| Abstract: | Randomly sheared DNA fragments from HTLV-III proviral DNA were cloned into an E. coli open reading frame (ORF) expression vector. The inserted ORF DNA was expressed in E. coli transformants as a polypeptide fused to the lambda CI protein at the amino terminus and to beta-galactosidase at the carboxyl terminus. The reactivity of the recombinant peptides with antibodies from sera of AIDS patients was determined by the Western blot technique. The coordinates of the DNA inserts of the immunoreactive clones were then determined by DNA sequencing. A clone, ORF 628, was found to contain a short DNA segment located between the sor and env genes (nucleotide positions 5367 to 5597), a region previously thought to be noncoding. Inspection of the DNA sequences of this clone and of other HTLV-III isolates revealed the presence of a small ORF located between nucleotide position 5411 and 5625, capable of encoding a polypeptide of 72 amino acids. The biosynthesis of the polypeptide of ORF 628 initiates from an ATG codon within the HTLV-III insert. The fusion protein of ORF 628 was partially purified by affinity chromatography on CH Sepharose 4B coupled to a beta-galactosidase ligand, and tested against a panel of sera from AIDS patients by Western blot analysis. Approximately 35% of the sera from patients with AIDS or ARC contained antibodies reactive with the peptide. The DNA region spanned by ORF 628 is now thought to be the major functional element of the trans-activator gene, tat. |
