Molecular mechanisms of HTLV-1 infection and pathogenesis

Human T cell leukemia virus type 1 (HTLV-1) is an etiological pathogen of several human diseases, including adult T-cell leukemia (ATL), HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), and inflammatory disorders such as uveitis and dermatitis. HTLV-1 spreads mainly through cell-to-cell transmission, induces clonal proliferation of infected T cells in vivo, and after a long latent period, a subset of HTLV-1 carriers develop ATL. Understanding the molecular mechanisms of infection and oncogenesis is important for the development of new strategies of prophylaxis and molecular-targeted therapies, since ATL has a poor prognosis, despite intensive chemotherapy. In this review, we will summarize recent progress in HTLV-1 research, and especially novel findings on viral transmission and leukemogenic mechanisms by two viral oncogenes, HBZ and tax.     

Common human T cell leukemia virus type 1 (HTLV-1) integration sites in cerebrospinal fluid and blood lymphocytes of patients with HTLV-1-associated myelopathy/tropical spastic paraparesis indicate that HTLV-1 crosses the blood-brain barrier via clonal HTLV-1-infected cells

In the spinal cord of patients with human T cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), infiltrating CD4(+) lymphocytes seem to be the major reservoir for the virus. Little, however, is known about the mechanisms by which HTLV-1 crosses the blood-brain barrier. An oligoclonal proliferation of HTLV-1-infected CD4 lymphoid T cells is present in the peripheral blood of all HTLV-1-infected individuals. Here, such oligoclonal distribution of HTLV-1-infected cells is evidenced in the cerebrospinal fluid (CSF) derived from 5 patients with HAM/TSP. Furthermore, clonal populations of HTLV-1-infected lymphocytes sharing the same HTLV-1 proviral flanking sequences (i.e. , integration sites in the cellular DNA), and thus derived from a single HTLV-1-infected progenitor, were found, for a given patient, in both the CSF and the peripheral blood. These data demonstrate that HTLV-1 crosses the blood-brain barrier by migration of HTLV-1-infected lymphocytes in vivo.     

Dual cytoplasmic and nuclear localization of HTLV-1-encoded HBZ protein is a unique feature of adult T-cell leukemia

Adult T-cell leukemia-lymphoma (ATL), is a highly malignant T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1), characterized by poor prognosis. Two viral proteins, Tax-1 and HTLV-1 basic-zipper factor (HBZ) play important roles in the pathogenesis of ATL. While Tax-1 can be found in both the cytoplasm and nucleus of HTLV-1 infected patients, HBZ is exclusively localized in the cytoplasm of HTLV-1 asymptomatic carriers and in patients with the chronic neurologic disease HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HBZ is only localized in the nucleus of ATL cell lines, suggesting that the nuclear localization of HBZ can be a hallmark of neoplastic transformation. In order to clarify this crucial point, we investigated in detail the pattern of HBZ expression in ATL patients. We made use of our monoclonal antibody 4D4-F3, that at present is the only reported reagent, among the few described, able to detect endogenous HBZ by immunofluorescence and confocal microscopy in cells from asymptomatic carriers, HAM/TSP and ATL patients. We found that HBZ is localized both in the cytoplasm and nucleus of cells of ATL patients irrespective of their clinical status, with a strong preference for the cytoplasmic localization. Also Tax-1 is localized in both compartments. As HBZ is exclusively localized in the cytoplasm in asymptomatic carriers and in non-neoplastic pathologies, this finding shows that neoplastic transformation consequent to HTLV-1 infection is accompanied and associated with the capacity of HBZ to translocate to the nucleus, which suggests a role of cytoplasmic-to-nuclear translocation in HTLV-1- mediated oncogenesis.     

Development of peripheral T-cell lymphoma not otherwise specified in an HTLV-1 carrier

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) after a long latency period of about 60 years. As the mature T-cell neoplasms that emerge in patients infected with HTLV-1 are often ATL, T-cell neoplasms developing in such patients tend to be diagnosed simply as ATL without further investigation. However, not all T-cell neoplasms that develop in HTLV-1-infected cases are ATL. Mature T-cell malignancies other than ATL should be carefully excluded in patients infected with HTLV-1, as these sometimes closely resemble ATL in their clinical, morphological, and histological features. Here, we present a case of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) in an HTLV-1 carrier. Confirmation of monoclonal integration of the virus with Southern blotting leads to a definite diagnosis of ATL. Although we did not detect the monoclonal integration band of HTLV-1 in this case, the high HTLV-1 proviral load complicated the diagnosis. Multicolor flow cytometric analysis clearly showed that HTLV-1 was not integrated in the tumor cells, and facilitated discrimination of PTCL-NOS from ATL.     

High frequencies of Th1-type CD4(+) T cells specific to HTLV-1 Env and Tax proteins in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

CD4(+) T cells are critical for inducing and maintaining efficient humoral and cellular immune responses to pathogens. The CD4(+) T-cell response in human T-lymphotropic virus 1 (HTLV-1) infection has not been studied in detail. However, CD4(+) T cells have been shown to predominate in early lesions in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We present direct estimates of HTLV-1 Env- and Tax-specific CD4(+) T-cell frequencies in patients infected with HTLV-1. We first showed that there was a strong bias toward the Th1 phenotype in these HTLV-1-specific CD4(+) T cells in patients with HAM/TSP. We then demonstrated significantly higher frequencies of HTLV-1-specific Th1-type CD4(+) T cells in HAM/TSP patients than in asymptomatic HTLV-1 carriers. The majority of these HTLV-1-specific CD4(+) T cells did not express HTLV-1 Tax and were therefore unlikely to be infected by HTLV-1. High frequencies of activated HTLV-1-specific CD4(+) T cells of the Th1 phenotype might contribute to the initiation or pathogenesis of HAM/TSP and other HTLV-1-associated inflammatory diseases.     

Bronchopneumonopathy in HTLV-1 associated myelopathy (HAM) and non-HAM HTLV-1 carriers]

Human T-cell leukemia virus type 1 (HTLV-1) causes not only adult T-cell leukemia (ATL), but also HTLV-1 associated myelopathy, a recently described slowly progressive spastic paraparesis, in its carrier state. Pulmonary involvement has been reported in HAM patients. Based on bronchoalveolar lavage or histological examination, the pulmonary involvement has been characterized by accumulation of T-cells, especially activated T-cells, in the lung. We reviewed our data on pulmonary involvement in HAM, which suggested that the characteristic pulmonary involvement observed in HAM was not restricted to HAM patients, but was also observed in non-HAM HTLV-1 carriers. Based on the data, we report that HAM is a systemic disease and that HTLV-1 causes characteristic pulmonary involvement, which we termed HTLV-1 associated bronchopneumonopathy (HAB).     

Hypoxanthine-guanine phosphoribosyltransferase reporter gene mutation for analysis of in vivo clonal amplification in patients with HTLV type 1-associated Myelopathy/Tropical spastic paraparesis

We tested a surrogate selection approach utilizing mutation at a reporter gene [hypoxanthine-guanine phosphoribosyltransferase (hprt)] as a probe for in vivo cell division, for detection of clonal T cell expansion in human T lymphotropic (HTLV-1) carriers. Peripheral blood samples from HTLV-1-infected individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) were tested to determine the hprt mutant frequency (Mf). Wild-type and hprt mutant T cell clones were isolated, and clonal identity determined by multiplex PCR and DNA sequencing of T cell receptor (TCR) variable region beta-chain (TCR BV) and third complementarity determining regions (CDR3). Seven samples from HAM/TSP patients were tested, and Mfs were within the normal range for adults (mean 11.3 x 10(-6), max 22.4 x 10(-6), min 5.6 x 10(-6)). The frequency of HTLV-1 infection in wild-type and hprt mutant T cells from HAM/TSP patients was determined to identify enrichment in the mutant fraction of cells. This analysis was performed on 196 isolates from 6 individuals with HAM/TSP. In each case, there is enrichment for virally infected cells in the hprt mutant fraction of isolates. Ten mutant and eight wild-type isolates from sample LS42A (Mf 8.4 x 10(-6)) were tested for clonality by TCR BV PCR and sequencing. Of the 10 hprt mutants, there were two in vivo-expanded clones (four isolates with two identical TCRs, or 80% unique TCR sequences). These studies may provide new insights into the precise mechanism of HTLV-1 leukemogenesis, and aid in the study of mutator phenotypes generated by a combination of Tax-mediated in vivo expansion and mutagenesis.     

Impaired function of human T-lymphotropic virus type 1 (HTLV-1)-specific CD8+ T cells in HTLV-1-associated neurologic disease

Despite abundant activated virus-specific cytotoxic T lymphocytes (CTLs), patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) showed a significantly higher frequency of infected T cells than did healthy virus carriers (HVCs). Here, we demonstrate that at a given proviral load, the frequency of CD8(+) T cells that are negative for specific costimulatory molecules was significantly higher in HAM/TSP than in age-matched HVCs and uninfected healthy controls (HCs), whereas the frequency of intracellular perforin-positive CD8(+) T cells was significantly lower in both HAM/TSP and HVCs than in HCs. An inverse correlation between HTLV-1 proviral load (PVL) and percent perforin-positive CD8(+) T cells were observed only in disease-protective allele HLA-A*02-positive HVCs, but not in HAM/TSP patients, whether HLA-A*02 positive or negative, nor in HLA-A*02-negative HVCs. Significantly lower perforin expression was observed in HTLV-1-specific than in cytomegalovirus-specific CD8(+) T cells. Majority of HTLV-1-specific CD8(+) T cells in HVCs showed a CD28(-)CD27(+) phenotype, whereas HAM/TSP showed a CD28(-)CD27(-) phenotype. HTLV-1-specific CD8(+) T cells from HAM/TSP patients showed significantly lower degranulation than HVCs by CD107a mobilization assay. These findings suggest that an impaired function of HTLV-1-specific CTLs is associated with failing antiviral control and disease HAM/TSP.     

Degenerate specificity of HTLV-1-specific CD8+ T cells during viral replication in patients with HTLV-1-associated myelopathy (HAM/TSP)

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurologic disease caused by HTLV-1 infection, in which HTLV-1-infected CD4(+) T cells and HTLV-1-specific CD8(+) T cells may play a role in the disease pathogenesis. Patients with HAM/TSP have high proviral loads despite vigorous virus-specific CD8(+) T-cell responses; however, it is unknown whether the T cells are efficient in eliminating the virus in vivo. To define the dynamics of HTLV-1-specific CD8(+) T-cell responses, we investigated longitudinal alterations in HTLV-1 proviral load, amino acid changes in an immunodominant viral epitope, frequency of HTLV-1-specific T cells, and degeneracy of T-cell recognition in patients with HAM/TSP. We showed that the frequency and the degeneracy of the HTLV-1-specific CD8(+) T cells correlated well with proviral load in the longitudinal study. The proviral load was much higher in a patient with low degeneracy of HTLV-1-specific T cells compared to that in a patient with comparable frequency but higher degeneracy of the T cells. Furthermore, in a larger number of patients divided into 2 groups by the proviral load, those with high proviral load had lower degeneracy of T-cell recognition than those with low proviral load. Sequencing analysis revealed that epitope mutations were remarkably increased in a patient when the frequency and the degeneracy were at the lowest. These data suggest that HTLV-1-specific CD8(+) T cells with degenerate specificity are increased during viral replication and control the viral infection.     

Dual targeting of transformed and untransformed HTLV-1-infected T cells by DHMEQ, a potent and selective inhibitor of NF-kappaB, as a strategy for chemoprevention and therapy of adult T-cell leukemia

Human T-cell leukemia virus type I (HTLV-1) causes adult T-cell leukemia (ATL), a fatal T-cell leukemia resistant to chemotherapy, after more than 50 years of clinical latency from transmission through breast-feeding. Polyclonal expansion of virus-infected T cells predisposes them to transformation. Constitutive activation of nuclear factor-kappaB (NF-kappaB) in the leukemic cells is essential for their growth and survival. Blocking NF-kappaB has been shown to be a potential strategy to treat ATL. We tested this approach using a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), and also examined its application to chemoprevention by selective purging of the HTLV-1-infected cells. DHMEQ inhibited NF-kappaB activation in primary ATL cells and cell lines derived from them and induced apoptotic cell death. NF-kappaB inhibition down-regulated expression of genes involved in antiapoptosis or cell-cycle progression. DHMEQ protected severe combined immunodeficiency (SCID) mice inoculated with HTLV-1-transformed cells from death. In addition, DHMEQ selectively targeted HTLV-1-infected cells in the peripheral blood of virus carriers in vitro, resulting in a decreased number of infected cells. We conclude that NF-kappaB is a potential molecular target for treatment and prevention of ATL. As a potent NF-kappaB inhibitor, DHMEQ is a promising compound allowing the translation of this strategy into clinical medicine.     

Molecular Studies of HTLV-1 Replication: An Update

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus discovered. Studies on HTLV-1 have been instrumental for our understanding of the molecular pathology of virus-induced cancers. HTLV-1 is the etiological agent of an adult T-cell leukemia (ATL) and can lead to a variety of neurological pathologies, including HTLV-1-associated-myelopathy/tropical spastic paraparesis (HAM/TSP). The ability to treat the aggressive ATL subtypes remains inadequate. HTLV-1 replicates by (1) an infectious cycle involving virus budding and infection of new permissive target cells and (2) mitotic division of cells harboring an integrated provirus. Virus replication initiates host antiviral immunity and the checkpoint control of cell proliferation, but HTLV-1 has evolved elegant strategies to counteract these host defense mechanisms to allow for virus persistence. The study of the molecular biology of HTLV-1 replication has provided crucial information for understanding HTLV-1 replication as well as aspects of viral replication that are shared between HTLV-1 and human immunodeficiency virus type 1 (HIV-1). Here in this review, we discuss the various stages of the virus replication cycle—both foundational knowledge as well as current updates of ongoing research that is important for understanding HTLV-1 molecular pathogenesis as well as in developing novel therapeutic strategies.     

Effects of valproate on Tax and HBZ expression in HTLV-1 and HAM/TSP T lymphocytes

A determinant of human T-lymphotropic virus-1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development is the HTLV-1-infected cell burden. Viral proteins Tax and HBZ, encoded by the sense and antisense strands of the pX region, respectively, play key roles in HTLV-1 persistence. Tax drives CD4(+)-T cell clonal expansion and is the immunodominant viral antigen recognized by the immune response. Valproate (2-n-propylpentanoic acid, VPA), a histone deacetylase inhibitor, was thought to trigger Tax expression, thereby exposing the latent HTLV-1 reservoir to immune destruction. We evaluated the impact of VPA on Tax, Gag, and HBZ expressions in cultured lymphocytes from HTLV-1 asymptomatic carriers and HAM/TSP patients. Approximately one-fifth of provirus-positive CD4(+) T cells spontaneously became Tax-positive, but this fraction rose to two-thirds of Tax-positive-infected cells when cultured with VPA. Valproate enhanced Gag-p19 release. Tax- and Gag-mRNA levels peaked spontaneously, before declining concomitantly to HBZ-mRNA increase. VPA enhanced and prolonged Tax-mRNA expression, whereas it blocked HBZ expression. Our findings suggest that, in addition to modulating Tax expression, another mechanism involving HBZ repression might determine the outcome of VPA treatment on HTLV-1-infected-cell proliferation and survival.     

T CD4+ cells count among patients co-infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1): high prevalence of tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM)

INTRODUCTION: HIV positive patients co-infected with HTLV-1 may have an increase in their T CD4+ cell counts, thus rendering this parameter useless as an AIDS-defining event. OBJECTIVE: To study the effects induced by the co-infection of HIV-1 and HTLV-1 upon CD4+ cells. MATERIAL AND METHODS: Since 1997, our group has been following a cohort of HTLV-1-infected patients, in order to study the interaction of HTLV-1 with HIV and/or with hepatitis C virus (HCV), as well as HTLV-1-only infected asymptomatic carriers and those with tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). One hundred and fifty HTLV-1-infected subjects have been referred to our clinic at the Institute of Infectious Diseases "Emílio Ribas", S?o Paulo. Twenty-seven of them were also infected with HIV-1 and HTLV-1-infection using two ELISAs and confirmed and typed by Western Blot (WB) or polymerase chain reaction (PCR). All subjects were evaluated by two neurologists, blinded to the patient's HTLV status, and the TSP/HAM diagnostic was based on the World Health Organization (WHO) classification. AIDS-defining events were in accordance with the Centers for Disease Control (CDC) classification of 1988. The first T CD4+ cells count available before starting anti-retroviral therapy are shown compared to the HIV-1-infected subjects at the moment of AIDS defining event. RESULTS: A total of 27 HIV-1/HTLV-1 co-infected subjects were identified in this cohort; 15 already had AIDS and 12 remained free of AIDS. The median of T CD4+ cell counts was 189 (98-688) cells/mm(3) and 89 (53-196) cells/mm(3) for co-infected subjects who had an AIDS-defining event, and HIV-only infected individuals, respectively (p = 0.036). Eight of 27 co-infected subjects (30%) were diagnosed as having a TSP/HAM simile diagnosis, and three of them had opportunistic infections but high T CD4+ cell counts at the time of their AIDS- defining event. DISCUSSION: Our results indicate that higher T CD4+ cells count among HIV-1/HTLV-1-coinfected subjects was found in 12% of the patients who presented an AIDS-defining event. These subjects also showed a TSP/HAM simile picture when it was the first manifestation of disease; this incidence is 20 times higher than that for HTLV-1-only infected subjects in endemic areas.     

Ex vivo analysis of human T lymphotropic virus type 1-specific CD4+ cells by use of a major histocompatibility complex class II tetramer composed of a neurological disease-susceptibility allele and its immunodominant peptide

HLA-DRB1*0101 is associated with susceptibility to human T lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Here, we used a synthetic tetramer of DRB1*0101 and its epitope peptide to analyze HTLV-1-specific CD4(+) T cells ex vivo. The frequency of tetramer(+)CD4(+) T cells was significantly greater in patients with HAM/TSP than in healthy HTLV-1 carriers (HCs) at a given proviral load and correlated with HTLV-1 tax messenger RNA expression in HCs but not in patients with HAM/TSP. These cells displayed an early to intermediate effector memory phenotype and were preferentially infected by HTLV-1. T cell receptor gene analyses of 2 unrelated DRB1*0101-positive patients with HAM/TSP showed similar Vbeta repertoires and amino acid motifs in complementarity-determining region 3. Our data suggest that efficient clonal expansion of virus-specific CD4(+) T cells in patients with HAM/TSP does not simply reflect higher viral burden but rather reflects a rapid turnover caused by preferential infection and/or in vivo stimulation by major histocompatibility complex-peptide complexes.     

Human T-Cell Leukemia Virus Type 1 Envelope Protein: Post-Entry Roles in Viral Pathogenesis

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL), an aggressive and fatal CD4⁺ T-cell malignancy, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic neurological disease. Disease progression in infected individuals is the result of HTLV-1-driven clonal expansion of CD4⁺ T-cells and is generally associated with the activities of the viral oncoproteins Tax and Hbz. A closely related virus, HTLV-2, exhibits similar genomic features and the capacity to transform T-cells, but is non-pathogenic. In vitro, HTLV-1 primarily immortalizes or transforms CD4⁺ T-cells, while HTLV-2 displays a transformation tropism for CD8⁺ T-cells. This distinct tropism is recapitulated in infected people. Through comparative studies, the genetic determinant for this divergent tropism of HTLV-1/2 has been mapped to the viral envelope (Env). In this review, we explore the emerging roles for Env beyond initial viral entry and examine current perspectives on its contributions to HTLV-1-mediated disease development.