Initial cleavage of the human immunodeficiency virus type 1 GagPol precursor by its activated protease occurs by an intramolecular mechanism

Abstract

Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism.

FIG. 1.

Schematic models of intermolecular (cis) versus intramolecular (trans) processing for the initial processing of the GagPol precursor. Previous results indicated that initial cleavage of the GagPol precursor by the activated GagPol PR occurs at two sites: p2/NC site (M377/M378) and TF F440/L441 (34). Cleavage of these GagPol sites could conceivably occur by either an intermolecular mechanism (left) or an intramolecular mechanism (right). Note that processing of the Gag precursor can occur only by an intermolecular mechanism since the Gag precursor lacks an embedded protease.

FIG. 2.

Initial cleavages of the GagPol precursor in vitro after activation of the protease. Full-length GagPol was expressed in vitro as described in Materials and Methods. (A) Schematic for the ordered processing of the GagPol precursor after protease activation. Initial cleavage occurred at the p2/NC site (M377/M378) followed by rapid cleavage of the TF F440/L441 site. Significant cleavage of the other GagPol sites was not observed with wild-type precursor in vitro. The observed protein products with their calculated molecular masses based on sequence are shown. (B) Identification of the location of the initial cleavages and the effect of site-specific blocking mutation on ordered processing of GagPol by the activated protease. The full-length GagPol precursor was generated in vitro. Aliquots were removed at the indicated time and separated by SDS-PAGE. Processing of the wild-type precursor (GPfs) by the embedded protease is shown. Inactivation of the protease in GagPol with D25A (GPfs-PR) prevents processing of the Gag precursor. The effect of inhibiting cleavage at the p2/NC or the TF F440/L441 site with site-specific P1 Ile substitutions is shown. Major products of the GagPol precursor are denoted by square dots. The composition and calculated molecular masses (in kilodaltons) of the products based on published sequences are on the right. Products are presented in abbreviated form by their N- and C-terminal domains only according to accepted nomenclature (24). Numbers at left are molecular masses in kilodaltons. WT, wild type.

FIG. 3.

Comparison of the effects of the competitive inhibitor ritonavir on activated GagPol protease and trans protease. (A) Wild-type GagPol was translated in vitro for 2 h in duplicate reactions containing increasing concentrations of ritonavir (ABT-538) to monitor the effects of the drug on activity of GagPol protease. The protease within GagPol activates and cleaves the precursor at the primary p2/NC and secondary TF F440/L441 sites. The concentration of GagPol in the reaction mixture is approximately 1 nM. The concentration of ritonavir is given above the lanes. (B) Effect of ritonavir on trans-cleavage of the GagPol precursor in vitro. A 325 nM concentration of mature recombinant protease monomers was added in trans to PR D25A mutated GagPol (160 pM) with various concentrations of ritonavir (above). Reactions shown were stopped at 10 min of incubation. The gels shown are representative of triplicate experiments. Products are presented in abbreviated form by their N- and C-terminal domains only. Numbers at left of each panel are molecular masses in kilodaltons.

FIG. 4.

Comparison of the susceptibilities of cleavage at the p2/NC and TF sites to inhibition for cis versus trans cleavage. Plots show the percent inhibition for the individual p2/NC and TF F440/L441 sites for GagPol protease (A) or trans-protease (B) with increasing concentrations of ritonavir. Plots were derived from densitometric analysis of SDS-polyacrylamide gels as described in Materials and Methods. The estimated IC₅₀s for the inhibition of the individual sites for the GagPol protease are 189 nM for TF F440/L441 and 5.17 μM for p2/NC. The estimated IC₅₀s for the trans protease are 18 nM for TF F440/L441 and 64 nM for p2/NC. Curves were determined as explained in Materials and Methods with a minimum of three independent replicates. Error bars indicate standard errors of the means.

FIG. 5.

Cleavage of the p2/NC and TF F440/L441 sites by the activated GagPol protease occurs by an intramolecular mechanism rather than an intermolecular mechanism. GagPol or equal amounts of two GagPol species were expressed in vitro, and aliquots were taken at the indicated time prior to SDS-PAGE. (A) Wild-type GagPol-containing protease inactivated at the catalytic aspartate (D25A). (B) Wild-type GagPol. (C to G) trans-complementation test in which equal amounts of two GagPol species were cotranslated as shown above the panel. (C) Expression of wild-type and PR D25A protease. Efficient trans-complementation would be expected to result in 75% inhibition of GagPol precursor processing as shown by the persistence of the full-length precursor (75%) and reduced amounts of the 42-kDa MA-p2 product (25%). (D and E) A mutation that blocks cleavage of the p2/NC site (M377I) was placed on either the GagPol with an active PR monomer (D) or the GagPol with an inactive PR monomer (E). Processing of the p2/NC site, as shown by the 42-kDa product, is observed only when the unblocked site is located on the same precursor as the active protease, indicating an intramolecular cleavage mechanism (E). (F and G) A mutation that blocks cleavage of the TF F440/L441 site (F440I) was placed on either the GagPol with an active PR monomer (F) or the GagPol with an inactive PR monomer (G). Generation of the 113-kDa L441-IN product is seen only when the unblocked site is located on the same precursor as the activated protease, indicating that cleavage occurs by an intramolecular mechanism. Numbers at left are molecular masses in kilodaltons.

FIG. 6.

Cotranslation of the Gag and GagPol precursors. pGag1 (A) or Gag and GagPol (B to G) were cotranslated, and aliquots were taken at the indicated times prior to SDS-PAGE. (A) Translation of the Gag and Pol open reading frames (pGag1) in RRL resulted in the expression of pr55 Gag and pr160 GagPol by a translational frameshift mechanism (14). The Gag/GagPol ratio is approximately 20:1. Cleavage of the p2/NC site is evident by the generation of the 42-kDa MA-CA-p2 product. (B) Cotranslation of Gag and GagPol in a 20:1 ratio via separate plasmids. (C and D) A mutation that blocks cleavage of the p2/NC site (M377I) was placed on either Gag (C) or GagPol (D) and expressed at a 20:1 ratio. Processing of the p2/NC site, as shown by the 42-kDa product, is observed only when the unblocked site is located on the GagPol precursor (E). (F and G) Gag and GagPol were expressed in a 1:1 ratio in vitro. A mutation that blocks cleavage of the p2/NC site (M377I) was placed on either Gag (F) or GagPol (G). Cleavage of the p2/NC site, as shown by the 42-kDa MA-CA-p2 product, was observed only when the unblocked site was located on the GagPol precursor (G). Numbers at left are molecular masses in kilodaltons.