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The DNA fragments of the 5' and 3' halves of the putative env gene predicted from the DNA sequence of human T-cell leukemia virus (HTLV) provirus were inserted into expression vectors pORF2 and pORF1, respectively, and two hybrid proteins composed of env polypeptides and beta-galactosidase were efficiently produced in Escherichia coli. The hybrid proteins containing the NH2-terminal (EH9) and COOH-terminal (EA1) halves were both immunologically reactive with sera from adult T-cell leukemia patients, demonstrating the utility of the hybrid proteins for diagnosis of HTLV infection. Rabbit antisera against these hybrid proteins detected the two glycoproteins gp62 and gp46, which were previously identified as HTLV env gene products. With these rabbit antisera, two properties of the env gene products were studied. (i) The antisera inhibited syncytia formation of cat S+L- cells induced by HTLV, suggesting that one or both of the env gene products of HTLV, gp62 and gp46, are involved in induction of cell fusion. (ii) The env product gp62 or gp46 or both products are exposed on the surface of HTLV-infected cells and might modulate the proliferation of HTLV-infected T cells in the host because the antisera against the hybrid proteins were cytotoxic on HTLV-producing cell lines. The latter conclusion also is supported by the fact that adult T-cell leukemia patients and healthy HTLV carriers have antibodies to the env gene products.

Human T-cell leukemia virus type I (HTLV-I) is an etiological agent of adult T-cell leukemia and has a unique sequence, pX, that contains four possible open reading frames, I-IV. p40x was previously identified as the gene product of frame IV (x-lor) and was suggested to mediate transcriptional trans-activation of the viral long terminal repeats. We have identified two pX gene products, p27x-III and p21x-III, encoded by frame III, which mostly overlapped frame IV. These proteins were detected with rabbit antiserum against the synthetic peptide predicted from the 3' end of frame III. p27x-III is phosphorylated in cultured cells, and the phosphoprotein (pp27x-III) is localized in nuclei; some pp27x-III was tightly bound to nuclear components. p27x-III was detected in a number of cell lines that express other viral antigens, including a cell line previously reported to express only p40x as a viral protein. The function(s) of p27x-III and p21x-III is not known, but the tight binding of pp27x-III to nuclear components suggests that it is associated with regulation of viral gene expression.

Promoter function for gene expression of the long terminal repeat (LTR) of human T-cell leukemia virus type I (HTLV-I) was studied by constructing plasmids containing the LTR sequence. The gene encoding chloramphenicol acetyltransferase (CATase) was linked to an HTLV-I LTR sequence (pLTR-CAT) by replacing the simian virus 40 promoter in plasmid pSV2-CAT with the LTR sequence. The transient CATase activities of cells transfected with the plasmids were compared. The results are summarized as follows: The HTLV LTR was active even in an epithelial cell line, with efficiency similar to that of the simian virus 40 promoter. pLTR-CAT expressed high CATase activity, 40-200 times that expressed by pSV2-CAT, in HTLV-I-infected T-cell lines, such as the human cell lines MT-2 and HUT-102, or in HTLV-I-infected rat cell lines. This enhanced activity of the LTR seems to be associated with HTLV gene expression, since only low activity of pLTR-CAT was observed in the HTLV-infected cell line MT-1, in which only a small percent of cells express viral antigens. In HTLV-infected rat cell lines, the pX-encoded protein p40x was the only viral protein detected. Thus, we suggest that p40x is the factor associated directly or indirectly with the enhanced activity of the LTR.