Mutations in the C‐terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process <i>in vitro</i>

Moreau, Karen; Fauré, Christine; Violot, S.; Verdier, Gérard; Ronfort, Corinne

doi:10.1046/j.1432-1033.2003.03833.x

Cited by 16 publications

(23 citation statements)

References 60 publications

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“…Consequently, we conclude that the primary role of the core-core interface is in assembly of a tetrameric synaptic complex, which can catalyze the concerted joining of two 3Ј viral DNA ends into a target DNA (33,34). This interpretation is supported by previously published effects of other substitutions in this core-core interface (55).…”

Section: Discussionsupporting

confidence: 81%

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

Bojja

Andrake

Weigand

et al. 2011

Journal of Biological Chemistry

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We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly. Retroviral integrase (IN)3 catalyzes the insertion of viral DNA into the DNA of the infected host cell. IN is one of three retrovirally encoded enzymes that are essential for retroviral replication and therefore is an important target for drugs to treat HIV/AIDS. One IN active site-directed inhibitor, raltegravir, is approved by the Food and Drug Administration for this purpose, and a second, elvitegravir, is in advanced clinical trials.

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Section: Discussionsupporting

confidence: 81%

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

Bojja

Andrake

Weigand

et al. 2011

Journal of Biological Chemistry

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“…As our recombinant HIV-1 IN was not able to catalyze concerted integration from a blunt ended DNA substrate, we used a two LTRs pre-cleaved substrate mimicking the 3′ processed viral DNA and carrying 18 bp from U3 and U5 along with a two bases overgang TA at each 5′end (Figure 1A). This shows that this enzyme was less efficient for performing the whole integration reaction than the purified preintegration complex (PIC) or INs from other retroviral sources (3,13). …”

Section: Resultsmentioning

confidence: 99%

“…The volumetric determination of IN–DNA complexes as determined by atomic force microscopy revealed substrate-induced assembly of a tetramer (28). Moreover, avian IN mutants, in which tetramer formation is altered, are less efficient at performing two-ended concerted DNA integration (13). Tetramers and dimers have been reported in Mu transposase.…”

Section: Discussionmentioning

confidence: 99%

HIV-1 integrase crosslinked oligomers are active in vitro

Faure

2005

Nucleic Acids Research

174

208

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The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors.

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“…1). The isolated products were ligated and subsequently used to transform MC1061/P3 cells (Invitrogen) (41,42). The donor-target junctions of individually isolated recombinants were sequenced using HIV-1 U3 and U5 sequence-specific primers (46).…”

Section: Methodsmentioning

confidence: 99%

Recombinant Human Immunodeficiency Virus Type 1 Integrase Exhibits a Capacity for Full-Site Integration In Vitro That Is Comparable to That of Purified Preintegration Complexes from Virus-Infected Cells

Sinha

Grandgenett

2005

J Virol

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Retrovirus preintegration complexes (PIC) in virus-Integration of the retrovirus linear DNA genome into host chromosomes is essential for replication of human immunodeficiency virus type 1 (HIV-1). Preintegration complexes (PIC) are formed in the cytoplasm after reverse transcription of the viral RNA. Integrase (IN) in the PIC catalyzes the excision of two nucleotides adjacent to the phylogenetically conserved CA dinucleotide from the 3Ј-OH blunt ends of the viral DNA (7). After nuclear transport of the PIC, the two viral DNA ends are inserted by IN into the host genome in a concerted fashion, here termed "full-site integration" (8,25,44). The unpaired dinucleotides at the 5Ј ends of the inserted viral DNA are removed, and the single-stranded gaps are repaired by a host cell DNA repair pathway (35). This process results in a short duplication of cell DNA ranging in size from 4 to 6 bp depending on the retrovirus species.Significant progress in our understanding of retrovirus fullsite integration was initiated by isolating PIC from virus-infected cells (8). Purified HIV-1 and murine leukemia virus (MLV) PIC are capable of incorporating ϳ15 to 50% of their viral DNA into an exogenously supplied DNA target as fullsite integration products after 45 to 90 min of incubation at 37°C (6,11,13,21,39,48). One report using HIV-1 PIC showed an ϳ95% incorporation of viral DNA into a target substrate where incorporation was essentially over after 45 min of incubation at 37°C (21). Cellular factors like barrier-toautointegration factor (BAF) appear to play a role in maintaining stable MLV and HIV-1 PIC structures, thus promoting full-site integration by preventing integration of the viral DNA into itself, termed "autointegration" (12,33,48,55).Simplified integration assays using model linear retrovirus DNA with terminal U5 and U3 long terminal repeat (LTR) sequences, purified recombinant IN, and a DNA target were also developed (9,16,32,45). These studies established that IN was the only viral protein necessary for both 3Ј-OH processing and DNA strand transfer activities.Further progress in reconstituting the HIV-1 full-site integration process was made using IN derived from nonionic detergent lysates of virus particles (10,22,23). The 3Ј-OH recessed ends containing attachment (att) site sequences from two different viral donors (480 bp) were integrated by IN in a concerted manner into a circular DNA target (bimolecular

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Mutations in the C‐terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro

Cited by 16 publications

References 60 publications

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

HIV-1 integrase crosslinked oligomers are active in vitro

Recombinant Human Immunodeficiency Virus Type 1 Integrase Exhibits a Capacity for Full-Site Integration In Vitro That Is Comparable to That of Purified Preintegration Complexes from Virus-Infected Cells

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