Structure-based modeling of the functional HIV-1 intasome and its inhibition

Abstract

The intasome is the basic recombination unit of retroviral integration, comprising the integrase protein and the ends of the viral DNA made by reverse transcription. Clinical inhibitors preferentially target the DNA-bound form of integrase as compared with the free protein, highlighting the critical requirement for detailed understanding of HIV-1 intasome structure and function. Although previous biochemical studies identified integrase residues that contact the DNA, structural details of protein-protein and protein-DNA interactions within the functional intasome were lacking. The recent crystal structure of the prototype foamy virus (PFV) integrase-viral DNA complex revealed numerous details of this related integration machine. Structures of drug-bound PFV intasomes moreover elucidated the mechanism of inhibitor action. Herein we present a model for the HIV-1 intasome assembled using the PFV structure as template. Our results pinpoint previously identified protein-DNA contacts within the quaternary structure and reveal hitherto unknown roles for Arg20 and Lys266 in DNA binding and integrase function. Models for clinical inhibitors bound at the HIV-1 integrase active site were also constructed and compared with previous studies. Our findings highlight the structural basis for HIV-1 integration and define the mechanism of its inhibition, which should help in formulating new drugs to inhibit viruses resistant to first-in-class compounds.

Fig. 1.

Architecture of the HIV-1 intasome. (A) The inner subunits of the IN tetramer, comprising residues 1–270 and engaged with viral DNA, are green and cyan; outer IN CCDs (residues 56–202) are blue and yellow. The reactive and nontransferred DNA strands are magenta and orange, respectively. Red sticks, side chains of inner monomer active site residues; red and gray spheres, Mn and Zn ions, respectively. (B) Resection of the upper IN dimer from A highlights the part of the model analogous to the PFV crystallographic asymmetric unit. Locations of canonical IN domains are indicated.

Fig. 2.

IN DNA binding and 3′ processing activities. (A) DNA binding assay. Left: Representative gel loaded with reactions conducted in the absence of IN or containing WT, D64A, or K156E/K159E (EE) IN and U5 or sequence nonspecific DNA; Right: quantified results of n = 4 experiments. (B) 3′ Processing (black) and DNA-binding (gray) activities of indicated mutants expressed as percentage WT function for two (3′ processing) or three (DNA binding) experimental replicates.

Fig. 3.

INSTI interactions with the HIV-1 intasome. (A) View of the IN active site in the committed, drug-free state. Amino acid side chains and nucleotide bases within 4 Å of both drugs are labeled with the same coloring scheme used in Fig. S3A. (B and C) RAL and EVG binding models, respectively, with interacting side chains and nucleotides labeled. Drug atoms are colored: yellow, C; blue, N; red, O; gray, F; green, Cl. The 3′-OH of the terminal adenosine is labeled. Red spheres, Mg ions.