Triggering HIV polyprotein processing by light using rapid photodegradation of a tight-binding protease inhibitor

Abstract

HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro. Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time.

Figure 1. A photolabile inhibitor of HIV-1 PR and its degradation triggered by light.

(a) HIV-1 protease inhibitor Ritonavir; (b) Photodegradable inhibitor of HIV-1 PR (compound 1) and products resulting from photolysis (compound 2 and coumarin derivative). Inhibition constants determined as in Fig. 3a are shown for each compound.

Figure 2. Comparison of binding mode of compounds 1 and 2 to HIV-1 PR.

(a) Two views of the HIV-1 PR-1 complex (PDB code 4U7Q). The protein is shown in cartoon representation with a transparent surface, while the inhibitor atoms are represented by spheres. The coumarin moiety protrudes from the enzyme active site cavity. (b) Superposition of 1 (pink carbon atoms) and RTV (ritonavir; grey carbon atoms) bound in the HIV-1 PR active site. (c) Superposition of 1 with 2 (in green, PDB code 4U7V) bound to HIV-1 PR (PDB code 1HXW (ref. 28)). (b,c) Residues interacting with 1, 2 and RTV are indicated in the corresponding colours for individual enzyme subsites. Residues forming polar interactions are highlighted in bold italics. To identify non-polar interactions, the cut-off for distance between any atom of residue and any atom of inhibitor was 4 Å. For polar interaction, the cut-off for distance between hydrogen bond donor and acceptor was 3.5 Å. Active site aspartates are shown in stick representation.

Figure 3. Kinetic analysis of HIV-1 PR reactivation by inhibitor photodegradation.

(a) A non-linear fit of Morrison equation of inhibition of HIV-1 PR by compound 1. The activity of purified recombinant HIV-1 PR was determined in vitro as described in the experimental section in the presence of the indicated inhibitor concentrations. Two independent experiments yielded very similar results; (b,c) Reactivation of purified recombinant HIV-1 PR in buffer (100 mM sodium acetate, 300 mM NaCl, 4 mM EDTA, pH 4.7) by photodegradation of 1 using either the cuvette set up (b) or the capillary set up (c): (b) Purified recombinant HIV-1 PR (8 nM) incubated with compound 1 at the indicated concentrations was irradiated with two 405 nm lasers (combined output of 300 mW) for various time intervals. The PR activity was then measured using a chromogenic substrate. The plot shows relative PR activity as a function of time. (c) Purified recombinant HIV-1 PR (160 nM) incubated with 2 μM compound 1 was pumped at different flow rates through a thin glass capillary onto which two 405 nm lasers (combined output of 300 mW) were focused (for set up see Supplementary Fig. 4). Relative PR activity was determined as in b after 20-fold dilution into cleavage buffer using the same chromogenic substrate (for details, see Experimental section) and plotted against the flow rate of the sample through the capillary. Flow rate 0 represents a non-irradiated sample. The graph shows mean values and s.d. from three independent experiments.

Figure 4. Inhibition of HIV-1 Gag processing by compound 1.

(a) Schematic representation of the 55 kDa HIV-1 Gag polyprotein and its cleavage products. (b) Inhibition of HIV-1 Gag processing by compound 1. HIV-1 particles were produced in HEK293T cells in the presence of the indicated inhibitor concentrations. The experiment was performed in duplicate and a representative result is shown. Molecular mass standards are shown on the left; Gag and its respective cleavage products are identified on the right. CA, capsid; MA, matrix; NC, nucleocapsid; p6, p6 protein; SP1, spacer peptide 1; SP2, spacer peptide 2.

Figure 5. Photoinduced Gag processing in the context of the assembled virion.

(a) Schematic illustration of the irradiation experiment to trigger HIV-1 maturation using photoinactivation of compound 1. HEK293T cells were transfected with a proviral HIV-1 plasmid and particles were produced in the presence of 2 μM compound 1. At 44 h post transfection, tissue culture supernatant was harvested and either subjected to ultracentrifugation (b,d) or used directly (c,e). In both cases, samples were then pumped through the capillary set up shown in Supplementary Fig. 4 either with or without ultraviolet irradiation. Subsequently, samples were incubated for various lengths of time. (b,c) Immunoblot analysis of Gag processing products. Samples were separated by SDS–PAGE, and products of Gag processing were detected by quantitative immunoblot (LiCor) using antiserum raised against recombinant HIV-1 CA. The figures show samples incubated for the indicated times without prior irradiation or following irradiation, respectively. Positions of Gag-derived proteins are indicated. (d,e) Quantitative analysis of the experiments shown in b or c, respectively. Anti-CA reactive bands from the immunoblots shown and from corresponding blots from irradiated mature control virus produced in the absence of inhibitor (not shown here) were quantified using Image Studio Light. The graphs show the proportion of mature CA relative to the sum of all anti-CA reactive bands in the respective lane. Filled triangles, irradiated control virus; open circles, inhibitor-treated virus, not irradiated; filled circles, inhibitor-treated virus, irradiated. Curves through data from inhibitor-treated samples represent fits to a single exponential equation. The results are representative of several independent experiments with a slight variation in the half-time of Gag polyprotein processing between 20 and 30 min. CA, capsid; MA, matrix.