Retroviral intasome assembly and inhibition of DNA strand transfer

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.

Figure 1. Architecture of the PFV intasome

a, Views along (left) and perpendicular (right) to the crystallographic two-fold axis. The inner subunits of the IN tetramer, engaged with viral DNA, are blue and green; outer IN chains are yellow. The reactive and non-transferred DNA strands are magenta and orange, respectively. Side chains of Asp128, Asp185 and Glu221 active site residues are red sticks; gray spheres are Zn atoms. Locations of the canonical IN domains (NTD, CCD and CTD) are indicated; b, inner (green) IN chain with domains and linkers indicated. The orientation is the same as in the right panel of a.

Figure 2. Sequence-specific protein-DNA interactions

a, View of the IN active site with bound DNA. Amino acid side chains discussed in text, DNA bases and the main chain carbonyl of Gly218 are shown as sticks; b, interactions of the same DNA molecule with the NTD and NTD-CCD and CCD-CTD linkers from the opposing (blue) IN subunit. Note the widening of the minor groove due to insertion of Arg313 and Asn106 side chains. The Arg69 side chain additionally packs into the major groove, forming hydrogen bonds with guanine 10 of the non-transferred strand.

Figure 3. PFV IN active site in committed and drug-bound states

Views 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.

Figure 4. Predicted target DNA binding orientation

Cartoon 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 binding, 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 activity.