Human immunodeficiency virus integration in a cell-free system

Ellison, Viola; Abrams, H; Roe, Taiyun; Lifson, Jeffrey D.; Brown, Patrick O.

doi:10.1128/jvi.64.6.2711-2715.1990

Cited by 173 publications

(75 citation statements)

References 16 publications

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“…The PIC is transported through the nuclear pore to the nucleus where the strand transfer occurs. During this second step, IN transfers both newly exposed 3 0 extremities of the viral DNA into the target DNA by a one step transesterification [Brown et al, 1989;Ellison et al, 1990].…”

Section: Introductionmentioning

confidence: 99%

Structural effects of amino acid variations between B and CRF02‐AG HIV‐1 integrases

Malet

Soulié

Tchertanov

et al. 2008

Journal of Medical Virology

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HIV-1 integrase is one of the three essential enzyme required for viral replication and has a great potential as a novel target for anti-HIV drugs. The sequence variability of the entire integrase (IN) was examined in HIV-1 subtype B and CRF02-AG antiretroviral naïve infected patients for the presence of naturally occurring polymorphisms IN gene sequences and protein structures from both subtypes were compared. The phylogenetic analysis showed a total concordance between the 3 pol gene sequences for patients identified as subtype B whereas 3% of patients identified as CRF02-AG showed a mixture of subtypes. The analysis of IN aa sequences showed that 13 positions (K/R14, V/I31, L/I101, T/V112, T/A124, T/A125, G/N134, I/V135, K/T136, V/I201, T/S206, L/I234, and S/G283) differed between subtypes B and CRF02-AG. As observed in the 3D model of the preintegration complex, these differences may impact the functional property of IN. The fact that most variations were grouped suggests that some of them are linked together through compensatory mechanisms. This comparison allowed us to identify several variations of amino acids in HIV-1 IN subtype CRF02-AG that could have a putative impact on anti-integrase sensitivity. In particular, the region formed by Thr125, Thr124, Val31 contains at least one residue, T125, which variation has been involved in eliciting resistance to the naphtyridine carboxamide L870,810 IN inhibitor. In conclusion, virological response to anti-integrase should be studied carefully, according to the subtype, in clinical trials.

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Section: Introductionmentioning

confidence: 99%

Structural effects of amino acid variations between B and CRF02‐AG HIV‐1 integrases

Malet

Soulié

Tchertanov

et al. 2008

Journal of Medical Virology

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“…Several hours after infection, the linear viral cDNA can be extracted from infected cells as part of a nucleoprotein complex that is competent to integrate the viral DNA into an added target DNA in vitro (6,12,13). Such active complexes, operationally defined as preintegration complexes, contain the viral DNA, the viral IN protein, and possibly other viral proteins (8,9,15).…”

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Human immunodeficiency virus type 1 preintegration complexes containing discontinuous plus strands are competent to integrate in vitro

Miller

Wang

Bushman

1995

J Virol

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Despite intensive study, the mechanism by which many retroviruses complete reverse transcription has remained unclear. Most retroviruses and all lentiviruses fail to synthesize a full-length second strand of the viral cDNA (plus strand) efficiently in infected cells. For human immunodeficiency virus type 1, we find in synchronous infection experiments that full-length plus strands are rare (<1% of products) at times when integration is likely taking place. Subviral nucleoprotein complexes containing such discontinuous cDNA can be extracted from infected cells and used to generate integration products in vitro. Analysis of such integration products using two-dimensional gel electrophoresis revealed that the discontinuous viral DNA was efficiently integrated into an added target DNA. These data support a model in which the discontinuities in the plus strand need not be sealed until after integration, potentially by the enzymes that are already thought to repair DNA gaps at the junctions between host and viral DNA.

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“…This step, termed 3Ј-end processing, generates a 2-nucleotide 5Ј-end overhang and 3Ј ends that terminate with the phylogenetically conserved CA dinucleotide (4,24,39). In the nucleus, a concerted cleavage and ligation joins the processed 3Ј viral DNA ends to 5Ј staggered sites on opposite strands of the host chromosomal DNA (3,4,16,20,24). This strand transfer reaction results in a gapped intermediate in which the viral DNA 5Ј ends and the host chromosomal DNA 3Ј ends are unjoined.…”

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confidence: 99%

“…Purified recombinant IN has been shown both to remove two nucleotides from the 3Ј end of model viral DNA substrates and to join the recessed 3Ј ends to target DNA in vitro (6, 8, 13, 30-32, 35, 36, 43, 46, 49, 50, 52). Both the 3Ј-end processing and strand transfer steps of integration proceed via one-step transesterification reactions (17) that require no exogenous energy source (3,16,20). The strand transfer reaction has been shown to be reversible in vitro by using a substrate that mimics a strand transfer intermediate (10).…”

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Characterization of recombinant murine leukemia virus integrase

Dotan

Scottoline

Heuer

et al. 1995

J Virol

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Retroviral integration involves two DNA substrates that play different roles. The viral DNA substrate is recognized by virtue of specific nucleotide sequences near the end of a double-stranded DNA molecule. The target DNA substrate is recognized at internal sites with little sequence preference; nucleosomal DNA appears to be preferred for this role. Despite this apparent asymmetry in the sequence, structure, and roles of the DNA substrates in the integration reaction, the existence of distinct binding sites for viral and target DNA substrates has been controversial. In this report, we describe the expression in Escherichia coli and purification of Moloney murine leukemia virus integrase as a fusion protein with glutathione S-transferase, characterization of its activity by using several model DNA substrates, and the initial kinetic characterization of its interactions with a model viral DNA substrate. We provide evidence for functionally and kinetically distinct binding sites for viral and target DNA substrates and describe a cross-linking assay for DNA binding at a site whose specificity is consistent with the target DNA binding site.Integration of the retroviral genome into host cell chromosomal DNA is required for viral replication. Genetic studies have demonstrated that retroviral integration requires two viral functions: the viral IN protein, encoded by the 3Ј end of the pol gene (14,28,38,41), and att sites located at the termini of the viral long terminal repeats (11,12,37; for reviews, see references 2,25,26,48,53). Integration proceeds in three steps. After reverse transcription in the cytoplasm of the infected cell, the 3Ј ends of each of the termini of the linear viral DNA are cleaved to remove the terminal two nucleotides. This step, termed 3Ј-end processing, generates a 2-nucleotide 5Ј-end overhang and 3Ј ends that terminate with the phylogenetically conserved CA dinucleotide (4, 24, 39). In the nucleus, a concerted cleavage and ligation joins the processed 3Ј viral DNA ends to 5Ј staggered sites on opposite strands of the host chromosomal DNA (3,4,16,20,24). This strand transfer reaction results in a gapped intermediate in which the viral DNA 5Ј ends and the host chromosomal DNA 3Ј ends are unjoined. The integration process is completed by repair of the gapped intermediate. This step generates short direct repeats that flank the integrated provirus. The proteins involved in this final step remain to be identified.The end-processing and strand transfer steps in integration require the activity of only a single protein, the virally encoded IN. Purified recombinant IN has been shown both to remove two nucleotides from the 3Ј end of model viral DNA substrates and to join the recessed 3Ј ends to target DNA in vitro (6, 8, 13, 30-32, 35, 36, 43, 46, 49, 50, 52). Both the 3Ј-end processing and strand transfer steps of integration proceed via one-step transesterification reactions (17) that require no exogenous energy source (3,16,20). The strand transfer reaction has been shown to be reversible in vitro b...

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Human immunodeficiency virus integration in a cell-free system

Cited by 173 publications

References 16 publications

Structural effects of amino acid variations between B and CRF02‐AG HIV‐1 integrases

Structural effects of amino acid variations between B and CRF02‐AG HIV‐1 integrases

Human immunodeficiency virus type 1 preintegration complexes containing discontinuous plus strands are competent to integrate in vitro

Characterization of recombinant murine leukemia virus integrase

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