Identification of the catalytic and DNA-binding region of the human immunodeficiency virus type I integrase protein

Vink, Cornelis; Groeneger, Antoinette A. M. Oude; Plasterk, Ronald H.A.

doi:10.1093/nar/21.6.1419

Cited by 263 publications

(279 citation statements)

References 29 publications

Supporting

Mentioning

266

Contrasting

Order By: Relevance

“…Models proposing either independent DNA binding sites (13,18) or a single binding site (19) for viral DNA and target DNA have been suggested. Our results are most consistent with the existence of clearly distinct binding sites for viral DNA and target DNA.…”

Section: Discussionmentioning

confidence: 99%

A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro.

Ellison

Brown

1994

Proc. Natl. Acad. Sci. U.S.A.

150

145

View full text Add to dashboard Cite

Retroviral replication depends on integration of the viral genome Into a chromosome of the host cell. The steps in this process are orchestrated in vivo by a large nucleoprotein complex and are catalyzed by the retroviral enzyme integrase. Several (5,6). The product of this highly regulated, precise set of reactions is a mature provirus flanked by direct repeats of short sequences from the target site. The first two steps are apparently executed in vivo by large, fast-sedimenting, nucleoprotein complexes (5, 7). These integration complexes, when isolated from infected cells, can faithfully reproduce the in vivo integration reaction in vitro and are stable through multiple steps of purification. Thus far, only a subset of viral proteins have been identified as constituents of the integration complex along with viral genomic DNA (7-9).Although purified integrase can mediate the catalytic steps of the reaction in vitro (10-12), the role played by integrase in other key properties of the integration complex has not been rigorously investigated. In this study, we employ a modified version of the standard in vitro integration assay to identify and characterize a stable, functional complex between human immunodeficiency virus type 1 (HIV-1) integrase and viral DNA ends. After this stable complex is formed, the two catalytic steps in the reaction occur processively. Thus, the ability to form a stable complex with viral DNA is intrinsic to HIV-1 integrase. MATERIALS AND METHODSOligonudeotides and Preparation of Substrates. Oligonucleotides were purchased from Operon Technologies (Alameda, CA) and purified by electrophoresis through a denaturing 15% polyacrylamide gel. The standard integration substrate consisted of the terminal 20 bp from the U5 end of viral DNA and was prepared by using the oligonucleotides C220 (5'-ATGTGGAAAATCTCTAGCAGT-3') and C120 (5'-ACTGCTAGAGATTTTCCACAT-3'). C220 was phosphorylated with T4 polynucleotide kinase (New England Biolabs) and [.-32P]ATP (ICN) and the specific activity (typically =700,000 cpm/pmol) was determined. The radiolabeled oligonucleotide was then annealed to C120 and the unincorporated nucleotide was removed by centrifugation through a Sephadex G-15 column with a 1-ml bed volume. The 5' deletion (AAC) viral substrate was prepared as described above by annealing C220 to B1-1 (5'-TGCTA-GAGATTTTCCACAT-3').To prepare the competitor/target DNA, a duplex molecule without free 5' or 3' ends, the oligonucleotide 5'-AGCTGG-CTAACGGCCCTTGGGCCGTTAGCCAGCTATAGACG-GCGCTTGCGCCGTCTAT-3' was phosphorylated and then incubated at 120 nM in 50 mM NaCl to favor intramolecular annealing. The DNA was then ligated by T4 DNA ligase (New England Biolabs) under standard conditions, extracted with phenolchloroform (50:50, vol/vol), and then purified in a denaturing 10% polyacrylamide gel.In Vitro Integration Assays. HIV integrase was expressed in Escherichia coli using the T7 expression system and was purified as described (13). In a typical experiment, 100 nM integrase (-70 7316The publicatio...

show abstract

Section: Discussionmentioning

confidence: 99%

A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro.

Ellison

Brown

1994

Proc. Natl. Acad. Sci. U.S.A.

150

145

View full text Add to dashboard Cite

show abstract

“…In the case of retroviral integrase, the core domain can carry out a reaction called disintegration which mimics the reversal of the normal strand transfer step, although the normal reaction requires the intact protein (Chow et al 1992;Drelich et al 1992;Bushman et al 1993;Vink et al 1993). The cause of the difference in specificity of the reaction direction between the core domain and the intact integrase is currently not well Polynucleotidyl transfer reactions in site-specific DNA recombination…”

Section: K Mizuuchimentioning

confidence: 99%

Polynucleotidyl transfer reactions in site‐specific DNA recombination

1997

View full text Add to dashboard Cite

Site-specific DNA rearrangement reactions are widespread among organisms. They are used, for example, by vertebrates to boost immune response diversity, and in turn by parasitic organisms to evade the host immune system by surface antigen switching. Parasitic genetic elements ubiquitous to most organisms invade new host genomic sites by a variety of types of site-specific recombination. Polynucleotidyl transfer reactions are central to these DNA recombination reactions. The recombinase of each reaction system that 'catalyses' such chemical reactions at specific DNA sites are apparently designed to accomplish unique DNA geometrical specificity, or delicate control over the extent or direction of the reaction, with the sacrifice of protein turnover. Here we discuss our current understanding of several issues that relate to the polynucleotidyl transfer steps in several of the better studied site-specific recombination reactions.

show abstract

“…Furthermore, the core domain was more sensitive to alterations in the conserved A/T and C/G bps than was full-length integrase, despite their similar activity on wild-type substrates (either a U5 or U3 viral DNA end; data shown for the U5 end only). The hypersensitivity of the core domain to mutations in these bps may be a result of the loss of the nonspecific DNA binding activity of the COOH terminus (33)(34)(35), making the core domain more dependent on the remaining specific contacts with the CA/TG bps.…”

Section: (Panel a A/c) Or A Base (Panel A A/a) Than From A Wild-typmentioning

confidence: 99%

The Core Domain of HIV-1 Integrase Recognizes Key Features of Its DNA Substrates

Gerton¹,

Brown²

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

We investigated which features of the substrate specificity of human immunodeficiency virus type 1 (HIV-1) integrase could be assigned to the central domain of the 288-residue HIV-1 integrase protein, composed of amino acids 50 -212. This domain contains the active site and shares structural homology with a large family of polynucleotidyl transferases. Using model substrates with defined alterations in critical features we found that this domain alone is sufficient for recognition of: 1) the phylogenetically conserved CA/TG base pairs near the viral DNA end; 2) the 5-terminal dinucleotide that is left unpaired after end processing; and 3) target DNA flanking the site of joining. Future efforts aimed at identifying specific amino acids involved in recognition of these key substrate features can now be targeted at this domain.Integration of a double-stranded DNA copy of the retroviral genome into a host cell chromosome is essential for viral replication. Genetic and biochemical studies have shown that retroviral integration requires two viral components, integrase and the U3 and U5 att sites, which are phylogenetically conserved sequences at the ends of viral DNA. Analysis in vivo and in vitro has revealed that integrase processively catalyzes two chemical steps. In 3Ј-end processing, integrase cleaves two nucleotides from the 3Ј-ends of the double-stranded viral DNA, leaving a phylogenetically invariant CA dinucleotide sequence at the 3Ј-termini of the recessed viral DNA ends. In the second chemical step, integrase catalyzes the joining of the two 3Ј-recessed viral DNA ends to sites on opposite strands of a target DNA, separated by 5 base pairs (bps) 1 in the case of HIV-1. The gaps that flank this product of the joining reaction are repaired by a pathway that remains uncharacterized. The chemical reactions catalyzed by integrase can be studied in vitro using model oligonucleotide substrates and purified recombinant protein.We evaluated the specificity of the isolated core domain of HIV-1 integrase. A minimal catalytically active peptide has been identified by deletion analysis, comprised of amino acids 50 -186 (1). To ensure reasonable dynamic range in our assays, we used a slightly larger, more active polypeptide, consisting of amino acids 50 -212 (1). The three-dimensional structure of this domain has been solved, and it reveals striking structural homology to a larger family of polynucleotidyl transferases (2). This domain contains the phylogenetically conserved DD35E motif that defines the active site (3-8). Using model substrates with defined alterations in features known to be critical for the catalytic specificity of full-length integrase, we assessed the ability of the isolated core domain to distinguish between altered and wild-type substrates.Integrase can carry out a concerted cleavage-ligation reaction, termed disintegration, on a model substrate that mimics the product of integration of a single viral DNA end, yielding a free cleaved viral DNA end and a ligated target DNA strand (9). A divalent meta...

show abstract

Identification of the catalytic and DNA-binding region of the human immunodeficiency virus type I integrase protein

Cited by 263 publications

References 29 publications

A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro.

A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro.

Polynucleotidyl transfer reactions in site‐specific DNA recombination

The Core Domain of HIV-1 Integrase Recognizes Key Features of Its DNA Substrates

Contact Info

Product

Resources

About