Human T-cell Leukemia Virus Type I Env Research Articles

Human T-cell leukemia viruses are highly unstable over a wide range of temperatures

The biological properties of human T-cell leukemia virus type I (HTLV-I) and HTLV type II (HTLV-II) are not well elucidated as cell-free viruses. We established new assay systems to detect the infectivity of cell-free HTLVs and examined the stability of cell-free HTLVs at different temperatures. HTLVs lost infectivity more rapidly than did bovine leukemia virus (BLV), which is genetically related to HTLVs. The half-lives of three HTLV-I strains (two cosmopolitan strains and one Melanesian strain) at 37 °C were approximately 0.6 h, whereas the half-life of a BLV strain was 8.5 h. HTLV-I rapidly lost infectivity unexpectedly at 0 and 4 °C. We examined the stability of vesicular stomatitis virus pseudotypes with HTLV-I, HTLV-II or BLV Env proteins, and the Env proteins of HTLVs were found to be more unstable at 4 and 25 °C than the Env proteins of the BLV. Over the course of the viral life cycle, heat treatment inhibited HTLV-I infection at the phase of attachment to the host cells, and inhibition was more marked upon entry into the cells. The HTLV-I Env surface (SU) protein (gp46) was easily released from virions during incubation at 37 °C. However, this release was inhibited by pre-treatment of the virions with N-ethylmaleimide, suggesting that the inter-subunit bond between gp46 SU and gp21 transmembrane (TM) proteins is rearranged by disulfide bond isomerization. HTLVs are highly unstable over a wide range of temperatures because the disulfide bonds between the SU and TM proteins are labile.

Using a sensitive luciferase immunoprecipitation system (LIPS) to detect HTLV-I/II seroindeterminates in Jamaica

Human T-cell Leukemia Virus type I (HTLV-I) infects an estimated 15-20 million persons worldwide. A number of diseases have been associated with this virus including adult T-cell leukemia/lymphoma (ATLL), HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), HLTV-I uveitis, and HTLV-I–associated infective dermatitis. The screening process for HTLV-I among blood banks includes detection by an enzyme immunoassay (EIA) followed by a confirmatory western blot (WB). Seropositive results are defined by the presence of all core bands (p19, p24, and p53) as well as the band specific for recombinant HTLV-I Env glycoprotein. However, numerous cases have been documented in which a positive response was detected by ELISA, but WB displayed an incomplete banding pattern. These samples were then categorized as seroindeterminates. Using the LIPS assay, 60% of the 53 seroindeterminate samples previously screened by ELISA and WB displayed anti-HTLV-I Gag, Env or Tax antibody responses (based on 2 standard deviations above the mean value for the HTLV-I negative group). The LIPS assay was also able to detect anti-HTLV-I antibody responses in 6% of the 167 samples determined to be HTLV-I ELISA negative. A number of these samples were subsequently found to have an indeterminate WB pattern. Although the significance of these HTLV-I/ II seroindeterminates is unclear, it may suggest a much higher prevalence of exposure to HTLV-I/II than previously estimated, as seroindeterminate samples may indicate exposure to the virus. The LIPS assay is a sensitive and high throughput antibody detection assay which may prove to be a useful tool in HTLV-I related clinical investigations.

HTLV-I env protein acts as a major antigen in patients with HTLV-I-associated arthropathy.

Our objective was to investigate the pathological mechanisms of HTLV-I (human T-cell leukemia virus type I)-associated chronic arthritis (HAAP) with respect to T-cell response to HTLV-I viral proteins. We examined T-cell clonality and the antigen recognized by T cells from the inflamed synovium of patients with HAAP by using histology, a single-strand conformation polymorphism (SSCP) analysis and T cell receptor (TCR) sequencing. The SSCP analysis showed oligoclonal expansion of T cells in the synovium, suggesting an antigen-mediated stimulation. In contrast, there was less clonal expansion in peripheral blood lymphocytes (PBL). The expression of HTLV-1 env and tax mRNA was detected in the affected synovium as well as in PBL. A number of T-cell clones in the synovium recognized HTLV-I env and tax proteins. Twenty-seven (24.9%) of 109 examined T-cell clones in the joints were HTLV-I env reactive, and 7 clones (6.4%) were HTLV-I tax reactive. Junctional sequence analysis of synovial T cells showed a lack of highly conserved amino acid motifs in the complementarity-determining region 3 (CDR3) of HTLV-I env and tax reactive T cells, suggesting that these cells recognized multiple T-cell epitopes on HTLV-I antigen. These findings suggest that HTLV-I env protein acts as a major antigen and may play a role in the development of arthropathy in patients with HAAP.

In Vitro Induction of HIV-1 Replication in Resting CD4+ T Cells Derived from Individuals with Undetectable Plasma Viremia upon Stimulation with Human T-Cell Leukemia Virus Type I

Microbial coinfections have been associated with transient bursts of human immunodeficiency virus (HIV) viremia in patients. In this study we investigated whether human T-cell leukemia virus type I (HTLV-I), another human retrovirus that is prevalent among certain HIV-infected populations, can induce HIV-1 replication in patients who had been successfully treated with highly active antiretroviral therapy. We demonstrate that supernatants from HTLV-I-producing MT-2 cells can induce in vitro replication of HIV-1 from highly purified, resting CD4+ T cells obtained from individuals with undetectable plasma viremia. Depletion of proinflammatory cytokines from the supernatants reduced, but did not abrogate, the ability to induce HIV-1 replication, indicating that other factors such as HTLV-I Tax or Env also have a role. The HTLV-I-mediated effect does not require productive infection: exposure to heat-inactivated HTLV-I virions, purified Tax protein, or HTLV-I Env glycoprotein also induced expression of HIV-1. Furthermore, we demonstrate that coculture of resting CD4+ T cells with autologous CD8+ T cells markedly inhibits the HTLV-I-induced virus replication. Our results suggest that coinfection with HTLV-I may induce viral replication in the latent viral reservoirs; however, CD8+ T cells may play an important role in controlling the spread of virus upon microbial stimulation.

Unusual CD4+CD8+ phenotype in a Greek patient diagnosed with adult T-cell leukemia positive for human T-cell leukemia virus type I (HTLV-I)

We describe the first Greek patient diagnosed with Adult T cell leukemia (ATL) characterized by an expansion of CD4+CD8+ double positive lymphocytes. Low levels of plasma antibodies against HTLV-I Env and Gag proteins were detected. Analysis of the the patient's DNA revealed that she was infected by a cosmopolitan strain of HTLV-I. Since HTLV-I usually leads to the expansion of CD4+ cells, this patient illustrates a rare immunophenotype, which suggests that the HTLV-I-induced proliferative response may occur in a pre-T cell stage.

Host Range of Human T-cell Leukemia Virus Type I Analyzed by a Cell Fusion-Dependent Reporter Gene Activation Assay

We constructed a sensitive and quantitative assay system to examine human T-cell leukemia virus type I (HTLV-I) envelope (env) glycoprotein-mediated cell fusion in which T7 RNA polymerase in donor cells coexpressing env glycoproteins activates a reporter gene in recipient cells upon cell fusion. An efficient expression of HTLV-I env glycoproteins (gp46 and gp21) was observed in 293T cells transfected with an expression plasmid by both immunoblot and immunofluorescence analyses. The cells expressing env glycoproteins also exhibited self-fusion. By cocultivating the donor cells with recipient cells transfected with a reporter plasmid possessing the luciferase gene under the T7 promoter, the expression of luciferase was observed upon cell fusion. The activation of the luciferase gene was inhibited by either anti-env neutralizing antibody or synthetic peptide corresponding to env gp21, thus indicating the cell fusion to be specifically mediated by the HTLV-I env glycoproteins expressed in the donor cells. A broad range of cell lines exhibited susceptibility to HTLV-I env-mediated cell fusion by this assay. This newly established assay system may thus provide an efficient way both to study the fusion mechanisms mediated by HTLV-I env glycoproteins and to identify the HTLV-I receptor(s).

Host Range of Human T-Cell Leukemia Virus Type I Analyzed by a Cell Fusion-Dependent Reporter Gene Activation Assay

We constructed a sensitive and quantitative assay system to examine human T-cell leukemia virus type I (HTLV-I) envelope (env) glycoprotein-mediated cell fusion in which T7 RNA polymerase in donor cells coexpressing env glycoproteins activates a reporter gene in recipient cells upon cell fusion. An efficient expression of HTLV-I env glycoproteins (gp46 and gp21) was observed in 293T cells transfected with an expression plasmid by both immunoblot and immunofluorescence analyses. The cells expressing env glycoproteins also exhibited self-fusion. By cocultivating the donor cells with recipient cells transfected with a reporter plasmid possessing the luciferase gene under the T7 promoter, the expression of luciferase was observed upon cell fusion. The activation of the luciferase gene was inhibited by either anti-env neutralizing antibody or synthetic peptide corresponding to env gp21, thus indicating the cell fusion to be specifically mediated by the HTLV-I env glycoproteins expressed in the donor cells. A broad range of cell lines exhibited susceptibility to HTLV-I env-mediated cell fusion by this assay. This newly established assay system may thus provide an efficient way both to study the fusion mechanisms mediated by HTLV-I env glycoproteins and to identify the HTLV-I receptor(s).

Evidence for autoantigens of Env/Tax proteins in human T cell leukemia virus type I Env-pX transgenic mice.

To examine T cell clonotypes infiltrating into arthritic joints and to investigate whether human T cell leukemia virus type I (HTLV-I) env-pX gene products act as autoantigens in HTLV-I env-pX transgenic mice. Complementary DNA (cDNA) encoding the V-D-J (third complementarity-determining region [CDR3]) region of T cell receptor beta chain was amplified by Vbeta family polymerase chain reaction. T cell clonotypes were detected by a single-strand conformational polymorphism method, and sequence analysis of the CDR3 region was performed. Distinct oligoclonal T cell expansion was observed in arthritic joints, and a conserved amino acid motif in the CDR3 region was found in T cells infiltrating joints. Moreover, several intraarticular T cells recognized HTLV-I Env and Tax proteins. Our results suggest that HTLV-I Env and Tax proteins act as autoantigens that are recognized by autoreactive T cells in inflamed arthritic lesions in the HTLV-I env-pX transgenic mouse. Thus, some T cells infiltrating the joint recognize Env or Tax protein. These cells may trigger chronic arthritis in HTLV-I env-pX transgenic mice.

Evidence for autoantigens of Env/Tax proteins in human T cell leukemia virus type I Env‐pX transgenic mice

Objective To examine T cell clonotypes infiltrating into arthritic joints and to investigate whether human T cell leukemia virus type I (HTLV-I) env-pX gene products act as autoantigens in HTLV-I env-pX transgenic mice. Methods Complementary DNA (cDNA) encoding the V-D-J (third complementarity-determining region [CDR3]) region of T cell receptor β chain was amplified by Vβ family polymerase chain reaction. T cell clonotypes were detected by a single-strand conformational polymorphism method, and sequence analysis of the CDR3 region was performed. Results Distinct oligoclonal T cell expansion was observed in arthritic joints, and a conserved amino acid motif in the CDR3 region was found in T cells infiltrating joints. Moreover, several intraarticular T cells recognized HTLV-I Env and Tax proteins. Conclusion Our results suggest that HTLV-I Env and Tax proteins act as autoantigens that are recognized by autoreactive T cells in inflamed arthritic lesions in the HTLV-I env-pX transgenic mouse. Thus, some T cells infiltrating the joint recognize Env or Tax protein. These cells may trigger chronic arthritis in HTLV-I env-pX transgenic mice.

Expression and immunogenicity in rats of recombinant adenovirus 5 DNA plasmids and vaccinia virus containing the HTLV-I-env gene

The complete human T-cell leukemia virus type I (HTLV-I) env gene was inserted into an expression cassette containing the adenovirus 5 major late promoter (Ad5-MLP). Recombinant Ad5-HTLV-I-env was obtained by homologous recombination in 293 cells simultaneously transfected by the expression cassette and the genomic DNA of Ad5. In vitro expression of the HTLV-I-env gene in the recombinant vector was detected by immunofluorescence and Western blotting. Functional expression of HTLV-I-env was confirmed by syncitium formation specifically in HeLa cells infected with Ad5-HTLV-I-env. Two immunization regimens against HTLV-I were tested in WKY and Fischer F-344 rats. The first involved WKY rats primed with Ad5-HTLV-I-env or naked DNA plasmids containing the HTLV-I-env gene and boosted with Ad5 containing the HTLV-I-env gp46 gene or with baculovirus-derived recombinant gp46. No antibody against HTLV-I was detected, while HTLV-I-specific cytotoxic T lymphocytes were recovered from all immunized groups but not from controls. The second approach involved Fischer F-344 rats primed and boosted with recombinant vaccinia virus containing the HTLV-I-env gene. Such rats developed antibodies against the HTLV-I env gp21 and gp46 (non-neutralizing). After challenge with human HTLV-I-producing cells (MT-2), both immunization regimens were found to induce partial protection.

Constitutive expression of the HTLV-I pX and env regions in Jurkat T-cells induces differential activation of SRE, CRE and NF kappa B pathways.

Human T-cell leukemia virus type I (HTLV-I) causes adult T-cell leukemia/lymphoma (ATLL). HTLV Tax, the viral transcriptional activator, can activate a variety of cellular genes. HTLV-mediated T-cell transformation, however, may involve additional viral proteins expressed from singly- as well as doubly-spliced viral mRNA. To determine the combined effect of these viral proteins on cellular gene expression in Jurkat T-cells, we derived stable transfectants that constitutively express the HTLV-I pX and env regions (J3.9). J3.9 cells show substantially increased mRNA levels of egr-1 and c-jun but no induction of either CD25 or GM-CSF by Northern blotting. This pattern corresponded to the activation of an egr-1 but not a GM-CSF promoter-driven reporter construct in transient gene expression assays. In DNA electrophoretic mobility shift assays (EMSA), nuclear extract from J3.9 cells has significantly increased binding to CRE and SRE but not nuclear factor kappa B (NF kappa B) DNA oligos, as compared to J-Neo cell extract. These results suggest that low level expression of pX and env region gene products in Jurkat T-cells stimulates persistent activation of CRE- and SRE- but not NF kappa B-induced cellular genes.

Isolation of Human T-Cell Leukemia Virus Type I From a Transformed T-Cell Line Derived Spontaneously From Lymphocytes of a Seronegative Egyptian Patient With Mycosis Fungoides

Mycosis fungoides (MF) is a rare form of cutaneous T-cell lymphoma that may be associated with human T-cell leukemia virus type I (HTLV-I) infection. Using the polymerase chain reaction, the HTLV-I pX region was constantly detected in the genomic DNA extracted from peripheral blood mononuclear cells (PBMCs) of an HTLV-I antibody-seronegative Egyptian MF patient enrolled in a study to isolate HTLV-I from North Africa. A CD4+ and interleukin-2 (IL-2) receptor-positive T-cell line was established when the phytohemagglutinin-stimulated PBMCs of that patient were maintained in IL-2-containing culture medium. The cell line (EMF) was initially IL-2 dependent and then became IL-2 independent after gradual withdrawal of the IL-2. The cells reacted positively with monoclonal antibodies specific for the HTLV-I Env or HTLV-I Gag proteins. Using the Southern blot analysis, HTLV-I provirus could be detected in the genomic DNA extracted from the EMF cells. Limited nucleotide sequence of the env region showed more than 95% homology between the EMF provirus and other known HTLV-I isolates. Western blot analysis of the cell lysates showed the expression of the HTLV-I structural proteins. These data imply that a transforming HTLV-I provirus may be present, at least in certain cases of MF, regardless of the presence or absence of the specific antibodies.

Expression of sequences homologous to HTLV-I tax gene in the labial salivary glands of Japanese patients with Sjögren's syndrome.

To address the question of whether the human T cell leukemia virus type I (HTLV-I) gene is associated with the etiology of Sjögren's syndrome (SS). RNA expression of HTLV-I gag, pol, env, and tax genes in labial salivary glands (LSGs) from SS patients who were seronegative for antibodies to HTLV-I was examined using reverse transcription and polymerase chain reaction techniques. The HTLV-I tax gene, but not the HTLV-I gag, pol, or env genes, was detected in LSG samples from 4 of 14 patients (29%). The nucleotide sequences of the HTLV-I pXIV region in these 4 patients' LSGs showed 100% homology to the HTLV-I pXIV gene from the MT-2 cell line. These findings suggest that products encoding sequences homologous to the HTLV-I pXIV gene in SS patients' LSGs might be candidates for self-antigen and/or lead to activation of autoreactive T lymphocytes through trans-acting transcriptional activation.

Immunogenicity of human T cell leukemia virus type-I (HTLV-I) antigens for cytotoxic T lymphocytes in the rat system.

We showed previously that WKA (rat MHC, RT-1k) and DA (RT-1a) rat CTL specific for human T cell leukemia virus type-I (HTLV-I) recognized the gag and pX gene-encoded Ag. In the present study, we explored HTLV-I Ag recognized by CTL from other MHC genotype (RT-1l) rats, LEW and F344, and examined whether HTLV-I Ag expressed by recombinant vaccinia viruses (rVV) could prime WKA and LEW rats for HTLV-I-specific CTL. Upon priming in vivo and repetitive stimulation in vitro with HTLV-I+ syngeneic T cells, HTLV-I-specific CD8+ CTL were demonstrated in LEW and F344 rat spleen cell cultures. Interestingly, these CTL were directed against HTLV-I env and pX gene products. Immunization of LEW and WKA rats with the env and gag gene-expressing rVV, respectively, resulted in generation of HTLV-I-specific CD8+ CTL. However, a pX-gene expressing rVV failed to prime either rat strain. In addition, HTLV-I-specific CD8+ CTL from F1 hybrid (WKA x LEW) rats generated by immunization and restimulation with HTLV-I+ syngeneic T cells showed gag and pX Ag specificity on WKA rat cells and env and pX Ag specificity on LEW rat cells. These results suggest that immunogenicity of HTLV-I gag and env Ag in induction of HTLV-I-specific CTL varies depending on MHC, and that the pX Ag expressed by rVV is not a potent immunogen for rats.

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.

We have generated a number of mAb against various epitopes on the external envelope glycoprotein, gp46, of human T cell leukemia virus type I (HTLV-I) from a WKA rat immunized with a recombinant vaccinia virus containing the HTLV-I env gene. Among these mAb, one group of mAb, represented by a mAb designated LAT-27, could neutralize the infectivity of HTLV-I, as determined by a HTLV-I-mediated cell fusion inhibition assay. LAT-27 also interfered with transformation of normal T lymphocytes by HTLV-I in vitro. An antibody-binding assay using overlapping synthetic oligopeptides showed that LAT-27 bound specifically to 10-mer peptides that contained the gp46 amino acid sequence 191-196 (Leu-Pro-His-Ser-Asn-Leu). Antibodies from HTLV-I+ humans interfered with the binding of LAT-27 to gp46 Ag. Sera from rabbits immunized with a LAT-27-reactive peptide, 190-199, conjugated with OVA, but not sera from OVA-immunized rabbits, reacted with gp46 Ag and neutralized infectivity of HTLV-I. These results show that the HTLV-I neutralization epitope recognized by LAT-27 locates to the gp46 amino acids 191-196, and that immunization with a peptide containing the LAT-27 epitope can elicit an HTLV-I neutralizing antibody response.

Recognition of human T cell leukemia virus type I (HTLV-I) gag and pX gene products by MHC-restricted cytotoxic T lymphocytes induced in rats against syngeneic HTLV-I-infected cells.

We established rat T cell lines expressing human T cell leukemia virus type I (HTLV-I) Ag from inbred strains of rats, WKA/H, DA, and F344, to study CTL response against the HTLV-I-infected cells. HTLV-I-specific Ag expressed in these rat cells were HTLV-I gag Ag, p19, p24, and p15, and pX Ag, p40tax and p27rex, but not env Ag, as determined by immunofluorescence and immunoblot assays. By immunization of rats with syngeneic HTLV-I-infected cells, CTL against syngeneic HTLV-I-infected cells and antibodies to HTLV-I Ag were generated in WKA/H and DA rats. The bulk CTL cultures from WKA/H and DA rats lysed specifically syngeneic SV40-transformed kidney cells infected with recombinant vaccinia viruses (RVV) expressing HTLV-I gag and pX Ag, but not those infected with RVV expressing HTLV-I env Ag or a control vaccinia virus. From WKA/H rat CTL cultures, four CTL clones reactive with syngeneic HTLV-I-infected cells were isolated, three of which were specific for p27rex/p21x, but the Ag recognized by the other CTL clone was not defined with any RVV used. These results indicate that HTLV-I gag and pX gene products are recognized by MHC-restricted rat CTL specific for syngeneic HTLV-I-infected cells.

Simultaneous infections with human T cell leukemia virus type I and the human immunodeficiency virus.

Four adults form four separate households were found to have simultaneous retroviral infections with human T cell leukemia virus type I (HTLV-I) and human immunodeficiency virus (HIV). These individuals were seropositive for the HTLV-I env transmembrane protein p21E, and all had antibodies to the HTLV-I core polypeptide p24. All four patients also had antibodies to the HIV env transmembrane polypeptide p41E and to the HIV core polypeptide p24. HTLV-I was isolated from peripheral blood lymphocytes of all four individuals, and both viruses were isolated from two of them. Evidence of HIV transmission was noted in the family contacts. Eight of 10 children of these four adults were seropositive for HIV, presumably because of perinatal transmission from infected mothers. Two of five spouses of these adults were examined; these spouses had antibodies to HIV and were positive for virus. No evidence of HTLV-I transmission was noted in these families.

Human monoclonal antibody directed against an envelope glycoprotein of human T-cell leukemia virus type I.

We report the production and characterization of a human monoclonal antibody reactive against the major envelope glycoprotein of human T-cell leukemia virus type I (HTLV-I), a virus linked to the etiology of adult T-cell leukemia. We exposed lymph-node cells derived from a patient with adult T-cell leukemia to the Epstein-Barr virus in vitro and obtained a B-cell clone (designated 0.5 alpha) by a limiting dilution technique. The secreted product of 0.5 alpha is a monoclonal antibody (also designated 0.5 alpha; that is IgG1 and has kappa light chains) that binds to the cell membrane of T-cells infected with HTLV-I and lyses them in the presence of complement. The antibody does not react with HTLV-I-negative T cells. In electroblot assays, the monoclonal antibody detects a 46-kDa glycoprotein in disrupted HTLV-I virions and a 34-kDa product following digestion of the viral protein with endoglycosidase F. These molecules have been reported to represent the HTLV-I env gene products. The antibody does not react with HTLV-II and HTLV-III virions. Glycoproteins of 61 and 68 kDa, which are known to be encoded at least in part by the env gene of HTLV-I, are precipitated by the antibody from endogenously radiolabeled HTLV-I-infected HUT 102-B2 and MT-2 cells, respectively. These results suggest that this human monoclonal antibody reacts with an env-encoded glycoprotein of HTLV-I. By using a competition assay with a biotin-labeled 0.5 alpha antibody, we observed that 15 out of 15 patients with adult T-cell leukemia had antibodies that block binding of the 0.5 alpha antibody to HTLV-I virions. This suggests that the antigen detected by 0.5 alpha antibody is a common epitope recognized in HTLV-I-infected individuals in vivo. This antibody, as well as the general strategy for making human monoclonal antibodies reactive against pathogenic retroviruses, may have diagnostic or therapeutic application.