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Retroviral intasome assembly and inhibition of DNA strand transfer.

Hare S, Gupta SS, Valkov E, Engelman A, Cherepanov P - Nature (2010)

Bottom Line: The structure shows the organization of the retroviral intasome comprising an integrase tetramer tightly associated with a pair of viral DNA ends.The binding of strand-transfer inhibitors displaces the reactive viral DNA end from the active site, disarming the viral nucleoprotein complex.Our findings define the structural basis of retroviral DNA integration, and will allow modelling of the HIV-1 intasome to aid in the development of antiretroviral drugs.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine, Imperial College London, St-Mary's Campus, Norfolk Place, London W2 1PG, UK.

ABSTRACT
Integrase is an essential retroviral enzyme that binds both termini of linear viral DNA and inserts them into a host cell chromosome. The structure of full-length retroviral integrase, either separately or in complex with DNA, has been lacking. Furthermore, although clinically useful inhibitors of HIV integrase have been developed, their mechanism of action remains speculative. Here we present a crystal structure of full-length integrase from the prototype foamy virus in complex with its cognate DNA. The structure shows the organization of the retroviral intasome comprising an integrase tetramer tightly associated with a pair of viral DNA ends. All three canonical integrase structural domains are involved in extensive protein-DNA and protein-protein interactions. The binding of strand-transfer inhibitors displaces the reactive viral DNA end from the active site, disarming the viral nucleoprotein complex. Our findings define the structural basis of retroviral DNA integration, and will allow modelling of the HIV-1 intasome to aid in the development of antiretroviral drugs.

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PFV IN active site in committed and drug-bound statesViews without drug (a) and with MK0518 (b) or GS9137 (c) bound. Protein and DNA in upper panels are cartoons, with A17, DNA bases and the side chains of indicated amino acids as sticks. Drug atoms are colored: yellow, C; blue, N; red, O; orange, P; gray, F; green, Cl. The complex is shown as a solvent accessible surface in lower panels, colored by atoms (light gray, C; red, O; blue, N). Gray spheres are Mn2+ (a, labeled A and B) or Mg2+ (b, c) ions.
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Figure 3: PFV IN active site in committed and drug-bound statesViews without drug (a) and with MK0518 (b) or GS9137 (c) bound. Protein and DNA in upper panels are cartoons, with A17, DNA bases and the side chains of indicated amino acids as sticks. Drug atoms are colored: yellow, C; blue, N; red, O; orange, P; gray, F; green, Cl. The complex is shown as a solvent accessible surface in lower panels, colored by atoms (light gray, C; red, O; blue, N). Gray spheres are Mn2+ (a, labeled A and B) or Mg2+ (b, c) ions.

Mentions: The reactive 3′ termini of the donor DNA molecules are positioned within close proximity of the Asp128, Asp185 and Glu221 active site carboxylates (Fig. 2a). Although the crystals could be grown in the presence of MgCl2, which considerably improved their diffraction limit (Supplementary Table 1), data resolution did not allow unambiguous visualization of Mg2+ cations in the active site. Fortuitously, similar to other retroviral INs1, PFV IN can efficiently utilize Mn2+, a more electron-rich element, as a metal ion cofactor (Supplementary Fig. 1d). A difference electron density map calculated using diffraction data collected on crystals soaked in the presence of MnCl2 revealed two strong positive peaks (9.4 and 12.4σ) within the active sites of the inner IN subunits. This result confirmed the expected two-metal binding mode of retroviral INs and revealed the positions of metal ion cofactors within the assembled active site, which could be refined at full occupancy (Fig. 3a, see also Supplementary Fig. 4a for metal atom omit and final electron density maps). Based on the current model for two-metal active site catalysis4, metal atom B, coordinated by the carboxylates of Asp128 and Glu221, is in place to activate the 3′ hydroxyl group of the pre-processed viral DNA for strand transfer while metal A, bound by Asp128 and Asp185, would be expected to destabilize the scissile phosphodiester group in target DNA. Superposition of the Cα atoms of the active site Asp and Glu residues revealed striking conservation between the metal and DNA substrate binding modes of the Tn5 synaptic complex27 and PFV intasome (Supplementary Fig. 5). In addition, the positions of the metal ions are nearly identical to those of Cd2+ and Zn2+ cations observed in structures of the avian sarcoma virus IN CCD28. Of note, soaking crystals in MgCl2 or MnCl2 did not change the organization of the intasome active site (Supplementary Fig. 6). Hence, the positioning of the 3′ end of viral DNA is independent of bound divalent metal ions.


Retroviral intasome assembly and inhibition of DNA strand transfer.

Hare S, Gupta SS, Valkov E, Engelman A, Cherepanov P - Nature (2010)

PFV IN active site in committed and drug-bound statesViews without drug (a) and with MK0518 (b) or GS9137 (c) bound. Protein and DNA in upper panels are cartoons, with A17, DNA bases and the side chains of indicated amino acids as sticks. Drug atoms are colored: yellow, C; blue, N; red, O; orange, P; gray, F; green, Cl. The complex is shown as a solvent accessible surface in lower panels, colored by atoms (light gray, C; red, O; blue, N). Gray spheres are Mn2+ (a, labeled A and B) or Mg2+ (b, c) ions.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2837123&req=5

Figure 3: PFV IN active site in committed and drug-bound statesViews without drug (a) and with MK0518 (b) or GS9137 (c) bound. Protein and DNA in upper panels are cartoons, with A17, DNA bases and the side chains of indicated amino acids as sticks. Drug atoms are colored: yellow, C; blue, N; red, O; orange, P; gray, F; green, Cl. The complex is shown as a solvent accessible surface in lower panels, colored by atoms (light gray, C; red, O; blue, N). Gray spheres are Mn2+ (a, labeled A and B) or Mg2+ (b, c) ions.
Mentions: The reactive 3′ termini of the donor DNA molecules are positioned within close proximity of the Asp128, Asp185 and Glu221 active site carboxylates (Fig. 2a). Although the crystals could be grown in the presence of MgCl2, which considerably improved their diffraction limit (Supplementary Table 1), data resolution did not allow unambiguous visualization of Mg2+ cations in the active site. Fortuitously, similar to other retroviral INs1, PFV IN can efficiently utilize Mn2+, a more electron-rich element, as a metal ion cofactor (Supplementary Fig. 1d). A difference electron density map calculated using diffraction data collected on crystals soaked in the presence of MnCl2 revealed two strong positive peaks (9.4 and 12.4σ) within the active sites of the inner IN subunits. This result confirmed the expected two-metal binding mode of retroviral INs and revealed the positions of metal ion cofactors within the assembled active site, which could be refined at full occupancy (Fig. 3a, see also Supplementary Fig. 4a for metal atom omit and final electron density maps). Based on the current model for two-metal active site catalysis4, metal atom B, coordinated by the carboxylates of Asp128 and Glu221, is in place to activate the 3′ hydroxyl group of the pre-processed viral DNA for strand transfer while metal A, bound by Asp128 and Asp185, would be expected to destabilize the scissile phosphodiester group in target DNA. Superposition of the Cα atoms of the active site Asp and Glu residues revealed striking conservation between the metal and DNA substrate binding modes of the Tn5 synaptic complex27 and PFV intasome (Supplementary Fig. 5). In addition, the positions of the metal ions are nearly identical to those of Cd2+ and Zn2+ cations observed in structures of the avian sarcoma virus IN CCD28. Of note, soaking crystals in MgCl2 or MnCl2 did not change the organization of the intasome active site (Supplementary Fig. 6). Hence, the positioning of the 3′ end of viral DNA is independent of bound divalent metal ions.

Bottom Line: The structure shows the organization of the retroviral intasome comprising an integrase tetramer tightly associated with a pair of viral DNA ends.The binding of strand-transfer inhibitors displaces the reactive viral DNA end from the active site, disarming the viral nucleoprotein complex.Our findings define the structural basis of retroviral DNA integration, and will allow modelling of the HIV-1 intasome to aid in the development of antiretroviral drugs.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine, Imperial College London, St-Mary's Campus, Norfolk Place, London W2 1PG, UK.

ABSTRACT
Integrase is an essential retroviral enzyme that binds both termini of linear viral DNA and inserts them into a host cell chromosome. The structure of full-length retroviral integrase, either separately or in complex with DNA, has been lacking. Furthermore, although clinically useful inhibitors of HIV integrase have been developed, their mechanism of action remains speculative. Here we present a crystal structure of full-length integrase from the prototype foamy virus in complex with its cognate DNA. The structure shows the organization of the retroviral intasome comprising an integrase tetramer tightly associated with a pair of viral DNA ends. All three canonical integrase structural domains are involved in extensive protein-DNA and protein-protein interactions. The binding of strand-transfer inhibitors displaces the reactive viral DNA end from the active site, disarming the viral nucleoprotein complex. Our findings define the structural basis of retroviral DNA integration, and will allow modelling of the HIV-1 intasome to aid in the development of antiretroviral drugs.

Show MeSH
Related in: MedlinePlus