Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

Rat models of human T-cell leukemia virus type 1 (HTLV-1)-related diseases such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis have been reported. However, these models do not completely reproduce human diseases partly because HTLV-1 replicates poorly in rats. We investigated here the possible reason for this. We found that the activity of Rex in rat cells is quite low compared to that in human cells. As Rex function depends largely on the CRM1 protein, whose human type (human CRM1 [hCRM1]) directly binds to Rex and exports it from the nucleus to the cytoplasm, we assessed whether rat CRM1 (rCRM1) could act as well as hCRM1 as a cofactor for Rex activity. We first cloned a cDNA encoding rCRM1 and found that both rCRM1 and hCRM1 could bind to and export Rex protein to the cytoplasm with similar efficiencies. However, unlike hCRM1, rCRM1 could hardly support Rex function because of its poor ability in inducing the Rex-Rex interaction required for RNA export into the cytoplasm. These observations suggest that the poor ability of rCRM1 to act as a cofactor for Rex function may be responsible for the poor replication of HTLV-1 in rats.     

A multifunctional domain in human CRM1 (exportin 1) mediates RanBP3 binding and multimerization of human T-cell leukemia virus type 1 Rex protein

Human CRM1 (hCRM1) functions in the Rex-mediated mRNA export of human T-cell leukemia virus type 1 (HTLV-1) as an export receptor and as an inducing factor for Rex multimerization on its cognate RNA. Although there are only 24 amino acid differences between hCRM1 and rat CRM1 (rCRM1), rCRM1 can hardly support Rex activity, suggesting a role for rCRM1 as a determinant restricting the host range of HTLV-1. Here, we used a series of mutants, which were generated by interchanging residues of these CRM1s, to examine the relationship of hCRM1 functions. The functions for Rex multimerization and binding to nuclear export signals are mapped to different amino acid residues, and these are separable, suggesting that CRM1 not only functions as an export receptor but also participates in the formation of the RNA export complex through higher-ordered interaction with Rex. The region for the interaction with RanBP3, comprising four residues (amino acids [aa] 411, 414, 474, and 481), and the region for Rex multimerization, including two residues (aa 411 and 414), form an overlapped domain. Our results provide the molecular basis underlying the species-specific ability of HTLV-1 to propagate in human cells.     

CRM1, an RNA transporter, is a major species-specific restriction factor of human T cell leukemia virus type 1 (HTLV-1) in rat cells

Rat ortholog of human CRM1 has been found to be responsible for the poor activity of viral Rex protein, which is essential for RNA export of human T cell leukemia virus type 1 (HTLV-1). Here, we examined the species-specific barrier of HTLV-1 by establishing rat cell lines, including both adherent and CD4(+) T cells, which express human CRM1 at physiological levels. We demonstrated that expression of human CRM1 in rat cells is not harmful to cell growth and is sufficient to restore the synthesis of the viral structural proteins, Gag and Env, at levels similar to those in human cells. Gag precursor proteins were efficiently processed to the mature forms in rat cells and released into the culture medium as sedimentable viral particles. An HTLV-1 pseudovirus infection system suggested that the released virus particles are fully infectious. Our newly developed reporter cell system revealed that Env proteins produced in rat cells are fully fusogenic, which is the basis for cell-cell HTLV-1 infection. Moreover, we show that the early steps in infection, from post-entry uncoating to integration into the host chromosomes, occur efficiently in rat cells. These results, in conjunction with reports describing efficient entry of HTLV-1 into rat cells, may indicate that HTLV-1 is unique in that its major species-specific barrier is determined by CRM1 at a viral RNA export step. These observations will enable us to construct a transgenic rat model expressing human CRM1 that is sensitive to HTLV-1 infection.     

Involvement of Human CRM1 (Exportin 1) in the Export and Multimerization of the Rex Protein of Human T-Cell Leukemia Virus Type 1

We investigated the role of human CRM1 (hCRM1) (exportin 1) in the function of Rex protein encoded by human T-cell leukemia virus type 1. hCRM1 promoted the export of Rex protein from the nucleus to the cytoplasm. A Rex protein with a mutation in the activation domain, RexM90, lost both the ability to bind to hCRM1 and the ability to multimerize. The overexpression of hCRM1 complemented the functional defects of RexM64, which contains a mutation in the multimerization domain of Rex. A dominant-negative mutant of Rex which sequesters cofactors of Rex abrogated multimerization as well as the activity of the wild-type Rex protein. These two functions were simultaneously restored by the overexpression of hCRM1. Taken together, these results suggest that hCRM1 plays important roles in the multimerization and export of Rex protein.     

Envelope is a major viral determinant of the distinct in vitro cellular transformation tropism of human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are related deltaretroviruses but are distinct in their disease-inducing capacity. These viruses can infect a variety of cell types, but only T lymphocytes become transformed, which is defined in vitro as showing indefinite interleukin-2-independent growth. Studies have indicated that HTLV-1 has a preferential tropism for CD4+ T cells in vivo and is associated with the development of leukemia and neurological disease. Conversely, the in vivo T-cell tropism of HTLV-2 is less clear, although it appears that CD8+ T cells preferentially harbor the provirus, with only a few cases of disease association. The difference in T-cell transformation tropism has been confirmed in vitro as shown by the preferential transformation of CD4+ T cells by HTLV-1 versus the transformation of CD8+ T cells by HTLV-2. Our previous studies showed that Tax and overlapping Rex do not confer the distinct T-cell transformation tropisms between HTLV-1 and HTLV-2. Therefore, for this study HTLV-1 and HTLV-2 recombinants were generated to assess the contribution of LTR and env sequences in T-cell transformation tropism. Both sets of proviral recombinants expressed p19 Gag following transfection into cells. Furthermore, recombinant viruses were replication competent and had the capacity to transform T lymphocytes. Our data showed that exchange of the env gene resulted in altered T-cell transformation tropism compared to wild-type virus, while exchange of long terminal repeat sequences had no significant effect. HTLV-2/Env1 preferentially transformed CD4+ T cells similarly to wild-type HTLV-1 (wtHTLV-1), whereas HTLV-1/Env2 had a transformation tropism similar to that of wtHTLV-2 (CD8+ T cells). These results indicate that env is a major viral determinant for HTLV T-cell transformation tropism in vitro and provides strong evidence implicating its contribution to the distinct pathogenesis resulting from HTLV-1 versus HTLV-2 infections.     

Altered expression of tyrosine kinases of the Src and Syk families in human T-cell leukemia virus type 1-infected T-cell lines

During the late phase of adult T-cell leukemia/lymphoma, a severe lymphoproliferative disorder caused by human T-cell leukemia virus type 1 (HTLV-1), leukemic cells no longer produce interleukin-2. Several studies have reported the lack of the Src-like protein tyrosine kinase Lck and overexpression of Lyn and Fyn in these cells. In this report we demonstrate that, in addition to the downregulation of TCR, CD45, and Lck (which are key components of T-cell activation), HTLV-1-infected cell lines demonstrate a large increase of FynB, a Fyn isoform usually poorly expressed in T cells. Furthermore, similar to anergic T cells, Fyn is hyperactive in one of these HTLV-1-infected T-cell lines, probably as a consequence of Csk downregulation. A second family of two proteins, Zap-70 and Syk, relay the signal of T-cell activation. We demonstrate that in contrast to uninfected T cells, Zap-70 is absent in HTLV-1-infected T cells, whereas Syk is overexpressed. In searching for the mechanism responsible for FynB overexpression and Zap-70 downregulation, we have investigated the ability of the Tax and Rex proteins to modulate Zap-70 expression and the alternative splicing mechanism which gives rise to either FynB or FynT. By using Jurkat T cells stably transfected with the tax and rex genes or inducibly expressing the tax gene, we found that the expression of Rex was necessary to increase fynB expression, suggesting that Rex controls fyn gene splicing. Conversely, with the same Jurkat clones, we found that the expression of Tax but not Rex could downregulate Zap-70 expression. These results suggest that the effect of Tax and Rex must cooperate to deregulate the pathway of T-cell activation in HTLV-1-infected T cells.     

Expansion of human T-cell leukemia virus type 1 (HTLV-1) reservoir in orally infected rats: inverse correlation with HTLV-1-specific cellular immune response

Adult T-cell leukemia (ATL) occurs in a small population of human T-cell leukemia virus type 1 (HTLV-1)-infected individuals. Although the critical risk factor for ATL development is not clear, it has been noted that ATL is incidentally associated with mother-to-child infection, elevated proviral loads, and weakness in HTLV-1-specific T-cell immune responses. In the present study, using a rat system, we investigated the relationships among the following conditions: primary HTLV-1 infection, a persistent HTLV-1 load, and host HTLV-1-specific immunity. We found that the persistent HTLV-1 load in orally infected rats was significantly greater than that in intraperitoneally infected rats. Even after inoculation with only 50 infected cells, a persistent viral load built up to considerable levels in some orally infected rats but not in intraperitoneally infected rats. In contrast, HTLV-1-specific cellular immune responses were markedly impaired in orally infected rats. As a result, a persistent viral load was inversely correlated with levels of virus-specific T-cell responses in these rats. Otherwise very weak HTLV-1-specific cellular immune responses in orally infected rats were markedly augmented after subcutaneous reimmunization with infected syngeneic rat cells. These findings suggest that HTLV-1-specific immune unresponsiveness associated with oral HTLV-1 infection may be a potential risk factor for development of ATL, allowing expansion of the infected cell reservoir in vivo, but could be overcome with immunological strategies.     

Establishment of a Seronegative Human T-Cell Leukemia Virus Type 1 (HTLV-1) Carrier State in Rats Inoculated with a Syngeneic HTLV-1-Immortalized T-Cell Line Preferentially Expressing Tax

Human T-cell leukemia virus type 1 (HTLV-1) causes T-cell malignancies in a small percentage of the population infected with the virus after a long carrier state. In the present study, we established a seronegative HTLV-1 carrier state in rats inoculated with a newly established HTLV-1-infected rat T cell line, FPM1. FPM1 originated from rat thymocytes cocultured with a human HTLV-1 producer, MT-2 cells, and expressed rat CD4, CD5, CD25, and HTLV-1 Tax. However, FPM1 scarcely expressed other major HTLV-1 structural proteins and failed to induce typical antibody responses against HTLV-1 in inoculated rats. In contrast, control rats inoculated with MT-2 cells generated significant levels of anti-HTLV-1 antibodies. HTLV-1 proviruses were detected in peripheral blood cells of syngeneic rats inoculated with FPM1 for more than 1 year. Analysis of the flanking region of HTLV-1 provirus integrated into host cells suggested that FPM1 cells remained in these animals over a relatively long period of time. However, a similar seronegative HTLV-1 carrier state was induced in the rats inoculated with mitomycin C-treated FPM1 cells and also in FPM1-inoculated allogeneic rats, suggesting that FPM1 could also transmit HTLV-1 into host cells in vivo. Our findings indicated that (i) HTLV-1-immortalized T cells which preferentially express HTLV-1 Tax persisted in vivo but failed to induce any diseases in immunocompetent syngeneic rats and that (ii) suboptimal levels of HTLV-1 for antibody responses allowed the establishment of persistent HTLV-1 infection.     

Prevention of adult T-cell leukemia-like lymphoproliferative disease in rats by adoptively transferred T cells from a donor immunized with human T-cell leukemia virus type 1 Tax-coding DNA vaccine

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. To dissect the mechanisms of the development of the disease, we have previously established a rat model of ATL-like disease which allows examination of the growth and spread of HTLV-1 infected tumor cells, as well assessment of the effects of immune T cells on the development of the disease. In the present study, we induced HTLV-1 Tax-specific cytotoxic T lymphocyte (CTL) immunity by vaccination with Tax-coding DNA and examined the effects of the DNA vaccine in our rat ATL-like disease model. Our results demonstrated that DNA vaccine with Tax effectively induced Tax-specific CTL activity in F344/N Jcl-rnu/+ (nu/+) rats and that these CTLs were able to lyse HTLV-1 infected syngeneic T cells in vitro. Adoptive transfer of these immune T cells effectively inhibited the in vivo growth of HTLV-1-transformed tumor in F344/N Jcl-rnu/rnu (nu/nu) rats inoculated with a rat HTLV-1 infected T cell line. Vaccination with mutant Tax DNA lacking transforming ability also induced efficient anti-tumor immunity in this model. Our results indicated a promising effect for DNA vaccine with HTLV-1 Tax against HTLV-1 tumor development in vivo.     

Repression of tax expression is associated both with resistance of human T-cell leukemia virus type 1-infected T cells to killing by tax-specific cytotoxic T lymphocytes and with impaired tumorigenicity in a rat model

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.     

Reduction of human T-cell leukemia virus type 1 (HTLV-1) proviral loads in rats orally infected with HTLV-1 by reimmunization with HTLV-1-infected cells

Human T-cell leukemia virus type 1 (HTLV-1) persistently infects humans, and the proviral loads that persist in vivo vary widely among individuals. Elevation in the proviral load is associated with serious HTLV-1-mediated diseases, such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, it remains controversial whether HTLV-1-specific T-cell immunity can control HTLV-1 in vivo. We previously reported that orally HTLV-1-infected rats showed insufficient HTLV-1-specific T-cell immunity that coincided with elevated levels of the HTLV-1 proviral load. In the present study, we found that individual HTLV-1 proviral loads established in low-responding hosts could be reduced by the restoration of HTLV-1-specific T-cell responses. Despite the T-cell unresponsiveness for HTLV-1 in orally infected rats, an allogeneic mixed lymphocyte reaction in the splenocytes and a contact hypersensitivity response in the skin of these rats were comparable with those of naive rats. HTLV-1-specific T-cell response in orally HTLV-1-infected rats could be restored by subcutaneous reimmunization with mitomycin C (MMC)-treated syngeneic HTLV-1-transformed cells. The reimmunized rats exhibited lower proviral loads than untreated orally infected rats. We also confirmed that the proviral loads in orally infected rats decreased after reimmunization in the same hosts. Similar T-cell immune conversion could be reproduced in orally HTLV-1-infected rats by subcutaneous inoculation with MMC-treated primary T cells from syngeneic orally HTLV-1-infected rats. The present results indicate that, although HTLV-1-specific T-cell unresponsiveness is an underlying risk factor for the propagation of HTLV-1-infected cells in vivo, the risk may potentially be reduced by reimmunization, for which autologous HTLV-1-infected cells are a candidate immunogen.     

Oligomannose-coated liposomes efficiently induce human T-cell leukemia virus-1-specific cytotoxic T lymphocytes without adjuvant

Human T-cell leukemia virus-1 (HTLV-1) causes adult T-cell leukemia/lymphoma, which is an aggressive peripheral T-cell neoplasm. Insufficient T-cell response to HTLV-1 is a potential risk factor in adult T-cell leukemia/lymphoma. Efficient induction of antigen-specific cytotoxic T lymphocytes is important for immunological suppression of virus-infected cell proliferation and oncogenesis, but efficient induction of antigen-specific cytotoxic T lymphocytes has evaded strategies utilizing poorly immunogenic free synthetic peptides. Here, we examined the efficient induction of an HTLV-1-specific CD8+ T-cell response by oligomannose-coated liposomes (OMLs) encapsulating the human leukocyte antigen (HLA)-A*0201-restricted HTLV-1 Tax-epitope (OML/Tax). Immunization of HLA-A*0201 transgenic mice with OML/Tax induced an HTLV-1-specific gamma-interferon reaction, whereas immunization with epitope peptide alone induced no reaction. Upon exposure of dendritic cells to OML/Tax, the levels of CD86, major histocompatibility complex class I, HLA-A02 and major histocompatibility complex class II expression were increased. In addition, our results showed that HTLV-1-specific CD8+ T cells can be efficiently induced by OML/Tax from HTLV-1 carriers compared with epitope peptide alone, and these HTLV-1-specific CD8+ T cells were able to lyse cells presenting the peptide. These results suggest that OML/Tax is capable of inducing antigen-specific cellular immune responses without adjuvants and may be useful as an effective vaccine carrier for prophylaxis in tumors and infectious diseases by substituting the epitope peptide.     

Induction of Adult T-Cell Leukemia-Like Lymphoproliferative Disease and Its Inhibition by Adoptive Immunotherapy in T-Cell-Deficient Nude Rats Inoculated with Syngeneic Human T-Cell Leukemia Virus Type 1-Immortalized Cells

Human T-cell leukemia virus type 1 (HTLV-1) has been shown to be the etiologic agent of adult T-cell leukemia (ATL), but the in vivo mechanism by which the virus causes the malignant transformation is largely unknown. In order to investigate the mechanisms of HTLV-1 leukemogenesis, we developed a rat model system in which ATL-like disease was reproducibly observed, following inoculation of various rat HTLV-1-immortalized cell lines. When previously established cell lines, F344-S1 and TARS-1, but not TART-1 or W7TM-1, were inoculated, systemic multiple tumor development was observed in adult nude (nu/nu) rats. FPM1 cells, newly established from a heterozygous (nu/+) rat syngeneic to nu/nu rats, caused transient tumors only at the injection site in adult nu/nu rats, but could progressively grow in newborn nu/nu rats and metastasize in lymph nodes. The derivative cell line (FPM1-V1AX) serially passed through newborn nu/nu rats acquired the potency to grow in adult nu/nu rats. These results indicated that only some with additional changes but not all of the in vitro HTLV-1-immortalized cell lines possessed in vivo tumorigenicity. Using the syngeneic system, we further showed the inhibition of tumor development by transferring splenic T cells from immunized rats, suggesting the involvement of T cells in the regression of tumors. This novel and reproducible nude rat model of human ATL would be useful for investigation of leukemogenesis and antitumor immune responses in HTLV-1 infection.     

Highly enhanced expression of CD70 on human T-lymphotropic virus type 1-carrying T-cell lines and adult T-cell leukemia cells

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). In Japan, the number of HTLV-1 carriers is estimated to be 1.2 million and more than 700 cases of ATL have been diagnosed every year. Considering the poor prognosis and lack of curative therapy of ATL, it seems mandatory to establish an effective strategy for the treatment of ATL. In this study, we attempted to identify the cell surface molecules that will become suitable targets of antibodies for anti-ATL therapy. The expression levels of approximately 40,000 host genes of three human T-cell lines carrying HTLV-1 genomes were analyzed by oligonucleotide microarray and compared with the expression levels of the genes in an HTLV-1-negative T-cell line. The HTLV-1-carrying T-cell lines used for experiments had totally different expression patterns of viral genome. Among the genes evaluated, the expression levels of 108 genes were found to be enhanced more than 10-fold in all of the T-cell lines examined and 11 of the 108 genes were considered to generate the proteins expressed on the cell surface. In particular, the CD70 gene was upregulated more than 1,000-fold and the enhanced expression of the CD70 molecule was confirmed by laser flow cytometry for various HTLV-1-carrying T-cell lines and primary CD4(+) T cells isolated from acute-type ATL patients. Such expression was not observed for primary CD4(+) T cells isolated from healthy donors. Since CD70 expression is strictly restricted in normal tissues, such as highly activated T and B cells, CD70 appears to be a potential target for effective antibody therapy against ATL.     

Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.     

Development of human T-cell leukemia virus type 1-transformed tumors in rats following suppression of T-cell immunity by CD80 and CD86 blockade

Host immunity influences clinical manifestations of human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we demonstrated that HTLV-1-transformed tumors could develop in immunocompetent rats by blocking a costimulatory signal for T-cell immune responses. Four-week-old WKA/HKm rats were treated with monoclonal antibodies (MAbs) to CD80 and CD86 and subcutaneously inoculated with syngeneic HTLV-1-infected TARS-1 cells. During MAb treatment for 14 days, TARS-1 inoculation resulted in the development of solid tumors at the site of inoculation, which metastasized to the lungs. In contrast, rats not treated with MAbs promptly rejected tumor cells. Splenic T cells from MAb-treated rats indicated impairment of proliferative and cytotoxic T-lymphocyte responses against TARS-1 in vitro compared to untreated rats. However, tumors grown in MAb-treated rats regressed following withdrawal of MAb therapy. Recovery of TARS-1-specific T-cell immune responses was associated with tumor regression in these rats. Our results suggest that HTLV-1-specific cell-mediated immunity plays a critical role in immunosurveillance against HTLV-1-transformed tumor development in vivo.