ROLE OF HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 P30(II) AND
SURFACE ENVELOPE AS DETERMINANTS OF IN VIVO PATHOGENESIS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Lee Silverman, DVM, DACVP
* * * * *
The Ohio State University 2005
Dissertation Committee: Approved by Dr. Michael Lairmore, Advisor
Dr. Kathleen Boris-Lawrie Advisor Dr. Natarajan Muthusamy Veterinary Biosciences Graduate Dr. Thomas Rosol Program ABSTRACT
Human T-cell leukemia virus type 1 (HTLV-1) is the first identified human retrovirus. It is the causative agent of adult T-cell leukemia/lymphoma and
HTLV-1-associated myelopathy/tropical spastic paresis, and it has been implicated in a variety of other immune-mediated disorders such as dermatitis, polymyositis, and uveitis. HTLV-1 exhibits high genetic stability in vivo. In addition to the canonical retroviral gag, pol, and env genes, HTLV-1 contains four open reading frames (ORF) in its pX region. ORF II encodes two proteins, p30II and p13II, both of which are incompletely characterized. p30II localizes to the nucleus/nucleolus and has distant homology to the transcription factors,
Oct-1, Pit-1, and POU-M1. p30II modulates cellular and viral gene expression at the transcriptional and post-transcriptional level.
Herein, we determine the in vivo significance of p30II by inoculating rabbits with cell lines expressing either a wild-type clone of HTLV-1 (ACH.1) or a clone containing a mutation in ORF II, which eliminated wild-type p30II expression (ACH.30.1). ACH.1-inoculated rabbits maintained higher HTLV-1- specific antibody titers than ACH.30.1-inoculated rabbits, and all ACH.1- inoculated rabbits were seropositive for HTLV-1, whereas only two of six
ACH.30.1-inoculated rabbits were seropositive. Provirus could be consistently
PCR amplified from peripheral blood mononuclear cell (PBMC) DNA in all
ii ACH.1-inoculated rabbits but in only three of six ACH.30.1-inoculated rabbits.
Quantitative competitive PCR indicated higher PBMC proviral loads in ACH.1- inoculated rabbits. Interestingly, sequencing of ORF II from PBMCs of provirus positive ACH.30.1-inoculated rabbits revealed a reversion to wild-type sequence with evidence of early coexistence of mutant and wild-type sequence.
Our data provide evidence that HTLV-1 must maintain key accessory genes to survive in vivo and that in vivo pressures select for maintenance of wild-type
ORF II gene products during the early course of infection.
Recent gene array analysis has indicated a role for p30II in modulating cellular expression of a number of apoptosis regulatory genes. Herein, we seek to determine if this translates into a functional role for p30II in modulating cellular apoptosis. ACH.1 or ACH.30.1 lymphocyte cell lines, 293T cells transiently expressing p30II, and Jurkat T cells expressing p30II were induced into apoptosis with camptothecin (targeting cells in the S phase of the cell cycle), etoposide (intrinsic apoptosis pathway), or TRAIL (extrinsic apoptosis pathway). Compared to ACH.1 cells, ACH.30.1 cells showed increased apoptosis induction following treatment with camptothecin, but no difference in apoptosis induction following treatment with etoposide or TRAIL. Transient p30II-expressing 293T cells and lentivirus p30II vector transduced Jurkat T cells showed no differential susceptibility to apoptosis inducing agents compared to empty vector transfected/infected controls. However, expression of p30II in
Jurkat T cells reduced cell proliferation by delaying onset of division. We conclude that although HTLV-1 p30II does not modulate susceptibility to
iii apoptosis in transformed epithelial cells or T lymphocytes, it does reduce cell proliferation and thereby promotes lymphocyte survival.
HTLV-1 Env protein (surface unit, SU, and transmembrane TM) is unique among retroviral Env proteins in that it maintains high amino acid sequence conservation. Previous in vitro assays indicate that HTLV-1 Env Ser75Ile,
Asn95Asp, and Asn195Asp SU mutants maintain the ability to replicate in and immortalize lymphocytes. Herein, we examine the effects of these critical Env mutants in rabbits inoculated with HTLV-1 immortalized ACH.75, ACH.95, or
ACH.195 cell lines (expressing full length molecular clones with the SU mutations) or ACH.1 cell line (expressing wild-type SU). All rabbits became infected, and the fidelity of the mutations was maintained for the 8 week duration of the study. The ACH.75- and ACH.95 –inoculated rabbits had a decreased overall antibody response to Gag and Env antigens. The ACH.195- inoculated rabbits had a selective decreased antibody response to the Env SU protein. One of four ACH.195 rabbits had detectable proviral loads in PBMCs in the absence of a detectable antibody response, and another of the ACH.195 rabbits mounted an antibody response against both HTLV-1 Gag and Env antigens and HTLV-2 SU protein. ACH.75 rabbits on average had higher
PBMC proviral loads compared to ACH.1 and ACH.95 rabbits. PBMC proviral loads, however, did not correlate with antibody responses to SU. Mutations in critical determinants of HTLV-1 SU, while altering proviral loads and antibody responses against Env, did not prevent virus replication in vivo.
iv
Dedicated to:
Sadie and Meyer Greenblatt Mary and Ralph Silverman Wilbur Silvermintz
v ACKNOWLEDGMENTS
It would not have been possible to complete this work without the support of numerous people on both an academic and personal level. I apologize in advance for forgetting to mention anyone. I acknowledge the following people:
Past and current members of the Lairmore laboratory, who were always available for support and advice. Special thanks to Andrew Phipps and Andy
Montgomery, with whom I learned how much more productive teamwork can be than working alone.
Kate Hayes, Tim Vojt, Soledad Fernandez, and Rick Meister, whose technical support was invaluable in completing these projects.
Michael Lairmore, who was the primary advisor on these projects, for continual advice and support and for allowing me to be independent. Special thanks to Michael Lairmore, Kathleen Boris-Lawrie, Natarajan Muthusamy, and
Thomas Rosol for serving on my Ph.D. committee. I appreciate their time and interest in my education.
K. Nasir Khan, whose intelligence, integrity, and kindness inspired me to embark upon this journey and who has continued to be a source of professional support.
vi Laura Rush, Rani Sellers, and Sarah Tannehill-Gregg, whose friendship at various stages in my training was invaluable in getting me through the day.
Steven E. Weisbrode, who significantly contributed to my understanding of the word “mentor” and whose integrity never once failed me.
Tetsuya and Chizuko Takei, with whom I always have a home and a family.
Loretta Silvermintz and family, Max and Betty Bridge and family, and
Albert and Sally Greenblatt and family, who have provided an example of what family should be for generations to come, and who are the foundation of the structure on which everything of meaning in my life stands.
Arthur and Sheryn Silverman and family, who have been the rock of my thirty-three years of existence, and without whom, completion of this work simply would not have been possible.
vii VITA
April 30, 1971…………………………Born – St. Louis, Missouri
1993……………………………………B.A. Mathematics and Japanese Washington University, St. Louis, MO
2000……………………………………D.V.M. University of Missouri, Columbia, MO
2000 - present……………………….Graduate Research Associate The Ohio State University, Columbus, OH
PUBLICATIONS
1. Silverman, Lee R., Andrew J. Phipps, Andy Montgomery, Lee Ratner and Michael D. Lairmore. (2004). Human T-Lymphotropic Virus Type 1 Open Reading Frame II Encoded p30II is Required for In Vivo Replication: Evidence of In Vivo Reversion. Journal of Virology, 78 (8), 3837-3845.
2. Seller, Rani S., Lee R. Silverman and K. Nasir Khan. (2004). Cyclooxygenase-2 Expression in the Cornea of Dogs with Keratitis. Veterinary Pathology 41 (2), 116-121.
viii 3. Marshall, J.L., Kristine M. Stanfield, Lee R. Silverman and K. Nasir Khan. 2004. Enhanced Expression of Cyclooxygenase-2 in Glaucomatous Eyes. Veterinary Ophthalmology 7 (1), 59-62.
4. Ye, Jianxin, Lee R. Silverman, Michael D. Lairmore and Patrick L. Green. 2003. HTLV-1 Rex is Required for Viral Spread and Persistence In Vivo but is Dispensable for Cellular Immortalization In Vitro. Blood 102 (12), 3963-3969.
5. Khan, K. Nasir, Lee R. Silverman, A. Logan and Richard K. Harris. (1999). Paratrichial Sweat Gland Adenocarcinoma in a Marmoset. Journal of Veterinary Diagnostic Investigation 11 (5), 478-480.
6. Khan, K. Nasir, Lee R. Silverman and David A. Baron. (1999). Cyclooxygenase-2 (COX-2) Expression in the Developing Canine Kidney. Veterinary Pathology 35 (5), 486.
7. Silverman, Lee R. and K. Nasir Khan. (1998). Nonsteroidal Anti- Inflammatory Drug-Induced Renal Papillary Necrosis in a Dog. Toxicologic Pathology 27 (2), 244-245.
FIELDS OF STUDY
Major Field: Veterinary Biosciences
ix TABLE OF CONTENTS
Page
Abstract ...... ii
Dedication ...... v
Acknowledgments ...... vi
Vita ...... viii
List of Tables ...... xiii
List of Figures...... xiv
Abbreviations...... xvi
Chapters:
1. Literature Review...... 1
1.1 HTLV-1 diseases and epidemiology...... 1
1.1.1 HTLV-1 epidemiology...... 1 1.1.2 Adult T-cell leukemia/lymphoma...... 2 1.1.3 HTLV-1 associated myelopathy/Tropical spastic paresis .... 4 1.1.4 Determinants of clinical course...... 5
1.2 Animal models of HTLV-1 infection ...... 8
1.2.1 Rabbit models of HTLV-1 infection...... 8 1.2.2 Rat models of HTLV-1 infection...... 11 1.2.3 Nonhuman primate models of HTLV-1 infection...... 12 1.2.4 Mouse models of HTLV-1 infection ...... 14
1.3 HTLV-1 structure and genomic organization ...... 15 1.4 Env determinants of infectivity and immune response ...... 18 1.5 Viral pX region: Tax and Rex...... 21
x 1.5.1 Tax ...... 21 1.5.2 Rex...... 23
1.6 Viral pX region: p30II and other accessory proteins...... 25
1.6.1 Introduction...... 25 1.6.2 pX ORF I p12I...... 26 1.6.3 pX ORF II p13II ...... 29 1.6.4 pX ORF II p30II ...... 30
1.7 HTLV-1 p30II influences on lymphocyte apoptosis ...... 35 1.8 Summary...... 37 1.9 References...... 37
2. Human T-Cell Lymphotropic Virus Type 1 Open Reading Frame II- Encoded p30II is Required for In Vivo Replication: Evidence of In Vivo Reversion ...... 75
2.1 Introduction...... 75 2.2 Materials and Methods ...... 78 2.3 Results ...... 82 2.4 Discussion ...... 88 2.5 References...... 93
3. Role of Human T-Lymphotropic Virus Type 1 p30II in Lymphocyte Apoptosis and Proliferation ...... 107
3.1 Introduction...... 107 3.2 Materials and Methods ...... 110 3.3 Results ...... 116 3.4 Discussion ...... 120 3.5 References...... 125
4. In Vivo Analysis of Replication and Immunogenicity of Proviral Clones of Human T-Lymphotropic Virus Type 1 with Selective Envelope Surface Unit Mutations...... 138
4.1 Introduction...... 138 4.2 Materials and Methods ...... 142 4.3 Results...... 146 4.4 Discussion ...... 150 4.5 References ...... 154
xi 5. Synopsis and Future Directions...... 168
5.1 Introduction...... 168 5.2 The requirement for p30II in establishment of HTLV-1 infection...... 170 5.3 The role of p30II in modulation of lymphocyte apoptosis...... 171 5.4 The role of p30II in modulation of cell cycle...... 172 5.5 Follow-up studies with HTLV-1 Env SU mutants ...... 174 5.6 ACH molecular clones in the rabbit model...... 176 5.7 References ...... 177
Bibliography...... 181
xii LIST OF TABLES
Table Page
1.1 Functional domains of HTLV-1 Env SU ...... 72
2.1 Rabbit groups and inocula for ACH.30.1 study...... 98
2.2 Western blot assay summary of antibody response to HTLV-1 antigens for ACH.30.1 study...... 99
2.3 Viral detection in PBMCs of rabbits by PCR for ACH.30.1 study...... 100
2.4 Quantification of provirus in PBMCs 8 weeks post-inoculation for ACH.30.1 study...... 101
4.1 Rabbit groups and inocula for in vivo Env study ...... 159
4.2 Western blot assay summary of antibody response to HTLV-1 antigens for in vivo Env study...... 160
xiii LIST OF FIGURES
Figure Page
1.1 HTLV-1 genome ...... 73
1.2 HTLV-1 p30II...... 74
2.1 ACH.p30II molecular clone and ACH.30.1 cell line ...... 102
2.2 HTLV-1-specific serologic response of inoculated rabbits for ACH.30.1 study ...... 104
2.3 Viral loads of inoculated rabbits determined by qcPCR for ACH.30.1 study ...... 105
2.4 Coamplification of ACH.1 wild-type and ACH.30.1 mutant sequence in ACH.30.1-inoculated rabbits ...... 106
3.1 Differential camptothecin-induced apoptosis in HTLV-1 immortalized cell lines ...... 131
3.2 HTLV-1 p30II does not modulate apoptosis in 293T cells (PARP cleavage)...... 133
3.3 HTLV-1 p30II does not modulate apoptosis in 293T cells (flow cytometry)...... 134
3.4 HTLV-1 p30II does not modulate apoptosis in Jurkat T lymphocytes... 136
3.5 HTLV-1 p30II does not alter proliferation rate but is associated with an initial lag in cell proliferation in Jurkat T lymphocytes ...... 137
4.1 Construction of ACH-envelope clones...... 162
4.2 ELISA absorbance values on serum samples for in vivo Env study .... 163
4.3 HTLV-1-specific antibody response by western blot for in vivo Env study...... 164
xiv 4.4 Semi-quantitative analysis of HTLV-1 SU and TM specific antibody response...... 165
4.5 Proviral loads in week 8 PBMCs from in vivo Env study...... 166
4.6 PBMC proviral loads do not correlate with antibody response to SU... 167
xv ABBREVIATIONS
ABP actin binding protein
ATL adult T cell leukemia/lymphoma
CBP CREB binding protein
CRE cAMP response element
CREB/ATF-1 cAMP response element binding protein/activating transcription
factor-1
CTL cytotoxic T lymphocyte
DNA deoxyribonucleic acid
ELISA enzyme-linked immunosorbent assay
Env envelope
ER endoplasmic reticulum
FPPS farnesyl pyrophosphate synthetase
HAM/TSP HTLV-1 associated myelopathy/tropical spastic paresis
HLA human lymphocyte antigen
IKK inhibitor of kappa kinase
LTR long terminal repeat
MAPK mitogen-activated protein kinase
MHC major histocompatibility complex mRNA messenger ribonucleic acid
xvi MTS mitochondrial targeting sequence
NES nuclear export sequence
NFAT nuclear factor of activated T cells
NK natural killer
NLS nuclear localization sequence
ORF open reading frame
PARP poly (ADP-ribose) polymerase
PBMC peripheral blood mononuclear cell
PCR polymerase chain reaction
PI propidium iodide qcPCR quantitative competitive PCR
RNA ribonucleic acid
RxRE Rex response element
SCID severe combined immunodeficiency siRNA small interfering RNA
SU surface
TM transmembrane
TNF tumor necrosis factor
TRAIL TNF related apoptosis inducing ligand
TRE Tax responsive element
TSP tropical spastic paresis
WKAH wistar, king, aptekman, hokudai
xvii CHAPTER 1
LITERATURE REVIEW
1.1 HTLV-1 diseases and epidemiology
1.1.1. HTLV-1 epidemiology
Human T-lymphtropic virus type 1 (HTLV-1) was first identified from T lymphoblast cell lines established from patients with cutaneous T cell lymphoma 243. This retrovirus was later shown to be the causative agent of adult T cell leukemia/lymphoma (ATL)311 and tropical spastic paresis
(TSP)82,233. HTLV-1 has also been shown to be associated with but not the sole causative agent of a number of other diseases, including arthritis, uveitis, dermatitis, and polymyositis164,171,194,217.
Fifteen to twenty-five million people are estimated to be infected with
HTLV-1 worldwide83. Endemic regions include Japan100, the Carribean islands19, central Africa261, Central and South America184,245, and the southwest
Pacific6. Transmission of HTLV-1 requires cell-to-cell contact and results in a lifelong chronic infection132. Predominant modes of transmission include sexual, blood contact (via intravenous drug use or blood transfusion), and
1 perinatal via breast feeding132. Less than 5% of infected individuals will develop clinical disease, and when clinical disease does occur, the onset is often decades after the initial infection306. Asymptomatic individuals retain the potential to transmit the virus.
1.1.2. Adult T Cell Leukemia/Lymphoma
ATL is an aggressive T cell leukemia that typically manifests in middle- aged to elderly patients, who were originally infected with the virus neonatally via breast feeding288,306. There are four clinically overlapping subsets of ATL.
In order of decreasing prognostic survival time, these include smoldering, chronic, lymphoma and acute269. The most severe form is the acute form. It is an aggressive T cell leukemia with a concomitant hypercalcemia, bone marrow involvement, generalized lymphadenopathy, skin lesions, and spleen and liver involvement200. Median survival time for acute ATL is less than six months, even with aggressive chemotherapy24. Circulating leukemic cells in ATL patients have a characteristic lobulated nuclear morphology and are most often
CD2+, CD3+, CD4+, CD8- T cells97,298.
What determines whether an HTLV-1-infected individual will progress to develop ATL is unclear. In the early course of HTLV-1 infection, integration of the provirus into host cell chromatin is random and polyclonal197. During the long premalignant phase, there is oligoclonal expansion of HTLV-1 infected cells27,69,304 . Somatic mutations are increased in peripheral blood mononuclear cell (PBMC) DNA from ATL patients compared to asymptomatic carriers197.
2 Somatic mutations are increased in both the provirus and within the proviral flanking sequences, and these mutations are thought to primarily arise during the clonal proliferative phase rather than during reverse transcription199.
Factors which impair the cellular immune response and promote T cell proliferation are thought to increase genetic instability of the virus and therefore increase the likelihood of development of ATL197. One such factor is the HTLV-
1 Tax protein. This protein is reviewed in section 1.5a of this introduction.
Briefly, expression of the viral Tax protein is known to both drive cellular proliferation and increase genetic instability while at the same time evoking a cellular immune response (reviewed in 310). Therefore, factors which allow for
Tax expression with evasion of the cellular immune response increase the likelihood for malignant transformation197. This is supported by investigations which have shown suppression of HTLV-1-specific cellular immune responses leading to development of ATL53,92,129,296. In general, one of the tenets of malignant progression is that it requires mutations in several genes, including oncogenes, tumor suppressor genes, DNA repair genes, and apoptosis- regulating genes. Tax has been demonstrated to regulate gene expression of
294 22 the apoptosis-regulating genes, bcl-xL and bax , the DNA repair genes,
PCNA247 and β-polymerase126, the cell cycle regulators, cyclin D2, cyclin E,
E2F1, CDK2, CDK4, CDK6, p19(INK4d) and p27 (Kip1)118, and the tumor suppressor, p5356. Therefore, Tax can promote malignant progression not only by increasing cellular proliferation, but also by altering the expression of genes known to have the potential to contribute to malignant progression. Other non-
3 viral factors may also promote T cell proliferation and thereby increase the likelihood for development of ATL. Strongyloides stercoralis infection has been clearly linked to decreased time to development of ATL in HTLV-1 infected individuals, and this is thought to be due to increased T cell proliferation259.
1.1.3. HTLV-1 Associated Myelopathy/Tropical Spastic Paresis
The degenerative central nervous system disorder caused by HTLV-1 was identified separately, first as tropical spastic paresis (TSP) by Gessain et al.82 in 1985 and then as HTLV-1-associated myelopathy (HAM) by Osame et al.233 in 1986. It was subsequently agreed in 1988 that that these two syndromes are identical, and the World Health Organization declared TSP and
HAM as the same disease, collectively known as HAM/TSP. HAM/TSP has recently been reviewed165. Clinical signs include paraparesis, urinary incontinence and impotence, and pathologic features include demyelination and lymphocytic meningomyelitis targeting the lower thoracic spinal cord200. The age of patients who develop HAM/TSP is typically younger than that seen with
ATL, and there is a clear association between risk of HAM/TSP and transfusion with HTLV-1 contaminated blood85,231. The risk of development of HAM/TSP has also been correlated with certain human lymphocyte antigen (HLA) haplotypes303. HLA-A*02 and Cw*08 are associated with lower patient HTLV-1 proviral loads and reduced risk of developing HAM/TSP, while HLA-B*5401 is associated with higher proviral loads and an elevated risk of developing
HAM/TSP127,128. HLA-DRB1*0101 in the absence of HLA-A*02 has also been
4 correlated with a higher risk for developing HAM/TSP128,303. Central nervous system lesions are thought to result from a direct or indirect immune-mediated pathogenesis103. Molecular mimicry and autoantigens are also implicated in pathogenesis173,174. Patients have high levels of CD8+ cytotoxic T lymphocytes, which are specific for the viral Tax protein, both peripherally and within their central nervous system120, as well as high-levels of anti-HTLV-1 antibodies within their cerebral spinal fluid81. CD4+ T cells and activated macrophages are also found within central nervous system lesions119. There is a progressive time course to the compositition of the inflammatory cells within the spinal cord. Equal numbers of CD4+ T cells and CD8+ T cells as well as B cells and foamy macrophages are present initially and up to 5 years after disease onset301. Later in the disease, CD8+ T cells predominate301.
1.1.4. Determinants of Clinical Course
Although both ATL and HAM/TSP are caused by HTLV-1, these diseases have vastly different pathologies and seldom occur in the same patient. What determines whether an individual develops ATL, HAM/TSP, or remains asymptomatic is the subject of ongoing investigation. Factors investigated include virus strain, HLA haplotype, route of infection and immune response12. Numerous studies have focused on identification of neuropathogenic or leukemic viral strains54,150,246. However, existence of purely neuropathogenic or purely leukemic viral strains has not been identified.
Despite this, both the Tax protein and the viral p121 protein have been
5 implicated as determinants of viral clinical outcome (p12I is reviewed in section
1.6b). Phylogenetic analysis has identified a subgroup of the Tax gene with higher incidence in HAM/TSP patients80, and differences in the Tax sequence have been found between HAM/TSP-affected and asymptomatic individuals within the same family208, suggesting that nucleotide differences within the Tax gene may influence the clinical outcome of HTLV-1 infection. Regarding p121, naturally occurring variants with a lysine at amino acid 88 as opposed to an arginine at amino acid 88 have been reported to be more commonly found in individuals with HAM/TSP291. p12I is thought to bind MHC I prior to its association with β2 microglobulin and target it for proteasome degradation, thereby decreasing surface expression of MHC I131,133. The lysine-88 p12I variant is less stable than the arginine-88 variant291. Therefore, it is thought that the less stable lysine-88 p12I variant allows for increased surface MHC I expression and a subsequent stronger viral-specific CTL response, thereby promoting the development of HAM/TSP. This is not proven, however, and asymptomatic individuals with the lysine-88 variant of p12I do exist180.
As mentioned previously, certain HLA haplotypes are correlated with increased or decreased risk for development of HAM/TSP. The same holds true for the development of ATL127,128,308. In particular, the influence of HLA haplotype on the development of a cytotoxic T cell lymphocyte (CTL) response is important12. HTLV-1-specific CTL response is high in HAM/TSP patients but low in ATL patients120,135,235. In ATL patients, it is thought that the diminished
6 CTL response allows for increased survival of the infected CD4+ T cells, resulting in increased probability of their eventual transformation.
The route of primary HTLV-1 infection is also correlated with the course of clinical disease. Specifically, most cases of ATL occur subsequent to mucosal exposure, whereas most cases of HAM/TSP occur subsequent to intravenous exposure136,232. The current thought about reasons for this are derived from experimental inoculation of rats. Oral inoculation of rats with
HTLV-1 generally results in a persistent infection with immune unresponsiveness139 whereas intravenous inoculation results in strong antibody and T cell responses136. Extrapolating from this, it is currently believed that intravenous exposure in humans also results in the strong immune response typical of HAM/TSP patients, whereas oral (mucosal) exposure allows for an initial diminished immune response and subsequent survival and outgrowth of infected CD4+ T cells. Intravenous exposure initially leads to infection of a large number of circulating T lymphocytes whereas mucosal exposure initially leads to infection of macrophage and dendritic cells and only a small number of
T lymphocytes (reviewed in 86). HTLV-1 has the ability to stimulate T lymphocytes to enter the cell cycle and promote high levels of gene expression, whereas mucosal dendritic cells and macrophages are post-mitotic and therefore not likely to produce high levels of virus following infection86. Because immune response correlates with virus production, the lower levels of viral production by macrophages and dendritic cells leads to an initially diminished immune response following oral exposure.
7 1.2 Animal Models of HTLV-1 Infection
1.2.1. Rabbit Models of HTLV-1 Infection
Since the initial discovery of the HTLV-1 virus, a variety of animal models of HTLV-1 infection have been reported. In cell culture, the virus infects cell types from several species280. However, in vivo, the virus only consistently infects rabbits1,166, some non-human primates207 and to a lesser extent, rats107,278. Among these in vivo models, the rabbit has been used extensively because of the ease and consistency of transmission of infection in this species. Infectivity for rabbits was first demonstrated in the mid-1980’s using intravenous inoculations of the MT-2 cell line1, a T cell leukemia cell line established from a patient with ATL, and with the Ra-1 cell line193, a rabbit lymphocyte cell line derived from cocultivation of rabbit lymphocytes with MT-2 cells. Early studies in rabbits verified routes of transmission (e.g., blood, semen, milk) for the virus infection102,117,138,159,299,300. Pioneering studies utilizing the rabbit model of HTLV-1 have provided important clues as to the number of cells capable of transmitting the virus infection138 and effective means to prevent the transmission of the virus138,191,260,284,286. These studies established that in the rabbit model, although passive immunization can be successful in preventing transmission of the virus, active immunization is less effectual.
The rabbit model has been extensively used to study the immune response to HTLV-1 infection. Early studies defined the sequential
8 development of antibodies against the virus infection in rabbits40 as well as methods to detect HTLV-1 proviral DNA in infected tissues40. Inoculation of rabbits with HTLV-1-infected cell lines derived from patients with ATL or
HAM/TSP have shown that there is heterogeneity in the biological response to
HTLV-1 infection, which is in part dependent on the strain of infecting virus166.
Immunization of rabbits with synthetic peptides has been key in determining immunodominant epitopes of the viral Env (envelope) protein. Early studies defined amino acids 175-199 and 242-246 of the Env protein as important in eliciting antibody responses168,287 and also defined regions of Env important for antibody dependent cell-mediated cytotoxicity30. Region 242-256 was subsequently shown to be an important B cell epitope; however immunization with the Env 242-256 peptide failed to protect rabbits from subsequent HTLV-1 challenge167. Soon after, it was demonstrated that peptide immunization with amino acids 190-199 of the Env protein could protect rabbits from subsequent
HTLV-1 challenge, opening the possibility for vaccine development286. More complex synthetic peptides, which use chimeric constructs that mimic native viral proteins, have also been generated and tested in rabbit models45,76.
Infectious molecular clones of HTLV-1 were first developed in the mid-
1990’s63,148,322. These molecular clones were used to immortalize human peripheral blood mononuclear cells to create the ACH cell line, which was then used to infect rabbits44. It was demonstrated that the lethally-irradiated ACH cell line successfully establishes infection in the PBMCs of rabbits44.
Subsequently, ACH-immortalized human PBMCs with mutations within the
9 open reading frames of the HTLV-1 accessory proteins, p12I, p13II, and p30II, were generated251, and inoculation of these molecular clones into rabbits has demonstrated the necessity of these accessory proteins for establishment of infection and maintenance of proviral loads14,43,272. The necessity of the Rex protein for in vivo infection has also recently been demonstrated in the rabbit model309.
Establishment of a rabbit model of clinical HTLV-1 disease has been more problematic. In the majority of studies, rabbit infection has paralleled the asymptomatic infection of humans. A few groups have reproduced an “ATL-like disease” via intraperitoneal or intravenous injection of HTLV-1 transformed cells; however this required a minimum of 1 x 108 cells in the inoculum, and death occurred within the first few weeks of inoculation222,267,323. Within these studies, it was not established if the leukemic cells were of inoculum cell or recipient cell origin, and it is likely that inoculation of such a large number of cells led to widespread dissemination of these cells with the appearance of leukemia without a true malignant transformation of the recipient lymphocytes.
Sporadic reports of clinical disease in HTLV-1 infected rabbits do exist. HTLV-1 related clinical diseases reported include uveitis283, cutaneous lymphoma149,273, and thymoma321. In each of these cases, clinical disease developed at least one year and usually several years after the initial infection.
10 1.2.2. Rat Models of HTLV-1 Infection
Experimental infection of rats with HTLV-1 was first established in
1991278. Although initial experimental infection was achieved with F344 rats, it was rapidly established that there are strain variations in the response to HTLV-
1 infection107,114,162. It was through studies on variable strain responses to
HTLV-1 that the Wistar-King-Aptekman-Hokudai (WKAH) rat strain emerged as a model of HAM/TSP. HTLV-1 infected WKAH rats develop spastic paraparesis with degenerative thoracic spinal cord and peripheral nerve lesions several months following inoculation114,162. The pathology of rat HAM/TSP differs from that seen in humans. Lesions in humans have a marked T cell infiltration of affected regions, whereas lymphocytes are not seen in the lesions in rats312.
Subsequent studies defined the time periods over which the pathologic changes occur225,226, and indicated that apoptosis of oligodendrocytes and Schwann cells is the primary event leading to demyelination225,226,312,313. Macrophages are seen in the lesions of rats in response to the demyelination. Production of
HTLV-1 pX mRNA, tumor necrosis factor (TNF) alpha mRNA, as well as altered expression of the apoptosis modifying genes, bcl-2, bax, and p53, have been identified within the lesions225,226,289. In addition, HTLV-1 provirus has been identified in microglial cells and macrophages associated with lesions137.
Development of rat models for clinical ATL has required the use of immunodeficient rats. Ohashi et al.223 demonstrated that an “ATL-like lymphoproliferative disease” could be established in adult nude (nu/nu) rats following inoculation of some, but not all, HTLV-1-immortalized cells lines. This
11 led to studies which examined methods of protection against tumor development, including adoptive transfer of T cells136 and Tax-specific peptide vaccines91. A protective effect was achieved with each of these systems. Most recently, a protective effect against tumor formation in nude rats was achieved with Tax-specific small interfering RNAs (siRNA)219.
1.2.3. Nonhuman primate models of HTLV-1 infection
Due to their phylogenetic similarity to humans, nonhuman primates are a natural choice for the study of human diseases. Experimental HTLV-1 infection in nonhuman primates was first established in the mid-1980s. Cynomolgus monkeys, squirrel monkeys, and Japanese monkeys were inoculated with MT-2 cells, Ra-1 cells, or autologous HTLV-1-infected cell lines. In each of these systems, seroconversion was demonstrated, but no signs of clinical disease occurred up to 2 years post-inoculation192,207,307. Following establishment of the squirrel monkey as an experimental model of HTLV-1 infection, PBMCs, spleen and lymph nodes were verified as major reservoirs for HTLV-1 virus during the early phase of infection141,142,144. Importantly, it was subsequently established that similar to the sequence of events which occurs in humans, HTLV-1 infection in squirrel monkeys is also a two phase process consisting of a transient phase of reverse transcription followed by clonal expansion of infected cells198.
Reports of disease associated with HTLV-1 in nonhuman primates do exist. There are at least two reports of finding antibodies specific for HTLV-1
12 membrane antigens in macaques with malignant lymphoma104,134. In another case report, an experimentally HTLV-1 inoculated rhesus macaque developed arthritis, uveitis, and polymyositis15. More recently, development of clinical disease was reported in pig-tailed macaques following inoculation with a pig- tailed macaque cell line persistently infected with the ACH HTLV-1 molecular clone181. In this report, pig-tailed macaques died naturally at 35 to 82 weeks post-inoculation with hypothermia and lethargy +/- lymphopenia, arthropathy, and diarrhea. Animals that survived showed various combinations of rash, diarrhea, lymphadenopathy and lymphopenia at the time of euthanasia (weeks
85-221 post-inoculation). Rash and lympadenopathy are clinical signs also seen in certain forms of ATL in humans.
Nonhuman primates have been utilized to study vaccine candidates for
HTLV-1. Immunization with viral envelope gene products was attempted as early as 1987206. More recently, successful passive immunization with hyperimmune globulin from healthy donors with high HTLV-1 antibody titers has also been reported2,204. Successful vaccination has also been reported with recombinant vaccinia virus expressing envelope or gag gene products as well as with naked DNA vaccines108,143.
13 1.2.4. Mouse models of HTLV-1 infection
HTLV-1 persistent carrier mice can be established by intraperitoneal inoculation of MT-2 cells into C3H/HeJ and Balb/c mice70,72,161. Within these mice, provirus was identified within CD4+ T cells, CD8+ T cells, B cells and granulocytes within the spleen72. Provirus was also identified within various tissues including thymus, lymph nodes, lung, liver, and kidney, although the cell type containing provirus in these organs was not identified, and the proviral loads were not quantitated70,161. Interestingly, neither viral mRNA production nor an HTLV-1 antibody response was found in the majority of these mice, indicative of a true carrier state70,161. Moreover, similar to the rabbit and rat models, progression to ATL has not been demonstrated in the immunocompetent mouse.
The SCID (severe combined immunodeficiency) mouse has been a successful model to investigate the proliferative and tumorigenic potential of
ATL cells74,115,154,224. SCID mice inoculated with ATL cells succumb to lymphomas, and tumor cells recovered from mice retain the phenotypic and genotypic characteristics of the original tumor cell inoculate74,109,154.
Interestingly, HTLV-1-infected cell lines of nonleukemic origin are not tumorigenic following SCID mouse engraftment73,109,297. Inoculation of HTLV-1- infected nonleukemic cell lines will form tumors in SCID mice only when natural killer (NK) cell activity has been suppressed by sublethal irradiation or by treatment of animals with antiserum which transiently abrogates NK activity
(anti-asialo-GM1)73. Murine NK cells directly mediate cytolysis of cells
14 harboring active HTLV-1 gene expression, suggesting that the absence of viral gene expression in ATL cells contributes to the ability of these cells to evade immune surveillance in humans277. The absence of viral gene expression in the HTLV-1 leukemias of SCID mice has been confirmed109,110. The inability of
HTLV-1-infected nonleukemic cell lines to induce tumorigenesis has also been recently demonstrated in SCID/bg and NOD/SCID mice175. Using a SCID/bg mouse model, Richard et al.248 were able to develop a model for ATL with associated humoral hypercalcemia of malignancy. Interestingly, elevation of parathyroid hormone related protein in this model was shown to be independent of Tax expression. SCID models of ATL have proven useful in examining treatment strategies for ATL. Variable success in tumor suppression has been achieved with the proteasome inhibitor, PS-341285, humanized anti-CD2 monoclonal antibody319, an NF-kappaB inhibitor64, and humanized anti-CD52 monoclonal antibody318.
1.3 HTLV-1 Structure and Genetic Organization
HTLV-1 is an enveloped retrovirus with a diploid, single-stranded RNA genome 9032 nucleotides in length262. Structure of the HTLV-1 genome is depicted in figure 1.1. The viral genome is contained as duplicate strands of positive sense RNA within the virion. Viral infection of the cell is reviewed in
Coffin et al.106. Viral infection of a cell begins with binding of the viral surface
Env glycoproteins to receptors on the cell’s outer membrane. This binding event triggers a conformational change in Env, which allows for fusion of the
15 lipid bilayers between the cellular and viral membranes. Fusion events are mediated by the viral Env transmembrane (TM) protein. Following fusion, the viral genome is released into the host cell cytoplasm, where it undergoes reverse transcription via a virally-encoded reverse transcriptase enzyme, which is an RNA-dependent DNA polymerase. Reverse transcription generates a double stranded DNA intermediate provirus. The provirus enters the host cell nucleus and randomly integrates into the host cell chromatin with the aid of the virally-encoded integrase enzyme. The provirus enters the nucleus as part of a pre-integration complex. Although the composition of the pre-integration complex for HTLV-1 is not completely characterized, for Human
Immunodeficiency Virus it is known to contain viral integrase, reverse transcriptase, matrix, and Vpr proteins146. Once integrated into the host cell nucleus, transcription of the proviral DNA is accomplished with the host cell
RNA polymerase II, giving rise to viral unspliced and spliced mRNAs, which serve as both the genomic RNA for new virions as well as translation templates for viral structural and non-structural proteins.
The reverse transcription process generates 5’ and 3’ long terminal repeats
(LTRs). The LTRs are divided into U3, R, and U5 regions88. They contain sequences which direct viral transcription as well as mRNA polyadenylation, splicing, and nuclear export17,38,156. The U3 region of the LTR contains viral transcriptional enhancer and promoter elements, including the TATA box and three imperfect 21-base pair repeats (Tax-responsive elements-1; TRE-1) required for transcriptional activation by the viral Tax protein21,151. Although
16 numerous cellular proteins are able to bind elements within the U3 region of the
LTR and modulate transcription, the interaction of the cAMP respose element binding protein/activating transcription factor-1 (CREB/ATF-1) with a cAMP response element (CRE) motif within the TRE-1 is thought to be the most important for Tax-mediated transcription122,132.
The viral structural genes include gag, pol, and env. The 55 kd Gag precursor is translated from an unspliced, full-length mRNA and cleaved by the virally-encoded protease to form the 19 kd matrix, 24 kd capsid, and 15 kd nucleocapsid structural proteins46,230. The viral protease is also translated from the Gag precursor mRNA via a ribosomal frameshift mechanism210. Ribosomal frameshifting also is responsible for translation of the pol gene, which encodes the viral reverse transcriptase, integrase, and RNase H proteins210. The viral
Env is translated from a singly spliced, 4.3 kb mRNA (Figure 1.1). The Env protein is reviewed in section 1.4.
As a complex retrovirus, HTLV-1 also encodes regulatory and accessory genes, which are encoded by the viral pX region, located between the 3’ end of env and the 3’ LTR262 (Figure 1.1). The viral pX region encodes four ORFs.
ORFs III and IV encode the Rex and Tax regulatory proteins, respectively153.
ORFs I and II encode the accessory proteins, p12I, p27I, p13II, and p30II
38,79,153,156,205,263. The function of the viral regulatory and accessory proteins is discussed in section 1.5.
17 1.4 Env Determinants of Infectivity and Immune Response
HTLV-1 Env is unique among retroviral envelope proteins in that when isolates from ATL or HAM/TSP patients are compared, there is a high degree of sequence conservation in the env region54,177,262,293, with variability ranging from
1 to at most 8%54,57,177,238,262,293. HTLV-1 Env is a 488 amino acid protein, whose synthesis is similar to that of other retroviral Env proteins. It is synthesized as a precursor protein (gp61) on the endoplasmic reticulum. It then becomes glycosylated in the golgi apparatus and is cleaved into a surface (SU) subunit (gp46) and a transmembrane (TM) subunit (gp21)238, which then translocate to the cell surface. Failure to cleave gp61 prevents transport to the cell surface239. The SU protein is an ectoprotein which noncovalently associates with the TM protein61. The TM protein anchors the Env complex to the cell surface and consists of an ectodomain, a membrane spanning domain, and an intracytoplasmic domain253. There are four potential glycosylation sites within SU and one glycosylation site within TM. Site directed mutagenesis studies have shown all five sites to be glycoslylated239. In addition, full glycosylation is required for functionality61. The C-terminal half of SU is very antigenic in humans, being recognized by antibodies in serum from over 95% of
HTLV-1 infected individuals238. SU amino acids 187-196 are a major target for neutralizing antibodies61.
Early studies of SU using site directed mutagenesis demonstrated functional domains associated with intracellular maturation, syncytium formation, and association with TM (Table 1.1)59,61,169,238,240. Similar site
18 directed mutagenesis studies with the TM protein indicated the existence of three functionally distinct regions within extracellular TM: 1) an N-terminal region important for fusion competence, 2) a central region critical for proper post-translational processing, and 3) a region critical for infectivity immediately
N-terminal to the membrane-spanning domain253. Later studies also identified domains within the intracytoplasmic TM critical for cell-to-cell transmission60.
One of the limitations of the early studies of Env proteins using site- directed mutatgenesis and syncytia assays was that formation of syncytia does not necessarily translate into viral infectivity. A breakthrough in the study of Env occurred with the development of a cell-to-cell transmission assay, which allowed for assessment of infectivity62. In this assay, an HTLV-1 molecular clone with the env region replaced by a neo resistance gene under an SV40 promoter and an env plasmid are cotransfected into COS-1 cells. Transfected cells are then cocultured with B5 cells, followed by G418 selection62. By separating fusion events from infectivity events, it was verified that cell-to-cell fusion and cell-to-cell transmission are independent events60,253.
Another breakthrough in the study of Env came with the development of
HTLV-1 molecular clones in the mid 1990’s148. Subsequent mutagenesis of wild-type HTLV-1 molecular clones allowed for characterization of mutations of key viral proteins in the context of the entire virus. Building off the information obtained by transient tranfections of mutated env plasmid, similar mutations were created in the ACH plasmid295. Mutations in SU amino acids 75, 81, 95,
105, and 195 but not 101 did not alter the ability of the virus to immortalize
19 PBMCs295. One of the questions that remained open from this study was whether or not the in vitro findings would correlate with how the virus behaved in vivo. Data in Chapter 4 of this thesis addresses this question.
One of the areas of HTLV-1 pathogenesis that has continued to elude investigators is the identification of the HTLV-1 receptor, which binds to the Env protein. Although HTLV-1 has a broad tropism in vitro227,280, in vivo the virus is primarily found in CD4+ T lymphocytes87. Many cell surface components have been implicated in HTLV-1 Env-mediated membrane fusion and viral transmission, including adhesion molecules99, heat shock cognate proteins256, lipids255, lipid rafts218, and heparan sulfate237. Breakthroughs in the study of the Env receptor came with the development of SU immunoadhesins. These are characterized by fusion of the SU protein to the Fc region of an immunoglobulin. Advantages of immunoadhesins include efficient expression in mammalian cells and ease of purification7. Several key features of the in vivo
HTLV-1 receptor were identified through the use of SU immunoadhesins. It was demonstrated that TM is not required for SU receptor binding and that sequences within SU are both necessary and sufficient for receptor binding of
Env121. Also, it was demonstrated that the putative HTLV-1 receptor is not expressed in quiescent T cells but is an early marker for T cell activation and its expression requires protein synthesis179,211. The first definitive report of identification of the HTLV-1 Env receptor, which mediates virus binding and fusion events, occurred in 2003, twenty years after the initial discovery of HTLV-
20 1. Manel et al.178 reported that the glucose transporter, GLUT-1, is a receptor for HTLV-1. Whether this finding holds true over time remains to be seen.
1.5 Viral pX region: Tax and Rex
1.5.1. Tax
The HTLV-1 viral transactivator, Tax (transcriptional activator of the pX region), is a 353 amino acid 40 kDa phosphoprotein, which is translated from a doubly spliced mRNA25,71,78,275,276. It is a predominantly nuclear phosphoprotein, which can shuttle into the cytoplasm using a nuclear export protein23,266. Tax is known to modulate transcriptional activity from both the viral
LTR21,123,265 as well as from cellular promoters, including those for IL-2, IL-13,
IL-15, IL-2R, c-Fos, and GM-CSF, among others125 . Gene array studies have demonstrated that Tax is capable of modulating expression of hundreds of cellular genes213. The modulation of cellular gene expression by Tax is thought to occur via four distinct cellular signaling pathways: 1) CREB/ATF, 2) NF-κB,
3) AP-1, and 4) SRF77,122,124,185,279.
Tax mediates viral expression from the three TRE-1s in the U3 region of the viral LTR16,234,320. Each TRE-1 contains a CRE that is flanked by 5’ and 3’
G/C rich sequences, and it is these G/C rich sequences that physically associate with Tax132. CREB/ATF dimers bind to the TRE, and an interaction between Tax and CREB stabilizes these dimers84,96,236,320. In the normal course of cell signaling, protein kinase A (PKA) phosphorylates CREB, which then binds the transcriptional cofactor CBP/p30036,163. Tax also binds CBP/p300,
21 thereby eliminating the requirement of CREB phosphorylation for the recruitment of CBP/p300 to the transcriptional complex for transcriptional activation, which results in constitutive activation of this pathway in HTLV-1 infected cells18. Recruitment of CBP/p300 associated factor (P/CAF) to this complex via binding with Tax is also required for constitutive transcriptional activation95,130.
When considering the pathogenesis of ATL, the ability of Tax to activate the NFκB pathway is intriguing. ATL cells are known to express numerous lymphokines and lymphokine receptors, which are in part regulated by the
NFκB pathway122. Tax is known to upregulate both IL-2 and IL-2Rα via NFκB, thereby driving T cell proliferation9,112,172,254,270. A caveat to implicating Tax activation of NFκB in the pathogenesis of ATL is that although there is constitutive NFκB activity in primary leukemic cells from ATL patients, Tax is not always expressed at significant levels within these cells196.
Modulation of NFκB signaling by Tax is thought to have both nuclear and cytoplasmic components. Within the nucleus, Tax has been shown to bind to the p50 and p52 subunits of NFκB203,281. However, the predominant modulation by Tax of NFκB signaling occurs via its cytoplasmic localization122. NFκB family members include p105, p100, p65, p52, p50, c-Rel and Rel B122. The most common NFκB form is the p50-p65 heterodimer, which is retained in the cytoplasm by interaction with IκB proteins, particularly IkBα and IkBβ8. Upon cellular activation, IκB proteins are phosphorylated and subsequently ubiquitinated and targeted for proteasome degradation (reviewed in122). This
22 releases the NFκB protein, which subsequently localizes to the nucleus and binds to promoters to activate gene transcription. There are two theories as to how Tax is able to modulate NFκB signaling within the cytoplasm. The first maintains that Tax physically associates with IκB proteins, thereby releasing
NFκB proteins to the nucleus. In fact, Tax has been shown to physically associate with IκBα101,282. The more current theory holds that Tax contributes to the phosphorylation of IκBs via an interaction in the IκB kinase (IKK) complex.
The IKK complex consists of IKKα, IKKβ, and IKKγ. Tax is thought to interact with IKKγ and recruit MAP3Ks to the complex, thereby promoting the phosphorylation of IκBs by the complex122.
1.5.2. Rex
The HTLV-1 Rex protein has recently been reviewed314. Rex is a 27 kDa phosphoprotein encoded by ORF III of the pX region which localizes to the nucleolus of infected cells111,205. Rex plays a critical role in viral replication by functioning as a post-transcriptional regulator, which increases the amount of singly spliced and unspliced viral mRNAs (env, gag, pol)98 exported from the nucleus to the cytoplasm at the expense of the doubly spliced tax/rex mRNA98,113. Rex regulates mRNA nuclear export through an interaction with the Rex response element (RxRE), a highly stable stem-loop structure in the
U3/R region of the 3’ LTR10,13,93,264,302. RxRE is present in all mRNAs, including doubly spliced mRNAs, pointing to a role of other cis-acting sequences in the
23 determination of Rex regulation of mRNA nuclear export. Rex is also reported to have a role in preventing degradation of unspliced transcripts in T cells89.
The Rex protein contains a nuclear localization sequence (NLS) in its N- terminus, which is responsible for both nuclear localization and RxRE binding as well as a nuclear export sequence (NES) in its mid-region220,274. It is currently thought that Rex mediates mRNA nuclear export via the
CRM1/exportin pathway20,90. Although the mechanisms for this have yet to be completely elucidated, it is thought that it resembles the mechanisms used by
HIV Rev, which also utilizes CRM1/exportin-mediated mRNA nuclear export48,244, and it has been shown that the HTLV Rex protein can act as a functional substitute for the HIV Rev protein116,249. Ye et al.309 have recently shown that although Rex is dispensable for in vitro immortalization of lymphocytes by HTLV-1, Rex is required for maintenance of infection in vivo.
In addition to the 27 kDa form of Rex, a truncated form, p21Rex, has been detected in multiple HTLV-1 infected cell lines28,79,228. p21Rex can be generated from both the doubly spliced and the singly spliced message (Figure 1.1). The singly spliced message is highly expressed in primary cells isolated from asymptomatic carriers, ATL patients and HAM/TSP patients79,229. This protein maintains the NES sequence but lacks the NLS sequence and has been shown to repress the shuttle function of full length Rex while increasing its nuclear accumulation in vitro160. The exact function of p21Rex in vivo remains to be determined.
24 1.6 Viral pX region: p30II and other accessory proteins
1.6.1. Introduction
In addition to ORF III and IV, which encode the Rex and Tax proteins, respectively, the HTLV-1 pX region also contains ORF I and II, which encode the accessory proteins. Four proteins are encoded within pX ORF I and II, including p12I, p27I, p13II, and p30II. The alternative splicing patterns which generate these accessory protein mRNAs were originally describe by Ciminale et al.38 and Koralnik et al.156 and are reviewed in Albrecht et al.5. The first exon of all pX mRNA is encoded from nucleotides (nt) 1-119 in the R region of the viral 5’ LTR. For doubly spliced messages, the first exon is spliced to either nt
4641 or 4658. The second exon ends at nt 4831 and is spliced to various splice acceptor sites in the pX region. A splice acceptor at nt 6383 is used to generate the pX ORF I proteins, p27I (doubly spliced) and p121 (singly spliced). p12I can also be translated from the p27I message via initiation at an internal methionine codon. It is thought that p12I is preferentially expressed from the p27I message156. However, CTLs specific for p27I have been demonstrated in
HTLV-1 infected individuals, demonstrating that p27I is produced in the course of the in vivo infection241. Similar to pX ORF I, pX ORF II proteins are also produced from two alternatively spliced mRNAs. The larger protein, p30II, is encoded by a doubly spliced mRNA with a pX splice acceptor site at nt 6478.
The smaller p13II protein consists of the carboxy terminal 87 amino acids of p30II and is the product of a singly spliced mRNA with pX splice acceptor at nt
25 6875. p13II can also be translated from an internal methionine codon within p30II.
Although the importance of the accessory proteins in the course of in vivo infection was originally questioned, emerging data points to a critical role for these proteins in viral pathogenesis. pX ORF I and II mRNAs are detected within HTLV-1 infected cell lines, cells transfected with HTLV-1 proviral clones, and primary cells from HTLV-1 infected asymptomatic carriers as well as ATL patients17,28,38,79,156. Analogous gene regions are highly conserved among delta retroviruses including HTLV-2 and simian T lymphotropic virus (STLV)37,258,268.
Importantly, both antibodies and CTLs specific for the accessory proteins have been demonstrated in HTLV-1 infected individuals including asymptomatic carriers, ATL patients, and HAM/TSP patients31,58,241, providing evidence that these proteins are produced in the course of natural infection.
1.6.2. pX ORF I p12I
HTLV-1 p12I has recently been reviewed5,187. It is a 99 amino acid hydrophobic protein which consists of 32% leucine and proline residues156. p12I also contains two transmembrane domains extending from amino acids 12-
30 and 48-67 and four SH3-binding motifs75,156,291. The SH3-binding motifs have a minimal core of PXXP26,158. Additional motifs include two leucine zipper motifs, which form alpha helices, and a dileucine motif. The transmembrane domains and the leucine zipper motifs contribute to dimerization and membrane localization of p12I 291. SH3 binding motifs are common features of signal
26 transduction molecules that modulate the Ras/mitogen-activated protein kinase
(MAPK) and PI3-K signaling pathways212. The function of the dileucine motif is unknown. However, dileucine motifs in other viral proteins such as Nef are involved in directing protein trafficking through endosomal compartments47.
Recent data has shown a PSLP(I/L)T sequence in p12I which is highly homologous to the the PIXIXIT calcineurin-binding motif of nuclear factor of activated T cells (NFAT)147. This motif was found to modulate calcineurin binding by p12I. A ubiquitination motif surrounds lysine 88, and substitution of arginine for the lysine at amino acid 88 enhances the stability of p12I 291.
In transient expression assays, HTLV-1 p12I localizes to the endoplasmic reticulum (ER) and cis-Golgi compartments66,155,157. At its ER localization, in addition to its binding to calcineurin, p12I also associates with calreticulin and calnexin, both of which are calcium binding proteins66. Calcineurin regulates transcriptional activation of NFAT by triggering its dephosphorylation and subsequent nuclear localization183,324. All of these binding activities point to a role for p12I in modulating cellular calcium homeostasis and NFAT signaling. In fact, recent work from our laboratory has demonstrated a role for p12I in upregulating NFAT transcriptional activity and IL-2 in T cells via a calcium- dependent mechanism4,65,67,147. p12I has also been shown to be required for viral infectivity of quiescent but not activated T lymphocytes3. This points to a role for p12I in activating quiescent T lymphocytes early in the course of infection. Additionally, it has been demonstrated that in the absence of p12I, the virus is not infective in vivo43.
27 Other evidence exists which points to a role for p12I in modulating T cell signaling activity. Transient transfection assays show p12I interacting with the
IL-2 receptor β and γ chains, resulting in decreased surface expression201.
Further studies showed that via its binding to IL-2Rβ chain, p12I increases
STAT5b transcriptional activity while simultaneously decreasing the T cell requirement for IL-2 for proliferation216. This is particularly significant because the switch from IL-2 dependent to IL-2 independent growth of HTLV-1 infected
T cells in vitro frequently correlates with the acquisition of constitutive activation of the JAK/STAT pathway189,305. However, other work has shown that in peripheral blood-derived lymphocyte cell lines immortalized by transfection with
HTLV-1 infectious molecular clones with selected elimination of pX ORF I, IL-
2R signaling pathways are intact, and STAT3, STAT5, JAK1, and JAK3 have similar phosphorylation status to wild-type HTLV-1 immortalized PBMCs, indicating that p12I is not necessary for activation of these IL-2-associated JAKs and STATs in long term immortalized T cell lines42.
The role of p12I in down-regulating MHC I surface expression was previously discussed in the review of HAM/TSP in section 1.1. In contrast to this, T lymphocytes immortalized with wild type and p12I mutant molecular clones expressed similar levels of MHC I, indicating that if p12I does have a role in down regulation of MHC I expression, it is likely to be important early in the course of infection42. Whether or not p12I alters MHC I expression in the in vivo infection remains to be determined.
28 1.6.3. pX ORF II p13II
HTLV-1 p13II is an 87 amino acid protein which has been shown to localize to both the nucleus and to mitochondria39,155. Localization of p13II to the mitochondria has been shown to be specifically to the inner mitochondrial membrane51. The mitochondrial targeting sequence (MTS) of p13II is localized between amino acids 22 and 31. It is predicted to fold into an amphipathic α helix with hydrophobic residues on one side and positively charged residues on the other, which is typical of the MTS found in proteins targeted to the mitochondrial matrix52. Interestingly, the p13II MTS differs from the canonical
MTS in that it is shorter in length, it is not cleaved upon import, and it does not require positively charged residues39.
Accumulation of p13II in mitochondria has been shown to cause alteration from the normal highly interconnected network of string-shaped mitochondria to isolated clusters of round, swollen mitochondria39.
Furthermore, it has been shown that a subset of p13II-positive mitochondria show a disruption in the mitochondrial inner membrane potential with altered mitochondrial conductance to Ca2+ and K+. These effects suggest that p13II may alter mitochondrial functions such as energy production, redox status, and apoptosis39. However, p13II induction of mitochondrial cytochrome c release or the permeability transition pore, two events known to occur in some types of apoptosis, has not been demonstrated39,51. Despite this, the role of p13II in modulating lymphocyte apoptosis has recently been confirmed (Hiraragi et al. submitted).
29 Several proteins with which p13II interacts have been identified. Using a yeast two-hybrid assay, Hou et al.105 identified two proteins, designated C44 and C254, as binding partners of p13II. C254 was identified as rabbit actin binding protein 280 (ABP-280). ABP-280 functions in the modulation of cell shape and polarity and is essential for migration of certain cell types in cell culture49,68,190. It also plays a role in the insertion of adhesion molecules into cell membranes186. C44 was identified to have structural similarities to archeal adenylate kinases. Interestingly, C44 was expressed in proliferating but not quiescent PBMCs105. The significance of the interaction of p13II with these two proteins has yet to be elucidated.
Recent studies have also identified farnesyl pyrophosphate synthetase
(FPPS) as a binding partner of p13II 170. FPPS is involved in the mevalonate/squalene pathway and in the synthesis of FPP, which is required for prenylation of Ras. Interestingly, Hiraragi et al. recently showed that p13II modulation of apoptosis is dependent on Ras (Hiraragi et al. submitted).
Recent studies have also identified a role for p13II in reducing cellular proliferation and enhancing responses to calcium-mediated stimuli, suggesting a role for p13II as a negative regulator of cell growth and a potential link between mitochondria, calcium signaling, and tumorigenicity271.
1.6.4. pX ORF II p30II
HTLV-1 p30II is a 241 amino acid 30 kDa protein, of which the carboxy- terminal 87 amino acids comprise the p13II protein (Figure 1.2). It was
30 originally detected as mRNA in HeLa cells transfected with the HTLV-1 molecular clone CS-HTLV-1 and in chronically infected lymphocyte cell lines
MT-2, C91PL, and HTLV-1LAF as well as in PBMC of HTLV-I infected individuals38,156. Early studies identified that p30II accumulates in the the nucleoli/nuclei38,155. Amino acid sequence analysis shows that p30II contains serine and threonine-rich regions with similarities to serine-rich activation domains found in transcriptional activators such as Oct-1, Oct-2, Pit-1, and
POU-M1, suggesting a role for p30II as a transcription factor38. Studies which came slightly later identified a bipartite arginine-rich nuclear localization signal at amino acids 73-78 and 91-9850 (Figure 1.2). Interestingly, a study which soon followed supported the role of p30II as a regulator of mRNA expression50.
In this study, it was shown that the NLS of Tof could replace the NLS of Rex in mediating interaction with the RxRE50. Additionally, it was shown that p30II mRNA requires Rex for efficient expression at both the mRNA and protein levels, suggesting that p30II may regulate viral or cellular mRNA expression during the later stages of viral replication. However, in contrast to Rex, a known regulator of viral mRNA expression, p30II does not influence Env, Tax, or Rex protein expression, and p30II cannot replace Rex in Rex-dependent protein expression systems252. Also in contrast to Rex, whereas Rex can shuttle between the nucleus and cytoplasm, p30II is known to be retained in the nucleus50,214.
As research on p30II has evolved over the past decade, the two lines of thought of p30II as both a transcriptional regulator and a regulator of mRNA
31 expression have both been supported. There have been two reports in support of p30II as a regulator of viral mRNA expression. The first, by Nicot et al.214, provided evidence that p30II acts as a negative regulator of viral gene expression by retaining the doubly spliced Tax/Rex mRNA in the nucleus.
Because Tax and Rex are known positive regulators of viral gene expression, retention of Tax/Rex mRNA in the nucleus would result in decreased Tax and
Rex protein expression and subsequent decreased viral gene expression.
Interestingly, and in direct contrast to subsequently described studies, this study also reported that p30II neither acts as a transcriptional factor nor directly affects
Tax-mediated transcription. In support of their contention that p30II acts to retain Tax/Rex mRNA in the nucleus, it was shown that: 1) p30II down-regulates
Tax-mediated transcription when Tax is expressed from a full-length molecular clone but not from a cDNA, 2) p30II expression results in decreased Tax/Rex mRNA accumulation in the cytoplasm, and 3) p30II specifically interacts with
Tax/Rex splice junctions214. These findings were further confirmed via in vitro analysis by Younis et al.315, who also extended these findings to the HTLV-2 p28II protein.
Despite the above reports that p30II does not modulate transcriptional activity, there are several reports which contradict this. Zhang et al.317 showed that p30II can act as a direct transcriptional activator and mapped the transcriptional activity to a central core region between amino acids 62 and 220.
Additionally, this report also demonstrated that at low concentrations, p30II is able to repress transcription from reporter genes driven by a CRE promoter
32 while enhancing LTR-driven reporter activity. At higher concentrations, p30II caused decreased LTR-driven reporter activity. Subsequent work sought to define mechanisms by which p30II regulates transcriptional activity. Zhang et al. went on to show that the transcriptional activity of p30II is enhanced by the transcriptional cofactors CBP/p300 and that addition of proteins that bind p300 to a transient expression system decrease p30II-mediated transcription316. In the same study, pull-down assays demonstrated a direct interaction between p30II and CBP/p300 and localized the CBP/p300-binding domain to a highly conserved KIX region. DNA binding assays confirmed the interference of p30II with the assembly of CREB-Tax-CBP/p300 on TRE oligonucleotides in vitro.
Because CBP/p300 is present at limiting concentrations in the cell nucleus11,41, these data are suggesting that p30II differentially regulates CREB-responsive promoters via the sequestration of cellular CBP/p300.
It is interesting that as research into the function of p30II has evolved, the question of whether p30II is a transcriptional or a post-transcriptional regulator has remained. It may be that this is not an either/or question but that p30II is able to regulate cellular gene activity along multiple parts of the gene expression pathway. What is not in doubt is that p30II does have the ability to modulate and often repress gene expression at some level. This has recently been confirmed via a gene array analysis of p30II 188 .
It was originally thought that p30II is not necessary for the in vivo viral infection. Chou et al.35 described a case of ATL in which the leukemic cells had a prematurely truncated ORF II. In an in vitro setting, ORFII is not required for
33 expression of Tax, Rex, Gag, or Env252. Robek et al.251 showed that p30II is not required for immortalization of PBMC by HTLV-1 in vitro. Despite this, evidence points to the necessity of ORF II protein for in vivo infection. In a study conducted by Bartoe et al.14, rabbits were intravenously inoculated with a T cell line immortalized by either a wild-type molecular clone of HTLV-1 (ACH) or a clone containing mutations in pX ORF II which eliminated expression of both wild-type p13II and p30II. Compared to ACH-inoculated rabbits, ACH.30/13- inoculated rabbits showed weaker antibody responses and had weak and transient ex vivo p19 production from their PBMC cultures. While all ACH- inoculated rabbits became infected as early as 2 weeks post-inoculation, some of the ACH.30/13-inoculated rabbits failed to become infected. Additionally, those ACH.30/13-inoculated rabbits that became infected showed markedly reduced proviral loads in their PBMCs compared to their ACH-inoculated counterparts. This data suggests that pX ORF II is necessary for the maintenance of proviral loads in vivo. However, it does not separate the role of p30II from that of p13II in the context of the in vivo infection. This question is addressed in this thesis. In Chapter 2, we utilize an ACH.30 molecular clone, which is predicted to produce a truncated mutant of p30II while maintaining wild- type p13II expression. This molecular clone was used to develop an immortalized T cell line. Via inoculation of this cell line into rabbits, we show that p30II is required for maintenance of the in vivo infection.
34 1.7 HTLV-I p30II influences on lymphocyte apoptosis
Apoptosis is an important innate antiviral defense that can result in aborted infection and elimination of infected cells182,221,292. In response, many viruses encode proteins that suppress host cell apoptosis, and suppression of apoptosis is believed to be critical for virus replication and in vivo pathogenesis182,221,292. Gene expression array studies have shown a variety of apoptotic regulatory genes to have altered expression in HTLV-1-infected cell lines as well as ATL and HAM/TSP patients55,94,242. Not surprisingly, these studies have demonstrated a trend towards down-regulation to complete shut- off of apoptosis accelerators and an upregulation of apoptosis inhibitors94,242.
While it is clear that apoptosis regulatory genes are dysregulated in the course of HTLV-1 infection, how this occurs is not clear. There have been many studies focusing on the role of Tax in dysregulation of apoptosis regulatory genes. While it is agreed that Tax modulates cellular apoptosis, whether that effect is pro-apoptotic or anti-apoptotic is subject to much debate, and ample in vitro evidence exists to support both arguments. The effect of
Tax on apoptosis has recently been reviewed145. Data to support both Fas-
FasL dependence and Fas-FasL independence in Tax-mediated apoptosis induction exists29,32,34,250. TNF-related apoptosis-inducing ligand (TRAIL) has also been implicated in Tax-mediated apoptosis induction250. Nicot et al.215 demonstrated caspase-dependent Tax-mediated apoptosis via the Tax interaction with CBP/p300. Also implicated in Tax-mediated apoptosis are oxidative stress33,176 and TNF-α and NFκB152.
35 Inhibition of apoptosis by Tax has been shown to be mediated via NFκB and CREB signaling, with this effect being in part mediated by upregulation of
140,195,209,294 the antiapoptotic Bcl-xL gene . Upregulation of Bcl-xL via NFκB and
CREB pathways has been demonstrated in the in vivo infection195. Saggioro et al.257 show Tax mediating resistance to growth factor withdrawal induced apoptosis via inhibition of mitochondrial cytochrome c release. This inhibition of apoptosis was subsequently shown to be dependent on the phosphorylation status of CREB290. Tax-mediated interference with p53 transcriptional activity has also been demonstrated202. Other factors implicated in the inhibition of apoptosis by Tax include down-regulation of the apoptosis-inducing protein,
Bax22. Resolution of the apparent paradox of Tax of having both pro- and anti- apoptotic functions was recently attempted by de la Feunte et al.56, who via a gene array analysis reported that whether Tax exerts a pro- or anti-apoptotic effect following stress signals depends on the particular stage of the cell cycle.
Data examining the potential role for other HTLV-1 viral proteins in the regulation of lymphocyte apoptosis is scant. Silec-Benussi et al.271 and Hiraragi et al. (submitted) have recently provided the first evidence supporting p13II as an apoptosis regulator. Regarding p30II, only one study has addressed the role of p30II as a potential regulator of apoptosis188. This study was a gene array study, and it identified 19 apoptosis regulatory genes which had altered expression in a lentiviral infected p30II- expressing cell line. To date, there are no studies addressing the functional role of p30II in modulating cellular apoptotic activity. In Chapter 3 of this thesis, the question of whether or not the altered
36 apoptotic regulatory gene expression by p30II translates into a functional role for p30II in modulation of apoptosis is addressed.
1.8 Summary
Data presented in this thesis expand our use of the rabbit as an animal model of HTLV-1. We utilize the rabbit to determine the significance of pX
ORFII p30II to the in vivo infection. This is followed up with in vitro studies addressing the functional role of p30II in viral pathogenesis by examining the role of p30II in modulating cellular apoptosis. Finally, we return to the rabbit model to show how point mutations in the Env SU affect the viral infectivity in vivo. Specifically, we are the first to perform any type of in vivo study with Env mutants in the context of the whole virus, and we show that in vitro findings regarding Env mutations and infectivity do in fact translate to the in vivo infection.
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71
Amino Acids Function Assay Author
25-68 Intracellular maturation Radioimmunoprecipitationb Delamarre 1997
75-101 Syncytium formation Syncytium formationa Delamarre 1994
109-178 Intracellular maturation Radioimmunoprecipitationb Delamarre 1994
170 Association between Radioimmunoprecipitationb Delamarre 1994 SU and TM
181-208 Syncytium formation Syncytium formationa Delamarre 1994 Pique 1990
238-295 Intracellular maturation Radioimmunoprecipitationb Delamarre 1997
Table 1.1. Functional domains of HTLV-1 Env SU a Syncytium formation assay consisting of coculture of COS-1 transfected envelope expressing cells with indicator XC or HeLa cells59,62,240. b Radioimmunoprecipitation of gp62, gp46, and gp21 following transient transfection of envelope plasmids into COS-1 cells59,62.
72
Figure 1.1. Diagram of HTLV-1 genome and alternatively spliced mRNA and protein species from pX open reading frames (ORFs). Numbers correspond to nucleotide positions of exon splice acceptor and donor sites with respect to the full length HTLV-1 genome. Lines indicate the mRNA while the boxes below the lines indicate the coding regions of each protein. ORF from which each protein is produced is indicated on the right.
73
p30II 73-78 91-98 155 1 241
II NLS p13
Figure 1.2. Diagram of HTLV-1 p30II. There is a bipartite nuclear localization sequence (NLS) from amino acids 73-78 (RRCRSR) and 91-98 (GPRRSRPR). HTLV-1 p13II constitutes the carboxy-terminal 87 amino acids of p30II with its initiatior methionine at amino acid 155 of p30II.
74 CHAPTER 2
HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 OPEN READING FRAME II
ENCODED P30II IS REQUIRED FOR IN VIVO REPLICATION: EVIDENCE OF
IN VIVO REVERSION
2.1 Introduction
Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus causally linked with adult T-cell leukemia/lymphoma (ATLL), HTLV-1 associated myelopathy/tropical spastic paresis (HAM/TSP), and a variety of other immune- mediated disorders31. Compared to other members of the retroviridae family of viruses, HTLV-1 exhibits high genetic stability in vivo. Across geographically separate HTLV-1 infected populations, there is <10% divergence of viral nucleic acid sequences, and within a single patient, the variability is < 0.5%27. Along with the typical gag, pol, and env retroviral gene products, the HTLV-1 genome contains various regulatory and accessory genes encoded in the pX region between env and the 3’ long terminal repeat (LTR). The pX region contains four open reading frames (ORFs). ORF III and IV encode the well- characterized Rex and Tax proteins, respectively17. Tax is a 40-kDa nuclear phosphoprotein, which increases viral transcription from the HTLV-1 LTR. The
75 ability of HTLV-1 to cause T cell transformation is linked to dysregulation of cellular gene expression and cell cycle checkpoints by Tax16,18,25 . Rex is a 27- kDa nucleolar phosphoprotein, which increases the cytoplasmic accumulation of nonspliced and singly spliced viral RNA17 .
In contrast to the extensive knowledge of Tax and Rex structure and function, less is known about the role of pX ORF I- and II-encoded proteins in the replication cycle and pathogenesis of HTLV-1. The ORF I accessory protein p12I has recently been been reviewed4. p12I is a 99 amino acid protein that localizes to the endoplasmic reticulum and cis-golgi, where it induces increased cytoplasmic calcium to enhance the activation of nuclear factor of activated T cells3,14,15,21. Recent work has shown that it targets the major histocompatibility complex class I heavy chain for degradation20. It has also been shown to enhance STAT5 activation and decrease the interleukin-2 requirement for proliferation of primary human peripheral blood mononuclear cells28. In addition, we are the first to identify a functional role for pX ORF I in establishment of infection in an animal model10.
ORF II is spliced to the first Tax coding exon and encodes two proteins, a full-length p30II and an internally initiated p13II. The smaller protein, p13II, is derived from initiation at the first internal methionine codon in ORF II and represents the carboxy-terminal 87 residues of p30II. The p30II and p13II proteins were initially found to localize to the nucleolus and nucleus23, respectively, and p13II was subsequently identified as also localizing to mitochondrial membranes9. The cellular segregation of the ORF II gene
76 products suggests specific roles for these proteins in the regulation of HTLV-1 expression or as mediators of virus-cell interactions. The p30II protein contains serine- and threonine-rich regions with distant homology to the transcription factors Oct-1, Pit-1 and POU-M18. Work from our laboratory has demonstrated that p30II differentially regulates CREB-responsive element and Tax-responsive element mediated transcription through an interaction with CREB binding protein/p30033,34. Localization of p13II to mitochondria is associated with mitochondrial clustering and energy-dependent swelling via a permeability transition pore independent mechanism and without release of cytochrome c, suggesting altered mitochondrial respiratory activity9,12. We have recently reported that mutations in the ACH.p30II/p13II viral clone, which destroy the initiator methionine of the mRNA encoding p13II and insert an artificial termination codon in the mRNA encoding p30II, prevent the virus from obtaining normal proviral loads in rabbits5.
In this study, we utilize the ACH.p30II viral clone in order to examine the role of p30II in viral infectivity and replication in vivo. ACH.p30II was constructed by cloning an insert with an artificial termination codon in the mRNA encoding p30II while leaving wild-type p13II intact30. Absence of p30II does not influence the ability of ACH.p30II to infect and immortalize PBMC in vitro and does not effect the function of Tax and Rex30. Human T-cell lines were immortalized with either a wild-type HTLV-1 viral clone (ACH.1) or with the ACH.p30II viral clone
(ACH.30.1). Lethally γ-irradiated ACH.1 and ACH.30.1 producing cell lines were inoculated into rabbits. Prior to inoculation, the fidelity of ORF II was
77 confirmed by both diagnostic restriction endonuclease digestion and sequencing. Both cell lines elicited anti-HTLV-1 antibodies; however, responses in ACH.30.1-inoculated animals were inconsistent, and overall, these animals had lower titers and less reactivity to specific viral epitopes. Viral replication was confirmed by detection of proviral DNA in all ACH.1-inoculated rabbits by PCR from PBMC-extracted DNA. However, provirus was detected in only four of six ACH.30.1-inoculated rabbits, and one of these was only transiently positive. Quantitative competitive PCR (qcPCR) analysis showed higher proviral loads in ACH.1-inoculated rabbits compared to ACH.30.1- inoculated PCR-positive rabbits. Sequencing data showed that the PBMC of all
ACH.30.1-inoculated PCR-positive rabbits contained only wild-type sequence by week 6 post-inoculation, with evidence of the co-presence of both wild-type and mutant sequence apparent as early as week 2 post-inoculation. Taken together, our data indicates that in vivo pressure selected a reversion to wild- type ORF II gene product and that this reversion is necessary to maintain infection following inoculation with an HTLV-1 p30II mutant clone. Our data provide evidence in an animal model that this highly cell-associated virus must maintain its key accessory genes to survive in vivo. Importantly, this is the first time in vivo reversion to wild-type has been demonstrated with HTLV-1.
2.2 Materials and Methods
Viral clones and cell lines. The derivation and infectious properties of the full- length ACH viral clone have been reported elsewhere11,22. The ACH.p30II clone
78 was produced by creating a mutation in ACH30. A 24-bp linker inserted at a
SacII site located 291 bp into the pX ORF II encoding p30II results in an artificial termination codon 16 bp downstream from the SacII site.
ACH.1 and ACH.30.1 cell lines were obtained from the outgrowth of immortalized PBMC previously transfected with the ACH and ACH.p30II clones, respectively11,30. PBMC were isolated from normal human donors by Ficoll-
Hypaque (Pharmacia, Peapack, N.J.) centrifugation. Cells were maintained in
RPMI 1640 supplemented with 15% fetal bovine serum, L-glutamine (0.3 mg/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), and recombinant IL-2
(10 U/ml) (complete medium).
Detection of viral p19 matrix antigen. To compare virus production between
ACH.1 and ACH.30.1 cell lines, duplicate samples of 106 cells from each line were washed and seeded in a 24-well plate in 1 mL of complete RPMI. Culture samples were collected at 72 h, serially diluted 10-fold, and tested for HTLV-1 p19 matrix antigen by a commercially available ELISA (Zeptometrix
Corporation, Buffalo, NY).
Detection of proviral sequences. For detection of provirus in cell lines and rabbit PBMC, genomic DNA was harvested by salt purification (Gentra,
Minneapolis, MN) and examined for the presence of HTLV-1 sequences following PCR amplification. Five hundred nanograms of DNA was amplifed by using a primer pair specific for the HTLV-1 pX ORF II region (7047, 5’-TGC
CGA TCA CGA TGC GTT TC-3’; 7492-5’-AGC CGA TAA CGC GTC CAT CG-
3’), which yielded a 445-bp product from the wild-type ACH.1 cell line and a
79 469-bp product from ACH.30.1. The ACH.30.1 amplicon included an XbaI site at nucleotide 712830. ACH plasmid was used as a positive control. After an initial 10-min incubation at 94˚C to activate the Taq polymerase (AmpliTaq
Gold; Applied Biosystems, Foster City, CA), 40 cycles of PCR were performed with the following cycle parameters: denaturation at 94˚C for 1 min, annealing at 60˚ for 1 min, and extension at 72˚C for 45 s, followed by a final extension at
72˚C for 5 min. The amplified products were separated in a 10% polyacrylamide gel.
HTLV-1-specific PCR products resulting from the 7047-7492 pX primer pair were sequenced to further confirm specificity. PCR products were purified
(Qiagen, Valencia, CA) and sequenced by the automated dye terminator cycle sequencing method (3700 DNA Analyzer; Applied Biosystems, Foster City, CA;
Big Dye Terminator Cycle Sequencing Chemistry) using the 7047 primer.
Titrations of ACH.1 cell line DNA in ACH.30.1 cell line DNA were performed to determine the sensitivity of the PCR assay in detecting the purity of the
ACH.30.1 inoculum. Detection of as little as 1 ng of ACH.1 DNA per 99 ng of
ACH.30.1 DNA was achieved.
Quantitative competitive PCR. Estimates of in vivo viral loads were determined with qcPCR as previously described2 . DNA was extracted from rabbit PBMC at 8 weeks post-inoculation. Primers SG 166 and SG 296 were used to amplify a 272 bp segment of the HTLV-1 gag region. The competitor
StyI∆28, which contains nucleotide sequences identical to that of the 272-bp gag amplicon with the addition of a 28-bp linker, was varied in concentration
80 over 2 orders of magnitude while genomic DNA remained constant. Aliqouts of the reactions were separated on 10% polyacrylamide gels, stained with ethidium bromide, and analyzed under UV light. Equivalence points were determined by plotting regression curves of copy number versus band intensity as measured by densitometry. From the equivalence points, the amount of provirus per cell was calculated by a conversion of 5 amol of competitor ≈ 3 x
106 copies. A single qcPCR reaction was run per rabbit sample. Samples were rerun if the R2 value of the regression curve was less than 0.90.
Rabbit inoculation. To test the in vivo replication capacity of each viral clone,
12-week-old female specific pathogen free New Zealand White rabbits (Harlan,
Indianapolis, IN) were inoculated via the lateral ear vein. Inocula were equilibrated by viral p19 protein production assayed by ELISA as described above. 1 x 107 ACH.1 cells (n = 2) or ACH.30.1 cells (n=6) were inoculated. 1 x 107 uninfected PBMC (n=1) were inoculated as a negative control. All cells were gamma-irradiated (7500 R) prior to injection to prevent outgrowth of the cellular inoculum in vivo but allow virus transmission10.
Serologic and clinical analysis. Plasma antibody response to HTLV-1 in inoculated rabbits was determined by use of a commercial ELISA (BioMerieux
Inc., Durham, N.C.), which was adapted for use with rabbit plasma by substitution of horseradish peroxidase conjugated goat anti-rabbit immunoglobulin G (1:3000 dilution; Chemicon, Temecula, CA). Plasma was diluted 1:12,000 to obtain values in the linear range of the assay, and data were expressed as absorbance values. Reactivity to specific viral antigenic
81 determinants was detected using a commercial HTLV-1 western blot assay
(GeneLabs Diagnostics, Singapore) adapted for rabbit plasma by use of alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (1:1000 dilution; BioMerieux, Inc. Durham, N.C.). Plasma showing reactivity to Gag
(p24 or p19) and Env (p21 or gp46) antigens was classified as positive for
HTLV-1 seroreactivity. Rabbits were regularly evaluated for any overt signs of clinical disease. Rabbits were euthanized for necropsy at post-inoculation interval of 8 weeks.
2.3 Results
In vitro analysis of viral clones. We have reported that a mutation within the
ACH clone designed to selectively eliminate p30II protein expression does not affect Gag and Env protein compositions of virus particles (evidence of Rex function)30. In addition, the mutated clones maintain in vitro viral infectivity of human PBMC and have functional Tax activity30. To further investigate the role of p30II, we developed immortalized human T-cell lines which continually produce either wild-type HTLV-1 (ACH.1) or HTLV-1 containing a mutation in
ORF II, which is predicted to produce a severely truncated p30II (ACH.30.1). As we have reported, the cell lines were representative of the phenotype of T cells immortalized by HTLV-1 and had typical expression profiles of CD3, CD4, CD8, and CD2530. The ACH.p30II viral clone was constructed by the insertion of a
24-bp linker into a SacII site of the p30II ORF, which adds an XbaI site (Figure
2.1A). This insert was designed to not alter the sequence of p12I, the coding
82 sequence of which spans this SacII site. To ensure that the mutation was present prior to inoculation, a region of ORF II containing the mutation site was amplified by PCR from the ACH.30.1 line. The product was then digested with
XbaI. As expected, XbaI digestion of DNA amplified from the ACH.30.1 cell line yielded fragments of 386 and 81 bp (Figure 2.1B). ACH.1 cell line DNA was analyzed concurrently, using the same primer pair. As expected for wild-type provirus, XbaI failed to cut the amplified DNA5. To further confirm the fidelity of the mutation, the amplified fragment was sequenced. Sequencing results confirmed in frame insertion of the 24-bp linker in ACH.30.1 (Figure 2.1C).
In order to confirm the absence of p30II expression in the ACH.30.1 cell line, we previously performed an in vitro transcription/translation with the p30II
ORF amplified by PCR from the ACH and ACH.p30II plasmids and from the genomic DNA from ACH.1- and ACH.30.1-immortalized cell lines. The p30II
ORF derived from ACH wild-type plasmid or genomic DNA expressed a 30 kd product, whereas a 30 kd product was not expressed from the p30II ORF derived from ACH.p30II plasmid or genomic DNA30. The insertion of the artificial stop codon into the p30II reading frame is predicted to produce a truncated p30II mutant of approximately 13kb. This truncated mutant was also not detected from the p30II ORF derived from the ACH.p30II plasmid or genomic
DNA, and it may be that this mutant is not stable30.
Serologic response of rabbits to viral clones. To evaluate the function of
HTLV-1 p30II in vivo, we compared the abilities of ACH.1 and ACH.30.1 cell lines to establish and maintain infection in our rabbit model. To ensure
83 comparable infection potential, inocula were equilibrated by HTLV-1 p19 antigen production on a per cell basis (Table 2.1). Prior to inoculation, the fidelity of the ORF II mutation was confirmed by both restriction enzyme analysis with XbaI and by sequencing. To determine the sensitivity of the PCR assay in detecting the purity of the ACH.30.1 inoculum, titrations of ACH.1 cell line DNA in ACH.30.1 cell line DNA were performed. Detection of as little as 1 ng of ACH.1 DNA per 99 ng of ACH.30.1 DNA was achieved.
Serologic response of the rabbits to the inocula was determined by measuring titers of antibody directed against inactivated HTLV-1 viral antigens and recombinant envelope protein by ELISA (Figure 2.2A). Responses were assayed at 2, 4, 6, and 8 weeks post-inoculation. Both ACH.1-inoculated rabbits (R1 and R6) developed positive antibody titers by week 4, whereas the earliest time point at which a positive antibody titer developed in ACH.30.1- inoculated rabbits was week 6. Antibodies in the negative control rabbit (R11) and in 2 of 6 ACH.30.1-inoculated rabbits (R8 and R9) remained below the positive cutoff point (absorbance ≥ .183) for all time points assayed. ACH.1- inoculated rabbit titers were significantly higher (week 8 mean absorbance, 0.60
± 0.25; P = 0.01; Student t test) than the levels seen for ACH.30.1-inoculated rabbits (week 8 mean absorbance, 0.20 ± 0.06).
Reactivity to specific HTLV-1 antigens was confirmed at 2-week intervals throughout the study by western blot analysis (Table 2.2); band intensity was evaluated visually and correlated with ELISA titers (Figure 2.2B). All ACH.1- inoculated rabbits were considered strongly seropositive for HTLV-1 (strong
84 reactivity to both Gag and Env antigens), while rabbits inoculated with
ACH.30.1 were either weakly seropositive (weak reactivity to both Gag and Env antigens; 2 of 6 rabbits), indeterminate (seropositive for Env antigens, seronegative for Gag antigens; 2 of 6 rabbits), or seronegative (no reactivity to either Gag or Env antigens; 2 of 6 rabbits). Control rabbits failed to seroconvert to any HTLV-1 specific antigens. As expected, all rabbits reacted to cellular antigens in the assay.
Detection of provirus from rabbit PBMC. To detect the presence of HTLV-1 provirus in rabbits, we attempted to amplify HTLV-1-specific ORF II proviral sequences from rabbit PBMC DNA by PCR using an ORF II specific primer pair. Provirus was detected in ACH.1-inoculated rabbits by 2 weeks post- inoculation; however, results in the ACH.30.1-inoculated rabbits were variable.
Provirus was not detected throughout the study in two of the six rabbits (R8 and
R9). Provirus was transiently detected in one of the rabbits at week 4 (R3). In two of the rabbits, provirus was detected at weeks 2, 6, and 8, but not at week 4
(R4 and R5). Provirus was consistently detected by week 2 in only one of the
ACH.30.1-inoculated rabbits (R10). The control rabbit (R11) was HTLV-1- negative by PCR throughout the duration of the study (Table 2.3).
Quantitative competitive PCR. To measure the ability of each HTLV-1 clone to maintain viral loads in vivo, we determined the number of proviral copies per cell (rabbit PBMC) by qcPCR for the 5 rabbits which were PCR positive for provirus at 8 weeks post-inoculation(R1, R4 to R6, and R10). This included both ACH.1-inoculated rabbits and three of six ACH.30.1-inoculated rabbits.
85 Band intensities were evaluated and regression curves were plotted to determine equivalence points (Figure 2.3). Viral load was calculated from the resultant equivalencies (Table 2.4). ACH.1-inoculated rabbit PBMC contained an average of 0.432 proviral copies per cell. ACH.30.1-inoculated rabbit PBMC contained proviral loads at the lower limit of our assay detection sensitivity and were two to three orders of magnitude lower than that seen in ACH.1-inoculated rabbits. However, the sample size was too small to achieve statistical significance in proviral loads between the ACH.1- and the ACH.30.1-inoculated groups.
Sequence and restriction enzyme analysis. We sequenced the PCR products derived from the ORF II sequence specific primer pair to ensure that there were no unexpected mutations or reversions within ORF II. Interestingly, all ACH.30.1-inoculated rabbits which were PCR positive at week 6 had eliminated the 24 bp insert, reverting back to wild-type sequence. In order to determine how early this reversion occurred, DNA from PBMC harvested 2 weeks post-inoculation was more closely examined. In the 2 week post- inoculation samples from 2 of 3 ACH.30.1-inoculated rabbits (R5 and R10),
PCR amplification yielded a product with multiple bands consistent with coamplification of the original 469 bp region from the ACH.30.1 inoculum and the 445 bp region expected to be amplifed from wild-type ACH.1. In R5, the
PCR product consisted of only these two bands. In R10, there was also a third band (Figure 2.4A). XbaI digestion of 2 week post-inoculation samples from
R5 completely eliminated the ACH.30.1 469 bp band and yielded the expected
86 386 bp product. XbaI digestion of 2 week post-inoculation samples from R10 partially eliminated the upper bands and yielded the expected 386 bp product
(Figure 2.4B). Sequencing of 2 week post-inoculation PBMC DNA from
ACH.30.1-inoculated rabbits (R4, R5 and R10) showed the wild-type sequence, consistent with a predominance of wild-type sequence by 2 weeks post- inoculation. This is supported by the stronger intensity of the 445 bp band as compared to the 469 bp band in the week 2 post-inoculation PCR products and the more frequent amplification of a single 445 bp ACH.1 band versus multiple bands in the week 2 post-inoculation samples. Similar amplification of multiple bands in 6 week post-inoculation samples from R5 and R10 were not seen
(Figure 2.4C), and as mentioned above, sequencing of ORF II from 6 week post-inoculation PBMC DNA of R5 and R10 indicated a reversion to wild-type
ACH.1 sequence.
One of the ACH.30.1-inoculated rabbits (R3) was only transiently positive for provirus by PCR analysis of PBMC DNA at week 4. Repeated attempts to
PCR amplify the proviral DNA yielded only slight quantities of DNA and we could not obtain sufficient quantities for restriction enzyme digestion or sequencing. However, gel analysis of the PCR product revealed a 469 bp fragment, consistent with the ORF II mutant sequence (data not shown).
Thus, within the group of ACH.30.1-inoculated rabbits, only those which exhibited reversion to wild-type ORF II sequence were able to maintain proviral loads for the eight week duration of this study. These represented 50% of the
87 ACH.30.1-inoculated group. The other 50% did not have detectable proviral loads by the end of the study.
2.5 Discussion
To date, a function for the HTLV-1 ORF II protein, p30II, remains elusive. Our group has demonstrated that selective mutations of the ACH clone designed to eliminate p30II expression do not effect in vitro viral infectivity of HTLV-1 in human PBMC or influence Tax function in transfected cell lines30. ORF II is dispensable for in vitro replication and immortalization of primary T lymphocytes13. However, mRNA, serum antibodies and cytotoxic CD8+ T cells specific for p30II have been demonstrated in HTLV-1-infected individuals6-8,29.
Moreover, it would be unique among retroviruses for HTLV-1 to retain highly conserved sequences of DNA, which serve no purpose in viral propagation or alteration of the host cell environment. These data suggest a significant role for p30II in the survival of the virus in vivo. It would not be unprecedented for an
HTLV-1 accessory protein to be dispensable for in vitro viral infectivity but required for in vivo viral infectivity. Our laboratory has demonstrated that HTLV-
1 p12I is dispensable for infection of activated lymphocytes but is necessary for in vivo viral replication1,10. Additionally, it has recently been demonstrated that in the absence of the regulatory protein, Rex, HTLV-1 virus is still capable of in vitro replication, albeit at significantly reduced levels, but is absolutely required for in vivo infectivity 32.
88 HTLV-1 inoculation of the rabbit has been established as an appropriate model of the persistent asymptomatic infection in humans26. We and others have used this animal model extensively to investigate the mechanisms of transmission, antiviral immune responses, and the role of ORF I and II in viral expression in vivo 5,10,11,19,24. Here, we used the rabbit model to test the influence of mutations in HTLV-1 p30II on viral replication in vivo.
We confirmed the integrity of the p30II mutation prior to exposing the rabbits to ACH.1 and ACH.30.1 cell lines by both restriction enzyme digestion and sequencing. The mutation added a diagnostic restriction endonuclease site, which proved to be intact upon digestion with XbaI. To test the effects of this mutation in vivo, we inoculated rabbits with lethally irradiated ACH.1 and
ACH.30.1 cell lines. Inocula were equilibrated by p19 production. As expected, the wild-type ACH.1 cell line induced a vigorous and continuous humoral immune response against major viral antigenic determinants; however, the response to the ACH.30.1 cell line varied from weakly positive or indeterminate to no response. Previously, we have shown ACH.1 to be consistently infectious in rabbits5,10,11. Similarly, in this study we were able to consistently PCR amplify
HTLV-1 specific sequences from all ACH.1-inoculated rabbits beginning at 2 weeks post-inoculation. In contrast to the ACH.1-inoculated rabbits, in
ACH.30.1-inoculated rabbits we could amplify HTLV-1-specific sequences at all time points in only one of six rabbits, and we were unable to amplify HTLV-1- specific sequences at any time point in two of six rabbits. Only three of six
ACH.30.1-inoculated rabbits were PCR positive at weeks 6 and 8. Quantitative
89 competitive PCR analysis of proviral loads within PBMC of these three rabbits at week 8 indicated lower proviral loads as compared with ACH.1-inoculated rabbits.
A rather unexpected result was the finding that week 6 post-inoculation provirus in PBMC DNA from ACH.30.1-inoculated rabbits had reverted to wild- type sequence. Further analysis of the week 2 post-inoculation PBMC DNA from these three rabbits revealed the presence of at least two ORF II sequence variations as evidenced by multiple band amplifications within a single PCR reaction. Sequencing data indicated that the predominant ORF II sequence was that of wild-type. However, restriction enzyme analysis showed partial to complete digestion of the ACH.30.1 469 bp band, indicating the coexistence of mutant and wild-type sequence within the rabbits at week 2 post-inoculation.
The week 2 post-inoculation PBMC DNA PCR product from one of the
ACH.30.1-inoculated rabbits (R5) consisted of a doublet, the upper band of which completely digested with XbaI, thereby representing the coexistence of the mutant ACH.30.1 and the ACH.1 wild-type sequence. The week 2 post- inoculation PBMC DNA PCR product from another of the ACH.30.1-inoculated rabbits (R10) consisted of multiple bands which only partially digested with
XbaI, representing the coexistence of multiple intermediate forms of ORF II, including wild-type ACH.1 and mutant ACH.30.1. By week 6, only the 445 bp
ACH.1 ORF II PCR product could be amplified from ACH.30.1-inoculated rabbits, demonstrating a complete reversion to wild-type. Interestingly, one of the six ACH.30.1-inoculated rabbits was only transiently positive for the provirus
90 at week 4 post-inoculation. Gel analysis showed that the proviral isoform present at that time was that of the ORF II mutant. These data clearly demonstrate that following inoculation with the ACH.30.1 proviral clone, there is an in vivo reversion to wild-type sequence that subsequently accounts for the proviral load observed in the infected animals.
An alternative explanation for these data is that our samples were contaminated with wild-type plasmid or proviral DNA. We do not believe this to be the case because of the following: 1) DNA from all samples was isolated in a retrovirus-free laboratory. In running the PCR amplifications, sample DNA was never in the same laboratory as positive control DNA or known ACH.1 cell line DNA until placement in the PCR machine. Additionally, all PCR reactions were run with appropriate negative controls, 2) Antibody responses correlated well with the presence of provirus, i.e. rabbits with stronger antibody responses had higher proviral loads, and rabbits without proviral loads did not show an antibody response, 3) Quantitative competitive PCR showed levels of provirus similar to what we have previously reported5. In the event of contamination, it is likely that the values for proviral loads would have been higher. We also recognize the possibility that the original inoculum may have contained small numbers of cells harboring the ACH wild-type sequence at undetectable levels.
While we think it is unlikely that the ACH.30.1 inoculum contained any ACH.1- immortalized cells, in the event that this was the case, the above data indicates a clear preference for selection of ACH.1 over the ACH.30.1 mutant in an in vivo setting.
91 The implications of the above data are interesting on several fronts. First and foremost, this is the first time an in vivo reversion of HTLV-1 has been demonstrated. In this study, the 24 bp insertion used to generate the ORF II mutation was constructed to be highly homologous to the 24 bases immediately
3’ of the inserted linker so as to not disrupt the coding sequences of overlapping reading frames (see figure 2.1C). The first 15 nucleotides of sequence of the insert were identical to the 15 nucleotides of sequence following the insert. This may have facilitated the precise excision of the insert during the process of reverse transcription. It would be interesting to see if a similar reversion would occur or if the virus would survive in vivo in the face of a different type of mutation to eliminate p30II (i.e. alteration of a splice site).
The second implication of our data is that HTLV-1 ORF II p30II is an absolute requirement for successful HTLV-1 survival in vivo. Previous work from our laboratory demonstrated that simultaneous ablation of both ORF II p30II and p13II resulted in reduced proviral loads in our rabbit model5. That study did not attempt to separate the in vivo effects of p30II versus p13II ablation. Additionally, sequencing of proviral DNA from PBMC isolated from rabbits was not done in that study, leaving open the possibility that those rabbits that did become infected in fact had reverted to a wild-type infection.
One of the questions not addressed in this study is the effect of the mutation on mRNA splicing. Methods to quantify HTLV-1 accessory protein transcripts are currently being developed in our laboratory and others. This has proven to be a difficult task because of the markedly low levels of accessory
92 protein transcripts compared to those of other viral structural and regulatory proteins. We are confident that both p12I mRNA and protein are being produced by the ACH.30.1 cells, because previous work has demonstrated that in the absence of a p12I message, the virus is not infectious within the rabbit model10 . The 24 base pair linker used to create the ACH.30.1 mutant cell line was not inserted into a region known to modulate accessory protein splicing.
Therefore, although we cannot exclude the possibility that the mutation altered the balance of accessory protein transcripts, we think it highly unlikely. A future study using wild-type ACH and small interfering RNAs to selectively eliminate translation of transcripts would control for any imbalances in transcripts created by sequence alterations.
HTLV-1 continues to be a significant problem in endemic regions around the world, and as of yet, a successful vaccine has not been generated.
Continued work from our laboratory and others has demonstrated an inability for mutations in the pX region to be maintained in an in vivo setting. This work opens the door to the possibility of creating vaccines based on pX mutants which allow an antibody response to be mounted followed by elimination of the virus. A more detailed understanding of the process of in vivo wild-type reversion will be necessary to pursue this.
2.5 References
1. Albrecht, B., N. D. Collins, M. T. Burniston, J. W. Nisbet, L. Ratner, P. L. Green, and M. D. Lairmore. 2000. Human T-lymphotropic virus
93 type 1 open reading frame I p12(I) is required for efficient viral infectivity in primary lymphocytes. J. Virol. 74:9828-9835.
2. Albrecht, B., N. D. Collins, G. C. Newbound, L. Ratner, and M. D. Lairmore. 1998. Quantification of human T-cell lymphotropic virus type 1 proviral load by quantitative competitive polymerase chain reaction. J Virol Meth 75:123-140.
3. Albrecht, B., C. D. D'Souza, W. Ding, S. Tridandapani, K. M. Coggeshall, and M. D. Lairmore. 2002. Activation of nuclear factor of activated T cells by human T-lymphotropic virus type 1 accessory protein p12(I). J Virol 76:3493-3501.
4. Albrecht, B. and M. D. Lairmore. 2002. Critical role of human T- lymphotropic virus type 1 accessory proteins in viral replication and pathogenesis. Microbiol. Mol. Biol. Rev. 66:396-406, table.
5. Bartoe, J. T., B. Albrecht, N. D. Collins, M. D. Robek, L. Ratner, P. L. Green, and M. D. Lairmore. 2000. Functional role of pX open reading frame II of human T- lymphotropic virus type 1 in maintenance of viral loads in vivo. J. Virol. 74:1094-1100.
6. Berneman, Z. N., R. B. Gartenhaus, M. S. Reitz, W. A. Blattner, A. Manns, B. Hanchard, O. Ikehara, R. C. Gallo, and M. E. Klotman. 1992. Expression of alternatively spliced human T-lymphotropic virus type 1 pX mRNA in infected cell lines and in primary uncultured cells from patients with adult T-cell leukemia/lymphoma and healthy carriers. Proc. Natl. Acad. Sci. USA 89:3005-3009.
7. Chen, Y. A., S. Chen, C. Fu, J. Chen, and M. Osame. 1997. Antibody reactivities to tumor-suppressor protein p53 and HTLV-I TOF, REX, and TAX in HTLV-I-infected people with differing clinical status. Int. J. Cancer 71:196-202.
8. Ciminale, V., G. N. Pavlakis, D. Derse, C. P. Cunningham, and B. K. Felber. 1992. Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: Novel mRNAs and proteins produced by HTLV type I. J. Virol. 66:1737-1745.
9. Ciminale, V., L. Zotti, D. M. Dagostino, T. Ferro, L. Casareto, G. Franchini, P. Bernardi, and L. Chiecobianchi. 1999. Mitochondrial targeting of the p13(II) protein coded by the x-II ORF of human T-cell leukemia/lymphotropic virus type I (HTLV-I). Oncogene 18:4505-4514.
10. Collins, N. D., G. C. Newbound, B. Albrecht, J. L. Beard, L. Ratner, and M. D. Lairmore. 1998. Selective ablation of human T-cell
94 lymphotropic virus type 1 p12I reduces viral infectivity in vivo. Blood 91:4701-4707.
11. Collins, N. D., G. C. Newbound, L. Ratner, and M. D. Lairmore. 1996. In vitro CD4(+) lymphocyte transformation and infection in a rabbit model with a molecular clone of human T-cell lymphotropic virus type 1. J. Virol. 70:7241-7246.
12. D'Agostino, D. M., L. Ranzato, G. Arrigoni, I. Cavallari, F. Belleudi, M. R. Torrisi, M. Silic-Benussi, T. Ferro, V. Petronilli, O. Marin, L. Chieco-Bianchi, P. Bernardi, and V. Ciminale. 2002. Mitochondrial alterations induced by the p13II protein of human T-cell leukemia virus type 1. Critical role of arginine residues. J Biol. Chem. 277:34424-34433.
13. Derse, D., J. Mikovits, and F. Ruscetti. 1997. X-I and X-II open reading frames of HTLV-I are not required for virus replication or for immortalization of primary T-cells in vitro. Virology 237:123-128.
14. Ding, W., B. Albrecht, R. E. Kelley, N. Muthusamy, S. J. Kim, R. A. Altschuld, and M. D. Lairmore. 2002. Human T-cell lymphotropic virus type 1 p12(I) expression increases cytoplasmic calcium to enhance the activation of nuclear factor of activated T cells. J Virol 76:10374-10382.
15. Ding, W., B. Albrecht, R. Luo, W. Zhang, J. R. Stanley, G. C. Newbound, and M. D. Lairmore. 2001. Endoplasmic reticulum and cis- Golgi localization of human T-lymphotropic virus type 1 p12(I): association with calreticulin and calnexin. J Virol 75:7672-7682.
16. Franchini, G. 1995. Molecular mechanisms of human T-cell leukemia/lymphotropic virus type I infection. Blood 86:3619-3639.
17. Green P.L. and I. S. Y. Chen. 2000. Human T-cell leukemia virus types 1 and 2, p. 1941-1969. Lippincott, Williams & Wilkins.
18. Hollsberg, P. 1999. Mechanisms of T-cell activation by human T-cell lymphotropic virus type I. Microbiol. Mol. Biol. Rev. 63:308-333.
19. Iwahara, Y., N. Takehara, R. Kataoka, T. Sawada, Y. Ohtsuki, H. Nakachi, T. Maehama, T. Okayama, and I. Miyoshi. 1990. Transmission of HTLV-1 to Rabbits via Semen and Breast Milk from Seropositive Healthy Persons. Int. J. Cancer 45:980-983.
20. Johnson, J. M., C. Nicot, J. Fullen, V. Ciminale, L. Casareto, J. C. Mulloy, S. Jacobson, and G. Franchini. 2001. Free Major Histocompatibility Complex Class I Heavy Chain Is Preferentially Targeted for Degradation by Human T-Cell Leukemia/Lymphotropic Virus Type 1 p12(I) Protein. J. Virol. 75:6086-6094. 95 21. Kim, S. J., W. Ding, B. Albrecht, P. L. Green, and M. D. Lairmore. 2003. A conserved calcineurin-binding motif in human T lymphotropic virus type 1 p12I functions to modulate nuclear factor of activated T cell activation. J Biol. Chem. 278:15550-15557.
22. Kimata, J. T., F. Wong, J. Wang, and L. Ratner. 1994. Construction and characterization of infectious human T-cell leukemia virus type 1 molecular clones. Virology 204:656-664.
23. Koralnik, I. J., J. Fullen, and G. Franchini. 1993. The p12, p13 and p30 proteins encoded by human T-cell leukemia/lymphotropic virus type- 1 open reading frames I and II are localized in three different cellular compartments. J. Virol. 67:2360-2366.
24. Lairmore, M. D., B. Roberts, D. Frank, J. Rovnak, M. G. Weiser, and G. L. Cockerell. 1992. Comparative biological responses of rabbits infected with human T-lymphotropic virus Type I isolates from patients with lymphoproliferative and neurodegenerative disease. Int. J. Cancer 50:124-130.
25. Mesnard, J. M. and C. Devaux. 1999. Multiple control levels of cell proliferation by human T- cell leukemia virus type 1 tax protein. Virology 257:277-284.
26. Miyoshi, I., S. Yoshimoto, I. Kubonishi, M. Fujishita, Y. Ohtsuki, M. Yamashita, K. Yamato, S. Hirose, H. Taguchi, and K. Niiya. 1985. Infectious transmission of human T-cell leukemia virus to rabbits. Int. J. Cancer 35:81-85.
27. Mortreux, F., A. S. Gabet, and E. Wattel. 2003. Molecular and cellular aspects of HTLV-1 associated leukemogenesis in vivo. Leukemia 17:26- 38.
28. Nicot, C., J. C. Mulloy, M. G. Ferrari, J. M. Johnson, K. Fu, R. Fukumoto, R. Trovato, J. Fullen, W. J. Leonard, and G. Franchini. 2001. HTLV-1 p12(I) protein enhances STAT5 activation and decreases the interleukin-2 requirement for proliferation of primary human peripheral blood mononuclear cells. Blood 98:823-829.
29. Pique, C., A. Uretavidal, A. Gessain, B. Chancerel, O. Gout, R. Tamouza, F. Agis, and M. C. Dokhelar. 2000. Evidence for the chronic in vivo production of human T cell leukemia virus type I Rof and Tof proteins from cytotoxic T lymphocytes directed against viral peptides. J. Exp. Med. 191:567-572.
30. 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 96 dispensable for the immortalization of primary lymphocytes. J. Virol. 72:4458-4462.
31. Uchiyama, T. 1997. Human T cell leukemia virus type I (HTLV-I) and human diseases. Annu. Rev. Immunol. 15:15-37:15-37.
32. Ye, J., L. Silverman, M. D. Lairmore, and P. L. Green. 2003. HTLV-1 Rex is required for viral spread and persistence in vivo but is dispensable for cellular immortalization in vitro. Blood. 102(12):3963-9.
33. 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.
34. 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.
97
Rabbit Inoculum Typea No. of cells
R1 ACH.1 1x107
R6 ACH.1 1x107
R3 ACH.30.1 1x107
R4 ACH.30.1 1x107
R5 ACH.30.1 1x107
R8 ACH.30.1 1x107
R9 ACH.30.1 1x107
R10 ACH.30.1 1x107
R11 Uninfected PBMC 1x107
Table 2.1. Rabbit groups and inocula a Twelve-week-old specific-pathogen-free New Zealand White rabbits were inoculated via the lateral ear vein as described in Material and Methods. The ACH.1 cell line was obtained by outgrowth of immortalized PBMC previously transfected with the full-length HTLV-1 molecular clone ACH11. The ACH.30.1 cell line was obtained as described above and contains a select mutation in ORF II of ACH30.
98
Antibody response at wk:a
Inoculum Rabbit 0 2 4 6 8
ACH.1 R1 - +++ +++ +++ +++
ACH.1 R6 - * +++ +++ +++
ACH.30.1 R3 - * * * *
ACH.30.1 R4 - * * + +
ACH.30.1 R5 - * * * *
ACH.30.1 R8 - - - - -
ACH.30.1 R9 - - - - -
ACH.30.1 R10 - * + + +
PBMC R11 - - - - -
Table 2.2. Western blot assay summary of antibody response to HTLV-1 antigens
a – indicates no response; +++ indicates a strong response (strong reactivity to both Gag and Env antigens); + indicates a weak response (weak reactivity to both Gag and Env antigens); * indicates an indeterminate response (reactivity to only one of Gag or Env antigens)
99
PCRa at wk:
Inoculum Rabbit 0 2 4 6 8
ACH.1 R1 - + + + +
ACH.1 R6 - + + + +
ACH.30.1 R3 - - + - -
ACH.30.1 R4 - + - + +
ACH.30.1 R5 - + - + +
ACH.30.1 R8 - - - - -
ACH.30.1 R9 - - - - -
ACH.30.1 R10 - + + + +
PBMC R11 - - - - -
Table 2.3. Viral detection in PBMC of rabbits by PCR a Amplification of HTLV-1 ORF II-specific proviral sequence (+), sensitivity estimated to be one viral copy per 5000 cells5
100
Inoculum Rabbit Copies per cella ACH.1 R1 .1922
ACH.1 R6 .6709
ACH.30.1 R4 <.0006
ACH.30.1 R5 <.0015
ACH.30.1 R10 ≤.0074
Table 2.4. Quantification of provirus in PBMC 8 weeks post-inoculation
a Proviral copy number per cell was calculated from the equivalency points determined at 8 weeks post-inoculation as described in Materials and Methods
101
Figure 2.1. A mutation in ORF II of the full-length HTLV-1 molecular clone ACH adds a diagnostic restriction endonuclease site. (A) The top schematic drawing represents the organization of the HTLV-1 provirus, including the four ORFs (ORF I and II, tax and rex) located in the pX region between env and the 3’ LTR. The lower schematic demonstrates the mutation created in ORF II of ACH, which is present in the ACH.30.1 cell line. A 24-bp linker, including a novel XbaI site, was inserted into a SacII site, producing a premature stop codon in the doubly spliced p30II transcript. (B) PCR amplification with the primer pair 7047-7492, specific for ORF II, produced a fragment of 445 bp from ACH plasmid and 469 bp from ACH.p30II plasmid. Lane 2 demonstrates an absence of sensitivity to XbaI digestion for the ACH plasmid. Lane 4 demonstrates XbaI digestion of the ACH.p30II plasmid. Lanes 5 and 6 demonstrate the 469 bp product isolated from the ACH.30.1 cell line and the digestion of this product with XbaI. (C) Sequence alignment of ACH.1 and ACH.30.1 showing the 24 bp insert used to introduce a stop codon into the p30II reading frame. The stop codon introduced into the p30II reading frame is underlined with a solid line. Notice that the first 15 nucleotides of the insert are of identical sequence to the fifteen nucleotides following the insertion site. This preserves wild-type p12II sequence. The p12II in-frame stop codon is underlined with a dashed line.
102
Figure 2.1
103
Figure 2.2. HTLV-1-specific serologic response of inoculated rabbits. Rabbit R1 was inoculated with the ACH.1 cell line and represents a group of two animals. Six animals were injected with the ACH.30.1 cell line; data for rabbits R5, R8, and R10 are shown from the group. Control animal, R11, was inoculated with uninfected PBMC. Data shown are absorbance (Abs) values from plasma samples diluted 1:12,000 and determined by anti-HTLV-1 antibody ELISA (A) or specific reactivity to HTLV-1 epitopes measured by Western blot analysis (B). * serum control band; ** cellular antigen
104
Figure 2.3. Viral loads of inoculated rabbits determined by qcPCR. HTLV- 1-specific sequences (rabbit 1 [R1]) were amplified from genomic DNA extracted from the PBMC of rabbits inoculated with ACH.1 or ACH.30.1 cells in the presence of increasing competitor (C) concentrations. (A) Representative gel from PBMC collected at 8 weeks post-inoculation from R1. (B) Regression curve for the gel in figure A. The log of the band intensity of the sample DNA (log IR1) divided by the log of the band intensity of the competitor DNA (log IStyI∆28) was plotted against the log of the band intensity of the competitor DNA (log CO (StyI∆28)). Equivalence was determined to be at the point where the y- axis value = 1.
105
Figure 2.4. Coamplification of ACH.1 wild-type and ACH.30.1 mutant sequence in ACH.30.1-inoculated rabbits. R5 and R10 are ACH.30.1 inoculated rabbits. (A) PCR product isolated 2 weeks post-inoculation from R5 and R10. Note that the PCR product contains multiple bands. (-) negative control; (+) positive control. (B) 2 week post-inoculation PBMC DNA from R5 and R10 was digested with XbaI to check for the presence of the ORF II mutation. Note that XbaI digestion completely (R5) or partially (R10) digests the upper band of the PCR product, indicating the presence of the ORF II mutation. (-) undigested (C) PCR product isolated 6 weeks post-inoculation from R5 and R10. Note that the PCR product is a single band of 445 bp similar to the PCR product amplified from the wild-type ACH plasmid. Sequencing of this PCR product indicated the wild-type ACH.1 sequence. (-) negative control; (+) positive control
106
CHAPTER 3
ROLE OF HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1) P30II IN
LYMPHOCYTE APOPTOSIS AND PROLIFERATION
3.1 Introduction
Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus causally linked with adult T-cell leukemia/lymphoma (ATL), HTLV-1 associated myelopathy/tropical spastic paresis (HAM/TSP), and a variety of other immune- mediated disorders45. Along with typical gag, pol, and env retroviral gene products, the HTLV-1 genome contains various regulatory and accessory genes encoded in the pX region between env and the 3’ long terminal repeat (LTR).
The pX region contains four open reading frames (ORFs). ORFs III and IV encode the well-characterized Rex and Tax proteins, respectively18. Tax is a
40-kDa nuclear phosphoprotein that increases viral transcription from the
HTLV-1 LTR. The ability of HTLV-1 to cause T-cell transformation is linked to dysregulation of cellular gene expression and cell cycle checkpoints by
Tax15,20,28. Rex is a 27-kDa nucleolar phosphoprotein that increases the cytoplasmic accumulation of nonspliced and singly spliced viral RNA18,50.
107 In contrast to the extensive knowledge about Tax and Rex structure and function, less is known about the role of pX ORF I- and II-encoded proteins in the replication cycle and pathogenesis of HTLV-1. p12I is a 99-amino acid protein that localizes to the endoplasmic reticulum and cis-Golgi, where it induces increased cytoplasmic calcium to enhance the activation of nuclear factor of activated T cells2,13,14,24,29. p12I has also been shown to enhance
STAT5 activation and decrease the interleukin-2 (IL-2) requirement for proliferation of primary human peripheral blood mononuclear cells (PBMCs)35, and pX ORF I is required for infection in an animal model8.
ORF II mRNA is spliced to the first Tax coding exon and encodes two proteins, a full-length p30II and an internally initiated p13II 6. The smaller protein, p13II, is derived from initiation at the first internal methionine codon in
ORF II and represents the carboxy-terminal 87 residues of p30II. The p30II and p13II proteins were initially found to localize to the nucleolus and nucleus, respectively26, and p13II was subsequently demonstrated to localize predominantly to mitochondrial membranes7,10. Localization of p13II to mitochondria is associated with mitochondrial clustering and energy-dependent swelling via a permeability transition pore independent mechanism without release of cytochrome c, suggesting altered mitochondrial respiratory activity7,10. Recent studies have also identified a role for p13II in reducing cellular proliferation and enhancing CREB-responsive element (CRE) responses to calcium-mediated stimuli, suggesting a role for p13II in mitochondrial-mediated cell signaling42.
108 HTLV-1 p30II is a 241 amino acid protein, which contains serine- and threonine-rich regions with distant homology to the transcription factors Oct-1,
Pit-1, and POU-M16. In vivo studies have demonstrated that p30II is necessary for establishment and maintenance of the HTLV-1 infection5,43. Although the role of p30II in viral pathogenesis is largely unknown, emerging evidence indicates a role for p30II in the regulation of gene expression at both the transcriptional and the post-transcriptional level. Nicot et al.33 and Younis et al.51 have demonstrated that p30II acts as a negative regulator of viral gene expression by retaining the doubly spliced Tax/Rex mRNA in the nucleus. We have demonstrated that p30II differentially regulates CRE and Tax-responsive element (TRE)-mediated transcription through an interaction with CREB binding protein/p30053,54. In a recent gene array study, Michael et al.30 confirmed the role of p30II as a selective regulator of cellular gene expression.
Apoptosis is an important innate antiviral defense that can result in aborted infection and elimination of infected cells27,36,44. In response, many viruses encode proteins that suppress host cell apoptosis, and suppression of apoptosis is believed to be critical for virus replication and virus-induced pathogenesis27,36,44. Gene expression array studies have shown a variety of apoptosis regulatory genes to have altered expression in HTLV-1-infected cell lines, as well as in ATL and HAM/TSP patients11,19,37. These studies have demonstrated a trend towards down-regulation to complete shut-off of apoptosis accelerators and an upregulation of apoptosis inhibitors19,37. While it is clear that apoptosis regulatory genes are dysregulated in the course of
109 HTLV-1 infection, how this occurs is not clear. There have been many studies focusing on the role of Tax in dysregulation of apoptosis regulatory genes
(recently reviewed in 23). While it is agreed that Tax modulates cellular apoptosis, whether this effect is pro-apoptotic or anti-apoptotic is subject to much debate and ample in vitro evidence exists to support both arguments23.
Data examining a potential role for other HTLV-1 viral proteins in the regulation of lymphocyte apoptosis is scant. Silic-Benussi et al.42 have recently provided the first evidence supporting p13II as an apoptosis regulator. Regarding p30II, only one study has addressed the role of p30II as a potential regulator of apoptosis30. This study was a gene array study, and it identified 19 apoptosis regulatory genes, which had altered expression in p30II-expressing Jurkat T cells. Herein, we address the question of whether or not the altered apoptosis regulatory gene expression by p30II translates into a functional role for p30II in modulation of cellular apoptosis. Our data indicate that p30II expression does not alter the susceptibility of transformed epithelial cells or Jurkat T cells to apoptotic stimuli, but does reduce cell proliferation in Jurkat T cells, which may lead to enhanced cell survival.
3.2 Materials and Methods
Cell lines. ACH.1 and ACH.30.1 cell lines were obtained from outgrowth of immortalized PBMCs previously transfected with the ACH or ACH.p30II clones, respectively9,38. PBMCs were isolated from normal human donors by Ficoll-
Hypaque (Pharmacia, Peapack, N.J.) centrifugation. Cells were maintained in
110 RPMI 1640 supplemented with 15% fetal bovine serum, L-glutamine (0.03 mg/mL), penicillin (100 µg/mL), streptomycin (100 µg/mL), and recombinant IL-2
(10 U/mL) (complete medium). The 293 cell line is a human embryonic kidney epithelial cell line (catalog number 1573, American Type Culture Collection).
293T is the 293 cell line which stabile expresses the simian virus 40 (SV40) T antigen (obtained from G. Franchini, National Insititute of Health). 293T cells were maintained in modified Dulbecco’s eagle medium containing 10% fetal bovine serum and 1% streptomycin and penicillin. Jurkat T cells (clone E6-1;
American Type Culture Collection catalog number TIB-152) were maintained in
RPMI 1640 supplemented with 15% fetal bovine serum, penicillin (100 µg/mL), streptomycin (100 µg/mL), and L-glutamine (0.03 mg/mL).
Lentiviral vectors and other plasmids. Generation of the pWPT-p30IIHA-
IRES-GFP and pWPT-IRES-GFP lentiviral vectors has been previously described30. The pME-p30IIHA and pME-18S plasmids have been previously described30.
Transient transfections. Sixty percent confluent 293T cells were transfected with 15 µg of either pME-p30IIHA or pME-18s using Superfect (Qiagen,
Valencia, CA) according to manufacturer’s protocol. After 48 h, transfected cells were washed with PBS and lysed with RIPA buffer (150 mM NaCl, 0.01 M sodium pyrophosphate, 10 mM EDTA, 10 mM sodium fluoride, 50 mM Tris (pH
8.0), 0.1% SDS, 12.8 mM deoxycholic acid, 10% glycerol, 1% NP-40) containing one complete mini protease inhibitor cocktail tablet (Roche Applied
Science, Indianapolis, IN). Cell suspensions were incubated on ice for 20 min,
111 and the lysates were centrifuged at 14,000 rpm for 20 min at 4° C. Supernatant was stored at -80° C.
Recombinant lentivirus production and transduction of Jurkat T lymphocytes. Production of recombinant lentivirus and transduction of Jurkat
T lymphocytes with pWPT-IRES-GFP and pWPT-p30IIHA-IRES-GFP has been previously described30. Briefly, Jurkat T cells (5 x 106 cells in 3 mL) were spin infected (2700 rpm, 30° C, 1 h) with pWPT-IRES-GFP or pWPT-p30IIHA-IRES-
GFP vector at multiplicity of infection (MOI) of 3 in the presence of polybrene at
5 µg/mL (Sigma, St. Louis, MO). The cells were left in the virus-containing media for 24 h, then fed with and maintained in fresh media. Cells were analyzed for p30II expression by lysing as described for 293T cells, followed by western immunoblotting.
Apoptosis induction. For lymphocyte cell lines (ACH.1, ACH.30.1, Jurkat T cells), apoptosis was induced with camptothecin (10 µM, 4 h)39, etoposide (12
µg/mL, 6 h)46, and TRAIL (TNF-related apoptosis inducing ligand) (1 µg/mL, 2 h)17. 293T cells were induced into apoptosis with camptothecin (10 µM, 24 h)52 and etoposide (12 µg/mL, 24 h)40. Drug doses were optimized for maximal apoptosis induction prior to each experiment.
Flow cytometry. ACH.1, ACH.30.1, and Jurkat T cells were prepared for flow cytometry by labeling with Annexin V Alexa Fluor® 488 conjugage (Molecular
Probes, Eugene, OR) and propidium iodide (PI) (Molecular Probes) or Annexin
V Alexa Fluor® 647 conjugate (Molecular Probes) according to manufacturer’s protocol. In brief, the cells were collected, washed once with PBS, and re-
112 suspended at 1 x 106 cells/mL in 100 µL of annexin-binding buffer (Molecular
Probes), followed by incubation with 5 µL annexin V conjugate solution and 1
µL 100 µg/mL PI for 15 min at room temp. After the incubation period, 400 µL of annexin-binding buffer was added, and samples were kept on ice. The samples were analyzed by flow cytometry (Coulter Epics Elite, Beckman
Coulter Inc., Fullerton, CA) and data were analyzed using Coulter Flow Center software (Beckman Coulter Inc.). For each sample, 10,000 gated cells were examined for annexin V and PI staining, and the percentage of cells in early apoptosis was defined by high annexin V- and low PI-staining cell population.
All annexin V assays were performed in a minimum of three independent experiments. Nonparametric Wilcoxon rank sum test was used for statistical analysis of significant apoptosis induction and comparison of apoptosis induction between cell lines.
To estimate apoptosis events in the adherent cell line, 293T, flow cytometry was performed on fixed cells following PI staining34. Following apoptosis induction, transfected 293T cells were incubated 30 min with 50
µg/mL PI. Detached cells were collected with the culture supernatant, pelleted by centrifugation, and washed with PBS. Adherent cells were washed twice with PBS, harvested by standard trypsinization, and pooled with the pellet of detached cells. One mL of the total cell suspension (1 x 106 cells/mL) was fixed with 10 ml 70% EtOH on ice for 2 h. Cells were centrifuged for 10 min, supernatant was removed, and cells were suspended in 100 µL DNA extraction buffer (0.2 M phosphate citrate buffer, pH 7.8) and incubated at 37° for 1 h with
113 gentle agitation. Cells were centrifuged at 2600 rpm and resuspended in 1 mL
PI staining solution (10 mL PBS, 200 µL DNase-free RNase A (10 mg/mL), 200
µL PI (1 mg/mL), 10 µL Triton X-100) for 30 min and analyzed for DNA content with flow cytometry (Coulter Epics Elite Analzyer, Beckman Coulter Inc.) using a doublet discrimination protocol (Coulter Flow Center software, Beckman Coulter
Inc.) in order to calculate the percentage of cells with subgenomic DNA content, which is reflective of apoptosis. All analysis results were based on data from a minimum of three independent experiments. Nonparametric Wilcoxon rank sum test was used for statistical analysis of significant apoptosis induction and comparison of apoptosis induction between cell lines.
Western blot assays. The expression of p30II in 293T cells and Jurkat T cells was analyzed by western blot assay. Protein concentrations in cell lysates were determined by bicinchoninic acid (BCA) assay (micro-BCA Protein Assay,
Pierce, IL). 50 µg of 293T cell lysates or total lysate from 1 x 107 Jurkat T cells were separated by SDS-10% polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose membranes. Membranes were blocked with 5% non- fat dry milk and 10% fetal bovine serum in Tris-buffered saline with 0.1% Tween
(TBST) for 2 h at room temp., then incubated with primary antibody overnight at
4° C. For HA detection, mouse anti-HA monoclonal Ab (1:1000) (clone 16B-12)
(Covance Research Products, Princeton, NJ) was used. For the C-terminus of cleaved Poly (ADP-ribose) polymerase (PARP) detection, mouse monoclonal anti-PARP Ab (1:1000) (clone C-2-10) (Oncogene Research Products, Boston,
MA) was used. For β-actin detection, mouse monoclonal anti-β-actin Ab
114 (1:4000) (clone AC-74) (Sigma) was used. Western blots were developed with horseradish peroxidase-labeled secondary Ab (1:1000) and enhanced chemiluminescence reagent (Cell Signaling Technology, Beverly, MA).
Cell proliferation assay. For comparison of short term (1-24 h) cell growth curves, MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt] CellTiter 96® cell proliferation assay kit
(Promega, Madison, WI) was used according to manufacturer’s protocol. 6.25 x
104, 1.25 x 105, or 2.5 x 105 cells/mL pWPT-IRES-GFP or pWPT-p30IIHA-IRES-
GFP infected Jurkat T cells were seeded in triplicate in 100 µL of fresh media in a 96 well plate. After 1 , 6 , 12 , 18.5, and 24 h, 20 µL of MTS reagent was added to each well and incubated for an additional 2 h. A photospectrometric microplate reader (Beckmann Coulter Inc.) was used to measure optical density at 490 nm. A mixed model was used for statistical analysis to compare replication rates between cell lines.
For comparison of long term (1-5 d) growth curves, a trypan blue staining assay was used. A total of 2 x 105 cells was plated in 3 mL of RPMI in a six well plate in triplicate. Cells were counted every 24 h. Briefly, cells were pelleted, washed with PBS, and resuspended in 100 µL PBS. Viable cells were counted with trypan blue staining. Each sample was counted 3 times. A regression model with repeated measures that also included time as a quadratic effect in addition to cell line effect was used to study the change in proliferation over time and between cell lines. A nonparametric Wilcoxon rank sum test was used to compare total cell numbers between cell lines.
115 3.3 Results
Wild type and p30II mutant HTLV-1 immortalized cell lines respond differently to apoptosis induction. Features of the ACH.1 and ACH.30.1 cell lines have been previously described9,38,43. Immortalized human T-cell lines that continually produce either wild-type HTLV-1 (ACH.1) or HTLV-1 containing a mutation in ORF II, which is predicted to produce a severely truncated p30II protein (ACH.30.1), were developed. As we have reported, the cell lines were representative of the phenotype of T cells immortalized by HTLV-1 and had typical expression profiles of CD3, CD4, CD8, and CD259,38. PCR amplification across ORF II of DNA extracted from the cell lines followed by XbaI digestion of the PCR product and sequencing analysis confirmed the fidelity of the mutation in the ACH.30.1 cell line43.
To determine if the ACH.1 and ACH.30.1 cell lines would display differential sensitivity to apoptotic stimuli, we performed annexin V/PI flow cytometry on the cell lines following treatment with various apoptosis inducing agents. Camptothecin, etoposide, and TRAIL were used to induce apoptosis.
Camptothecin is a topoisomerase I inhibitor, which induces apoptosis in cells in the S phase of the cell cycle (reviewed in 22). Etoposide is a topoisomerase II inhibitor, which induces apoptosis via the intrinsic apoptosis induction pathway25,48. TRAIL is a member of the TNF ligand family, which induces apoptosis through activating the death receptors (reviewed in 41). Following treatment with etoposide, there was no significant difference in the degree of apoptosis induction between ACH.1 and ACH.30.1 cell lines (nonparametric
116 Wilcoxon rank sum test, p-value 0.248) (Figure 3.1). Neither the ACH.1 cell line nor the ACH.30.1 cell line was susceptible to TRAIL-mediated apoptosis
(nonparametric Wilcoxon rank sum test, p-value 0.593 and 0.414, respectively)
(Figure 3.1). In three to five independent trials, camptothecin induced apoptosis in the ACH.30.1 cell line to a greater degree than in the ACH.1 cell line
(nonparametric Wilcoxon rank sum test, p-value 0.025) (Figure 3.1). Increased susceptibility to camptothecin-induced apoptosis in the ACH.30.1 cell line suggests that within this cell line, cells are more likely to be in the S phase of the cell cycle compared to the ACH.1 cell line.
HTLV-1 p30II does not modulate apoptosis in 293T cells. To examine the role of p30II in modulating cellular apoptosis independent of other viral proteins, we expressed p30II in 293T cells using the pME-p30IIHA vector and compared the percentage of cells induced into apoptosis with 293T cells transfected with an empty vector control (pME-18S) after being left untreated or following treatment with camptothecin or etoposide. 293T cells were not susceptible to
TRAIL-mediated apoptosis (data not shown). Analysis for apoptosis was done via immunoblot assay for the 89 kDa fragment of cleaved PARP47 (Figure 3.2). p30II expression did not induce apoptosis in untreated cells. Additionally, although both camptothecin and etoposide induced apoptosis in 293T cells, a differential degree of PARP cleavage was not seen between p30II-expressing cells and negative control cells.
Because immunoblot analysis is not quantitative and would not detect subtle differences in apoptosis induction, we repeated these experiments using
117 flow cytometry for DNA content to detect a sub-G1 peak, which is indicative of apoptosis34 (Figure 3.3). p30II expression in 293T cells did not induce 293T cells into apoptosis (nonparametric Wilcoxon rank sum test, p-value 0.824).
Additionally, although both camptothecin and etoposide induced apoptosis in
293T cells, there was no percentage difference in apoptotic cells between p30II- expressing cells and negative control cells for either camptothecin or etoposide
(nonparametric Wilcoxon rank sum test, p-value 0.369 and 1.00, respectively).
HTLV-1 p30II expression does not modulate apoptosis in Jurkat T cells.
Because HTLV-1 is a virus which infects T lymphocytes, we next used a lentivirus expression system30 to express p30II in Jurkat T cells, which are a non-HTLV-1-infected immortalized T cell line. A corresponding mock cell line was generated by spin-infecting Jurkat T cells with the empty lentiviral vector virus, pWPT-IRES-GFP. The expression of p30II was verified by western blot assay (Figure 3.4B). Expression of p30II in Jurkat T cells did not result in increased apoptosis when left untreated compared to mock infected cells (data not shown). Following establishment of a p30II-expressing Jurkat T cell line and a mock infected cell line, the cell lines were treated with camptothecin, etoposide, or TRAIL and assayed for apoptosis via annexin V flow cytometry
(Figure 3.4A). Although the infected cells were induced into apoptosis following treatment with either camptothecin, etoposide, or TRAIL, there was not a significant difference in the percentage of apoptotic cells between p30II- expressing Jurkat T cells and mock infected Jurkat T cells for any of the
118 treatment groups (nonparametric Wilcoxon rank sum test, p values: camptothecin 0.827, etoposide 0.513, TRAIL 0.127).
HTLV-1 p30II does not alter proliferation rate but causes delay in cell division in Jurkat T lymphocytes. Our data indicated that wild-type HTLV-1 immortalized human T lymphocytes (ACH.1) and human T-lymphocytes immortalized with a p30II-mutant HTLV-1 molecular clone (ACH.30.1) responded differently to apoptosis induction with camptothecin, with ACH.30.1 cells having a greater percentage of cells induced into apoptosis compared to
ACH.1 cells (Figure 3.1). Because camptothecin displays specificity for cells in the S phase of the cell cycle, these data suggested that at any given point in time, a greater percentage of ACH.30.1 cells are in the S phase of the cell cycle compared to the ACH.1 cells. This is consistent with what we see when culturing these cells, in that the ACH.30.1 cells clearly replicate faster than the
ACH.1 cells (data not shown). There are two possible explanations for this.
One is that p30II is modulating the cell cycle, with an absence of p30II leading to a faster replication rate. The other explanation is that differences in the ACH.1 and the ACH.30.1 cells in terms of site of integrated provirus or number of integrated proviral copies manifest as differential replication rates. These cell lines have been previously shown to produce equivalent amounts of p19 on a per cell basis43. In order to address the effect of p30II on modulating cellular replication, we compared replication rates in the p30II infected Jurkat T cells with the mock infected Jurkat T cells using a short term (<24 h) MTS cell proliferation assay. Data were collected with seeding of cells at 3 different
119 concentrations (6.75 x 104, 1.25 x 105, and 2.5 x 105 cells per ml) in triplicate at
1, 6, 12, 18.5, and 24 h post-seeding in 2 independent experiments. Using a mixed model to take into account that data were correlated across time and two different experiments were performed, we found that there were no significant differences between the mock cell line and the p30II-expressing cell line replication rate after adjusting for concentration and time (p-value 0.418)
(Figure 3.5A).
To examine p30II modulation of cell proliferation over a more extended time period (1-5 d), we next compared the growth of the p30II-expressing Jurkat
T cell line and the mock infected Jurkat T cell line by a trypan blue exclusion assay (Figure 3.5B). Interestingly, following seeding of 2 x 105 cells, the growth curve of the p30II-expressing Jurkat T cells differed from that of the mock infected Jurkat T cells (p-value 0.018 after adjusting for time in a quadratic model) due to an initial lag in the p30II-expressing Jurkat T cell growth rate compared to that of the mock infected Jurkat T cells, and at day 5 there were reduced numbers of p30II-expressing Jurkat T cells compared to mock infected
Jurkat T cells (nonparametric Wilcoxon rank sum test, p-value 0.050).
3.4 Discussion
Although data supports that HTLV-1 p30II is necessary for maintenance of the virus in vivo5,43, the definitive role of p30II in viral pathogenesis is unknown. Several groups have reported that p30II is able to modulate viral and cellular gene expression at either the transcriptional or post-transcriptional
120 level30,33,51,53,54. One of these studies was a gene array study, which implicated p30II in modulation of expression of a variety of cellular genes, including many apoptosis regulatory genes30. In this study, we sought to determine if this translated into a functional role for p30II in modulation of cellular apoptosis.
We began by exposing the wild-type HTLV-1 immortalized cell line,
ACH.1, and the p30II-mutant HTLV-1 immortalized cell line, ACH.30.1, to various inducers of apoptosis and then quantifying the percentage of cells induced into apoptosis. For induction of apoptosis, we used camptothecin, a topoisomerase I inhibitor with specificity for cells in the S phase of the cell cycle22, etoposide, a topoisomerase II inhibitor that induces apoptosis via the intrinsic pathway25,48, and TRAIL, a member of the TNF ligand family that induces apoptosis via the extrinsic pathway41. Through these studies, we found that the p30II-mutant ACH.30.1 cell line consistently responded to camptothecin treatment with a greater percentage of cells being induced into apoptosis compared to the wild-type ACH.1 cells. No differential apoptosis induction was seen following treatment with either etoposide or TRAIL. These data suggested that within the ACH.30.1 cell line, more cells are in the S phase of the cell cycle at any given point in time compared to the ACH.1 cell line, pointing to a potential role for p30II in modulation of cell cycle. We then proceeded to examine the effects of p30II in modulating cellular apoptosis independent of other viral proteins. We first utilized a transient transfection system to express p30II in the human embryonic kidney epithelial cell line, 293T. In this system, we were unable to demonstrate a role for p30II in modulation of apoptosis by
121 either the more qualitative approach of immunoblot for PARP cleavage or by the more quantitative approach of flow cytometry for DNA content analysis.
Because HTLV-1 is a virus which naturally infects lymphocytes, we then established a p30II-expressing Jurkat T cell line and a mock infected Jurkat T cell line and compared apoptosis induction in these cell lines following treatment with camptothecin, etoposide, and TRAIL. We did not find evidence for a role for p30II in modulating apoptosis either before or after treatment with apoptosis inducing agents in these lymphocyte cell lines. Because we had found that camptothecin induces increased apoptosis in HTLV-1-infected lymphocytes immortalized with the p30II mutant molecular clone, ACH.30.1, and camptothecin exhibits specificity for cells in the S phase of the cell cycle, we compared the proliferation of the p30II-expressing Jurkat T cell line with that of the mock infected cell line. Our data indicated that p30II reduces overall cell numbers in lymphocyte cultures due to an initial lag in cell proliferation but does not alter cell proliferation rate.
Cellular apoptosis is an important antiviral defense used to abort infection and eliminate infected cells27,36,44. Viral anti-apoptotic proteins have been identified in a wide range of viruses including lymphotropic γ herpesviruses, α and β herpesviruses, poxviruses, papovaviruses, adenoviruses, African swine fever virus, and baculoviruses (reviewed in 27,44).
Various studies have indicated that HTLV-1 infection protects cells from undergoing apoptosis4,49, and gene array studies have demonstrated a trend towards decreased expression of pro-apoptotic genes and increased
122 expression of anti-apoptotic genes in HTLV-1 transformed T cells19,37. How
HTLV-1 protects lymphocytes from undergoing apoptosis is less clear. There have been numerous studies pointing towards a role for Tax in modulating lymphocyte apoptosis, with evidence to support both a pro-apoptotic effect and an anti-apoptotic effect (reviewed in 23). Gene array studies of Tax expression in lymphocytes confirm that Tax expression induces both pro- and anti- apoptotic gene expression12,32, and there is data to suggest that which set of genes expressed is in part dependent on the stage of the cell cycle12. The mitochondrial-localizing p13II has been associated with increased susceptibility to apoptosis following Ca2+ influx into mitochondria in rat fibroblast cell lines42.
Regarding p30II, our study is the first to address the functional role of p30II in modulation of cellular apoptosis.
Because HTLV-1 p30II has been evolutionarily conserved, we believe it plays a critical role in HTLV-1 pathogenesis. Previous studies have indicated
II an inability for the HTLV-1 virus to establish and maintain infection without p30 in in vivo systems5,43. There is not a large body of literature addressing the functional role of p30II in viral pathogenesis. As such, we believe it is important to report studies which help to rule out functional effects for p30II. Although data in this paper points against a role for p30II in modulating cellular apoptosis, there are caveats to drawing this as an absolute conclusion. HTLV-1 is a virus which infects quiescent primary lymphocytes, whereas the experiments done in this study utilized either immortalized or transformed cells. Studies exist in which an HTLV-1 viral protein modulates an effect in quiescent primary
123 lymphocytes but not in activated primary lymphocytes and not in immortalized cell lines1. We are currently working on developing a system for expressing p30II in primary lymphocytes, but to date, we have been unable to do so.
Therefore, until some of these studies are repeated using primary lymphocytes, a role for p30II in modulating lymphocyte apoptosis cannot be entirely excluded.
We set out to determine if p30II has a role in modulation of cellular apoptosis and in the process collected data which points to a role for p30II in modulating cellular proliferation. The differential response of ACH-derived cell lines to camptothecin suggested a modulation of cellular proliferation by p30II, and proliferation studies on a p30II-expressing Jurkat cell line showed that p30II expression leads to reduced cell numbers in longer term lymphocyte cultures due to an initial lag in cell proliferation. These data suggest a role for p30II in modulating the cell cycle parameters of T lymphocytes. In this regard, p30II may play a similar role as HIV Vpr, which modulates the cell cycle of T lymphocytes by causing G2 arrest leading to enhanced virus replication
(reviewed in 3). There is abundant data to support a role for Tax in modulating cell proliferation and cell cycle (reviewed in16,21,31). Although there is gene array data to suggest a role for p30II in modulating cell cycle30, further studies on the role of p30II in modulation of cell proliferation and cell cycle have not yet been reported. Ongoing work in our laboratory is examining these questions in hopes of shedding light on the mechanism of p30II modulation of cell proliferation and cell cycle.
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130
Figure 3.1. Differential camptothecin-induced apoptosis in HTLV-1 immortalized cell lines. ACH.1 and ACH.30.1 cell lines were exposed to various apoptosis inducing agents and assayed for percentage of cells induced into apoptosis via annexin V flow cytometry. Data represents the results of at least three independent experiments. Jurkat T cells were used as a positive control. A) Representative result of annexin V flow cytometry following apoptosis induction with camptothecin (10 µM, 4 h). Apoptotic fraction is seen in the lower right quadrant by FACS analysis. B) Following treatment with camptothecin (10 µM, 4 h), a greater percentage of ACH.30.1 cells was induced into apoptosis compared to ACH.1 cells (nonparametric Wilcoxon rank sum test, p-value 0.025). ACH.30.1 cells and ACH.1 cells were induced into apoptosis to an equal degree following treatment with etoposide (12 µg/mL, 6 h) (nonparametric Wilcoxon rank sum test, p-value 0.248). Neither ACH.1 nor ACH.30.1 cells were induced into apoptosis following treatment with TRAIL (1 µg/mL, 2 h) (nonparametric Wilcoxon rank sum test, p-value 0.593 and 0.414, respectively). C) As a positive control, apoptosis was induced in Jurkat T cells with all apoptosis inducing agents. * Statistically significant apoptosis induction; ** Statistically more apoptosis induction in ACH.30.1 cells compared to ACH.1 cells following treatment with camptothecin.
131
Figure 3.1
132
Figure 3.2. HTLV-1 p30II does not modulate apoptosis in 293T cells (PARP cleavage). 293T cells were transiently transfected with either pME-p30IIHA or the empty pME-18S vector. Cells were untreated or treated with camptothecin (10µM, 24 h) or etoposide (12 µg/mL, 24 h). Cell lysates were harvested and 50 µg of lysate was separated by SDS-PAGE. Apoptosis was assayed via immunoblot for the 89 kDa fragment of cleaved PARP. Expression of p30II was verified via immunoblot for HA. Expression of β-actin was verified as a loading control. – cells transfected with empty vector control; + cells transfected with pME-p30IIHA.
133
Figure 3.3. . HTLV-1 p30II does not modulate apoptosis in 293T cells (flow cytometry). 293T cells were transiently transfected with either pME-p30IIHA or the empty pME-18S vector. Cells were untreated or treated with camptothecin (10µM, 24 h) or etoposide (12 µg/mL, 24 h). Cells were analyzed for apoptosis by flow cytometry for DNA content. Data represents the results of three independent experiments. A) A representative example of p30II-expressing 293T cells before and after treatment with etoposide. The apoptotic cell fraction is seen as the sub-G1 peak. B) Results of three independent experiments show that p30II does not modulate cellular apoptosis in 293T cells when assayed via flow cytometry (nonparametric Wilcoxon rank sum test; untreated cells, p-value 0.824; camptothecin, p-value 0.369; etoposide, p-value 1.00).
134
Figure 3.3
135
Figure 3.4. HTLV-1 p30II does not modulate apoptosis in Jurkat T lymphocytes. A) p30II-expressing Jurkat T cells or mock infected Jurkat T cells were treated with camptothecin (10 µM, 4 h), etoposide (12 µg/mL, 6 h), or TRAIL (1 µg/mL, 2 h) and assayed for apoptosis induction via annexin V flow cytometry. Data represents the results of three independent experiments. Although camptothecin, etoposide, and TRAIL induced both cell lines into apoptosis, a differential degree of apoptosis induction was not seen between the two cell lines (nonparametric Wilcoxon rank sum test, p values: camptothecin 0.827, etoposide 0.513, TRAIL 0.127). B) Expression of HA- epitope tagged p30II in Jurkat T cells with a lentiviral infection system demonstrated by western immunoblot assay seventeen days post-infection. Jurkat T cells were infected with p30II-expressing (pWPT p30II) or mock (pWPT) lentiviral vectors, each at multiplicity of infection of three. The cell lysates were subject to SDS-PAGE followed by western immunoblotting using mouse monoclonal anti-HA antibody.
136
Figure 3.5. HTLV-1 p30II does not alter proliferation rate but is associated with an initial lag in cell proliferation in Jurkat T lymphocytes. A) p30II- expressing Jurkat T cells or mock infected Jurkat T cells were assayed for proliferation rate using an MTS cell proliferation assay. No difference in replication rate was seen between the two cell lines (Mixed model, p-value 0.418). B) p30II-expressing Jurkat T cells or mock infected Jurkat T cells were assayed for growth using a trypan blue exclusion assay. The p30II-expressing Jurkat T cell line growth curve differed from that of the mock infected Jurkat T cell line (p-value 0.018 after adjusting for time in a quadratic model) due to an initial lag in the p30II-expressing Jurkat T cell growth rate compared to that of the mock infected Jurkat T cells. By day 5, p30II- expressing Jurkat T cell cultures had fewer total cell numbers compared to mock infected Jurkat T cell cultures (nonparametric Wilcoxon rank sum test, p-value 0.050)
137
CHAPTER 4
IN VIVO ANALYSIS OF REPLICATION AND IMMUNOGENICITY OF
PROVIRAL CLONES OF HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 WITH
SELECTIVE ENVELOPE SURFACE UNIT MUTATIONS
4.1 Introduction
Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus etiologically linked with adult T-cell leukemia/lymphoma (ATL), HTLV-1- associated myelopathy/tropical spastic paresis (HAM/TSP), and a variety of other immune-mediated disorders23. HTLV-1 Env is unique among retroviral envelope proteins in that when isolates from ATL or HAM/TSP patients are compared, there is a high degree of sequence conservation in the env region, with variability ranging from 1 to at most 8%7,8,24,32,36,41. HTLV-1 Env is a 488 amino acid protein. It is synthesized as a polyprotein precursor (gp62), which is subsequently glycosylated and cleaved into two proteins, surface unit gp46
(SU) and transmembrane gp21 (TM)15,21. SU is required for entry into the target cell by mediating specific attachment to a cellular receptor, which was recently reported to be the glucose transporter, GLUT-125. The TM supports fusion between viral and cellular membranes to allow viral entry.
138 HTLV-1 SU is a 312 amino acid protein. The C-terminal half of SU is very antigenic in humans, being recognized by antibodies in serum from over
95% of HTLV-1 infected individuals32. Amino acids 187-196 of SU are a major target for neutralizing antibodies as evidenced by the use of monoclonal antibodies to inhibit syncytium formation or infectivity as well as ELISA peptide binding assays and in vivo peptide immunization studies1,2,10,19,30,32,39. Early studies using site directed mutagenesis demonstrated functional domains within
SU involved in intracellular maturation, syncytium formation, and the association between SU and TM9,10,20,32,33. Subsequent development of a cell- to-cell transmission assay allowed for separation of fusion events from infectivity events and verified that cell-to-cell fusion and cell-to-cell transmission are independent events11,34.
A major breakthrough in HTLV-1 research came with the development of the ACH HTLV-1 molecular clone in the mid 1990’s18. Subsequent mutagenesis of the wild-type ACH HTLV-1 molecular clone allowed for characterization of mutations of key viral proteins in the context of the entire virus3,5,38. Wild-type and mutant HTLV-1 molecular clones were used to create immortalized human peripheral blood mononuclear cell (PBMC) lines, which were inoculated into rabbits to define how mutations in key viral proteins alter viral infectivity in vivo3,5,38,44. It is through such studies that the necessity of Rex as well as the viral accessory proteins p121 and p30II for establishment and maintenance of infection in vivo has been determined.
139 Based on earlier studies using transient transfections of HTLV-1 env plasmids mutated in key determinants of Env, Tsukahara et al.42 created identical mutations in the Env region of the ACH molecular clone. Specifically, point mutations were generated to alter key amino acids 75, 81, 95, 101, 105, and 195 of the SU protein to create the full length proviral plasmids ACH.75,
ACH.81, ACH.95, ACH.101, ACH.105, and ACH.195, respectively. Transient transfections of these molecular clones into 293T cells showed similar levels of
Gag (p55Prgag, p24, p19) and Env (gp62, gp46, gp21) production compared to wild-type ACH, with the exception of ACH.81, which produced slightly lower levels of gp4642. Cell-free and cell-to-cell transmission assays showed these clones to be equally infectious compared to the wild-type ACH molecular clone, with the exception of ACH.81 infection, which resulted in slightly decreased levels of p19 production42. With the exception of ACH.101, these molecular clones were then shown to be capable of immortalizing human PBMCs in vitro42.
Unresolved is the question of whether these in vitro findings correlate with virus replication events in vivo. Specifically, given that there is little sequence variation in the HTLV-1 Env protein in the natural infection, would mutations in Env that are maintained in in vitro immortalization assays be tolerated in the in vivo infection? Would these mutations alter the antibody responses in the in vivo infection? In this study, we address these questions by comparing the antibody responses and proviral loads between rabbits inoculated with the wild-type ACH.1 cell line to rabbits inoculated with the
140 ACH.75, ACH.95, and ACH.195 cell lines. These mutations all fall within regions predicted to be important for binding of SU to the viral receptor based on syncytium assays, and the ACH.195 mutation falls within a region, which is a major target for neutralizing antibody responses1,2,10,19,30,39. Our data indicate that following infection with these cell lines, rabbits became infected, and the fidelity of the mutations was maintained through 8 weeks of infection. The
ACH.75 and ACH.95 mutation caused a decreased antibody response as measured by whole virus ELISA. Interestingly, when assayed by western blot, the ACH.195 mutation was associated with a decreased antibody response to the SU protein, and in one ACH.195 rabbit, we were able to detect an antibody response to the HTLV-2 SU protein. In another ACH.195 rabbit, we were able to demonstrate provirus in the PBMCs in the absence of a detectable antibody response. Comparison of PBMC proviral loads between inoculation groups indicated that the ACH.75 group had higher proviral loads than the ACH.1 and
ACH.95 groups. However, correlation analysis did not demonstrate a relationship between PBMC proviral loads and the SU antibody responses.
Our data confirm that earlier results obtained with ACH Env mutants via in vitro assays correlate with in vivo findings. Additionally, our data support previous reports of the importance of the region in SU between amino acids 187-196 for immunogenicity in vivo. Our study is the first to directly demonstrate that mutations in HTLV-1 SU, while altering proviral loads and antibody responses against HTLV-1 Env, did not prevent virus replication in vivo.
141 4.2 Materials and Methods
Viral clones and cell lines. The derivation and infectious properties of the full- length ACH viral clone have been reported6,18. The derivation and in vitro infectious properties of ACH.75, ACH.95, and ACH.195 viral clones have also been reported42. ACH point mutants contain the env genes of HTLV-1 envelope mutants from the HTE series9 inserted between the SphI site at position 5121 and the NsiI site at position 6565 in the ACH clone18.
ACH.1, ACH.75, ACH.95, and ACH.195 cell lines were obtained from the outgrowth of immortalized PBMCs previously transfected with the ACH,
ACH.75, ACH.95, and ACH.195 clones, respectively6,42. PBMCs were isolated from normal human donors by Ficoll-Hypaque (Pharmacia, Peapack, New
Jersey) centrifugation. Cells were maintained in RPMI 1640 supplemented with
15% fetal bovine serum, L-glutamine (0.3 mg/mL), penicillin (100 U/mL), streptomycin (100 µg/mL), and recombinant IL-2 (10 U/mL) (complete medium).
Detection of viral p19 matrix antigen. To estimate and compare levels of virus production between the ACH.1, ACH.75, ACH.95, and ACH.195 cell lines, duplicate samples of 106 cells from each cell line were washed and seeded in a
24-well plate in one mL of complete RPMI. Culture samples were collected at
72 h, serially diluted 10-fold, and tested for HTLV-1 p19 matrix antigen by a commercially available enzyme-linked immunosorbent assay (ELISA)
(Zeptometrix Corporation, Buffalo, New York). According to manufacturer, sensitivity of detection is 25 pg/mL.
142 Detection of proviral sequences. For detection of provirus in cell lines and rabbit PBMCs, genomic DNA was harvested by affinity column (Qiagen,
Valencia, California) and examined for the presence of HTLV-1 sequences following PCR amplification. For cell lines, 1 µg of DNA was amplified using a primer pair specific for the HTLV-1 env region (5321, 5’-
CAGCAGATCAGGCCCTACAG-3’; and 5885, 5’-
GGAGAGTATAGGACGTGCCAAG-3’). For sequencing of provirus in rabbit
PBMC, 1 µg of DNA was amplified using a primer pair specific for the HTLV-1 pX env region (ACH.75: 5321 and 5885; ACH.95: 5321 and 5580, 5’-
CTTCCAGTAGGGGCTGGAGA-3’; ACH.195: 5718, 5’-
CAGCCAACTGCCTCCCACCG-3’ and 5885). After an initial 10 min incubation period at 94°C to activate the Taq polymerase (AmpliTaq Gold; Applied
Biosystems, Foster City, California), 40 cycles of PCR were performed with the following cycle parameters: denaturation at 94°C for 1 min, annealing for 1 min at 60° (cell lines - 5321, 5885), 64.8° ( PBMC – 5321,5885), or 55° (PBMC –
5718, 5885; PBMC – 5351, 5580), and extension at 72°C for 45 s, followed by a final extension at 72°C for 5 min. The amplified products were separated in a
10% polyacrylamide gel and visualized with ultraviolet light following ethidium bromide staining.
HTLV-1-specific PCR products were sequenced to further confirm specificity. PCR products were purified (Qiagen) and sequenced by the automated dye terminator cycle sequencing method (3700 DNA analyzer and
143 Big Dye terminator cycle sequencing chemistry; Applied Biosystems) using the
5’ primer used for the PCR amplification.
Real time PCR. The technique used for real time PCR has been previously reported26. DNA was extracted from the PBMCs with the Qiagen Blood Mini Kit according to manufacturer’s protocol (Qiagen). Quantity and quality of the DNA were assessed by NanoDrop spectroscopy (NanoDrop Technologies,
Wilmington, Delaware). HTLV-1 tax gene primers at final concentration of 300 nM each of forward and reverse primers and 200 nM of dual labeled probe, as well as the real time PCR conditions, were previously described26. Final concentrations of each forward and reverse 18S DNA primers were 900 nM, and final concentration of dual labeled probe was 200 nM. Sequences of the
18S primers were forward: 5’-CGGCTACCACATCCAAGGAA-3’; reverse: 5’-
GCTGGAATTACCGCGGCT-3’; probe: 5’-VIC-
TGCTGGCACCAGACTTGCCCTC-TAMRA-3’. Standard curves of the HTLV-1 tax gene or 18S DNA endogenous control were included on the same optical plate as the unknown test specimen samples. For each run, a standard curve was generated from triplicate samples of log 10 dilutions of purified plasma in
DNAse/RNAse free water. Senstivity of detection was estimated to be 82 copies per microgram of DNA.
Rabbit inoculation. To test the in vivo replication capacity of each cell line, 12- week-old female specific-pathogen-free New Zealand White rabbits (Harlan,
Indianapolis, Indiana) were inoculated via the lateral ear vein. Inocula were equilibrated by comparing viral p19 protein production per cell measured by
144 ELISA as described above. Inoculations were performed in two experiments.
In the first, a total of 107 ACH.1 cells (n=2), ACH.95 cells (n=2), or uninfected
PBMC (n=1) were inoculated (Table 4.1). In the second experiment, a total of
107 ACH.1 cells (n=1), 107 ACH.75 cells (n=4), 1.43 x 107 ACH.95 cells (n=2), or 1.35 x 107 ACH.195 cells (n=4) were inoculated (Table 4.1). All cells were gamma irradiated (7,500 R) prior to injection to prevent outgrowth of the cellular inoculum in vivo but allow virus transmission5.
Serologic and clinical analyses. The plasma antibody response to HTLV-1 in inoculated rabbits was determined with a commercial ELISA with solid phase consisting of purified HTLV-1 viral lysate and recombinant HTLV-1 TM (p21) antigen (BioMerieux, Inc., Durham, North Carolina), which was adapted for use with rabbit plasma by substitution of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:3,000 dilution; Chemicon, Temecula,
California). Plasma was diluted 1:12,000 to obtain values in the linear range of the assay, and data were expressed as absorbance values. Absorbance values greater than or equal to three standard deviations above the average absorbance value for all week 0 samples were considered to indicate a positive antibody titer. Estimated specificity according to manufacturer is greater than
99%. Reactivity to specific viral antigenic determinants was detected with a commercial HTLV-1 western blot assay (Genelabs Diagnostics, Singapore) adapted for rabbit plasma by use of alkaline phosphatase-conjugated goat anti- rabbit immunoglobulin G (1:1000 dilution; BioMerieux, Inc.). Plasma showing reactivity to Gag (p24 or p19) and Env (p21 or gp46) antigens was classified as
145 positive for HTLV-1 seroreactivity. For determination of positive and negative antibody responses, plasma was diluted at 1:100. In order to quantitate antibody responses, densitometry was performed on western blots using a commercial software package (AlphaEase, Alpha Innotech Corporation, San
Leandro, California). Because plasma dilutions of 1:100 saturated the western blot detection system, western blots were run at plasma dilutions of 1:2000 for densitometry analysis. Rabbits were regularly evaluated for any overt signs of clinical disease. Rabbits were euthanized and necropsied at a post-inoculation interval of 8 weeks.
4.3 Results
In vitro analysis of viral clones. We have reported that the amino acid point mutations Ser75Ile, Asn95Asp, and Asn195Asp within the Env SU protein do not affect the ability of the virus to immortalize PBMCs in vitro42. ACH Env SU point mutants contain the env genes of HTLV-1 envelope mutants from the HTE series9 inserted between the SphI site at position 5121 and the NsiI site at position 6565 in the ACH clone18,42 (Figure 4.1). To further investigate the effects of these point mutations, we developed immortalized human T-cell lines that continually produce either wild-type HTLV-1 (ACH.1) or HTLV-1 containing point mutations Ser75Ile, Asn95Asp, or Asn195Asp in the Env SU protein
(ACH.75, ACH.95, ACH.195, respectively). As we have reported, these cell lines were representative of the phenotype of T cells immortalized by HTLV-1 and had typical expression profiles of CD4 and CD86,42. To ensure that the
146 mutation was present prior to inoculation, a region of env containing the mutation site was amplified by PCR from each of the ACH.1, ACH.75, ACH.95, and ACH.195 cell lines. The amplified fragment was sequenced. Sequencing results confirmed the presence of the expected mutation in each cell line.
Serologic response of rabbits to viral clones. To evaluate the effects of point mutations within Env SU in vivo, we compared the abilities of the ACH.1,
ACH.75, ACH.95, and ACH.195 cell lines to establish and maintain infection in our rabbit model. To ensure comparable infection potentials, inocula were equilibrated by HTLV-1 p19 antigen production on a per-cell basis (Table 4.1).
Prior to inoculation, the fidelity of the mutations was confirmed by sequencing.
Three rabbits were inoculated with the ACH.1 cell line. For each of the ACH.75,
ACH.95, and ACH.195 cell lines, a group of four rabbits was inoculated. One rabbit was inoculated with uninfected PBMCs as a negative control.
Serologic response of rabbits to the inocula was determined by measuring titers of antibody directed against purified HTLV-1 viral lysate and recombinant TM (p21) protein by ELISA (Figure 4.2A). Responses were assayed from each rabbit’s serum at 0, 2, 4, and 8 weeks post-inoculation. By week 4, a positive antibody titer was detected in one of two ACH.1 inoculated rabbits, and three of four rabbits in each of the ACH.75, ACH.95, and ACH.195 inoculated groups. All rabbits developed a positive antibody titer by week 8 except one ACH.95 inoculated rabbit and one ACH.195 inoculated rabbit. Pair- wise comparisons between each inoculum group using a Bonferroni adjustment for multiple comparisons with an α level of 0.05 did not reveal a statistically
147 significant difference in ELISA absorbance values between any two inoculum groups at week 4. Using a similar analysis on week 8 samples, pair-wise comparisons showed statistically significant lower absorbance values in the
ACH.95 group compared to the ACH.1 group (p-value 0.034) and borderline statistically significant lower absorbance values in the ACH.75 group compared to the ACH.1 group (p-value 0.107) (Figure 4.2B).
Reactivity to specific HTLV-1 antigens was confirmed at 0, 2, 4, and 8 weeks post-inoculation by western blot analysis with plasma dilution of 1:100
(Table 4.2). With the exception of one ACH.195 inoculated rabbit (R21), all rabbits showed strong antibody responses by week 4 post-inoculation. In R21, we were able to detect a weak antibody response to the TM protein transiently at week 2. Interestingly, in another ACH.195 inoculated rabbit (R20), in addition to strong antibody responses against HTLV-1 Gag and Env TM proteins and a weak antibody response to HTLV-1 Env SU protein, we were also able to detect a strong antibody response to HTLV-2 SU (Figure 4.3). Because the plasma dilution of 1:100 saturated our detection system, we repeated the western blots with a 1:2000 plasma dilution followed by densitometry analysis in order to detect differences in reactivity to Env proteins between the inoculation groups
(Figure 4.4). Interestingly, results showed that compared to other inoculation groups, the ACH.195 inoculation group mounted a weaker antibody response to the SU protein as evidenced by both a lower SU average pixel density and a lower SU/TM average pixel density ratio.
148 Proviral loads. To measure the ability of each HTLV-1 clone to maintain viral loads in vivo, we determined the number of provirus copies per cell in rabbit
PBMCs by real time PCR for all rabbits at 8 weeks post-inoculation. Average proviral loads for ACH.1, ACH.75, ACH.95, and ACH.195 were 0.0039, 0.0290,
0.0071, and 0.0143 copies per cell, respectively (Figure 4.5). The negative control rabbit inoculated with uninfected PBMCs had no detectable proviral loads. The overall t test (ANOVA test) yielded a significant group effect (p- value < 0.0001). Pairwise comparisons using Bonferroni to adjust for multiplicity were performed using an overall α-level of 0.05. The ACH.75 inoculated rabbits had statistically significant higher proviral loads compared to
ACH.1 and ACH.95 inoculated rabbits (p-value 0.009 and 0.037, respectively).
There was no statistically significant difference in proviral loads between
ACH.95, ACH.195, and ACH.1 inoculated rabbits. The ACH.195 inoculated rabbit, R21, which did not have a detectable antibody response to HTLV-1 structural antigens, had low but detectable proviral loads (0.0052 copies per cell). Interestingly, other rabbits such as ACH.95-inoculated R16 and R17 and
ACH.1-inoculated R5 and R10 had similar or lower proviral loads as R21 but mounted strong antibody responses (Figure 4.5).
In order to determine if there is a relationship between the antibody response to SU and PBMC proviral loads, we performed correlation analysis of
PBMC proviral loads against the western blot densitometry values obtained for the SU protein (Figure 4.6). No correlation was found between PBMC proviral loads and SU densitometry values (Spearman correlation coefficient 0.19).
149 Sequence and restriction enzyme analysis. For each rabbit week 8 PBMC sample, we extracted DNA and sequenced the PCR products derived from env sequence specific primer pairs across the region spanning the mutations to ensure that there were no unexpected mutations or reversions within env.
Sequence analysis showed that all env mutations were maintained in all rabbits through the 8 week post-inoculation time point.
4.4 Discussion
Although numerous studies have addressed the effects of point mutations in the HTLV-1 env gene in an in vitro setting4,9,10,20,32,33,42, no studies to date have correlated in vitro findings to the replication capacity or immunogenicity of the virus in vivo. In this study, we expanded the findings of
Tsukahara et al.42, who demonstrated that the amino acid point mutations
Ser75Ile, Asn95Asp, and Asn195Asp within the Env SU protein do not alter the ability of HTLV-1 to infect and immortalize lymphocytes in vitro. At 2 months post-exposure, all ACH.75, ACH.95, ACH.195, and wild-type ACH.1 inoculated rabbits were infected with HTLV-1 with the fidelity of the mutation maintained.
ACH.75 and ACH.95 rabbits had decreased antibody response to purified
HTLV-1 viral lysate and recombinant TM (p21) by ELISA compared to wild-type
ACH.1 rabbits. With the exception of one ACH.195 rabbit, all rabbits mounted strong antibody responses by 8 weeks post-inoculation. Interestingly, one
ACH.195 rabbit had no detectable antibody response by ELISA and western blot, but proviral loads were comparable to rabbits, which mounted strong
150 antibody responses. Western blot and densitometry analysis indicated that the
ACH.195 group mounted a weaker antibody response to the SU protein compared to all other groups. Within the ACH.195 inoculated group, a single rabbit also mounted an antibody response to the HTLV-2 SU protein. ACH.75 rabbit PBMCs had higher proviral loads than the ACH.1 and the ACH.95 groups. Interestingly, correlation analysis did not reveal a relationship between
PBMC proviral loads and the antibody response to HTLV-1 SU.
In this study, we confirmed that mutations in HTLV-1 SU, which were tolerated by the virus in in vitro lymphocyte immortalization assays, do not alter the ability of HTLV-1 to replicate, adding validity to in vitro lymphocyte immortalization assays. The ACH.195 inoculation group was the most variable in their antibody responses, which is consistent with data from previous in vitro syncytium and infectivity inhibition studies, peptide binding ELISA studies, and in vivo peptide immunization studies, which implicate this amino acid to be in a highly immunogenic region1,2,10,19,30,39. Serological studies of HTLV-1 infected humans have clearly implicated amino acid 195 to lie within a region known to elicit neutralizing antibodies during HTLV-1 infection1,2,19,30. In addition, in vivo vaccine studies in animals have indicated that synthetic peptides spanning the region containing amino acid 195 have the potential to protect against HTLV-1 infection in rabbits39,40. However, in this study, we found no correlation between anti-Env antibody responses and proviral loads, indicating that a robust initial antibody response to Env did not correlate with increased protection from infection. Within humans, during the clinically asymptomatic HTLV-1 infection,
151 proviral loads in PBMCs range from 0.04 to 0.001 copies per cell12,14,16,29,37.
With the exception of one ACH.75 inoculated rabbit, all rabbit PMBC proviral loads in our study were within this range, indicating that our rabbit model closely mimics the natural asymptomatic infection common in most HTLV-1 infected humans.
Two rabbits within the ACH.195 inoculation group were of particular interest in our study. Rabbit R21 had a PBMC proviral load consistent with that seen within asymptomatic HTLV-1 infected individuals. However, with the exception of a transient antibody response to the TM protein at week 2, we were unable to detect an antibody response in this rabbit. It is likely that this animal did mount an antibody response, but that it was below the level of our assay detection sensitivity. The serologic and PCR results in this animal are consistent with reports of rare HTLV-1 infected individuals that are serologically negative while PCR positive for the virus in their PBMCs22,31,35. Another rabbit of interest in the ACH.195 group was the R20 rabbit, which mounted an antibody response not only to HTLV-1 Gag and Env proteins, but also to the
HTLV-2 SU protein. In this rabbit, the antibody response to HTLV-2 SU was stronger than that mounted against HTLV-1 SU. That a single amino acid change in the HTLV-1 SU could result in a seropositive result for HTLV-2 in an animal that clearly was not infected with HTLV-2 has interesting implications for serologic screening in humans13,17,22.
The HTLV-1 env region maintains a high degree of sequence conservation during the in vivo infection, with variability ranging from 1-
152 8%7,8,24,32,36,41. There are various reports of single base pair changes in the env sequence in HTLV-1 isolates from infected individuals8,43. We are not aware that the amino acid changes made in this study have been demonstrated as common variants in the human infection. Given the low degree of env sequence variation found in humans, it leads one to predict that the mutations would not be tolerated in vivo. However, studies on the in vivo expansion of
HTLV-1 have demonstrated that HTLV-1 spreads in vivo mainly by the relatively error-free process of clonal expansion of infected lymphocytes rather than by the error-prone process of reverse transcription27,28, and it may be that the absence of sequence variation is due more to an absence of the need to undergo reverse transcription rather than an inability for sequence variants to maintain infection. The maintenance of single point mutations in Env over the 8 week course of our study in rabbits is more supportive of clonal expansion of infected lymphocytes versus cell-free infection.
Future studies are needed to further define how specific mutations (e.g.
ACH.195) alter the in vivo pathogenesis of HTLV-1. We believe that our current study adds validity to both the use of in vitro lymphocyte immortalization assays as a model to study HTLV-1 Env, as well as the rabbit as a model of HTLV-1 infection. Additionally, this study provides further evidence to implicate the region spanning Env amino acid 195 to be highly immunogenic during the in vivo infection.
153 4.5 References
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14. Hashimoto, K., I. Higuchi, M. Osame, and S. Izumo. 1998. Quantitative in situ PCR assay of HTLV-1 infected cells in peripheral blood lymphocytes of patients with ATL, HAM/TSP and asymptomatic carriers. J Neurol Sci 159:67-72.
15. Hattori, S., T. Kiyokawa, K. Imagawa, F. Shimizu, E. Hashimura, M. Seiki, and M. Yoshida. 1984. Identification of gag and env gene products of human T-cell leukemia virus (HTLV). Virology 136:338-347.
16. Kamihira, S., N. Dateki, K. Sugahara, T. Hayashi, H. Harasawa, S. Minami, Y. Hirakata, and Y. Yamada. 2003. Significance of HTLV-1 proviral load quantification by real-time PCR as a surrogate marker for HTLV-1-infected cell count. Clin. Lab Haematol. 25:111-117.
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155 18. Kimata, J. T., F. Wong, J. Wang, and L. Ratner. 1994. Construction and characterization of infectious human T-cell leukemia virus type 1 molecular clones. Virology 204:656-664.
19. Kuroki, M., M. Nakamura, Y. Itoyama, Y. Tanaka, H. Shiraki, E. Baba, T. Esaki, T. Tatsumoto, S. Nagafuchi, S. Nakano, and Y. Niho. 1992. Identification of New Epitopes Recognized by Human Monoclonal Antibodies with Neutralizing and Antibody-Dependent Cellular Cytotoxicity Activities Specific for Human T-Cell Leukemia Virus Type 1. J. Immunol. 149:940-948.
20. Le, B., I, M. P. Grange, L. Delamarre, A. R. Rosenberg, V. Blot, C. Pique, and M. C. Dokhelar. 2001. HTLV-1 structural proteins. Virus Res. 78:5-16.
21. Lee, T. H., J. E. Coligan, T. Homma, M. F. McLane, N. Tachibana, and M. Essex. 1984. Human T-cell leukemia virus-associated membrane antigens: identity of the major antigens recognized after virus infection. Proc. Natl. Acad. Sci. USA 81:3856-3860.
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23. Mahieux, R. and A. Gessain. 2003. HTLV-1 and associated adult T-cell leukemia/lymphoma. Rev. Clin. Exp. Hematol. 7:336-361.
24. Malik, K. T., J. Even, and A. Karpas. 1988. Molecular cloning and complete nucleotide sequence of an adult T cell leukaemia virus/human T cell leukaemia virus type I (ATLV/HTLV-I) isolate of Caribbean origin: relationship to other members of the ATLV/HTLV-I subgroup. J. Gen. Virol. 69 ( Pt 7):1695-1710.
25. Manel, N., F. J. Kim, S. Kinet, N. Taylor, M. Sitbon, and J. L. Battini. 2003. The ubiquitous glucose transporter GLUT-1 is a receptor for HTLV. Cell 115:449-459.
26. Miley, W. J., K. Suryanarayana, A. Manns, R. Kubota, S. Jacobson, J. D. Lifson, and D. Waters. 2000. Real-time polymerase chain reaction assay for cell-associated HTLV type I DNA viral load. AIDS Res. Hum. Retroviruses 16:665-675.
27. Mortreux, F., A. S. Gabet, and E. Wattel. 2003. Molecular and cellular aspects of HTLV-1 associated leukemogenesis in vivo. Leukemia 17:26- 38. 156 28. Mortreux, F., I. Leclercq, A. S. Gabet, A. Leroy, E. Westhof, A. Gessain, S. Wain-Hobson, and E. Wattel. 2001. Somatic mutation in human T-cell leukemia virus type 1 provirus and flanking cellular sequences during clonal expansion in vivo. J. Natl. Cancer Inst. 93:367- 377.
29. Nagai, M., K. Usuku, W. Matsumoto, D. Kodama, N. Takenouchi, T. Moritoyo, S. Hashiguchi, M. Ichinose, C. R. Bangham, S. Izumo, and M. Osame. 1998. Analysis of HTLV-I proviral load in 202 HAM/TSP patients and 243 asymptomatic HTLV-I carriers: high proviral load strongly predisposes to HAM/TSP. J Neurovirol. 4:586-593.
30. Palker, T., M. Tanner, R. Scearce, R. Streilen, M. Clark, and B. Haynes. 1989. Mapping of Immunogenic Regions of Human T-Cell Leukemia Virus Type 1 (HTLV-I) gp46 and gp21 envelope glycoproteins with Env-encoded synthetic peptides and a monoclonal antibody to gp46. J. Immunol. 142:971-978.
31. Pancake, B. A., D. Zucker-Franklin, M. Marmor, and P. M. Legler. 1996. Determination of the true prevalence of infection with the human T- cell lymphotropic viruses (HTLV-I/II) may require a combination of biomolecular and serological analyses. Proc. Assoc. Am. Physicians 108:444-448.
32. Pique, C. and M. C. Dokhelar. 1991. The HTLV-1 Envelope: An Overview, p. 499-510. In W. A. Haseltine and F. Wongstaal (eds.), Genetic Structure and Regulation of HIV. Raven Press,Ltd., New York.
33. Pique, C., T. Tursz, and M. C. Dokhelar. 1990. Mutations introduced along the HTLV-I envelope gene result in a non functional protein: a basis for envelope conservation ? EMBO J. 9:4243-4248.
34. Rosenberg, J. R., L. Delamarre, C. Pique, D. Pham, and M. C. Dokhelar. 1997. The ectodomain of the human T-cell leukemia virus type 1 TM glycoprotein is involved in postfusion events. J. Virol. 71:7180- 7186.
35. Segurado, A. A., C. M. Malaque, L. M. Sumita, C. S. Pannuti, and R. B. Lal. 1997. Laboratory characterization of human T cell lymphotropic virus types 1 (HTLV-1) and 2 (HTLV-2) infections in blood donors from Sao Paulo, Brazil. Am. J. Trop. Med. Hyg. 57:142-148.
36. Seiki, M., S. Hattori, Y. Hirayama, and M. Yoshida. 1983. Human adult T-cell leukemia virus: Complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc. Natl. Acad. Sci. 80:3618- 3622.
157 37. Shinzato, O., S. Ikeda, S. Momita, Y. Nagata, S. Kamihira, E. Nakayama, and H. Shiku. 1991. Semiquantitative analysis of integrated genomes of human T-lymphotropic virus type I in asymptomatic virus carriers. Blood 78:2082-2088.
38. 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.
39. Tanaka, Y., R. Tanaka, E. Terada, Y. Koyanagi, N. Miyanokurosaki, N. Yamamoto, E. Baba, M. Nakamura, and H. Shida. 1994. Induction of antibody responses that neutralize human T- cell leukemia virus type I infection in vitro and in vivo by peptide immunization. J. Virol. 68:6323- 6331.
40. Tanaka, Y., L. Zeng, H. Shiraki, H. Shida, and H. Tozawa. 1991. Identification of a Neutralization Epitope on the Envelope gp46 Antigen of Human T-Cell Leukemia Virus Type I and Induction of Neutralizing Antibody by Peptide Immunization. J. Immunol. 147:354-360.
41. Tsujimoto, A., T. Teruuchi, J. Imamura, K. Shimotohno, I. Miyoshi, and M. Miwa. 1988. Nucleotide sequence analysis of a provirus derived from HTLV-1-associated myelopathy (HAM). Mol. Biol. Med. 5:29-42.
42. Tsukahara, T., M. M. Wielgosz, and L. Ratner. 2001. Characterization of envelope glycoprotein mutants for human T-cell leukemia virus type 1 infectivity and immortalization. J. Virol. 75:9553-9559.
43. Yang, Y. C., T. Y. Hsu, M. Y. Liu, M. T. Lin, J. Y. Chen, and C. S. Yang. 1997. Molecular subtyping of human T-lymphotropic virus type I (HTLV- I) by a nested polymerase chain reaction-restriction fragment length polymorphism analysis of the envelope gene: Two distinct lineages of HTLV-I in Taiwan. J. Med. Virol. 51:25-31.
44. Ye, J., L. Silverman, M. D. Lairmore, and P. L. Green. 2003. HTLV-1 Rex is required for viral spread and persistence in vivo but is dispensable for cellular immortalization in vitro. Blood. 102(12):3963-9.
158
Rabbit Inoculum Typea No. of Cells R1 ACH.95 1 x 107
R2 ACH.95 1 x 107
R16 ACH.95 1.43 x 107
R17 ACH.95 1.43 x 107
R11 ACH.75 1 x 107
R12 ACH.75 1 x 107
R13 ACH.75 1 x 107
R14 ACH.75 1 x 107
R18 ACH.195 1.35 x 107
R19 ACH.195 1.35 x 107
R20 ACH.195 1.35 x 107
R21 ACH.195 1.35 x 107
R5 ACH.1 1 x 107
R10 ACH.1 1 x 107
R22 ACH.1 1 x 107
R9 PBMC 1 x 107
Table 4.1. Rabbit groups and inocula a Twelve-week-old specific pathogen-free New Zealand White rabbits were inoculated via the lateral ear vein as described in Material and Methods. ACH.95, ACH.75, ACH.195, and ACH.1 cell lines were obtained by outgrowth of immortalized PBMCs previously transfected with the full-length HTLV-1 molecular clones ACH.95, ACH.75, ACH.195, and ACH, respectively6,42. Inocula were equilibrated by p19 gag production on a per cell basis prior to inoculation.
159
Antibody response at wk:a Inoculum Rabbit 0 2 4 8
ACH.95 R1 - + +++ +++
ACH.95 R2 - + +++ +++
ACH.95 R16 - +++ +++ +++
ACH.95 R17 ∆ +++ +++ +++
ACH.75 R11 - +++ +++ +++
ACH.75 R12 - +++ +++ +++
ACH.75 R13 - + +++ +++
ACH.75 R14 - +++ +++ +++
ACH.195 R18 - +++ +++ +++
ACH.195 R19 - * +++ +++
ACH.195 R20 - + +++^ +++^
ACH.195 R21 - * - -
ACH.1 R5 - + +++ +++
ACH.1 R10 - * +++ +++
ACH.1 R22 - +++ +++ +++
PBMC R9 - - - -
Table 4.2. Western blot assay summary of antibody response to HTLV-1 antigens a – indicates no response; +++ indicates a strong response (strong reactivity to both Gag and Env antigens); + indicates a weak response (weak reactivity to 160 both Gag and Env antigens); * indicates an indeterminate response (reactivity to only one of Gag or Env antigens); ^ indicates an antibody response to HTLV- 2 SU; ∆ slight response to Env TM was seen and was interpreted as a cross reaction due to the high concentration of serum (1:100) in the reaction.
161
Figure 4.1. Construction of the ACH-envelope clones. A) ACH.75, ACH.95, and ACH.195 contain the env gene of the HTE mutants9 between an SphI site and an NsiI site in the ACH molecular clone. The point mutants are designated by the wild-type amino acid, the position (in comparison to initiator methionine), and the mutant amino acid. SU, surface protein; TM, transmembrane protein; CD, cytoplasmic domain. B) HTLV-1 SU protein. Schematic summary of functional domains of SU1,2,9,10,19,20,30,32,33,39. The position of amino acid mutants examined in this study is indicated in bold.
162
Figure 4.2. ELISA absorbance values on serum samples. A) ACH represents a group of two animals. ACH.75, ACH.95, and ACH.195 represent a group of four animals. PBMC represents a negative control rabbit inoculated with uninfected PBMCs. The data shown are absorbance values from plasma diluted 1:12,000 and determined by anti-HTLV-1 antibody ELISA. * indicates a positive antibody titer. B) The data shown are from the week 8 serum samples. A statistically significant lower antibody titer was seen in ACH.95 and ACH.75 inoculated rabbits compared to wild-type ACH.1 inoculated rabbits (pairwise comparisons with Bonferroni adjustment, p-value 0.034 and 0.107, respectively).
163
Figure 4.3. HTLV-1-specific antibody response by western blot. Data represents 1:2000 dilutions of week 8 post-inoculation plasma. R11, R12, R13, and R14 were inoculated with ACH.75. R1, R2, R16, and R17 were inoculated with ACH.95. R18, R19, R20, and R21 were inoculated with ACH.195. R5, R10, and R22 were inoculated with ACH.1. R9 was inoculated with uninfected PBMC as a negative control. *, serum control band; **, cellular antigen; ‡, HTLV-2 SU band.
164
Figure 4.4. Semi-quantitative analysis of HTLV-1 SU and TM specific antibody responses. Densitometry analysis was performed on the western blots shown in figure 4.3. A) SU average pixel density. The ACH.195 SU average pixel density was lower than that seen in other inoculation groups. B) TM average pixel density. The ACH.195 TM average pixel density was similar to that seen in other inoculation groups. C) SU average pixel density/TM average pixel density. ACH.195 rabbits mounted a lower SU antibody response relative to their TM antibody response compared to rabbits in other inoculation groups.
165
Figure 4.5. Proviral loads in week 8 PBMC. DNA was extracted from 8 week post-inoculation PBMCs of rabbits. Proviral loads were quantitated with real time PCR as described in Materials and Methods. Proviral loads in ACH.75 inoculated rabbits were statistically higher compared to ACH.1 and ACH.95 inoculated rabbits (pairwise comparisons with Bonferroni adjustment, p-value 0.009 and 0.037, respectively).
166
Figure 4.6. PBMC proviral loads do not correlate with antibody response to SU. PBMC proviral loads were analyzed for correlation with degree of antibody response to SU. No correlation was found (Spearman correlation coefficient 0.19).
167
CHAPTER 5
SYNOPSIS AND FUTURE DIRECTIONS
5.1 Introduction
Since the initial discovery of HTLV-1 over two decades ago, much progress has been made in our understanding of its epidemiology, associated diseases, and molecular pathogenesis. Despite this progress, there is still much which remains unknown. Within this thesis, we expand our knowledge of
HTLV-1 in several areas. In chapter 2, we utilized the rabbit model to determine the significance of ORF II p30II to the in vivo infection. Our data demonstrated that in the absence of functional p30II, HTLV-1 infection results in reduced antibody responses and proviral loads. Moreover, mutant p30II HTLV-
1 immortalized lymphocytes were not infectious in our rabbit model unless a reversion to wild-type sequence occurred. These data provide evidence that p30II is required for HTLV-1 to establish persistent infection. Moreover, this study further expands our use of the rabbit as a model of the asymptomatic
HTLV-1 infection.
In chapter 3, we expand our understanding of the functional role of p30II in HTLV-1 pathogenesis. With previous gene array data indicating a role for
168 p30II in modulation of apoptosis regulatory genes, we sought to determine if this translates into a functional role for p30II in regulation of cellular apoptosis.
Our hypothesis was that p30II enhances viral infectivity and in vivo persistence by suppressing lymphocyte apoptosis, thereby allowing the virus to be maintained in vivo. We were unable to demonstrate that p30II modulates cellular apoptosis, which was important because it helps to rule out a functional effect for this protein, about which so little is known. We did discover a potential cell cycle modulatory effect of p30II. Specifically, our data demonstrates that p30II reduces cell proliferation by delaying onset of division.
In chapter 4, we return to our rabbit model to examine the effects of point mutations in the Env SU protein at the initial stages of HTLV-1 infection. This chapter represents the first in vivo study of Env mutants in the context of the entire virus. Specifically, we are the first to demonstrate that despite the minimal Env sequence variation observed in the natural HTLV-1 infection, this virus does tolerate Env mutations in an in vivo setting. All rabbits exposed to lymphocytes immortalized by Env mutant HTLV-1 provirus became persistently infected, with proviral loads comparable to that which is seen in the natural human asymptomatic infection, which further validates the rabbit as an animal model of HTLV-1. Findings in this in vivo study were similar to previous findings in lymphocyte immortalization assays, further validating the use of lymphocyte immortalization assays as a means to study the Env SU protein.
Mutations in SU did alter antibody responses. The reduced antibody response to SU and the high degree of variability in the antibody response to SU within
169 the ACH.195 group further confirmed the importance of the region spanning amino acid 195 to the immune response.
As is the universal nature of science, with any new discovery, although some questions are answered, more questions arise. The studies in this thesis have opened the doors to numerous new questions, some of which are addressed in the ensuing sections.
5.2 The requirement for p30II in establishment of HTLV-1 infection
It has been previously shown that the ACH.p30II molecular clone, which is predicted to produce a truncated mutant of p30II, is capable of infecting and immortalizing lymphocytes in vitro24. In chapter 2, we demonstrate that infection with lymphocytes immortalized by this molecular clone do not establish infection in vivo unless a reversion to wild-type p30II sequence occurs. This result indicates that there is preferential selection towards expression of wild- type p30II during the initial stages of HTLV-1 infection. However, a question that remains open is whether or not p30II is an absolute requirement for HTLV-1 infection.
The ACH.p30II molecular clone was made by inserting a 24 base pair linker with a stop codon into the wild-type ACH molecular clone24. Moreover, this linker had high sequence homology to the nucleotide sequence immediately 3’ to its insert location. It is easy to envision how during the process of reverse transcription, such a linker could be eliminated as the reverse transcriptase enzyme moved between the two strands of the diploid
170 RNA genome (reverse transcription is reviewed in 5). It would be interesting to see if forced replication by reverse transcription through serial passage of lethally gamma irradiated cells with uninfected PBMCs in vitro resulted in a similar reversion event.
In order to address the question of whether p30II is preferred for establishment of in vivo infection or required for establishment of in vivo infection, it would be necessary to repeat the study with an immortalized lymphocyte cell line, which is not prone to reversion to wild-type p30II expression. Such a study has been done with the ORF I p12I mutant HTLV-1 immortalized lymphocyte cell line, ACH.12 6. In this study, expression of p12I was eliminated by mutation of the p12I splice junction, and rabbits exposed to the ACH.12 cell line did not become infected with HTLV-1. Although it would have been ideal to create a similar splice junction mutation to eliminate p30II expression, because of the high degree of overlap of coding sequences of accessory and regulatory genes within the pX region4,19, creation of such a mutation, which does not disrupt the coding sequence of other proteins in this region, is difficult. An alternate approach would be to use small interfering
RNAs (siRNA) to eliminate p30II expression following infection with the wild type
ACH.1 cell line.
5.3 The role of p30II in modulation of lymphocyte apoptosis
In chapter 3, our data indicates that p30II does not modulate lymphocyte apoptosis. Within this chapter, we utilized ACH lymphocyte cell lines, Jurkat T
171 lymphocyte cell lines, and 293T epithelial cell lines. Although use of immortalized cell lines in initial studies is a common technique due to the ease of working with cell lines, these cell lines do not mimic the natural HTLV-1 infection, which is specific for quiescent primary lymphocytes. Albrecht et al.1 demonstrated that ORF I p12I is required for establishment of HTLV-1 infection in quiescent primary lymphocytes but not in activated primary lymphocytes.
Similarly, it is possible that p30II modulates functional effects which are apparent in primary quiescent lymphocytes, but not in activated primary lymphocytes or in immortalized cell lines. Until the role of p30II in modulation of apoptosis in primary quiescent lymphocytes is examined, a role for p30II in modulating lymphocyte apoptosis in HTLV-1 infection cannot be excluded.
One approach to this would be to examine apoptosis events in cocultures of primary lymphocytes and lethally irradiated ACH.1 and ACH.30.1 cell lines.
Another approach would be to establish a p30II expression system in primary lymphocytes. The lentiviral expression system used in chapter 3 has been used to infect primary CD4+ lymphocytes, and expression of p30II mRNA was confirmed in this system23. However, confirmation of protein expression in this system has not yet been demonstrated.
5.4 The role of p30II in modulation of cell cycle
Work with the ACH cell lines in chapter 3 provides evidence for a role for p30II in modulation of cell cycle. The ACH.30.1 cell line responded to camptothecin treatment with a greater percentage of cells being induced into
172 apoptosis compared to the ACH.1 cell line. Because camptothecin selectively induces apoptosis in cells in the S phase of the cell cycle (reviewed in 15), this result suggested that within the ACH.30.1 cell line, more cells are in the S phase of the cell cycle compared to the ACH.1 cell line. Following up on this, we examined the role of p30II in modulation of cell proliferation and found that although p30II does not alter the replication rate of Jurkat T lymphocytes, it does reduce cell proliferation by delaying the onset of cell division. This result is particularly intriguing in light of what is known about the HIV Vpr protein. Vpr modulates the cell cycle of T lymphocytes by causing G2 arrest, which leads to enhanced virus replication (reviewed in 2). Modulation of cell cycle by Vpr is characterized by low levels of cyclin B1 and wee-1 kinase as well as inhibition of the phosphatase, cdc25c13,32. Further work is necessary to determine the mechanism of p30II-mediated delay of cell division. Ongoing work in our laboratory has demonstrated that similar to Vpr, p30II also modulates a delay in the G2/M transition (A. Datta, personal communication). Interestingly, previous gene array study has demonstrated that p30II expression in Jurkat T lymphocytes is associated with downregulation of cyclin B and wee-1 kinase genes23. Current work in our laboratory has indicated that p30II expression in
Jurkat T lymphocytes modulates the phosphorylation status of cdc25c, and p30II may directly bind to cdc25c (A. Datta, personal communication). A follow-up question that arises from these findings is what advantage does a delay in G2/M transition confer upon the virus during the course of infection. For
173 HIV, it is known that this delay allows for enhanced viral replication2. Whether or not the same holds true for p30II remains to be determined.
5.5 Follow-up studies with HTLV-1 Env SU mutants
Our data in chapter 4 is the first in vivo examination of Env mutants in the context of the entire virus. While the results are interesting, because of the low number of rabbits used, these results should be viewed as an initial screening rather than a study from which absolute conclusions can be drawn.
Whether or not these findings would hold up in a larger study with more statistical power remains to be determined. That a single rabbit inoculated with
ACH.195 mounted a strong antibody response to HTLV-2 SU was intriguing.
Equally intriguing was that another ACH.195 inoculated rabbit had detectable proviral loads in its PBMCs in the absence of a detectable immune response.
These two rabbits represented 50% of the ACH.195 inoculated group. If these results were repeatable in a larger study, the implications for serologic screening in humans would be highly significant14,16,22.
We were not able to demonstrate a relationship between the antibody response to HTLV-1 SU and PBMC proviral loads. These results suggested that an initial robust antibody response to SU was not protective against infection. Since its initial discovery in the 1980’s, there have been several in vivo rabbit studies addressing whether or not peptide immunization protects against infection, and results have varied12,21,26,27. Tanaka et al.27 reported that immunization with peptides spanning SU amino acid 195 (190-199 and 180-
174 204) protects rabbits against infection. Takehara et al.26 reported that immunization with SU peptide 175-196 is not protective against infection in rabbits. Frangione-Beebe et al.12 reported that immunization with a chimeric peptide consisting of SU 175-218 linked to a T-cell epitope from measles virus fusion protein was not protective against infection in rabbits. Our data supports the contention that antibody response to SU is not linked to protection against
HTLV-1 infection.
In humans, the efficiency of HTLV-1-specific CD8+ T cells is positively correlated with the proviral load of HTLV-1 in both asymptomatic carriers and
HAM/TSP patients20,30, and the CTL response to HTLV-1 is a key determinant of proviral loads29. The CTL response in the rabbit model has yet to be characterized. However, it would be interesting to characterize the CTL response to the ACH infection, determine if CTL response correlates with proviral loads, and determine if Env SU mutant clones altered the CTL response.
The lymphocyte cell lines used to inoculate rabbits in chapter 4 were developed based on results from previous in vitro transfection assays with env
SU plasmids10,28. In vitro, transfections with these mutant molecular clones in
293T cells were shown to produce similar levels of Gag and Env proteins compared to wild-type ACH, and they were equally infectious in cell-free and cell-to-cell transmission assays compared to ACH28. Another SU mutant molecular clone, ACH.81, was also made in these studies. This molecular clone showed reduced levels of Gag p19 production in cell-free and cell-to-cell
175 transmission assays28, and although capable of immortalizing lymphocytes, the
ACH.81 cell line was extremely difficult to grow compared to the other HTLV-1
SU mutant cell lines (T. Tsukahara, personal communication). It would be interesting to inoculate rabbits with this mutant cell line to see if these in vitro immortalization characteristics translate to the in vivo infection. If ACH.81 were less infectious in the rabbit model, it would add further validity to in vitro lymphocyte immortalization assays as a means to predict in vivo infectious properties.
5.6 ACH molecular clones in the rabbit model
The creation of the ACH HTLV-1 molecular clone in the mid-1990s18 opened the door for the study of selected HTLV-1 mutations in an in vivo setting3,6,7,25,31. Although much has been done with ACH immortalized lymphocyte cell lines in the context of the rabbit model, much remains that could potentially be explored. With the advent of IVIS imaging technology, it would be interesting to track the spread of HTLV-1 infected lymphocytes following infection with a luciferase-tagged ACH immortalized cell line in our rabbit model. Although previous studies have documented proviral loads within
PBMCs, a more in depth analysis of tissue distribution of the provirus could provide information as to where the virus is harbored during the asymptomatic infection. Moreover, it would answer the question of whether animals, which do not have provirus in their PBMCs, might be harboring provirus at other tissue locations. Numerous in vitro studies have been done with the p12I, p13I, and
176 p30II proteins to define motifs which are important for functionality8,9,11,17,33,34. It would be interesting to mutate these motifs in the context of the ACH molecular clone and use these mutants in in vivo studies to determine if in vitro functionality data translates into alteration in in vivo viral infectivity properties.
ACH.30.1 mutants, which would be interesting to characterize in vivo, include mutants in the nuclear localization signal domain, which would prevent p30II from trafficking to the nucleus, or mutants to eliminate KIX binding, which would prevent binding to CBP/p30033.
5.7 References
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