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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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Predicted target DNA binding orientationCartoon representation of the intasome as in Fig. 1a with active site side chains shown as red sticks and 〈2 helices, known to contribute to target DNA binding29, in cyan. The DNA molecule modeled in black and gray shows the most likely orientation for target DNA binding. Note that the CTD, juxtaposed to the target DNA in this model, is known to possess sequence non-specific DNA binding activity35.
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Figure 4: Predicted target DNA binding orientationCartoon representation of the intasome as in Fig. 1a with active site side chains shown as red sticks and 〈2 helices, known to contribute to target DNA binding29, in cyan. The DNA molecule modeled in black and gray shows the most likely orientation for target DNA binding. Note that the CTD, juxtaposed to the target DNA in this model, is known to possess sequence non-specific DNA binding activity35.

Mentions: The active sites of the inner IN subunits, engaged with the 3′ termini of the viral DNA, are located deep within the dimer-dimer interface. Therefore, the only mode of host chromosomal DNA (target DNA) binding that would not require dramatic rearrangement of the intasome or severe DNA bending is along the cleft between IN dimers (Fig. 4). This target DNA binding mode could not have been predicted based on previous partial IN structures, and starkly differs from what we recently proposed18. Modeling B-form DNA within the cleft results in near perfect alignment of the active sites with opposing target DNA phoshodiester bonds separated by 4 bp, the known spacing of concerted PFV integration17. It is easy to see how mutations within the α2 helix of the CCD, described by Katzman and colleagues29, would prevent target DNA binding (Fig. 4). We tentatively speculate that the NTDs and/or the CTDs of the outer IN subunits, disordered in our structures, could be involved in target DNA capture30. However, this target-binding model requires verification using mutagenesis or crystallographic approaches.


Retroviral intasome assembly and inhibition of DNA strand transfer.

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

Predicted target DNA binding orientationCartoon representation of the intasome as in Fig. 1a with active site side chains shown as red sticks and 〈2 helices, known to contribute to target DNA binding29, in cyan. The DNA molecule modeled in black and gray shows the most likely orientation for target DNA binding. Note that the CTD, juxtaposed to the target DNA in this model, is known to possess sequence non-specific DNA binding activity35.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Predicted target DNA binding orientationCartoon representation of the intasome as in Fig. 1a with active site side chains shown as red sticks and 〈2 helices, known to contribute to target DNA binding29, in cyan. The DNA molecule modeled in black and gray shows the most likely orientation for target DNA binding. Note that the CTD, juxtaposed to the target DNA in this model, is known to possess sequence non-specific DNA binding activity35.
Mentions: The active sites of the inner IN subunits, engaged with the 3′ termini of the viral DNA, are located deep within the dimer-dimer interface. Therefore, the only mode of host chromosomal DNA (target DNA) binding that would not require dramatic rearrangement of the intasome or severe DNA bending is along the cleft between IN dimers (Fig. 4). This target DNA binding mode could not have been predicted based on previous partial IN structures, and starkly differs from what we recently proposed18. Modeling B-form DNA within the cleft results in near perfect alignment of the active sites with opposing target DNA phoshodiester bonds separated by 4 bp, the known spacing of concerted PFV integration17. It is easy to see how mutations within the α2 helix of the CCD, described by Katzman and colleagues29, would prevent target DNA binding (Fig. 4). We tentatively speculate that the NTDs and/or the CTDs of the outer IN subunits, disordered in our structures, could be involved in target DNA capture30. However, this target-binding model requires verification using mutagenesis or crystallographic approaches.

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