrated that p30II functions as a
transcriptional regulator and differentially modulates cAMP responsive element (CRE)
and Tax-responsive element (TRE)-mediated transcription through its interaction with
CBP/p30052. At low concentrations, p30II activated the transcription from the HTLV-1
LTR. However, at higher concentrations, p30II repressed HTLV-1 LTR driven
transcription52. In addition, p30II regulates CRE and TRE-mediated transcription in the
presence or absence of Tax52. We have also reported that p30II interacts with cellular
transcriptional regulator, CBP/p300, through the KIX domain, similar to the transactivating protein, Tax, encoded in pX ORF-III51. More importantly, p30II inhibits the assembly of Tax-p300-CREB multiprotein complexes on the TRE promoter51.
Recently, using serial deletion mutants of p30II, we localized the domain of p30II that is
critical for its binding to CBP/p300 and in mediating its transcriptional activity. In
addition, we also demonstrated that p30II-mediated LTR repression can be rescued by
p300 51(Michael et al, 2004, submitted). Recently, Nicot et al 34 reported a post-
159 transcriptional role of HTLV-1 p30II in modulating LTR mediated transcription.
Therefore, p30II appears to be a multi-functional protein with transcriptional and post- transcriptional role in regulating viral gene expression.
Recent studies have provided insight into the mechanism and functional significance of the interactions between many viral proteins and CBP/p300. CBP and p300 bridge transcription factors to relevant promoters and through their intrinsic histone acetyl transferase (HAT) activity, acetylates lysine residues at the amino terminus of histones, which results in modification of chromatin structure to allow access of the transcriptional machinery. p300 also directly acetylates transcription factors such as p53 and GATA-1 and thus augment their sequence-specific DNA-binding activity43.
Additionally, CBP/p300 is known to form complexes with proteins such as p300/CBP- associated factor (P/CAF), which also exhibits HAT activity17. HTLV-1 Tax also
associates with the LTR through its interaction with CREB. These Tax-CREB promoter
complexes act as a high-affinity binding site to recruit multifunctional cellular
coactivators CBP, p300 and P/CAF to activate the expression of viral genes from the
viral promoter28.
Identifying the molecular mechanism and functional significance of the
interaction between p30II and p300 is crucial in understanding the pathogenesis and
replication of HTLV-1. p30II contains six highly conserved lysine residues, four of which
are part of the consensus acetylation sequence (G/SK motif)6, and thus represent potential
acetylation sites for CBP/p300. We have previously demonstrated that lysine 106 (K3) of
160 p30II is critical in LTR repression (Michael et al, 2004, manuscript submitted). Based on
this, we hypothesized that the intrinsic HAT activity of CBP/p300 is utilized to acetylate and potentially modulate the transcriptional regulatory function of p30II. Herein, we have
characterized the interaction between p30II and p300 and demonstrate that p300 HAT
mutants do not rescue p30II-mediated LTR repression as much as wild type p300. In
addition, we also show that p30II is acetylated and that the deacetylation by histone
deacetylase-1 (HDAC-1) enhances p30II-mediated HTLV-1 LTR repression. Moreover,
inhibition of deacetylation by trichostatin A decreases p30II-mediated HTLV-1 LTR repression. This is the first report to demonstrate that the HAT activity of p300 is crucial in modulating HTLV-1 gene expression from the LTR by p30II. Overall, our study
confirms the role of p30II as a regulator of viral gene transcription, in association with
p300. Taken together, our data indicates that HTLV-1, a complex retrovirus associated
with lymphoproliferative disorders, uses accessory genes to modulate viral gene
expression, in context with cellular-mediated acetylation. Thus, HTLV-1 regulates its
gene expression in concert with cellular transcriptional regulators to avoid immune
elimination from the host, and facilitate clonal expansion of infected cells and
maintenance of proviral loads in vivo.
161 MATERIALS AND METHODS
Cell lines.
293T cells were obtained from G. Franchini (National Cancer Institute, NIH,
Bethesda) and grown in modified Dulbecco’s eagle medium containing 10% fetal bovine
serum and 1% streptomycin and penicillin at 37°C. Cells were split and cultured in six-
well plates or 10 cm dishes to 50-60 % confluence 18 h before transfection.
Plasmids.
The pTRE-Luc plasmid and pRSV-B-Gal have been described previously51,52. pME-p30IIHA plasmid contains p30II sequence from HTLV-1 molecular clone, ACH upstream of influenza hemagglutinin (HA1). Site directed mutants K2, K3, K4 and K5
harbor single arginine substitution of lysines in pMEp30IIHA at amino acid position 103,
106, 123 or 143 respectively. Fidelity of these plasmids were confirmed by Sanger
sequencing and p30II HA protein expression was confirmed by western immunoblot
assay using monoclonal anti-HA antibody (Covance, Berkeley, CA ). ACH.30 plasmid
has a 24 bp insertion that causes an artificial termination codon in p30II40. pCMV-p300
expresses the full-length p300 protein from a CMV I/E promoter (Upstate USA Inc.,
Charlottesville, VA). HDAC-1 plasmid codes for HDAC-1 and was a kind gift from S.
Schreiber, Harvard University, Cambridge, MA. Wild type and HAT mutants of P/CAF
have been described previously21.
162 Cell transfection and reporter gene assay.
For each transfection, 0.3 µg of pTRE-Luc reporter plasmid was cotransfected with pME-p30IIHA plasmid and 0.4 µg ACH.30 plasmid when specified, using
Lipofectamine Plus (Invitrogen, Carlsbad, CA). When pCMV-p300 wild type or mutant
plasmids were used, it was also co-transfected at quantities specified. When HDAC-1
plasmid was used, it was also co-transfected at 0.0, 0.5, 1.0 or 1.5 µg concentrations.
When specified, 0.0, 0.6, 1.2 or 2.4 µg of wild type P/CAF plasmid was also co- transfected. When HAT mutant P/CAF plasmid was used, 2.4 µg of this plasmid was also co-transfected. As described previously12, TSA (Sigma, St. Louis, MO) was added to the
culture medium at 100 nm concentration, at 24 h posttransfection. As an internal control
for transfection efficiency, 0.1 µg of pRSV-βGal (Invitrogen) was also used in each
transfection. pME 18S was used as carrier DNA to equalize DNA concentrations for each
transfection. Transfected cells were lysed with 400 µl lysis buffer (Promega, Madison,
WI) per well for 25 min. Twenty microliters of each lysate was used to test luciferase
reporter gene activity using an Enhanced Luciferase Assay kit (Promega). To normalize
the transfection efficiency, cells were washed with PBS, stained with 5-bromo-4-chloro-
3-indolyl-beta-D-galactopyranoside (X-Gal) (Sigma) and βGal expressing cells were
counted using the microscope. Co-transfection of pME-p30IIHA had no effect on the X-
Gal staining (data not shown). Results were expressed as mean of optimized luciferase
activity (luciferase activity / percentage cells stained positive for β-Gal expression) in
arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate
experiments. Statistical analysis was done using Student’s t test, P < 0.05.
163 Immunoprecipitation of p30II with anti-acetyl lysine antibody.
Sixty percent confluent 293T cells were cotransfected with 15 µg of either pME-
p30IIHA alone or with 10 µg pCMV-p300 wild type or mutant plasmids using Superfect
(Gibco BRL). After 48 h, the transfected cells were washed with PBS and resuspended in
1 ml of lysis buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 µg of leupeptin per ml, and 1 µg of aprotinin per ml. Cell suspensions were incubated on ice for 20 min and the lysates were centrifuged at 14,000 rpm for 20 min at 4° C and 1.5 mg of the lysate was incubated for 2 hours at 4° C with 100 ng of anti-acetyl lysine antibody (Upstate USA Inc). After adding
70 µl of 100% of protein A/G agarose slurry (Pierce, Rockford, IL), the mixture was incubated overnight at 4° C. The immunoprecipitated complexes were washed twice with
10 volumes of lysis buffer. The components of the complexes were resolved on 15 %
SDS-polyacrylamide gels and detected by western immunoblot assay using monoclonal
HA antibody (Covance).
164 RESULTS
Histone acetyltransferase (HAT) activity of p300 modulates p30II-dependent
repression of LTR driven transcription
HTLV-1 Tax mediated transactivation of the viral LTR is dependent on
transcriptional co-adaptors CBP/p300. Tax is reported to bind p300 at the KIX domain,
the same domain that p30II binds51. Our previous reports suggested that p30II and Tax serve different roles in the regulation of transcription. Recently, we also reported that overexpressing p300 rescues p30II-mediated repression of LTR-driven reporter gene
expression, indicating the possibility that p30II represses LTR-driven reporter gene
activities as a consequence of competition for limited basal quantities of CBP/p300. Our
findings that the lysine residue at amino acid position 106 (K3) of p30II is critical for
repressing TRE-mediated transcription, suggested a potential role of acetylation in p30II- mediated LTR repression (Michael et al, 2004, manuscript submitted). To test the role of p300 HAT domain on p30II-dependent repression of LTR driven transcription, we
transiently co-transfected pME-p30IIHA and an LTR-luciferase reporter plasmid along
with either wild type or HAT mutant of p300 plasmids. In contrast to the wild type p300,
p300 HAT mutants only partially rescued the p30II-dependent repression of LTR-
luciferase reporter gene activity (Fig. 4.1A).
We then tested the role of histone acetyltransferase activity mediated by p300
plasmids on p30II-dependent repression of LTR driven transcription, in the presence of
the HTLV-1 provirus. We transiently co-transfected pME-p30IIHA, LTR-luc reporter 165 plasmid and ACH.30, a provirus deleted for p30II expression, along with either wild type
or HAT mutants of pCMV-p300, and tested the luciferase reporter activity. Interestingly, p300 HAT mutant plasmids failed to fully rescue p30II-dependent repression of LTR-
luciferase reporter gene activity, compared to the wildtype p300 plasmid, in the presence
of the HTLV-1 provirus as well (Fig. 4.1B). These data confirm that the HTLV-1 LTR
transcriptional regulatory effect of p30II is dependent on the HAT activity of p300.
Deacetylation by HDAC-1 enhances p30II-dependent repression of HTLV-1 LTR
driven transcription
Histone acetyl transferases and histone deacetylases (HDACs) form multiprotein
complexes with transcriptional factors and are known to regulate their transcriptional
activity12. In addition, HDAC-1 is known to physically and functionally interact with
HTLV-1 Tax and repress the LTR transactivation function of Tax12. To test if
deacetylation by HDAC-1 has a similar role on p30II-dependent repression of LTR
mediated transcription, we transiently co-transfected increasing concentrations of HDAC-
1 plasmid with constant concentration of pME-p30IIHA plasmid and LTR-luciferase
reporter plasmid and tested the luciferase reporter activity. Our data indicated that
HDAC-1 expression enhanced p30II-dependent repression of LTR-luciferase reporter gene activity, in a dose-dependent manner (Fig. 4.2A).
We then tested the role of deacetylation on p30II-dependent repression of LTR
mediated transcription, in the presence of the HTLV-1 provirus, by transiently co-
transfecting increasing concentrations of the HDAC-1 plasmid with constant 166 concentration of pME-p30IIHA plasmid, LTR-luciferase reporter plasmid and ACH.30, a
provirus deleted for p30II expression. Our data indicated that addition of HDAC-1
enhanced p30II-dependent repression of LTR-luciferase reporter gene activity, in a dose-
dependent manner, in the presence of the provirus (Fig. 4.2B). These data further support
our findings that the HTLV-1 LTR transcriptional modulatory function of p30II is dependent on deacetylation by HDAC-1.
Acetylation by trichostatin A (TSA) decreases p30II-dependent repression of HTLV-
1 LTR driven transcription
Reversible modification of the core histones is crucial in the regulation of gene
expression. While histone acetylation is typically associated with nucleosomal
decondensation necessary for transcriptional activation, histone deacetylation is
associated with nucleosomal condensation and subsequent transcriptional repression12.
Repression of Tax transactivation of HTLV-1 LTR by HDAC-1 can be restored when treated with an HDAC inhibitor, namely, trichostatin A12. Similarly, we tested the effect
of inhibition of deacetylation on p30II-dependent repression of LTR mediated
transcription. To determine the most effective concentration of TSA, we transiently co-
transfected 293T cells with 1.0 µg of HDAC-1 plasmid, 1.2 µg of pME-p30IIHA plasmid and 0.3 µg of LTR-luciferase reporter plasmid, and added either 0.0, 100, 200 or 400 nM
TSA at 24 h post-transfection. We then tested the luciferase reporter activity at 48 h post- transfection and compared the difference in luciferase activity in the presence of various concentrations of TSA and found that 100nM TSA had the highest effect in decreasing
167 p30II-dependent LTR repression (Fig. 4.3). Next, to determine the effect of TSA at
different concentrations of HDAC-1, we transiently co-transfected 293T cells with
increasing concentrations of a HDAC-1 plasmid with constant concentration of pME-
p30IIHA plasmid and LTR-luciferase reporter plasmid, and added 100 nM TSA at 24 h
post-transfection. We then tested the luciferase reporter activity at 48 h post-transfection
and compared the difference in luciferase activity in the presence or absence of TSA. Our
data demonstrated that TSA, an inhibitor of deacetylation decreases p30II-dependent
repression of LTR-luciferase reporter gene activity, at various doses of HDAC-1, in the
absence of the provirus (Fig. 4.4A).
We then tested the role of inhibition of deacetylation on p30II-dependent
repression of LTR mediated transcription, in the presence of the HTLV-1 provirus, by
transiently co-transfecting increasing concentrations of HDAC-1 plasmid with constant
concentration of pME-p30IIHA plasmid, LTR-luciferase reporter plasmid and ACH.30, a
provirus deleted for p30II expression and by adding 100 nM TSA at 24 h post-
transfection. We then tested the luciferase reporter activity at 48 h post-transfection and
compared the difference in luciferase activity in the presence or absence of TSA.
Interestingly, addition of TSA decreased p30II-dependent repression of LTR-luciferase
reporter gene activity, at various concentrations of HDAC-1, in the presence of the
provirus as well (Fig. 4.4B). These data confirm that the HTLV-1 LTR transcriptional
regulatory effect of p30II is dependent on deacetylation by HDAC-1 and inhibition of deacetylation by TSA.
168 P/CAF does not rescue p30II-dependent repression of HTLV-1 LTR driven
transcription
P/CAF is an important coactivator that mediates transcription through HAT
activity43. P/CAF acetylates various nonhistone transcription-related proteins, such as the
chromatin proteins HMG17 and HMG I(Y), activators p53, MyoD, human
immunodeficiency virus (HIV) Tat, and general transcription factors TFIIE and TFIIF43.
In addition, P/CAF is recruited to the Tax responsive element sites on the HTLV-1 LTR,
through direct interaction with Tax and enhance Tax-mediated HTLV-1 transcription, in a
HAT-independent manner21. We therefore tested if P/CAF has a role on p30II-dependent
repression of HTLV-1 LTR driven transcription. We transiently co-transfected increasing
concentrations of P/CAF plasmid with constant concentration of pME-p30IIHA plasmid
and LTR-luc reporter plasmid and tested the luciferase reporter activity. Our data
indicated that, unlike p300, P/CAF does not rescue p30II-dependent repression of HTLV-
1 LTR driven transcription (Fig. 4.5). In addition, to test if the HAT activity of P/CAF
influences p30II-dependent repression of HTLV-1 LTR driven transcription, we transiently co-transfected pME-p30IIHA plasmid and LTR-luc reporter plasmid along
with either wild type P/CAF plasmid or P/CAF HAT mutant plasmid, and tested the luc
reporter activity. We found that the P/CAF HAT mutant did not significantly alter p30II- dependent repression of HTLV-1 LTR driven transcription, compared to wild type
P/CAF (Fig. 4.5). These data confirm that the HTLV-1 LTR transcriptional regulatory effect of p30II is p300-dependent and not P/CAF-dependent.
169 HTLV-1 p30II is acetylated by transcriptional co-activator p300
To test if HTLV-1 p30II is acetylated by transcriptional co-activator p300, we
transiently transfected 293T cells with pMEp30IIHA alone or in the presence of either the
wild type p300 or p300 HAT mutants, and whole-cell extracts made at 48 h post-
transfection were used for immunoprecipitation assays. We used anti-acetyl lysine
antibody pull down and subsequent probing with monoclonal HA antibody.
Demonstration of acetylation was performed with a weak, but consistent band, presumed to be acetylation. We detected the presence of a 27 kDa protein in the immunoprecipitated samples containing p30II, which appeared more intense when
expressed in the presence of exogenous wild type p300 (Fig. 4.6). Moreover, in the
presence of p300 HAT mutants, we did not see an increase in the presence of the 27 kDa
band, suggesting that the HAT activity of p300 is critical in the acetylation of p30II(Fig.
4.6). Based on this data, it appears that HTLV-1 p30II is acetylated by the transcriptional co-activator p300. Moreover, we tested four arginine substitution mutants, carrying a mutation in the lysine residues, and found that there was a decrease in the intensity of the
27 kDa band in the K3 mutant, compared to wildtype or K2 or K4 or K5 mutants of p30II
(Fig. 4.6). Interestingly, we have previously demonstrated that K3 mutant that contains an arginine substitution of the lysine at amino acid 106 of p30II is critical for its repression
of LTR driven transcription (Michael et al, 2004, manuscript submitted). Our data
indicated that HTLV-1 p30II is acetylated at lysine 106 (K3) by the transcriptional co-
activator p300. Follow up experiments are being performed (a) using nuclear lysates (b)
at different time points and (c) using an in vitro transcription/translation system.
170 DISCUSSION
Our data is the first to demonstrate that the histone acetyl transferase activity of p300 is critical for modulation of HTLV-1 LTR mediated transcription by p30II. These findings confirm previous reports from our laboratory on the transcriptional regulatory function of HTLV-1 p30II51,52. We have previously reported that the amino acid domain
100-179 of p30II is critical for repression of HTLV-1 LTR, in the presence or absence of
the provirus. Amino acids 100-179 of HTLV-1 p30II contains 5 lysine residues, four of
which are part of a G/SK consensus acetylation sequence6 representing potential
acetylation sites for CBP/p300, and we have reported that the lysine residue at amino acid
position 106 (K3) of HTLV-1 p30II is critical for p30II-mediated repression of LTR
driven transcription in the presence or absence of the provirus (Michael et al, 2004,
manuscript submitted).
CREB binding protein (CBP) and p300 are highly related and evolutionarily
conserved transcriptional coactivators with similar nucleotide sequence, but divergent
functions. CBP and p300 are not interchangeable, however they share many functional
properties45, such as mediating the transcriptional control of various cellular and viral
DNA binding transcription factors9,16. There are several cellular and viral proteins that
interact with either CBP or p300, including steroid and retinoid hormone receptors,
CREB, c-Jun, c-Myb, Sap-1a, c-Fos, MyoD, p53, Stat-1/2, NF-κB, pp90rsk, TATA-
binding protein, TFIIB4,16,19,20, HTLV-1 Tax, adenovirus E1A, Kaposi’s sarcoma-
associated herpes virus (KSHV) viral interferon regulatory factor protein (vIRF), simian
171 virus 40 large T antigen, HIV-1 Tat, small delta antigen of hepatitis delta virus and
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and EBNA2, and herpes
simplex virion protein-16 (VP16) 1-3,10,23,27,29,44,46. Additionally, proteins such as papillomavirus E 2 protein employs novel mechanism of transcriptional activation, by
interacting with cellular protein, AMF-1 to form complexes with p30036.
Acetylation at lysine residues within nucleosomal histones is known to neutralize
the basic charge of lysine and thus loosen the integrity of nucleosomes. CBP and p300
bridge transcription factors to relevant promoters and through its intrinsic histone acetyl transferase (HAT) activity, acetylates lysine residues at the amino terminus of histones, which results in modification of chromatin structure to allow access of the transcriptional
machinery. Recently, the mechanism and functional significance of the interactions between many viral proteins and CBP/p300 are increasingly understood. For example, interaction between the adenovirus oncoprotein E1A and CBP/p300 is critical for E1A- mediated regulation of transcription, suppression of differentiation, and immortalization of cells in culture1,3,32. Modification of the HAT activity of CBP/p300 or the bridging of
the gap between DNA binding transcription factors and components of the general transcription machinery is the mechanism by which simian virus large T antigen regulates the expression of a group of cellular genes4,16. Acetylation of c-myb oncoprotein of the
avian retrovirus group by CBP/p300 at multiple lysine residues is considered to be the
mechanism by which CBP/p300 regulates the functions of c-myb10. Acetylation of
hepatitis δ virus small δ antigen by p300, at lysine 72 is thought to influence its cellular
localization and viral RNA synthesis33. HIV Tat is acetylated by p300 at lysine 50 and 51
in the TAR RNA binding domain, which promotes the dissociation of Tat from TAR 172 RNA during early transcription elongation24. Acetylation of Tat is also thought to
facilitate dissociation of the Tat cofactor cyclinT1 from TAR RNA, serving to transfer
Tat onto the elongating RNA polymerase II22. Overall, acetylation of Tat by p300/CBP is important for its transcriptional activation of the HIV promoter35. EBNA3C, which is
essential for EBV-dependent immortalization of human primary B lymphocytes, interacts
with p300 and acetylation is thought to be critical in its function11. In addition, EBV
nuclear protein protein EBNA2 and herpes simplex VP16 acidic domains utilize the
intrinsic HAT or scaffolding properties of p300 to activate transcription46. Interestingly,
KSHV vIRF interacts with and inhibits p300 HAT activity, resulting in drastic reduction of nucleosomal histone acetylation and alteration of chromatin structure and thus help the virus to circumvent the host antiviral immune response and to induce a global alteration of cellular gene expression29.
An array of transcriptional activators, coactivators, and histone deacetylases are
known to participate in the regulation of HTLV-1 transcription in infected T
lymphocytes28. Chromatin immunoprecipitation analysis investigating factor binding and
histone modification at the integrated HTLV-1 provirus in infected T lymphocytes,
demonstrated the presence of Tax, CBP, p300, histone deacetylases and a variety of
ATF/CREB and AP-1 family members such as CREB, CREB-2, ATF-1, ATF-2, c-Fos,
and c-Jun, at the HTLV-1 promoter28. CBP stimulates Tax-mediated HTLV-1 LTR
transcription initiation and reinitiation from a naked DNA template in vitro23 while p300
and P/CAF act as coactivators for Tax-dependent HTLV-1 LTR transcription21. Tax
directly interacts with both P/CAF and p300/CBP in a multi-histone acetyltransferase/
173 activator-enhancer complex 18. Histone H3 and H4 acetylation occurs at 3 regions within
the proviral genome, and histone H4 acetylation on the HTLV-1 promoter is known to
increase upon inhibition of histone deacetylases, leading to increase in viral RNA28.
Histone acetylation by CBP/p300 is critical in activation of HTLV-1 transcription from chromatin templates in vitro28. Furthermore, it is necessary that the coactivator HAT activity has to be tethered to the template by Tax and CREB, since a p300 mutant that
fails to interact with Tax was unable to facilitate transcription or acetylate histones31. p300 mediates HTLV-1 LTR expression by targeted nucleosomal acetylation of histones
H3 and H4, leading to RNAP II and TFIID recruitment to the chromatin template. CREB-
2, also known as ATF-4, is known to activate the HTLV-1 promoter, in association with
Tax15. CREB-2 is acetylated by CBP/p300, but not by P/CAF, however CREB-2 acetylation does not affect the Tax transactivation of HTLV-1 LTR14.
CBP and p300 mediate the activities of various transcription factors, however they are present only at limiting concentrations in cells. Even small reductions in the concentrations of these coactivator are damaging in many instances, such as the human
Rubinstein-Taybi syndrome where loss of a single CBP allele causes developmental defects37. Our study demonstrates that deacetylation by HDAC-1 enhances p30II- dependent repression of HTLV-1 LTR driven transcription while inhibition of deacetylation by TSA decreases p30II-dependent repression of HTLV-1 LTR driven transcription. Therefore, it appears that p30II acts as an activator of the LTR when
acetylated and as a repressor when deacetylated. Since the level of p300 is limited in
cells, we believe that the endogenous amount of p300 is sufficient to acetylate p30II when
174 provided at lower concentrations, but not at higher concentrations. This is consistent with
our earlier reports that at lower concentrations, p30II acts as an activator of HTLV-1 LTR
mediated transcription, and as a repressor of LTR mediated transcription at higher
concentrations52 (Michael et al, 2004, manuscript submitted).
CBP/p300 is known to form complexes with proteins such as P/CAF, which is
important for transcription as a coactivator and due to its ability to activate selective
promoters via intrinsic HAT activity8,26,38,39,17. P/CAF interacts with p300 and CBP49at
the same site as adenoviral oncoprotein E1A in a competitive manner, and acetylates
either free histones or nucleosomes49, primarily on lysine-14 of histone H3, and more
weakly on lysine-8 of histone H441. P/CAF is important for transcription and is required
as a HAT and coactivator43. Importantly, P/CAF has the ability to acetylate various nonhistone transcription-related proteins, such as the chromatin proteins HMG17 and
HMG I(Y), activators p53, MyoD and general transcription factors TFIIE and TFIIF43. In addition, P/CAF acetylates HIV-1 Tat at Lys28 in the activation domain,and enhances
Tat binding to the Tat-associated kinase, CDK9/P-TEFb24. P/CAF also interacts with and
acetylates adenoviral E1 B protein, and this is known to interfere with the physical
interaction between P/CAF and p53, preventing its activation and thus contributing to
viral transformation30. P/CAF interacts with and acetylates polyomavirus large T antigen, thus stimulate DNA replication when tethered near the polyomavirus origin47. Besides, interaction between human papillomavirus E7 oncoprotein and the acetyl transferase domain of P/CAF, result in reduction of its acetyltransferase activity and thereby, alteration of cellular gene expression and growth5. In EBV-infected cells that do not
175 express the EBV encoded oncoproteins EBNA2 or LMP1, p300 expression enhances the
ability of EBNA2 to up-regulate LMP1 expression and through its intrinsic HAT activity,
P/CAF can further potentiate this p300 effect46. Interestingly, P/CAF is recruited to the
Tax responsive element sites on the HTLV-1 LTR, through direct interaction with Tax
and enhance Tax-mediated HTLV-1 transcription, in a HAT-independent manner21.
However, based on our findings, HTLV-1 p30II-mediated repression of the LTR mediated
transcription appears to be dependent on p300, but independent of P/CAF.
Recently, it was reported that p30II modulates LTR mediated transcription, in the context of the entire provirus, by a post-transcriptional mechanism 34. However, we have
previously reported that p300 rescues LTR driven gene transcription, in a dose-dependent
manner, irrespective of the presence or absence of the provirus. This report provides
additional evidence and further elucidates the mechanism of p300-dependent LTR-
mediated transcriptional modulation by HTLV-1 p30II. Therefore, HTLV-1 p30II appears to be a multi-functional protein with transcriptional and post-transcriptional role in regulating gene expression.
Using deletion mutants of CBP/p300 in GST pull-down assays, previously, we localized the binding site of CBP/p300 for p30II to a highly conserved KIX region51.
Interestingly, KIX domain is the binding site of CBP/p300 for HTLV-1 Tax protein as well48. In addition, p30II was able to disrupt CREB-Tax-CBP/p300 complexes bound to
the viral 21-bp TRE repeats51. In circumstances where CBP/p300 levels are limiting,
proteins are known to compete for binding CBP/p300 and there could be selective
preference of one over the other, causing an exclusion of one of the proteins10. We have 176 previously reported that HTLV-1 p30II and Tax compete with each other in modulating
the transcriptional activity from the LTR, possibly through competitive CBP/p300
binding between p30II and Tax 52(Michael et al, 2004, manuscript submitted). Such an
environment of coactivator competition between transcription factors provides an
additional layer of regulated gene expression. Overall, we believe that the relative amounts of Tax and p30II may be critical in determining which protein binds to
CBP/p300 and the consequent regulation of LTR mediated transcription of viral genes
from the HTLV-1 viral promoter. At low concentrations, p30II and Tax might act
synergistically to enhance transcription from the viral promoter, but at higher
concentrations, we believe that p30II might antagonize the Tax transactivation of the
HTLV-1 promoter (Fig. 4.7). Information on the relative levels of various HTLV-1
proteins during various stages of the infection is lacking and it is possible that differences
in expression levels of various viral proteins, leading to differences in transcriptional
regulation might be the mechanism by which HTLV-1 infection/ disease progresses though various stages. This is likely to be in synergy with differential regulation of cellular gene regulation by Tax and/or p30II and thus changing the immune responses in
accordance with different stages of progression of HTLV-1 infection and disease. Our
present data can be supported by recent findings from our laboratory where an infectious
HTLV-1 molecular clone failed to maintain viral loads in vivo when p30II expression was
abolished42. Overall, our data indicates that HTLV-1, a complex retrovirus associated
with lymphoproliferative disorders, uses accessory genes to regulate viral gene expression from the LTR, avoid immune elimination by the host and facilitate clonal
expansion of infected cells and maintenance of proviral loads in vivo.
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182
Figure 4.1. p300 HAT mutants only partially rescue p30II-dependent repression of LTR driven transcription in the absence of the provirus (A) or the presence of the provirus (B). 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II HA, 2.4 µg of either the wild type p300 plasmid or one of the p300 HAT mutant plasmids, M3 or M7, +/- 0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. A plasmid containing CMV promoter was used as carrier DNA to equalize DNA concentrations for each transfection. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed, comparing to the values obtained in the presence of p30II and CMV empty plasmid, using Student’s t test. * indicates P < 0.05.
183
Figure 4.2. Addition of HDAC-1 enhances p30II-dependent repression of HTLV-1 LTR driven transcription in the absence of the provirus (A) or the presence of the provirus (B). 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II HA, 0.0, 0.5, 1.0 or 1.5 µg HDAC-1 plasmid and +/- 0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. A plasmid containing the same promoter as the HDAC-1 plasmid was used as carrier DNA to equalize DNA concentrations for each transfection. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed, comparing to the values obtained in the presence of p30II and no HDAC-1 plasmid, using Student’s t test. * indicates P < 0.05.
184
Figure 4.3. Addition of 100 nM Trichostatin A (TSA) causes a decrease in the enhancement of p30II-dependent repression of HTLV-1 LTR driven transcription by HDAC-1. 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II HA and 1.0 µg HDAC-1 plasmid. At 24 h post- transfection, 100 nM, 200 nM or 400 nM TSA (Sigma) was added to the culture medium, as described previously12 and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed, comparing to the values obtained in the absence of TSA, using Student’s t test. * indicates P < 0.05. In the presence of 200 nM TSA, P = 0.23. In the presence of 400 nM TSA, P = 0.34
185
Figure 4.4. Addition of Trichostatin A causes a decrease in the enhancement of p30II- dependent repression of HTLV-1 LTR driven transcription by HDAC-1 in the absence (A) or the presence of the provirus (B). 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II HA, 0.0, 0.5, 1.0 or 1.5 µg HDAC-1 plasmid and +/- 0.4 µg of ACH.30 proviral plasmid. A plasmid containing the same promoter as the HDAC-1 plasmid was used as carrier DNA to equalize DNA concentrations for each transfection. At 24 h post-transfection, 100 nM TSA (Sigma) was added to the culture medium, when specified, as described previously12 and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed, comparing between values obtained in the presence or absence of TSA, at the same concentration of HDAC-1, using Student’s t test. * indicates P < 0.05.
186
Figure 4.5. Expression of P/CAF does not rescue p30II-dependent repression of HTLV-1 LTR driven transcription. 293T cells were transiently cotransfected with 0.3 µg of pLTR- luciferase reporter plasmid, 1.2 µg pME-p30II HA and 0.0, 0.6, 1.2 or 2.4 µg wildtype P/CAF or 2.4 µg P/CAF HAT mutant plasmid, and the luciferase activity was measured. A plasmid containing the same promoter as the P/CAF plasmids (CMV promoter) was used as carrier DNA to equalize DNA concentrations for each transfection. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed, comparing to the values obtained in the presence of p30II alone, using Student’s t test. Statistical significance was also performed, to compare between pCAF wildtype and HAT mutant, using Student’s t test. * indicates P < 0.05.
187
Figure 4.6. HTLV-1 p30II appears to be acetylated by the transcriptional co-activator p300 at lysine 106 (K3). (A) p30II appears to be acetylated by the transcriptional co- activator p300. Demonstration of acetylation was performed with a weak, but consistent band, presumed to be acetylation. 293T cells were transiently cotransfected with 15 µg of pME-p30II HA wildtype alone or in the presence of 10 µg of either the wildtype p300 expression plasmid or p300 HAT mutant. Cell lysates were pulled down using anti-acetyl lysine antibody (Upstate USA Inc.) and subsequently immunopreciptated with monoclonal anti-HA antibody (Covance). Follow up experiments are being performed (a) using nuclear lysates (b) at different time points and (c) using an in vitro transcription/translation system (B). p30II appears to be acetylated at lysine 106 (K3). Demonstration of acetylation was performed with a weak, but consistent band, presumed to be acetylation. 293T cells were transiently cotransfected with 15 µg of pME-p30II HA wildtype or arginine substitution mutants K2, K3, K4 or K5. Cell lysates were pulled down using anti-acetyl lysine antibody (Upstate USA Inc.) and subsequently immunopreciptated with monoclonal anti-HA antibody (Covance). K2, K3, K4 and K5 represent single amino acid mutations in p30II, where arginine is substituted for lysine at amino acid position 103, 106, 123 or 143 respectively.
188
Figure 4.7. Schematic model diagram of the potential mechanism by which HTLV-1 p30II modulates LTR-driven transcription. Tax and p30II compete with each other in modulating the LTR-driven transcription, possibly through competitive CBP/p300 binding, since both the viral proteins bind CBP/p300 through the KIX domain. At low concentrations, p30II and Tax might act synergistically to enhance transcription from the viral promoter, but at higher concentrations, we believe that p30II antagonizes Tax transactivation of the LTR, by sequestering p300, and thus disrupting CREB-Tax- CBP/p300 complexes at the TRE repeats on the viral promoter.
189
CHAPTER 5
SYNOPSIS AND FUTURE DIRECTIONS
HTLV-1 is able to evade immune elimination by the host and facilitate
lymphocyte proliferation, the molecular mechanisms of which are still not completely
understood. Distinct features of HTLV-1 pathogenesis include HTLV-1 mediated T cell
activation and transformation, which has been extensively investigated with specific
focus on the role of HTLV-1 Tax12,14. Tax, is the viral transcriptional transactivator
protein, which is considered to be the major viral oncoprotein with critical role in HTLV-
1 mediated T cell activation and lymphocyte transformation12,14. Animal model studies and in vitro transformation assays have established the oncogenic potential of Tax 12,20,30.
Although Tax appears to be responsible for multiple events necessary for HTLV-1- mediated lymphocyte immortalization, it is not certain whether Tax aids the virus to establish persistent infection. Recent studies from our laboratory, using molecular clones of HTLV-1 with selective mutations of ORF I and II, demonstrated that the expression of the accessory proteins encoded by pX ORFs I and II, p12I and p13II/p30II are critical for
the efficient HTLV-1 infection and maintenance of viral loads in vivo in a rabbit model of infection, although potentially dispensable for viral replication under activation conditions in vitro1,4,34. Additionally, previous reports from our laboratory 42,43and data 190 presented in this thesis demonstrate that HTLV-1 pX ORF II encoding protein, p30II regulates viral gene expression from HTLV-1 LTR and may thus be critical for the virus to evade immune recognition and thereby facilitate the establishment of persistent infection in vivo. In addition, data presented in this thesis demonstrate that p30II plays an important role in regulating cellular gene expression and activates many key transcription factors involved in T cell activation, such as NFAT, NFκB and AP-1. These results not only add to our understanding of the role of HTLV-1 p30II in HTLV-1 pathogenesis, but
also shed light on the potential mechanism by which p30II modulates HTLV-1 viral gene
expression and cellular gene expression in T lymphocytes. Taken together, data presented herein, indicates that HTLV-1, a complex retrovirus associated with lymphoproliferative disorders, uses accessory genes products such as p30II to promote lymphocyte activation
to enhance clonal expansion of infected cells and maintain proviral loads in vivo.
5.1 Role of p30II in T cell signaling / activation and transformation
Gene array data presented in this thesis shed light on the possible role of p30II in
T cell signaling / activation. Expression of p30II was associated with decrease in mRNA
levels of CD28, a co-stimulatory molecule with a distinct role in T lymphocyte activation
5. Besides, p30II expression appeared to upregulate Vav-2 and CD72 while
downregulating CD46 and Lck tyrosine kinase, a member of the Src family protein
tyrosine kinases that participates in signal transduction pathways initiated by T cell
surface receptors such as TCR/CD3, CD2, CD4, CD8, and CD28 29. Additionally, p30II
expression correlated with decrease in the level of CHP, an endogenous calcineurin
inhibitor, indicating a likely increase in calcineurin activity, which would be predicted to
191 correlate with upregulation of NFAT expression by p30II. Moreover, p30II expression was associated with increased expression of c-Jun and c-Fos, suggesting activation of AP-1 mediated transcription. In our gene array studies, p30II expression was associated with decreased expression of protein kinase D (PKD), which negatively modulates JNK signaling pathway 18, mediates cross-talk between different signaling systems, and is critical in processes as diverse as cell proliferation, apoptosis, immune cell regulation and tumor cell invasion 37. Interestingly, p30II expression was also associated with decrease in epidermal growth factor (EGF), which stimulates calcium influx 41 and activates ETS domain transcription factor Elk-1 33. In Jurkat T lymphocytes expressing p30II, there were no detectable levels of IKKγ, which is important for NF-κB signaling in response to both
T cell activation signals and Tax 35. However p30II expression increased HPK-1
(Hematopoetic Progenitor Kinase-1), a known NF-κB activator3. p30II expression was associated with decreased Ras GRP2, a guanyl nucleotide exchange factor that increases
Ras-GTP, suggesting a decrease in the level of activated Ras (Ras-GTP). In addition, using luciferase reporter assays, we found that p30II enhanced NFAT, NF-κB and AP-1 driven transcription. Therefore, it is possible that p30II increases IL-2 production, cause T cell activation and lymphocyte transformation.
5.2 Identify the mechanism by which p30II activates NF-κB, NFAT and AP-1 mediated transcription
Herein, we demonstrate that p30II enhanced NFAT, NF-κB and AP-1 driven transcription. p30II increased the NFAT driven luciferase reporter gene activity by 2.2,
4.5, 7.7 and 10.7 fold, when left unstimulated, stimulated with PMA or ionomycin alone 192 or both respectively and 5.2, 2.4 and 5 fold in the presence of CD3 or CD28 alone or both
respectively, indicating that p30II effectively enhanced NFAT driven transcription, when
stimulated with ionomycin or CD3. HTLV-1 p30II increased the AP-1 driven luciferase reporter gene activity by 2.6, 5.2, 1.3, 4.9 fold, when left unstimulated, stimulated with
PMA or ionomycin alone or both respectively and only 1.2, 1 and 1 fold in the presence of CD3 or CD28 alone or both repectively, indicating that p30II effectively enhanced AP-
1 driven transcription, significantly when stimulated with PMA. HTLV-1 p30II increased
the NF-κB driven luciferase reporter gene activity by 11.4, 3.7, 3.1 and 3.8 fold, when
left unstimulated, stimulated with PMA or ionomycin alone or both respectively and 5.6,
6 and 3.6 fold in the presence of CD3 or CD28 alone or both respectively, indicating that
p30II is able to effectively enhance NF-κB driven transcription, significantly even under
unstimulated conditions.
Binding of transcription factors such as, NFAT, AP-1 and NF-κB is required for
the expression from the IL-2 promoter. While NFAT is vital to proliferation of peripheral
lymphocytes for HTLV-1 infection 38, AP-1 is linked to the dysregulated phenotypes of
HTLV-1 infected T cells 11 and malignant transformation 28. Activation of AP-1 occurs
through Tax-dependent and independent mechanisms in HTLV-1-infected T cells in vitro
and in leukemia cells in vivo 11. NF-κB is highly activated in many hematopoietic
malignancies, HTLV-1 infected T cell lines and in primary ATL cells, even when Tax expression levels are low 28 and due to its anti-apoptotic activity, it is considered to be a
key survival factor for several types of cancer.
193 In parallel, HIV-1 accessory proteins Nef and Vpr have been implicated in
regulation of cellular gene expression during HIV-1 infection. Vpr-induced modest
increase in NFAT, AP-1, NF-κB and LTR-dependent transcription is dependent on its
ability to cause G2 arrest25. While Nef stimulates calcium signaling to activate NFAT,
Vpr potentiates Nef-induced activation of NFAT-dependent transcription by functioning farther downstream25. Previous studies from our laboratory have indicated that p12I mediated NFAT activation is dependent on cytosolic calcium, similar to HIV-1 Nef8.
Similar to HIV-1 Nef and Vpr, it is possible that p12I and p30II act synergistically, to modulate NFAT driven transcription and subsequent T cell activation / signaling.
Data presented in this thesis demonstrates the first HTLV-1 accessory protein
with broad modulating activities on the transcriptional activity of NF-κB, NFAT and AP-
1. However, additional research is crucial in elucidating the mechanisms employed by
p30II to enhance NFAT, AP-1 and NF-κB mediated transcription. It will also be valuable
to determine the functional domains and specific amino acids within p30II, critical for the
activation of NF-kB, NFAT and AP-1 mediated transcription.
5.3 Determine if p30II has an effect on IL-2 production and lymphocyte proliferation
Although T lymphocyte proliferation is highly regulated, HTLV-1 has developed
numerous mechanisms to destabilize this control and induce T lymphocyte proliferation
in the absence of appropriate signals. High levels of IL-2 are necessary to promote the
progression of resting T cells through cell cycle, leading to T lymphocyte
194 proliferation16,17. Most of the HTLV-1 infected T lymphocytes require IL-2 to proliferate
during early stages of the infection, although the T lymphocyte proliferation becomes IL-
2 independent later in the infection 39. The role of Tax on the IL-2 pathway and
lymphocyte proliferation has been extensively investigated12. In addition, previous
studies from our laboratory showed that another HTLV-1 protein, namely, p12I enhances
IL-2 production in T lymphocytes9. Data presented in this thesis demonstrates that p30II enhanced NFAT, NF-κB and AP-1 driven transcription. Based on this, we hypothesize that p30II has a major effect on IL-2 production and lymphocyte proliferation.
5.4 Does p30II affect cell cycle progression in T lymphocytes?
Dysregulation of the cell cycle is a key concept in HTLV-1 leukemogenesis and
the ability of Tax to dysregulate the cell-cycle machinery to regulate DNA replication
and cell division has been a focus of a multitude of investigations. Interestingly, as
described in this thesis, we found that p30II expression altered the expression of multiple genes involved in the regulation of different stages of cell cycle. These include checkpoint suppressor 1, cytosolic branched-chain amino acid transaminase 1, histone deacetylase 6, cyclin B1, WEE1 kinase, CDC14A, Lck, JAK2, GAS7, BZAP45, Cullin,
Rab6 GTPase activating protein (downregulated) and TERF1, AKAP8, DDX11, MSH2
and JUN (upregulated). Another gene downregulated by p30II expression was MDM2,
which, interestingly is overexpressed in certain leukemias24 and capable of enhancing the
tumorigenic potential of cells by inhibiting p300 / PCAF mediated p53 acetylation 27.
195 In parallel, HIV-1 Vpr is known to cause G2/M cell cycle arrest, characterized by
low levels of Cyclin B1, by directly binding and inhibiting the in vitro activity of a phosphatase, Cdc25C13. In addition, the cell cycle regulatory kinase, WEE-1, is also
depleted following prolonged G2 arrest induced by Vpr40. Interestingly, overexpression
of WEE-1 suppresses Vpr-mediated apoptosis and is thought to regulate Vpr and γ
irradiation-mediated apoptosis and possibly serve as a general regulator linking the cell
cycle to some pathways of apoptosis40. Our gene array findings that p30II expression was
associated with decreased expression of cyclin B1 and WEE-1, indicate another
functional similarity between p30II and Vpr. Based on the clues available from our gene
array study, future studies are essential not only to verify the findings, but also to test the
functional and biological significance of p30II in HTLV-1 pathogenesis.
5.5 Does p30II affect cell-to-cell adhesion between T lymphocytes?
Lymphocytes naturally infected with HTLV-1 produce virtually no cell-free
infectious HTLV-1 particles and cell-to-cell contact is necessary for efficient
transmission of the virus between cells and between individuals7,10. However, until recently, there was little known about the mechanism of cell-to-cell spread of HTLV-1.
Recently, Igakura et al19 demonstrated that cell contact rapidly induces polarization of the
cytoskeleton of the HTLV-1 infected cell to the cell-cell junction. The molecules that
mediate this process are not elucidated, and HTLV-1 Env protein is thought to be
involved in this, because it is the only HTLV-1 protein expressed intact on the outside of
the virus infected cell. Nonetheless, HTLV-1 is known to upregulate the expression levels
of certain adhesion molecules such as integrins32,36, which would increase the chances of cell to cell adhesion. 196 Gene array data presented in this thesis shed light on the possible mechanisms by
which p30II functions in HTLV-1 pathogenesis and in leukemogenesis, including in cell- to-cell adhesion between T lymphocytes. Based on our findings, p30II expression
appeared to alter the expression of several genes involved in cell adhesion. These include
decrease in integrin (integrin β8) immunoglobulin (MADCAM1), a counter-receptor for
P-selectin (SELPLG), cadherin (desmocollin 3), protocadherin (PC-LKC) liprin
(PPF1BP1), CD84 / Ly-9, CD58, CD43 / sialophorin and glycosyl-phosphatidyl-inositol phospholipase D1. Expression of p30II correlated with increase in integrin receptor α1
subunit and KIT ligand. Additional studies are essential to confirm these findings and to test their functional and biological significance in HTLV-1 pathogenesis.
5.6 Determine the significance of critical functional domains and specific amino acids within p30II, in vivo in rabbit model of infection
Data presented in this thesis demonstrates that amino acids 100-179 of p30II are
critical in regulating LTR mediated transcription, irrespective of the presence or absence
of the provirus, in our LTR-luciferase reporter assays. We have also identified the
domain of p30II, which is critical in the interaction between p30II and Tax. By dissecting
the amino acid domain (100-179) of p30II critical for repressing LTR mediated transcription further, we demonstrated that the lysine residue at amino acid position 106
(K3) of HTLV-1 p30II is critical for its repression of TRE-mediated transcription. In vivo
experiments using infectious molecular clones with mutations in the motifs or specific
amino acids, critical for the functions in vitro are necessary to determine the biological
significance of these findings. Therefore, the findings from the deletion and site directed
197 mutational analysis, presented in this thesis, are valuable in designing experiments to
reintroduce these mutations into infectious molecular clones to test their functional
significance in vivo.
5.7 Does HTLV-1 p30II form complexes at the HTLV-1 promoter, to modulate LTR mediated transcription?
An array of transcriptional activators, coactivators, and histone deacetylases are known to participate in the regulation of HTLV-1 transcription in infected T cells26.
Chromatin immunoprecipitation analysis investigating factor binding and histone modification at the integrated HTLV-1 provirus in infected T cells demonstrated the presence of Tax, CBP, p300, histone deacetylases and a variety of ATF/CREB and AP-1 family members such as CREB, CREB-2, ATF-1, ATF-2, c-Fos, and c-Jun, at the HTLV-
1 promoter26. CBP stimulates Tax-mediated HTLV-1 LTR transcription initiation and
reinitiation from a naked DNA template in vitro22 while p300 and pCAF act as
coactivators for Tax-dependent HTLV-1 LTR transcription21. Tax directly interacts with
both pCAF and p300/CBP in a multi-histone acetyltransferase/ activator-enhancer
complex 15 and histone acetylation by CBP/p300 is critical in the activation of HTLV-1
transcription from chromatin templates in vitro26. p300 mediates HTLV-1 LTR
expression by targeted nucleosomal acetylation of histones H3 and H4, leading to RNAP
II and TFIID recruitment to the chromatin template26.
Our laboratory has previously demonstrated that p30II binds p300 at the KIX domain, which is the same domain that HTLV-1 Tax also binds to42. Data presented in this thesis demonstrates that p30II and Tax compete to modulate the HTLV-1 LTR 198 mediated transcription. In addition, our data suggests that p30II is acetylated and that the
deacetylation by Histone Deacetylase-1 (HDAC-1) enhances p30II-mediated HTLV-1
LTR repression while inhibition of deacetylation by trichostatin A decreases p30II-
mediated HTLV-1 LTR repression. Herein, we also demonstrate that the HAT domain of
p300 is crucial in modulating HTLV-1 gene expression from the LTR by p30II. It will be
important to identify if p30II interacts with proteins other than CBP/p300, present at the
HTLV-1 promoter, and thus form complexes at the HTLV-1 promoter, to modulate the
LTR mediated transcription.
5.8 Identify the functional domains and specific amino acids within p30II, which are
critical in nuclear retention of tax/rex mRNA and protein-protein interactions
Recently, Nicot et al 31 reported that p30II modulates LTR mediated transcription,
in the context of the entire provirus, by a post-transcriptional mechanism, involving the
nuclear retention of tax/rex mRNA. Identifying the functional domains and specific
amino acids within p30II, which are critical in its ability to retain tax/rex mRNA in the nucleus, will be of use in elucidating the mechanism of function of p30II further. On-
going studies in our laboratory are aimed at identifying various proteins that interact with
p30II. To elucidate the functional role of p30II in HTLV-1 pathogenesis, it will be useful
to identify the functional domains and specific amino acids within p30II, which are
critical in such protein-protein interactions. Various serial deletion and site directed
mutants of p30II, described in this thesis, can be valuable tools in such investigations.
199 5.9 Temporal expression pattern and interaction between HTLV-1 regulatory and
accessory proteins during various stages of the HTLV-1 infection
Based on recent reports from our own laboratory and data presented in this thesis,
molecular functions for pX ORF I-encoded p12I and ORF II encoded p30II appears to be
associated with HTLV-1-induced T cell activation, particularly in NFAT, NF-κB and
AP-1 pathways2,8,9,23. While the mechanism of p30II function awaits additional research,
p12I function has been established to be calcium-dependent8, independent of Tax and
possibly occurs before Tax is expressed during a natural infection. However, highly
activated T cells mediated by expression of p12I and/or p30II likely allow HTLV-1
provirus to integrate into host cell genome and permit the early viral infection. Since Tax
is also able to cause T cell activation, it appears to be redundant for HTLV-1 to use
multiple proteins to activate T lymphocytes. It is possible that these proteins act
coordinately or synergistically. By modulating the expression of various HTLV-1
proteins, the virus might employ selective use of different viral proteins during different
stages of the infection. Particularly, it will be important to test if p12I and p30II act synergistically or compete with each other, in modulating NFAT mediated transcription.
Similar studies to elucidate the interaction of p30II and Tax in modulating NF-κB
mediated transcription may also be valuable in further understanding HTLV-1
pathogenesis.
In addition, previous results from our laboratory demonstrated that HTLV-1 Tax
and p30II both bind cellular transcriptional co-activator CBP/p300 at the KIX domain42.
Recent studies from our laboratory indicated that p30II activate the HTLV-1 LTR 200 mediated transcription at lower concentrations, and repress the LTR mediated
transcription at higher concentrations43. Based on the data presented herein, HTLV-1 Tax
and p30II appears to compete with each other in modulating the HTLV-1 LTR mediated transcription. When its levels are low, p30II also enhances transcription from the viral
promoter. Under such circumstances, p30II and Tax might act synergistically. However, at higher concentrations, p30II antagonizes the Tax transactivation of the HTLV-1
promoter. In addition, the data presented in this thesis indicate that p30II modulates the
LTR mediated transcription in a p300 dependent manner. The amount of p300 is limited in cells and such an environment of coactivator competition between transcription factors
is known to provide an additional layer of regulated gene expression6. Interestingly, recent unpublished findings from our laboratory indicate that HTLV-1 p12I increases the
levels of p300. Therefore, it appears that the relative amounts of Tax, p30II and p12I may be critical in modulating LTR mediated transcription of viral genes from the HTLV-1 viral promoter. It is possible that differences in expression levels of various viral proteins leading to differences in transcriptional regulation, might be the mechanism by which
HTLV-1 infection/ disease progresses though various stages. This is likely to be in synergy with differential regulation of cellular gene regulation by Tax, p30II and p12I, and thus changing the immune responses in accordance with different stages of progression of HTLV-1 infection and disease. However, since information on the expression profile of HTLV-1 proteins during different stages of the infection is limited, additional studies designed to test the temporal expression patterns of HTLV-1 regulatory and accessory proteins during viral infection are required to explore this possibility.
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