Site-specific hydrolysis and alcoholysis of human immunodeficiency virus DNa termini mediated by the viral integrase protein

Vink, C.; Yeheskiely, Esther; Marel, G.A. van der; Boom, J. H. van; Plasterk, Ronald H.A.

doi:10.1093/nar/19.24.6691

Cited by 131 publications

(134 citation statements)

References 26 publications

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“…The 3=-processing substrates, which modeled the HIV-1 U5 vDNA end, were labeled by incorporating radionuclide T at the plus-strand terminus, allowing reactions to be monitored through formation of labeled pGT OH cleavage product in denaturing polyacrylamide gels (Fig. 1A and B) (41,121). IN WT activity was defined as the percent substrate converted to product, and mutant activities were quantified relative to IN WT values ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Correlation of Recombinant Integrase Activity and Functional Preintegration Complex Formation during Acute Infection by Replication-Defective Integrase Mutant Human Immunodeficiency Virus

Koh

Engelman

2012

J Virol

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Previous studies characterized two types of replication-defective human immunodeficiency virus type 1 (HIV-1) integrase mutants: class I, which are specifically blocked at the integration step, and class II, which harbor additional virion production and/or reverse transcription defects. Class I mutant enzymes supported little if any metal ion-dependent 3=-processing and DNA strand transfer activities in vitro, whereas class II enzymes displayed partial or full catalytic function in studies with simplified assay designs, suggesting that defective interaction(s) with heterologous integrase binding proteins might underlie the class II mutant viral phenotype. To address this hypothesis, class I and II mutant enzymes were interrogated under expanded sets of in vitro conditions. The majority failed to catalyze the concerted integration of two viral DNA ends into target DNA, highlighting defective integrase function as the root cause of most class II in addition to all class I mutant virus infection defects. One mutant protein, K264E, in contrast, could support the wild-type level of concerted integration activity. After accounting for its inherent reverse transcription defect, HIV-1 K264E moreover formed preintegration complexes that supported the efficient integration of endogenous viral DNA in vitro and normal levels and sequences of 2-long terminal repeat-containing circle junctions during acute infection. K264E integrase furthermore efficiently interacted in vitro with two heterologous binding partners, LEDGF/p75 and reverse transcriptase. Our results underscore the physiological relevance of concerted integration assays for tests of integrase mutant function and suggest that the K264E mutation disrupts an interaction with an intranuclear integrase binding partner that is important for HIV-1 integration.

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Section: Resultsmentioning

confidence: 99%

Correlation of Recombinant Integrase Activity and Functional Preintegration Complex Formation during Acute Infection by Replication-Defective Integrase Mutant Human Immunodeficiency Virus

Koh

Engelman

2012

J Virol

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“…It seems possible that the carboxylate groups of the invariant D and E residues of IN may complex with metal to establish a similar DNA-enzyme intermediate. Cleavage of DNA strands may then be accomplished by nucleophilic attack via a hydroxyl group of an amino acid side chain on the protein (18,21), the 3' OH of processed LTR end (12,25), or a water molecule (12,34). We hypothesize that the invariant acidic residues in the D,D(35)E region are critical for metal binding and positioning of the DNA substrate for nucleophilic attack.…”

Section: Discussionmentioning

confidence: 99%

Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

Kulkosky¹,

Jones²,

Katz³

et al. 1992

Mol. Cell. Biol.

550

512

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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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“…Analysis of the biochemical activities of deletion derivatives of integrase has shown that this domain alone can promote the disintegration reaction, demonstrating that it is sufficient for catalysis of polynucleotidyl transfer (Bushman et al, 1993). However, the 3' processing and DNA strand transfer reactions require in addition both the N-and C-terminal parts of the protein (Drelich et al, 1992;Bushman et al, 1993 (Engelman et al, 1991;Vink et al, 1991b). The reactions contained the same protein reactants as in panel A.…”

Section: Introductionmentioning

confidence: 99%

Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex.

Engelman¹,

Bushman²,

Craigie³

1993

The EMBO Journal

339

346

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HIV‐1 integrase protein possesses the 3′ processing and DNA strand transfer activities that are required to integrate HIV DNA into a host chromosome. The N‐, C‐terminal and core domains of integrase are necessary for both activities in vitro. We find that certain pairs of mutant integrase proteins, which are inactive when each protein is assayed alone, can support near wild type levels of activity when both proteins are present together in the reaction mixture. This complementation implies that HIV‐1 integrase functions as a multimer and has enabled us to probe the organization of the functional domains within active mixed multimers. We have identified a minimal set of functional integrase domains that are sufficient for 3′ processing and DNA strand transfer and find that some domains are contributed in trans by separate monomers within the functional complex.

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Site-specific hydrolysis and alcoholysis of human immunodeficiency virus DNa termini mediated by the viral integrase protein

Cited by 131 publications

References 26 publications

Correlation of Recombinant Integrase Activity and Functional Preintegration Complex Formation during Acute Infection by Replication-Defective Integrase Mutant Human Immunodeficiency Virus

Correlation of Recombinant Integrase Activity and Functional Preintegration Complex Formation during Acute Infection by Replication-Defective Integrase Mutant Human Immunodeficiency Virus

Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex.

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