Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

Abstract

BackgroundHIV protease (PR) is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing.ResultsWe here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR), and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region.ConclusionsWe suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

Binding of Clinical Inhibitors to a Model Precursor of a Rationally Selected Multidrug Resistant HIV-1 Protease Is Significantly Weaker Than That to the Released Mature Enzyme

“…In this context, it is worth noting that in vitro inhibition of the SP1/NC site of the wild type Gag-Pol precursor which was shown to precede the N-terminal autoprocessing sites, TFP/p6pol and p6pol/PR sites, is inhibited by RTV ~10,000-fold weaker than the corresponding mature protease, 1,45 and similarly, significantly higher concentrations of PIs exert inhibition of a chimeric wild type TFR-PR precursor autoprocessing in cell culture. 46 These observations are consistent with the much weaker inhibition (IC 50 of 1–2 μM) of autoprocessing of the wild type TFR-PR precursor by DRV and SQV during expression in E.coli , 16 drastic increases in IC 50 of 8 PIs with the drug resistant TFR-PR S17 precursor described in this study, and no inhibition as in extreme instances of drug resistance such as the PR20 precursor, up to 150 μM DRV or SQV. 16 …”

Inhibition of autoprocessing of natural variants and multidrug resistant mutant precursors of HIV-1 protease by clinical inhibitors

“…3A), and virtually complete inhibition occurs in the presence of 10 μM DRV. Similar inhibition by DRV observed on the autoprocessing of a chimeric precursor in mammalian cells monitored by immunoblotting (16) confirms that inhibition of the native TFR-PR constructs in E. coli provides a rapid and reliable prediction of the effect of PIs on autoprocessing. This effect is in the same range as the maximum concentrations (6-10 μM) of DRV in plasma (17) or of SQV in cells (18) achieved on administration of PIs to human subjects.…”

The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility

“…Transfection of HEK293T cells was previously described [32, 36]. In brief, cells were seeded onto 6-well, 12-well or 24-well plates the day before transfection.…”