Formation of a small ribonucleoprotein particle between Tat protein and trans-acting response element in human immunodeficiency virus-infected cells

Abstract

The trans-acting response element (TAR) within the long terminal repeat of human immunodeficiency virus (HIV) is present in all 5' termini of HIV mRNAs and is recognized by the viral Tat protein. Now we describe that the 59-nucleotide-long TAR-RNA exists as a ribonucleoprotein particle in polysomal and heterogeneous nuclear RNP fractions of HIV-1-infected HeLa-T4+ cells. Applying an immunoprecipitation technique this Tat.TAR complex could be isolated from total cell extracts as well as from polysomal or heterogeneous nuclear RNP fractions. The chain length and the identity of the TAR-RNA were established by RNase protection assays while the Tat protein was confirmed by Western blotting technique. The TAR-RNA in this complex was sequenced and found to comprise nucleotides +2 to +61 and hence includes the 3-nucleotide bulge (nucleotides +23 to +25) and the loop sequence of the TAR stem-and-loop structure. The Tat.TAR complex is present in cells at low abundance (12.5 x 10(3) copies/cell). In contrast to the TAR-containing mRNAs, which decay very rapidly after incubation of cells with actinomycin D (half-life of approximately 120 min) the half-life of TAR in the Tat.TAR complex is greater than 180 min. Alignment studies revealed that TAR-RNA (positive strand) has a potential binding ability to the U5 region within the long terminal repeat (DNA negative strand; nucleotides +107 to +147); a complementary binding with a continuous homology of 16 nucleotides was identified. It is proposed that the Tat.TAR complex functions as a small ribonucleoprotein particle during transcription initiation of HIV mRNA.