Influence of the amino-terminal sequence on the structure and function of HIV integrase

Abstract

Background: Antiretroviral therapy (ART) can mitigate the morbidity and mortality caused by the human immunodeficiency virus (HIV). Successful development of ART can be accelerated by accurate structural and biochemical data on targets and their responses to inhibitors. One important ART target, HIV integrase (IN), has historically been studied in vitro in a modified form adapted to bacterial overexpression, with a methionine or a longer fusion protein sequence at the N-terminus. In contrast, IN present in viral particles is produced by proteolytic cleavage of the Pol polyprotein, which leaves a phenylalanine at the N-terminus (IN 1F). Inspection of available structures suggested that added residues on the N-terminus might disrupt proper protein folding and formation of multimeric complexes.

Results: We purified HIV-1 IN 1F^1-212 and solved its structure at 2.4 Å resolution, which showed extension of an N-terminal helix compared to the published structure of IN^1-212. Full-length IN 1F showed increased in vitro catalytic activity in assays of coupled joining of the two viral DNA ends compared to two IN variants containing additional N-terminal residues. IN 1F was also altered in its sensitivity to inhibitors, showing decreased sensitivity to the strand-transfer inhibitor raltegravir and increased sensitivity to allosteric integrase inhibitors. In solution, IN 1F exists as monomers and dimers, in contrast to other IN preparations which exist as higher-order oligomers.

Conclusions: The structural, biochemical, and biophysical characterization of IN 1F reveals the conformation of the native HIV-1 IN N-terminus and accompanying unique biochemical and biophysical properties. IN 1F thus represents an improved reagent for use in integration reactions in vitro and the development of antiretroviral agents.

Keywords: Biophysics; HIV; Integrases; Protein structure; Retroviridae; X-ray crystallography.

Fig. 1

Structure of HIV-1 integrase with a native N-terminus (PDB 6VRG). a Structure of IN 1F_NTD–CCD. NTDs are colored in red and CCDs are colored in blue. Zn²⁺ (grey), K⁺ (purple), and phosphate (orange and red) atoms are shown as spheres. b Comparison of the ɑ1 helix between IN 1F and IN GSH (PDB 1K6Y). The IN 1F NTD adopts a helical structure starting from the carbonyl of F1. The IN GSH NTD shows a disruption of the ɑ1 helix. c View highlighting differences between IN 1F and IN GSH at the N-terminus, with a deviation of 4.6 Å in the peptide backbone at L2 and a 10.4 Å deviation in side chain position. This change is accompanied by a flip of ~ 180° in the orientation of the N-terminus. 2Fo-Fc electron density (d) and simulated annealing omit (e) maps contoured at 1.5 σ unambiguously demonstrate the N-terminal structure of IN 1F

Fig. 2

3′-processing activity in vitro. a Diagram of 3′-processing fluorescence polarization assay. The double stranded oligo containing a 3′ fluorescent label exhibits high fluorescence polarization. Cleavage and release of the terminal dinucleotide causes a decrease in fluorescence polarization. 5′-ends are designated by filled circles and the fluorophore is designated by the green star. b 3′-processing activity of IN 1F compared to IN GSH (left) and IN 1F compared to IN MF (right) in the presence of Mg²⁺. c 3′ processing activity of IN 1F compared to IN GSH (left) and IN 1F compared to IN MF (right) in the presence of Mn²⁺. Data are plotted as mean ± SD. ** Denotes P < 0.01 and **** denotes P < 0.0001

Fig. 3

Strand transfer activity in vitro. a Diagram of gel-based strand transfer assay. A pre-processed double-stranded oligo containing a 5′ fluorescent label is integrated into a supercoiled target plasmid (pUC19) in the presence of integrase and cofactor (Mg²⁺ or Mn²⁺). Single strand integration results in the formation of a tagged circle product accompanied by the relaxation of supercoiling. Concerted integration results in the formation of a 2 LTR coupled product, accompanied by the linearization of the plasmid. Reaction products are separated by agarose gel electrophoresis. b Example gels of IN 1F and IN MF strand transfer activity. The slowest-migrating band represents single strand integration events (tagged circle) with the linearized concerted integration product (2 LTR coupled) migrating further. The supercoiled target plasmid is visible in the ethidium bromide stain as the furthest-migrating band. Unintegrated fluorescent oligo is observed at the bottom of the gel. c Quantification of IN 1F and IN GSH strand transfer activity. Single strand integration events are shown in the top panels and concerted integration events are shown in the bottom panels. IN 1F shows greater concerted integration activity than IN GSH in the presence of either Mg²⁺ or Mn²⁺. d Quantification of IN 1F and IN MF strand transfer activity. Single strand integration events are shown in the top panels and concerted integration events are shown in the bottom panels. IN 1F shows greater concerted integration activity than IN MF in the presence of either Mg²⁺ or Mn²⁺. e Inhibition of strand transfer activity by raltegravir. Raltegravir more potently inhibits both the single strand (left) and concerted integration (right) activity of IN GSH and IN MF as compared to IN 1F. Data before normalization are plotted in Additional file 5: Figure S5. Data are plotted as mean ± SD of 3 replicates. * Denotes P < 0.05, ** denotes P < 0.01, and **** denotes P < 0.0001

Fig. 4

ALLINI-induced aggregation of IN 1F, IN GSH, and IN MF. a Aggregation of IN at 15 µM was induced by incubation with 30 µM of the ALLINIs BI-224436, BI-D, or CX04328 at a range of NaCl concentrations for 20 min. Aggregation was measured by light scattering at 405 nm. No aggregation was observed for any ALLINI at 1000 mM NaCl. At intermediate NaCl concentrations, ALLINIs induce aggregation of IN 1F more potently than IN GSH or IN MF. b Aggregation in the absence of ALLINIs. No aggregation was observed at NaCl concentrations of 300 mM or greater. Below 300 mM, IN 1F, and, to a lesser extent, IN MF and IN GSH spontaneously aggregate. * Denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001, **** denotes P < 0.0001

Fig. 5

Biophysical analysis of IN 1F and IN MF. a SEC-MALS analysis of IN 1F and IN MF. Both the M_w (weight-average molecular mass) from multiangle light scattering and retention times are consistent with mixtures of monomers and dimers for both IN 1F and IN MF (expected MW of monomer: 32 kDa). b Sedimentation velocity analysis of IN 1F and IN MF shows distinct populations of monomer and dimer in solution. c(S) distributions derived from the fitting of the Lamm equation are shown. c Sedimentation equilibrium analysis of IN 1F and IN MF indicates the presence of monomers and dimers in solution at 4 °C. Globally fit radial distributions for 8.9 μM (IN 1F) and 6 μM (IN MF) in a monomer–dimer model are shown. Table 2 provides the association properties derived from this analysis