Differential precipitation and zinc chelate chromatography purification of biologically active HTLV-I Tax1 expressed in E. coli.

A protocol which involves sequential ammonium sulfate precipitation and zinc chelate chromatography to purify the HTLV-I Tax1 protein expressed in E. coli is described. The final Tax1 product is greater than 90% pure and the yield is approximately 1 mg per liter of liquid culture. The purified Tax1 protein is biologically active in indirect in vitro DNA binding assays and cellular NF-kB induction experiments.

A case of classical mycosis fungoides associated with human T-cell lymphotropic virus type I.

A 72-year-old male patient from north-eastern Iran developed the typical clinical and histopathological features of mycosis fungoides with lymphadenopathy, but without any other systemic involvement. Human T-cell lymphotropic virus (HTLV-I) antibodies were detected in the patient's serum by two different ELISAs and by Western blot using purified viral particles from MT-2 culture supernatants. Cultured peripheral blood lymphocytes were positive for labelling with anti-HTLV-I serum. Southern blot hybridization of DNA extracted from a skin tumour and from an involved lymph node revealed integrated proviral DNA with identical restriction patterns. This case supports a relationship between mycosis fungoides and HTLV-I and may indicate a new region of endemic HTLV-I infection.

Immunodetection of human T-cell lymphotropic virus type I core protein in biological samples by using a monoclonal antibody immunoassay.

A monoclonal antibody-based enzyme immunoassay (EIA) has been developed for detection of human T-cell lymphotropic virus type I (HTLV-I) core protein. The monoclonal antibody (clone 6.11) specifically recognizes the p19 gag gene-encoded protein of the virus. The EIA was over 100 times more sensitive than reverse transcriptase measurement and was capable of responding to less than 500 pg of whole-virus lysate. The assay exhibited type specificity in that HTLV-II antigens failed to produce a positive signal. In addition, a panel of other viruses demonstrated no antigenic cross-reactivity. These included herpesviruses, measles virus, human immunodeficiency viruses, and others. Viral p19 was followed during the course of density gradient ultracentrifugation in the presence of detergent, where it was noted to associate with viral membrane proteins. In comparison, reverse transcriptase activity localized in fractions of higher density containing envelope-free cores. Of clinical interest, the EIA was used to detect HTLV-I antigen in the viral cultures of patients with HTLV-I-associated myelopathies and from symptom-free individuals with proviral integration.

Human T-lymphotropic virus type I (HTLV-I) and tropical spastic paraparesis or HTLV-I-associated myelopathy in Hawaii.

Tropical spastic paraparesis or human T-lymphotropic virus type I (HTLV-I)-associated myelopathy is a degenerative encephalomyelopathy with pyramidal tract dysfunction affecting the lower extremities. It is associated with HTLV-I infection and found primarily in the Caribbean region and in southwestern Japan. Five cases of tropical spastic paraparesis (or HTLV-I-associated myelopathy) in Hawaii are reported. All five patients were born in Hawaii; four are women. Each of the patients has parents who were from HTLV-I-endemic areas of Japan. Two of these patients had serum antibodies to HTLV-I. Five of six of the spouses and children of the seropositive patients were also seropositive. Viral cultures of lymphocytes from both seropositive patients and two of the three seropositive children were positive for HTLV-I. None of the five patients had a history of antecedent blood transfusion, multiple sexual partners, or intravenous drug use. There is no evidence of adult T-cell leukemia or lymphoma in any of the patients or their families. Given the increasing seroprevalence of HTLV-I in the United States, clinicians need to be alert to new cases of this disorder.

Novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG in human serum using recombinant gag-env hybrid protein as antigen.

A novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG (anti-HTLV-1 IgG) in human serum using recombinant gag(14-139)-env-(197-295) hybrid protein is described. Anti-HTLV-1 IgG in test serum was reacted with dinitrophenyl biotinyl bovine serum albumin-recombinant gag-env hybrid protein conjugate. The complex formed was trapped onto polystyrene balls coated with affinity-purified antidinitrophenyl group IgG. After washing to eliminate nonspecific IgG in the test serum, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with streptavidin. After washing, anti-HTLV-1 IgG in the complex trapped onto the streptavidin-coated polystyrene balls was reacted with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Peroxidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorometry. By transfer of the complex, the nonspecific binding of nonspecific human IgG was considerably reduced, and the detection limit of anti-HTLV-1 IgG in serum was lowered 30-300-fold compared with that by Western blotting, gelatin particle agglutination, and the conventional enzyme immunoassay, in which a recombinant gag-env hybrid protein-coated polystyrene ball was incubated with test serum and, after washing, with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Usefulness of the immune-complex-transfer enzyme immunoassay was demonstrated using 271 serum samples.

CML-T1: a cell line derived from T-lymphocyte acute phase of chronic myelogenous leukemia.

Most data suggest that malignant transformation in chronic myelogenous leukemia (CML) occurs in hematopoietic stem cell that is the progenitor of myelopoiesis and of B but not T lymphopoiesis. We established a T-lymphoid cell line (CML-T1) from a person with Ph-chromosome-negative CML in acute phase. Evidence of its T-lymphocyte origin includes the pattern cytochemical reactivity, reactivity with anti-T-cell monoclonal antibodies (MoAbs), and rearrangement of the beta-T-cell receptor (TCRB) gene. CML-T1 cells have features of type IV thymocytes. Cytogenetic analyses indicate a 47,XX, del(11), t(6;7)(q23;q24), +mar karyotype. CML-T1 cells exhibit molecular changes typical of CML, including translocation of the ABL protooncogene from chromosome 9 to 22, rearrangement of the BCR gene, and transcription of a chimeric BCR-ABL messenger RNA (mRNA). The ABL insertion on chromosome 22 appears interstitial, similar to other cases of Ph-chromosome-negative CML. These data clearly indicate that T cells can be involved in acute-phase CML. CML-T1 should be useful in studying this process as well as that underlying Ph-chromosome-negative CML.

Detection of human T-cell leukemia/lymphoma virus type I in a transfusion recipient with chronic myelopathy.

A white man with a progressive spastic paraparesis that began 15 months after sustaining severe trauma in a motor vehicle accident was positive for antibodies to human T-lymphotropic virus type I (HTLV-I) by enzyme-linked immunosorbent assay. Serum antibody to HTLV-I was confirmed by Western blot and radioimmunoprecipitation assay. We detected specific proviral DNA in peripheral blood lymphocytes by the polymerase chain reaction. Because the incidence of HTLV-I is generally restricted to Southern Japan and Caribbean black populations, the most likely source of HTLV-I infection in this patient was multiple intraoperative blood transfusions. The relatively short interval between transfusion and development of HTLV-I-associated myelopathy is consistent with the more rapid evolution of this clinical syndrome compared with adult T-cell leukemia.

An enzyme-linked immunosorbent assay for immune complex of HTLV-I.

A sandwich enzyme-linked immunosorbent assay (ELISA) for immune complexes of human T cell leukemia virus type I (HTLV-I) was developed using monoclonal antibody (MoAb) 3G1 which recognizes a different epitope on HTLV-I to that with which natural human anti-HTLV-I antibody binds. The assay was capable of titrating artificial immune complexes not only at antigen-antibody equivalence but also at antibody excess. Although the antigen-antibody ratios could not be determined in the individual sera from patients with overt ATL, the level of immune complexes in three out of four sera was estimated to be 250 +/- 36 ng/ml. Immune complexes of HTLV-I could not be identified in sera obtained from one patient with overt ATL, three healthy HTLV-I carriers and three normal human controls.

Isolation and characterization of the tax protein of HTLV-I.

The transactivator protein tax of the human T-cell leukemia virus type I, HTLV-I, is responsible for transactivation of gene expression of viral and cellular genes and is involved in the onset of adult T-cell leukemia, ATL. Genetic deletion studies have implicated a region of the HTLV-I LTR designated as tax-acceptor region, TAR, which is the target of the tax protein. Using antibodies against a tax carboxyterminal synthetic decapeptide the tax protein was purified from an HTLV-I immortalized human T-lymphocyte cell line by immunoaffinity chromatography. The tax protein, purified to apparent homogeneity binds to double-stranded DNA irrespective of its origin from either a nuclear or cytoplasmic fraction of the HTLV-I immortalized cell-line - both of which harbor similar quantities of tax protein. The tax protein binds less to single-stranded DNA and not to single-stranded RNA in vitro. It also binds to DNA-cellulose and heparin-Sepharose. Nuclease treatment of isolated nuclei does not release the tax protein under conditions which release known DNA-binding proteins, such as the myb protein. Transactivation by the tax protein presumably involves host-cell factors, since it does not recognize specific DNA sequences.

Mapping of immunogenic regions of human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins with env-encoded synthetic peptides and a monoclonal antibody to gp46.

Antigenic sites on human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins that are immunogenic in man were studied with envelope gene (env)-encoded synthetic peptides and a mAb to HTLV-I gp46 envelope glycoprotein. Antibodies in 78% of sera from HTLV-I seropositive subjects reacted with synthetic peptide 4A (amino acids 190 to 209) from a central region of HTLV-I gp46. Human anti-HTLV-I antibodies also bound to synthetic peptides 6 (29% of sera) and 7 (18% of sera) from a C-terminal region of gp46 (amino acids 296 to 312) and an N-terminal region of gp21 (amino acids 374 to 392), respectively. mAb 1C11 raised to affinity-purified HTLV-I gp46 reacted with gp46 external envelope glycoprotein and gp63 envelope precursor in immunoblot assay and also bound to the surface of HTLV-I+ cells lines HUT-102 and MT-2. Antibody 1C11 did not react with HTLV-II or HIV-infected cells or with a broad panel of normal human tissues or cell lines. In competitive RIA, anti-gp46 antibody 1C11 was inhibited from binding to gp46 either by antibodies from HTLV-I seropositive subjects or by HTLV-I env-encoded synthetic peptide 4A, indicating that 1C11 bound to or near a site on gp46 within amino acids 190 to 209 also recognized by antibodies from HTLV-I-seropositive individuals. When tested in syncytium inhibition assay, mAb 1C11 did not neutralize the infectivity of HTLV-I. Thus, HTLV-I infection in man is associated with a major antibody response to a region of gp46 within amino acids 190 to 209 that is on the surface of virus-infected cells.