Avian Sarcoma and Leukemia Virus (ASLV) integration in vitro: Mutation or deletion of integrase (IN) recognition sequences does not prevent but only reduces the efficiency and accuracy of DNA integration

Moreau, Karen; Charmetant, Julie; Gallay, Kathy; Fauré, Christine; Verdier, Gérard; Ronfort, Corinne

doi:10.1016/j.virol.2009.06.031

Cited by 4 publications

(8 citation statements)

References 41 publications

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“…We cloned and purified a recombinant PERV-A/C IN as we previously did for RAV-1 (Moreau et al, 2002(Moreau et al, , 2003(Moreau et al, , 2004(Moreau et al, , 2009Charmetant et al, 2011) and HIV-1 INs (Cellier et al, 2013;Trabaud et al, 2015). The PERV IN coding sequences were subcloned into the pET14b plasmid and expressed as an N-terminal His 6 -tagged protein in Escherichia coli.…”

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confidence: 99%

Porcine endogenous retrovirus-A/C: biochemical properties of its integrase and susceptibility to raltegravir

Demange

Yajjou-Hamalian

Gallay

et al. 2015

Journal of General Virology

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Porcine endogenous retroviruses (PERVs) are present in the genomes of pig cells. The PERV-A/C recombinant virus can infect human cells and is a major risk of zoonotic disease in the case of xenotransplantation of pig organs to humans. Raltegravir (RAL) is a viral integrase (IN) inhibitor used in highly active antiretroviral treatment. In the present study, we explored the potential use of RAL against PERV-A/C. We report (i) a three-dimensional model of the PERV-A/C intasome complexed with RAL, (ii) the sensitivity of PERV-A/C IN to RAL in vitro and (iii) the sensitivity of a PERV-A/C-IRES-GFP recombinant virus to RAL in cellulo. We demonstrated that RAL is a potent inhibitor against PERV-A/C IN and PERV-A/C replication with IC 50 s in the nanomolar range. To date, the use of retroviral inhibitors remains the only way to control the risk of zoonotic PERV infection during pig-to-human xenotransplantation.

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confidence: 99%

Porcine endogenous retrovirus-A/C: biochemical properties of its integrase and susceptibility to raltegravir

Demange

Yajjou-Hamalian

Gallay

et al. 2015

Journal of General Virology

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“…RAV-1 IN displays standard activities for 3′ processing, strand transfer reactions and concerted DNA integration in vitro [28], [37]–[39]. The CCD:CCD interface of IN with two pairs of facing α-helices (α5/α1, α1/α5) has been the only form observed until now.…”

Section: Discussionmentioning

confidence: 99%

A Crystal Structure of the Catalytic Core Domain of an Avian Sarcoma and Leukemia Virus Integrase Suggests an Alternate Dimeric Assembly

et al. 2011

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Integrase (IN) is an important therapeutic target in the search for anti-Human Immunodeficiency Virus (HIV) inhibitors. This enzyme is composed of three domains and is hard to crystallize in its full form. First structural results on IN were obtained on the catalytic core domain (CCD) of the avian Rous and Sarcoma Virus strain Schmidt-Ruppin A (RSV-A) and on the CCD of HIV-1 IN. A ribonuclease-H like motif was revealed as well as a dimeric interface stabilized by two pairs of α-helices (α1/α5, α5/α1). These structural features have been validated in other structures of IN CCDs. We have determined the crystal structure of the Rous-associated virus type-1 (RAV-1) IN CCD to 1.8 Å resolution. RAV-1 IN shows a standard activity for integration and its CCD differs in sequence from that of RSV-A by a single accessible residue in position 182 (substitution A182T). Surprisingly, the CCD of RAV-1 IN associates itself with an unexpected dimeric interface characterized by three pairs of α-helices (α3/α5, α1/α1, α5/α3). A182 is not involved in this novel interface, which results from a rigid body rearrangement of the protein at its α1, α3, α5 surface. A new basic groove that is suitable for single-stranded nucleic acid binding is observed at the surface of the dimer. We have subsequently determined the structure of the mutant A182T of RAV-1 IN CCD and obtained a RSV-A IN CCD-like structure with two pairs of buried α-helices at the interface. Our results suggest that the CCD of avian INs can dimerize in more than one state. Such flexibility can further explain the multifunctionality of retroviral INs, which beside integration of dsDNA are implicated in different steps of the retroviral cycle in presence of viral ssRNA.

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“…In the first assay, radiolabeled donor DNA was used and the integration products were separated on agarose gel and visualized by autoradiography. No integration products was observed when IN was absent (data not shown; refer to Moreau et al, 2003Moreau et al, , 2009. By contrast, three characteristic mixes of bands were revealed with the WT IN (Fig.…”

Section: Effects Of In Mutations On Concerted Dna Integrationmentioning

confidence: 90%

“…Hydrogen-bonds are shown by a dashed line. The in vitro retroviral concerted integration assay has been previously described for ASLV by others (Aiyar et al, 1996;Grandgenett, 2000, 2003;Grandgenett et al, 2009;Hindmarsh et al, 1999Hindmarsh et al, , 2001Vora et al, 2004;Vora and Grandgenett, 2001) and by us (Moreau et al, 2003(Moreau et al, , 2004(Moreau et al, , 2009 as well as for HIV-1, Simian Immunodeficiency Virus (SIV) and PFV. Briefly, the system is composed of a linear donor DNA (D), a plasmid acceptor DNA, and recombinant IN.…”

Section: Effects Of In Mutations On Concerted Dna Integrationmentioning

confidence: 99%

Functional analyses of mutants of the central core domain of an Avian Sarcoma/Leukemia Virus integrase

Charmetant¹,

Moreau²,

Gallay³

et al. 2011

Virology

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Integrase (IN) is the enzyme responsible for the integration of the retroviral genome into the host cell DNA. Herein, three mutants of conserved residues (V79, S85 and I146) of the central core domain (CCD) of an Avian Sarcoma/Leukemia Virus IN were analyzed in vitro. Our data revealed (i) the inability of S85T mutant to form dimers and tetramers in the absence of DNA and (ii) a slightly reduced ability of V79A IN in tetramers formation. Surprisingly, both mutants were still able to efficiently achieve concerted DNA integration. This could be explained by the ability of the two mutants to form complexes in the presence of DNA. These data suggest a strong structural role of the region encompassing V79 and S85 residues (β2/β3 turn-β3 strands) following binding to viral DNA and highlight the dynamic nature of IN.

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Avian Sarcoma and Leukemia Virus (ASLV) integration in vitro: Mutation or deletion of integrase (IN) recognition sequences does not prevent but only reduces the efficiency and accuracy of DNA integration

Cited by 4 publications

References 41 publications

Porcine endogenous retrovirus-A/C: biochemical properties of its integrase and susceptibility to raltegravir

Porcine endogenous retrovirus-A/C: biochemical properties of its integrase and susceptibility to raltegravir

A Crystal Structure of the Catalytic Core Domain of an Avian Sarcoma and Leukemia Virus Integrase Suggests an Alternate Dimeric Assembly

Functional analyses of mutants of the central core domain of an Avian Sarcoma/Leukemia Virus integrase

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