Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance

Abstract

The development of HIV integrase (IN) strand transfer inhibitors (INSTIs) and our understanding of viral resistance to these molecules have been hampered by a paucity of available structural data. We recently reported cocrystal structures of the prototype foamy virus (PFV) intasome with raltegravir and elvitegravir, establishing the general INSTI binding mode. We now present an expanded set of cocrystal structures containing PFV intasomes complexed with first- and second-generation INSTIs at resolutions of up to 2.5 Å. Importantly, the improved resolution allowed us to refine the complete coordination spheres of the catalytic metal cations within the INSTI-bound intasome active site. We show that like the Q148H/G140S and N155H HIV-1 IN variants, the analogous S217H and N224H PFV INs display reduced sensitivity to raltegravir in vitro. Crystal structures of the mutant PFV intasomes in INSTI-free and -bound forms revealed that the amino acid substitutions necessitate considerable conformational rearrangements within the IN active site to accommodate an INSTI, thus explaining their adverse effects on raltegravir antiviral activity. Furthermore, our structures predict physical proximity and an interaction between HIV-1 IN mutant residues His148 and Ser/Ala140, rationalizing the coevolution of Q148H and G140S/A mutations in drug-resistant viral strains.

Fig. 1.

Chemical structures of the INSTIs used in this work. The metal chelating heteroatoms are colored red and the halogenated benzene groups are blue.

Fig. 2.

Active site region of the PFV intasome bound to metal cofactors and INSTIs. With the exception of carbon atoms, standard atom coloring is used throughout: oxygen, red; nitrogen, blue; sulfur, yellow; fluorine, pale blue; chlorine, green; manganese, purple; magnesium, light green; phosphorus, orange. (A) The intasome active site in Mn²⁺-bound form. Protein is represented as a gray cartoon. Stick representations are shown for side chains mentioned in the text and DNA, colored by atom with gray used for carbon. Large purple spheres indicate Mn atoms and smaller red spheres show the water molecules coordinated to the metal atoms. (B) The Mg²⁺/MK2048-bound intasome active site with viewpoint and representation as in A. Here, MK2048 is also shown as a stick representation and Mg atoms as spheres. (C) Comparison of the different inhibitor-bound structures. Inhibitor molecules are shown as sticks with cyan carbon atoms. The black ovals highlight the conserved features of these drugs: 1, the metal chelating heteroatoms; and 2, the halogenated benzene ring. (D) An alternative view comparing drug binding, rotated 90° about the horizontal axis compared with C. Here, the side chain of Tyr212 is shown to highlight its involvement in binding different INSTIs; DNA is shown as sticks with carbon atoms colored orange.

Fig. 3.

Bar plot showing the IC₅₀ values of raltegravir and MK2048 against WT and mutant PFV INs. The mean values and the SDs were obtained from five independent measurements.

Fig. 4.

The effects of the S217H and N224H amino acid substitutions on the conformation of the PFV IN active site. (A–C) A ribbon representation is used for the protein backbone, metals are shown as spheres, and sticks are used to show relevant side chains and INSTI molecules. Standard atom coloring is used throughout except where noted. (A) Mn²⁺-bound active sites of WT, S217H, and N224H PFV intasomes. Carbon and manganese atoms are colored gray for WT, light blue for S217H, and green for N224H. The relevant side chains and mutations are indicated in black or gray according to the PFV or HIV-1 IN sequence, respectively. (B) Comparison of Mn²⁺- and Mg²⁺/MK2048-bound states of S217H active sites. Carbon atoms are colored gray for the Mn²⁺-bound structure and light blue for the MK2048 structure; carbon atoms of the MK2048 INSTI are magenta. Black arrows indicate side-chain movements necessary to accommodate INSTI binding and the red arrow indicates the required protein main-chain distortion. (C) Comparison of Mn²⁺- and Mg²⁺/MK2048-bound active sites in N224H intasome. Carbon atoms of Mn²⁺- and Mg²⁺/MK2048-bound structures are colored gray and green, respectively. Black arrows show the side-chain rearrangements required for INSTI binding, and the red “x” indicates the breaking of the His–phosphate interaction.