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.

Folding Regulates Autoprocessing of HIV-1 Protease Precursor

“…Moreover, they showed that competitive active site inhibition by the drug retonavir was 10,000-fold less for the protease embedded in the precursor than for the mature free protease (20). Earlier, kinetic studies on the model precursor system MBP-⌬TF-Protease-⌬RT showed that the protease maturation takes place in two steps.…”

“…Pettit et al (20) have recently shown, by co-expressing equivalent amounts of substituted Gag-Pol constructs, that the initial cleavage of the HIV-1 Gag-Pol precursor is intramolecular. Moreover, they showed that competitive active site inhibition by the drug retonavir was 10,000-fold less for the protease embedded in the precursor than for the mature free protease (20).…”

Functions of the Lentiviral Accessory Protein Nef During the Distinct Steps of HIV and SIV Replication Cycle

“…The first cleavage site is located between sp1 and NC, and the second is inside the p6* region of the GagPol precursor polyprotein, divinding p6* in an octapeptide (sometimes referred as Transframe Peptide, TFP) and a 48 amino acids region that is immediately upstream the PR. These two cleavages generate the processing intermediates MA-CA-sp1 (42KDa), NC-TFP (7,4KDa) from Gag and p6*-PR-RT-IN (113KDa) from Pol (Pettit et al, 2004). It is thought that at this point p6* acts as a zymogen, lying on the catalityc center and blocking further cleavages (Partin et al, 1991).…”

“…Other feature is that the cleavage sites recognized by HIV and SIV PR do not share amino acid identity on the cleavage sequence, or on its flanking regions (Hellen et al, 1989;Krausslich et al, 1988). Finally, the last characteristic that separate HIV and SIV PR from its cellular relatives is that, as for cellular PRs the whole catalytic machinery is pre-formed, and its activation lies mostly on the cleavage of a zymogen (Tang and Wong, 1987), for HIV and SIV PR the activation is extremely controlled by mechanisms involving protein folding, zymogen cleavage, PR context, interactions and pH (Gatlin et al, 1998;Partin et al, 1991;Pettit et al, 2004). These abundant regulatory mechanisms point out that the correct PR activation is essential for the formation of infectious particles, and that a premature activation must be avoided.…”

“…These abundant regulatory mechanisms point out that the correct PR activation is essential for the formation of infectious particles, and that a premature activation must be avoided. Such activation could lead to complete viral processing before the completion of budding, resulting in the diffusion of viral constituents on the cytosol and altering the protein ratio in the budding particle (Pettit et al, 2004).…”