Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions. On the basis of similarities with enzymes that catalyze analogous reactions, we propose that the invariant D and E residues may participate in coordination of the metal cofactor (Mn2+ or Mg2') required for the catalytic activities of IN. We further speculate that a metal-DNA complex may be necessary to position both LTR and target DNA substrates for nucleophilic attack during the cleavage and joining reactions.Retroviral integration is dependent upon the interaction of three macromolecular components: (i) a virus-encoded protein, integrase (IN), (ii) specific sequences at the long terminal repeat (LTR) termini of viral "donor" DNA, and (iii) a host DNA "target." Results from the study of several different retroviral systems reveal a stepwise pathway for the interaction of these components during integration in a natural virus infection: (i) removal of a dinucleotide from the 3' ends of linear viral DNA (4) in the cytoplasm, which we refer to as the processing reaction, (ii) migration of the viral DNA to the cell nucleus within a higher-order protein complex (2), (iii) staggered cleavage of host DNA generating 5'-strand extensions, likely coupled to single-strand joining of the processed 3' hydroxyl ends of viral DNA to the 5' phosphate ends of the host target DNA, and (iv) repair and ligation of the gapped unjoined opposite strands. This final step produces a short duplication of host seq...

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Prostratin is a unique phorbol ester that stimulates protein kinase C activity but is nontumor promoting. Remarkably, prostratin is also able to inhibit de novo human immunodeficiency virus type 1 (HIV-1) infection yet up-regulate viral expression from latent proviruses. Prostratin's lack of tumor promotion, coupled with its ability to block viral spread yet induce latent proviral expression, prompted studies to determine whether this compound could serve as an inductive adjuvant therapy for patients treated with highly active antiretroviral therapy (HAART).