Identifying and characterizing a functional HIV-1 reverse transcriptase-binding site on integrase

Abstract

Integrase (IN) from human immunodeficiency virus, type 1 (HIV-1) exerts pleiotropic effects in the viral replication cycle. Besides integration, IN mutations can impact nuclear import, viral maturation, and reverse transcription. IN and reverse transcriptase (RT) interact in vitro, and the IN C-terminal domain (CTD) is both necessary and sufficient for binding RT. We used nuclear magnetic resonance spectroscopy to identify a putative RT-binding surface on the IN CTD, and surface plasmon resonance to obtain kinetic parameters and the binding affinity for the IN-RT interaction. An IN K258A substitution that disrupts reverse transcription in infected cells is located at the putative RT-binding surface, and we found that this substitution substantially weakens IN CTD-RT interactions. We also identified two additional IN amino acid substitutions located at the putative RT-binding surface (W243E and V250E) that significantly impair viral replication in tissue culture. These results strengthen the notion that IN-RT interactions are biologically relevant during HIV-1 replication and also provide insights into this interaction at the molecular level.

FIGURE 1.

HSQC spectra of free IN 220–270 (blue) at a concentration of 200 μm, and of IN 220–270 mixed with RT at IN 220–270:RT molar ratios of 10:1 (red) and 5:1 (green). A representative subset of IN 220–270 signals is shown in the expanded view. Residues whose amide group signals show a significant change in intensity are labeled in red.

FIGURE 2.

A, ribbon diagrams illustrating two views of the putative IN 220–270 binding surface (shown in “red”) that interacts with RT. B, space-filling model with orientation similar to the leftmost representation in A. The positions of amino acids whose peak signals decreased significantly in intensity in the presence of RT are indicated.

FIGURE 3.

Sensorgrams showing binding events for IN 220–270, IN, and IN 220–270/K258A, as well as negative control experiments using BSA. The concentrations employed were 0–500 nm IN 220–270, IN, or IN 220–270/K258A, or 0–1 μm BSA. The red lines are experimental data, whereas the black lines are simulated binding curves employing a two-state conformational exchange model.

FIGURE 4.

Kinetic parameters (k_a1, k_d1, k_a2, and k_d2) and dissociation constants (K_D) obtained by employing a two-state conformational exchange binding model for IN 220–270, IN, and IN 220–270/K258A.

FIGURE 5.

Replication kinetics of wild-type and mutant HIV-1 viruses. CEM cells were infected by wild type (♦) or mutant HIV-1 bearing IN substitutions W243E (▴) or V250E (×). The infections with each mutant virus were repeated to confirm the replication-defective phenotype (W243E, n = 2; V250E, n = 3), and representative data for each viral clone are shown. Mock infections were performed using heat-inactivated wild-type virus (□).