Mutations Proximal to Sites of Autoproteolysis and the α-Helix That Co-evolve under Drug Pressure Modulate the Autoprocessing and Vitality of HIV-1 Protease

Abstract

N-Terminal self-cleavage (autoprocessing) of the HIV-1 protease precursor is crucial for liberating the active dimer. Under drug pressure, evolving mutations are predicted to modulate autoprocessing, and the reduced catalytic activity of the mature protease (PR) is likely compensated by enhanced conformational/dimer stability and reduced susceptibility to self-degradation (autoproteolysis). One such highly evolved, multidrug resistant protease, PR20, bears 19 mutations contiguous to sites of autoproteolysis in retroviral proteases, namely clusters 1-3 comprising residues 30-37, 60-67, and 88-95, respectively, accounting for 11 of the 19 mutations. By systematically replacing corresponding clusters in PR with those of PR20, and vice versa, we assess their influence on the properties mentioned above and observe no strict correlation. A 10-35-fold decrease in the cleavage efficiency of peptide substrates by PR20, relative to PR, is reflected by an only ∼4-fold decrease in the rate of Gag processing with no change in cleavage order. Importantly, optimal N-terminal autoprocessing requires all 19 PR20 mutations as evaluated in vitro using the model precursor TFR-PR20 in which PR is flanked by the transframe region. Substituting PR20 cluster 3 into TFR-PR (TFR-PR(PR20-3)) requires the presence of PR20 cluster 1 and/or 2 for autoprocessing. In accordance, substituting PR clusters 1 and 2 into TFR-PR20 affects the rate of autoprocessing more drastically (>300-fold) compared to that of TFR-PR(PR20-3) because of the cumulative effect of eight noncluster mutations present in TFR-PR20(PR-12). Overall, these studies imply that drug resistance involves a complex synchronized selection of mutations modulating all of the properties mentioned above governing PR regulation and function.

Figure 1

(A) Domain organization of the HIV-1 Gag-Pol polyprotein. Abbreviations: MA, matrix; CA, capsid; SP1, spacer peptide 1; NC, nucleocapsid; TFR, transframe region; PR, protease; RT, reverse transcriptase; RN, ribonuclease; IN, integrase. (B) Sequence alignment of wild-type protease, pseudo-wild-type protease (PR), and the multidrug resistant variant PR20. PR bears mutations Q7K, L33I, L63I, C66A, and C95A (colored black and underlined) to restrict autoproteolysis at sites indicated by green arrows and avoid cysteine-thiol oxidation. Introduced mutations common to PR and PR20 are Q7K, C67A, and C95A. The 19 mutations, both naturally occurring and selected under drug pressure, in PR20 are colored blue. Dots denote identical residues. Highly conserved regions in PR are highlighted in yellow, and regions where major drug resistance mutations (indicted by red asterisks; http:/hivdb.stanford.edu/cgi-bin/PIResiNote.cgi and ref) occur are highlighted in gray. The three clusters that contain mutations in PR20 next to sites of autoproteolysis in the wild type are indicated by red stripes. (C) List of PR, PR20, and derived mutants showing the number of cluster and noncluster mutations. (D) Superimposition of ribbon representations of PR20 in blue (PDB entry 3UCB) and PR in white (PDB entry 2IEN) bound to the inhibitor darunavir (stick representation) with the regions corresponding to the three clusters colored red as well as indicated by the red arrows corresponding to red stripes in panel B. Positions of mutations in cluster 1 (residues 30–37), cluster 2 (residues 60–67), and cluster 3 (residues 88–94) are labeled in white on red circles. The remaining eight of 19 mutations in PR20 are shown in white on black circles. Sites of autoproteolysis are denoted by the green arrows.

Figure 2

Time course of autoproteolysis of PR^PR20-123 and PR20^PR-123. Hydrolysis of the chromogenic substrate in 50 mM sodium acetate (pH 5.0) containing 250 mM sodium chloride as a function of time of incubation of (A) PR^PR20-123 and (B) PR20^PR-123 at room temperature (in hours, prior to assaying). Aliquots of (C) PR^PR20-123 and (D) PR20^PR-123 were withdrawn at the indicated times, mixed with gel loading buffer, subjected to SDS-PAGE, and stained. For comparison with parent constructs, PR and PR20, see Figure S1.

Figure 3

Processing of ΔGag by the protease. Schematic representation of the sequential cleavages of ΔGag with the order of cleavage (blue), cleavage-site positions, and calculated molecular weight (in kDa) of the products indicated. The four bottom panels show the time course of processing of ΔGag by mature protease monitored by SDS–PAGE on 18% Tris-glycine gels. Reaction mixtures contained 50 μM ΔGag and either 0.5 μM PR or 1 μM PR20^PR-123, PR20, and PR^PR20-123 as indicated. Cleavage of full-length 48.4 kDa ΔGag between SP1 and NC (step 1) yields MA-CA-SP1 and NC, followed by cleavage between MA and CA (step 2) to generate the mature MA and the intermediate CA-SP1. The spacer polypeptide SP1 is cleaved subsequently (step 3) from the C-terminus of CA-SP1, some of which is still detectable just above mature CA at up to 180 min. No full-length ΔGag remains after reaction for 20 min with PR at half the concentration of the other three constructs, and no MA-CA-SP1 is detectable after 35 min. SP1, because of its small size, is predicted to have run out of the gel. A faint band between the MA-CA-SP1 and CA-SP1 bands corresponds to CA-SP1-NC formed by a minor MA/CA cleavage pathway.

Figure 4

Expression and autoprocessing of TFR-PR precursors. (A) Identification of precursor constructs shown in panels B–I. Individual clusters and various combinations are shown with PR clusters in black and PR20 clusters in blue. (B–D) Expression and autoprocessing of precursor constructs containing one or more substituted clusters. Total cell extracts were analyzed by SDS–PAGE. Numbers above the lanes correspond to the designated construct shown in panel A, and lanes indicated by minus (−) and plus (+) represent total extracts of uninduced and induced cultures, respectively. (E–I) Time course of N-terminal autoprocessing of purified precursors in vitro. (F and I) TFR-PR^PR20-3 and TFR-PR20^PR-12 exhibit very slow or no discernible autoprocessing, respectively, as indicated in panel A. (J and K) Inhibition of autoprocessing of TFR-PR^PR20-123 and TFR-PR20^PR-123 conducted for 60 min in the presence of increasing concentrations of saquinavir. (L) Time course of autoproteolysis of mature PR20^PR-12. Lowercase letters a–d and p indicate full-length precursor, an intermediate cleavage product of TFR-PR, TFR^9–56-PR (cleavage occurs between residues 8 and 9 of TFR), mature PR, TFR and products of mature PR autoproteolysis, respectively. The pathway to release the minor intermediate product TFR^9–56-PR precedes the cleavage at the N-terminus of PR. We have shown previously that this cleavage is pH-dependent and becomes pronounced at pH 4. M denotes molecular weight markers in kilodaltons. SQV denotes saquinavir.