Efficient Concerted Integration by Recombinant Human Immunodeficiency Virus Type 1 Integrase without Cellular or Viral Cofactors

Sinha, Sapna; Pursley, Michael; Grandgenett, Duane P.

doi:10.1128/jvi.76.7.3105-3113.2002

Cited by 71 publications

(100 citation statements)

References 43 publications

(72 reference statements)

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“…In preliminary experiments we explored a wide range of reaction conditions (data not shown). As reported previously by Grandgenett and co-workers (20), the presence of polyethylene glycol in the reaction mixture was found to be critical for promoting the twoend integration reaction. The products of a typical reaction are shown in Fig.…”

Section: Resultssupporting

confidence: 51%

“…The major product of concerted integration corresponds to the product labeled concerted in Fig. 1; restriction analysis showed that, as reported by Grandgenett and co-workers (20), pairs of U5 ends were preferentially used compared with U3/U5 pairs or U3/U3 pairs (supplemental Fig. 1).…”

Section: Resultsmentioning

confidence: 70%

“…Although early work demonstrated that the integrase proteins of several retroviruses could accomplish concerted integration, the efficiency was extremely low and required genetic selection to detect the products (2,5,14,15). More recently, Grandgenett and co-workers (15)(16)(17)(18)(19)(20)(21)(22) have demonstrated that, under appropriate reaction conditions, both Rous sarcoma virus and HIV-1 integrase proteins alone are capable of much higher efficiencies of concerted integration allowing the products to be detected directly by physical assays. Other studies have suggested that viral or cellular proteins in addition to integrase may be involved in promoting concerted integration (23)(24)(25)(26).…”

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confidence: 99%

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Processing of Viral DNA Ends Channels the HIV-1 Integration Reaction to Concerted Integration*[boxs]

Craigie

2005

Journal of Biological Chemistry

126

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Retroviral DNA made by reverse transcription is blunt-ended, and the viral integrase protein must remove two nucleotides from each 3 end prior to integration into chromosomal DNA. Under most reaction conditions for integration in vitro, the majority of the reaction products are "half-site" products that result from integration of only one viral DNA end into one strand of the target DNA. Preprocessed DNA substrates are more efficient substrates for half-site reactions than are blunt-ended substrates, which require the removal of two nucleotides prior to integration. In contrast, we find that blunt-ended DNA is a better substrate for the biologically relevant reaction of concerted integration of pairs of viral DNA ends. The reaction pathway is channeled to concerted integration, and half-site integration products are reduced with blunt-ended DNA substrate that must first be processed by integrase. In addition, the terminal nucleotide requirements for concerted integration are more stringent than for the halfsite reaction. Longer DNA is more efficient for the concerted reaction than is shorter DNA that is capable of efficient half-site integration. This suggests that nonspecific interactions of integrase with viral DNA distant from the termini contribute to the assembly of a complex that is competent for concerted integration. Finally, differential effects of mutation of a residue in the C-terminal domain of integrase on concerted versus half-site integration implicate protein-protein interactions involving this domain as important for concerted integration.Integration of retroviral DNA into the host chromosomal DNA, an essential step in the retroviral replication cycle, involves two chemical steps (1). The newly synthesized bluntended viral DNA first undergoes 3Ј end processing. In this reaction, two nucleotides are removed from each 3Ј end. Next, the exposed hydroxyl groups attack a pair of phosphodiester bonds on opposite strands of the target DNA to complete the strand transfer step. For human immunodeficiency virus (HIV)-1, 1 the sites of attack are separated by 5 bp, resulting in a five-base duplication of target DNA sequence upon repair of the resulting integration intermediate. In the presence of a divalent metal ion, the viral integrase protein alone is able to carry out both 3Ј processing and DNA strand transfer with simple DNA substrates that mimic the viral DNA ends (2-4). However, under most reaction conditions the products of DNA strand transfer result from the integration of a single viral DNA end into one strand of the target DNA, a reaction that has been termed "half-site" as opposed to concerted integration. Were this to occur in the cell, the viral DNA would fail to integrate, and the viral replication cycle would be aborted. Following reverse transcription, the viral DNA remains associated with integrase and other viral and cellular proteins as part of the preintegration complex (PIC) (5-13). PICs isolated from infected cells efficiently integrate their DNA into a target DNA in vitro. In contras...

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Section: Resultssupporting

confidence: 51%

Section: Resultsmentioning

confidence: 70%

mentioning

confidence: 99%

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Processing of Viral DNA Ends Channels the HIV-1 Integration Reaction to Concerted Integration*[boxs]

Craigie

2005

Journal of Biological Chemistry

126

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“…Some IN residues located in the pocket at the p75-IN dimer interface are highlighted at the bottom of the three domain illustration, with those N-terminal to E152 in one monomer, and those C-terminal to E152 in the other monomer of the IN dimer. Although its exact oligomeric state and abundance in the PIC is still ambiguous, a wealth of in vitro enzymatic and virological complementation studies suggest IN functions to integrate both long terminal repeat (LTR) ends into host DNA in a coupled (concerted) fashion, and that it acts as a multimer, with a tetramer likely [73,154,157,158,[188][189][190][191][192]. In vitro, an IN dimer is sufficient for 3′ end processing but a tetramer appears needed for DNA strand transfer activity [154,157,158].…”

Section: Targeting Lentiviral Vectorsmentioning

confidence: 99%

Integrase, LEDGF/p75 and HIV replication

Poeschla

2008

Cell. Mol. Life Sci.

115

104

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HIV integrates a DNA copy of its genome into a host cell chromosome in each replication cycle. The essential DNA cleaving and joining chemistry of integration is known, but there is less understanding of the process as it occurs in a cell, where two complex and dynamic macromolecular entities are joined: the viral pre-integration complex and chromatin. Among implicated cellular factors, much recent attention has coalesced around LEDGF/p75, a nuclear protein that may act as a chromatin docking factor or receptor for lentiviral pre-integration complexes. LEDGF/p75 tethers HIV integrase to chromatin, protects it from degradation, and strongly influences the genome-wide pattern of HIV integration. Depleting the protein from cells and/or over-expressing its integrase-binding domain blocks viral replication. Current goals are to establish the underlying mechanisms and to determine whether this knowledge can be exploited for antiviral therapy or for targeting lentiviral vector integration in human gene therapy.

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“…S2 is however one possible scenario, and further experimental data elucidating protein-protein interfaces are needed to validate higher order interactions at play during the concerted integration of both viral DNA ends. Conditions for effective full-site integration by recombinant IN and twodimensional gel electrophoretic isolation of synaptic complexes have been reported (58,59). These techniques can be combined with MS surface topology analyses to better understand the protein-protein interactions essential for the twoended integration reaction.…”

Section: Residue Free In In(e152c)xdna(g2) In(e246c)xdna(a7)mentioning

confidence: 99%

Subunit-specific Protein Footprinting Reveals Significant Structural Rearrangements and a Role for N-terminal Lys-14 of HIV-1 Integrase during Viral DNA Binding

Zhao

McKee

Kessl

et al. 2008

Journal of Biological Chemistry

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To identify functional contacts between HIV-1 integrase (IN)and its viral DNA substrate, we devised a new experimental strategy combining the following two methodologies. First, disulfide-mediated cross-linking was used to site-specifically link select core and C-terminal domain amino acids to respective positions in viral DNA. Next, surface topologies of free IN and IN-DNA complexes were compared using Lys-and Arg-selective small chemical modifiers and mass spectrometric analysis. This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. The footprinting studies for the first time revealed the importance of a specific N-terminal domain residue, Lys-14, in viral DNA binding. In addition, a DNA-induced conformational change involving the connection between the core and C-terminal domains was observed. Site-directed mutagenesis experiments confirmed the importance of the identified contacts for recombinant IN activities and virus infection. These new findings provided major constraints, enabling us to identify the viral DNA binding channel in the active full-length IN multimer. The experimental approach described here has general application to mapping interactions within functional nucleoprotein complexes. HIV-1 integrase (IN)4 is commonly viewed as an important therapeutic target for the following reasons: its catalytic activities are required for viral replication, there is no closely related cellular equivalent of IN, and specific IN inhibitors are likely to be effective against viral strains resistant to currently available therapies targeting reverse transcriptase (RT), protease, and virus-cell fusion. Detailed structural information on functional IN-DNA complexes could aid drug design efforts. For example, the promising diketo acid class of inhibitors preferentially bind to the assembled IN-viral DNA complex rather than the free protein (1-4).The two chemical reactions catalyzed by HIV-1 IN, 3Ј processing and DNA strand transfer, have been characterized in detail (reviewed in Ref. 5). First, IN removes two nucleotides from each 3Ј-end of the viral DNA synthesized by reverse transcriptase. In the following step, concerted transesterification reactions covalently join the viral DNA ends into the host genome (6). In vivo, the enzyme acts in the context of a large nucleoprotein complex with a number of viral and host proteins contributing to the integration process (7-21).HIV-1 IN is composed of three distinct structural and functional domains: the N-terminal domain (NTD) (residues 1-50) that contains an HHCC zinc binding motif, the catalytic core domain (CCD) (residues 51-212) containing the DDE motif essential for coordinating catalytic divalent metals, and the C-terminal domain (CTD) (residues 213-288) that is thought to provide a platform for DNA binding. Crystallographic or NMR structural data are available for each of the individual domains (22-26). In addition, two-domain CCD/ CTD (27) and NTD/CCD (28) crystal structures have been determined. However,...

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Efficient Concerted Integration by Recombinant Human Immunodeficiency Virus Type 1 Integrase without Cellular or Viral Cofactors

Cited by 71 publications

References 43 publications

Processing of Viral DNA Ends Channels the HIV-1 Integration Reaction to Concerted Integration*[boxs]

Processing of Viral DNA Ends Channels the HIV-1 Integration Reaction to Concerted Integration*[boxs]

Integrase, LEDGF/p75 and HIV replication

Subunit-specific Protein Footprinting Reveals Significant Structural Rearrangements and a Role for N-terminal Lys-14 of HIV-1 Integrase during Viral DNA Binding

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