We have cloned and sequenced a cDNA encoding gp34, a novel glycoprotein expressed in cells bearing human T-cell leukemia virus type I (HTLV-I). HTLV-I has a trans-acting transcriptional activator, p40tax, that is thought to be implicated in leukemogenesis through the activation of cellular enhancers. With a subline (JPX-9) of the human T-celi line Jurkat, in which p40 is inducible, gp34 was shown to be of cellular origin and to be transcriptionally activated by p40. It was also demonstrated that two species of mRNA are generated from one copy of the gp34 gene and that these mRNAs encode the identical gp34 product and differ in the 3' untranslated region. Analysis of the deduced amino acid sequence of gp34 showed that it lacks typical signal peptides; however, it has a hydrophobic stretch for membrane anchoring and four possible N-linked glycosylation sites at the carboxy-terminal portion, indicating that it belongs to the family of membrane proteins whose carboxy-terminal portion protrudes out of the cell. The gp34 gene displayed relatively delayed induction compared with other genes activated by p40. Taken together with the observation of the dependence of gp34 expression on HTLV-I p40, unlike other p4Otax-dependent genes such as those for the interleukin-2 receptor a chain and c-fos, which are expressed or induced under physiological conditions, we predict that the mechanism involved in the induction of gp34 expression by p40 is distinct from and more intricate than those for the previously characterized genes.

Using a monoclonal antibody, Lt‐4, directed against human T cell leukemia virus type I (HTLV‐I) trans‐activator (tax1) antigen, we examined the expression of tax1 and related antigens in a variety of T cell lines bearing HTLV‐I and related retroviruses, simian T cell leukemia virus type I (STLV‐I) and HTLV‐II, by immunofluorescence and immunoblot assays. Lt‐4 reacted with all HTLV‐I‐bearing cell lines tested and five out of eight simian cell lines bearing STLV‐I, but not with an HTLV‐II‐bearing cell line. Lt‐4 detected 40 kd tax1 antigen molecules in most HTLV‐I‐bearing cell lines except one cell line that expressed 39 kd tax1 antigen. In the STLV‐I‐bearing T cell lines, tax1‐related antigen molecules detected by Lt‐4 were heterogeneous, having molecular weights in the range of 36–41 kd.

We prepared a monoclonal antibody (MAb), Lt-4, which recognizes a tax protein expressed in HTLV-I-infected cells. Indirect immunofluorescence staining showed that the antigen recognized by Lt-4 MAb located mainly in the nuclei of HTLV-I-infected T cell lines such as HUT102, TCL-As2, MT-1 and MT-2, and also weakly in the cytoplasm of MT-2 cell line. Lt-4 MAb did not react with two HTLV-I-uninfected T cell lines tested and fresh and PHA-activated peripheral blood lymphocytes (PBL) of several normal donors. Furthermore, Lt-4 MAb stained the nuclei of HeLa cells infected with a recombinant vaccinia virus encoding the tax but not with a wild type vaccinia virus. In Western blot (WB) and radioimmunoprecipitation analyses, Lt-4 MAb detected a 40 kDa molecule (p40) in HUT102 and TCL-As2 cells, and p40 and p68 in MT-2 cells. These results show that Lt-4 MAb is highly specific for the tax protein.

We have prepared two new mouse monoclonal antibodies (MAbs) named TARM-34 (IgM) and TAG-34 (IgG1), that react with surface antigens of lines of human lymphocytes bearing a human T-cell leukemia virus type-I (HTLV-I). The characters of these antibodies are compared with those of anti-HTLV-1 gp21 MAb (TA-21, IgG1), anti-HTLV-I p19 MAb (GIN-14, IgG1) and human antibodies from patients with adult T-cell leukemia (ATL). An indirect membrane immunofluorescence assay showed that TARM-34, TAG-34 and TA-21 all reacted specifically with cell-surface antigens of HTLV-I-positive T- and B-cell lines and cultured peripheral blood lymphocytes from HTLV-I-infected adults. Radioimmunoassay showed that serum antibodies from the ATL patients interfered with the binding of TA-21 antibody to cells of the HTLV-I-positive T-cell line MT-2, but not with the bindings of TARM-34 and TAG-34 antibodies. TARM-34 and TAG-34 both precipitated a 34-kd glycoprotein (gP34), while TA-21 precipitated gp21 from a lysate of 3H-glucosamine-labelled MT-2 cells. TARM-34 and TAG-34 also precipitated the 34-kd protein from lysates of MT-2 and HUT 102 cells labelled with 125I- or 35S-cysteine. Interestingly, TARM-34 and TAG-34 also precipitated 35-kd protein from a lysate of other HTLV-I-positive cells (F-Taj cell line) derived from an ATL patient. TA-21 precipitated the 21-kd protein from the lysates of 35S-cysteine-labelled HTLV-IMT-2 virions, but TARM-34 and TAG-34 did not precipitate any protein from this lysate. TARM-34 lysed HTLV-I-bearing cells in the presence of rabbit complement. These results indicate that TARM-34 and TAG-34 both recognize a glycoprotein antigen that is expressed on the surface of HTLV-I-infected cells.

A 42-kDa recombinant protein, PX141, consisting of the trans-activator protein encoded by human T-cell leukemia virus (HTLV-1) (tax1 antigen) and the amino-terminal fusion peptide of 12 amino acid residues of the alpha-peptide encoded by the plasmid pUC19 was produced. In order to investigate the immunogenicity of the tax1 antigen, mice were immunized with the purified PX141 and 4 anti-tax1 monoclonal antibodies (MAbs) designated TAXY-1, TAXY-6, TAXY-7 and TAXY-8 were generated, and their reactivity was characterized along with another anti-tax1 MAb, Lt-4. Immunoblot assays showed that all the MAbs reacted with the PX141, the native tax1 antigen expressed in various HTLV-1-infected cell lines and the gp68 of MT-2 cells expressing the tax1 amino acids 94-353. Immunoblot assays using recombinant, truncated tax1 antigens, XD59 (expressing amino acids 180-338) and XD128 (expressing amino acids 1-47 and 286-353) showed that: (1) TAXY-1 and Lt-4 did not react with either antigen; (2) TAXY-6 and TAXY-8 reacted with only XD128: and (3) TAXY-7 reacted with both. In addition, TAXY-1, but not the other MAbs, reacted with a putative tax antigen of an STLV-I-infected cell line, designated RfM26-I. Competitive binding assays showed that TAXY-6 and TAXY-8 did not compete against each other. Sera from HTLV-I-infected humans interfered with the binding of all of these anti-tax1 MAbs. These results indicate that the tax1 antigen and the PX141 express at least 5 distinct epitopes recognized by human and mouse antibodies.