An Amino Acid in the Central Catalytic Domain of Three Retroviral Integrases That Affects Target Site Selection in Nonviral DNA

Harper, Amy L.; Sudol, Malgorzata; Katzman, Michael

doi:10.1128/jvi.77.6.3838-3845.2003

Cited by 43 publications

(44 citation statements)

References 45 publications

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“…The catalytic domain also specifies the choice of target DNA sequences for integration in vitro, as shown by the identification of amino acid substitutions at residue 119 in HIV IN that alter the pattern of favored sites. Similar results have been obtained for several other retroviral IN proteins (1,24,25,31,32,41).The N-terminal domain (residues 1-50) contains a conserved HHCC motif within a helix-turn-helix-like fold and appears to contribute to DNA binding (28). The C-terminal domain (residues 220-270) has an SH3-like fold and also contributes to DNA binding (16,18,28).…”

supporting

confidence: 69%

Division of Labor within Human Immunodeficiency Virus Integrase Complexes: Determinants of Catalysis and Target DNA Capture

Diamond

Bushman

2005

J Virol

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Following the completion of reverse transcription, the human immunodeficiency virus integrase (IN) enzyme covalently links the viral cDNA to a host cell chromosome. An IN multimer carries out this reaction, but the roles of individual monomers within the complex are mostly unknown. Here we analyzed the distribution of functions for target DNA capture and catalysis within the IN multimer. We used forced complementation between pairs of IN deletion derivatives in vitro as a tool for probing cis-trans relationships and analyzed amino acid substitutions affecting either catalysis or target site selection within these complementing complexes. This allowed the demonstration that the IN variant contributing the active catalytic domain was also responsible for recognition of the integration target DNA. We were further able to establish that a single monomer is responsible for both functions by use of assay mixtures containing three different IN genotypes. These data specify the ligands bound at the catalytically relevant IN monomer and allow more-specific modeling of the mechanism of inhibitors that also bind this surface of IN.Many DNA-modifying enzymes act as homomultimers, in which different monomers contribute separate functions during the overall reaction cycle (3, 10). For example, during phage Mu transposition, different monomers within a tetramer of the Mu A transposase carry out covalent chemistry and binding of the Mu B cofactor (2, 38). In the tyrosine recombinase family, single monomers of the Cre enzyme contribute both the active-site tyrosine and the activating catalytic residues (23), whereas for the related Flp recombinase, the two functions are contributed by two separate monomers in trans (35). Here we investigate a related issue for the human immunodeficiency virus (HIV) integrase (IN) complex, i.e., whether the catalytic residues and the target site selection function are contributed by the same or different IN monomers within the active IN multimer.The retroviral IN enzyme carries out the initial DNA-breaking and -joining reactions involved in integration (3, 9). After reverse transcription of the viral RNA to produce doublestranded cDNA, IN cleaves two nucleotides from each 3Ј end of the viral cDNA (terminal cleavage; Fig. 1A, part 1). The recessed 3Ј hydroxyl groups are then joined to phosphodiester bonds in the cellular target DNA (strand transfer; Fig. 1A, part 2). In vitro, recombinant IN is able to carry out the terminal cleavage and strand transfer reactions (3, 9). IN is also able to carry out disintegration, a reversal of the strand transfer reaction (Fig. 1A, part 3 ) (8).Retroviral IN proteins are composed of three domains (Fig. 1B). Structures of each IN domain have been determined in isolation (11,21,22), as have the structures of two-domain fragments (6,7,45,46). However, the structures of the entire Based on their catalytic domain sequences, the retroviral INs can be grouped into a large family of polynucleotide phosphotransferase enzymes that include RuvC, bacterial and retroviral R...

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confidence: 69%

Division of Labor within Human Immunodeficiency Virus Integrase Complexes: Determinants of Catalysis and Target DNA Capture

Diamond

Bushman

2005

J Virol

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“…The catalytic core domain (CCD) (ϳ162 residues) contains the highly conserved acidic D, D-35-E motif (27) that is involved in coordinating Mg 2ϩ for 3Ј-OH processing and strand transfer activities (4, 13). The catalytic core is also involved in target binding for strand transfer (3,20,26,40). The C-terminal domain (ϳ35 residues) binds to the viral DNA ϳ6 to 9 bp from the long terminal repeat (LTR) end (15); the C-terminus and CCD are also involved in IN multimerization (1, 24, 29a).…”

mentioning

confidence: 99%

Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate

Pandey

Bera

Zahm

et al. 2007

J Virol

124

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The human immunodeficiency virus type 1 (HIV-1) life cycle involves the reverse transcription of the viral RNA genome into cDNA and subsequent integration of the viral DNA by integrase (IN) into the host chromosomes. Although the amino acid sequences of INs differ significantly between viruses, INs share three conserved structural domains and their associated functions (13). The N-terminal domain (ϳ50 residues) contains a zinc-binding region (7), promotes multimerization (46), and is necessary for 3Ј-OH processing and strand transfer. The catalytic core domain (CCD) (ϳ162 residues) contains the highly conserved acidic D, D-35-E motif (27) that is involved in coordinating Mg 2ϩ for 3Ј-OH processing and strand transfer activities (4, 13). The catalytic core is also involved in target binding for strand transfer (3,20,26,40). The C-terminal domain (ϳ35 residues) binds to the viral DNA ϳ6 to 9 bp from the long terminal repeat (LTR) end (15); the C-terminus and CCD are also involved in IN multimerization (1, 24, 29a).IN, along with the reverse transcriptase and protease, is an antiretroviral target (2,35,38). Highly active antiretroviral therapy, consisting of various combinations of reverse transcriptase and protease inhibitors, has significantly decreased HIV-1 replication in humans. The emergence of multidrugresistant HIV-1 mutants and undesirable side effects associated with certain drug combinations necessitates continuing efforts to develop novel and effective combinational therapies. The addition of inhibitors of HIV-1 IN function would enhance highly active antiretroviral therapy. Raltegravir (MK-0518), an analog of the strand transfer inhibitor L-870,810 used in this report, is in phase III human clinical trials (18,33).Oligonucleotide-based assays in vitro have identified a large number of compounds that inhibit HIV-1 IN activities in vitro (25), the majority of which are ineffective at preventing HIV-1 replication in cell culture. The "strand transfer inhibitors" were identified as being effective against recombinant IN, suppressed HIV-1 replication in cell culture and in vivo, and were so named because of their selectivity in both cases towards inhibiting strand transfer over 16,21,22,38). The first generation of strand transfer inhibitors possessed a 1,3-diketo acid (DKA) pharmacophore, which served as a template in the development of the naphthyridine carboxamide inhibitors. They are structurally analogous to and function identically to DKA inhibitors but exhibit improved metabolic and pharmacokinetic properties and are represented here by compounds L-870,810 and L-870,812 (21, 23). DKA-mediated inhibition is accomplished by the contact of the DKA moiety with the divalent metal ion in the CCD of IN (19,38), and efficient inhibitor binding occurs only with IN bound to the viral DNA substrate (14,22,38

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“…Integrase itself has only a weak nucleotide sequence preference 12 so it is much more likely that it is the DNA associated proteins.…”

Section: What Guides the Integration Event?mentioning

confidence: 99%

Vector integration: Location, location, location

Mok

Lever

2004

Gene Ther

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An Amino Acid in the Central Catalytic Domain of Three Retroviral Integrases That Affects Target Site Selection in Nonviral DNA

Cited by 43 publications

References 45 publications

Division of Labor within Human Immunodeficiency Virus Integrase Complexes: Determinants of Catalysis and Target DNA Capture

Division of Labor within Human Immunodeficiency Virus Integrase Complexes: Determinants of Catalysis and Target DNA Capture

Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate

Vector integration: Location, location, location

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