HIV-1 protease autoprocessing liberates the free mature protease from its Gag-Pol polyprotein precursor through a series of highly regulated autoproteolysis reactions. Herein, we report the development and validation (Z’ ≥ 0.50) of a cell-based functional assay for high-throughput screening (HTS) of autoprocessing inhibitors using fusion precursors in combination with AlphaLISA (amplified luminescent proximity homogeneous assay ELISA). Through pilot screening of a collection of 130 known protease inhibitors, the AlphaLISA assay confirmed all 11 HIV protease inhibitors in the library capable of suppressing precursor autoprocessing at low micromolar concentrations. Meanwhile, other protease inhibitors had no impact on precursor autoprocessing. We next conducted HTS of ~23,000 compounds but found no positive hits. Such high selectivity is advantageous for large-scale HTS campaigns and as anticipated based on assay design because a positive hit needs simultaneously to be nontoxic, cell permeable, and inhibiting precursor autoprocessing. Furthermore, AlphaLISA quantification of fusion precursors carrying mutations known to cause resistance to HIV protease inhibitors faithfully recapitulated the reported resistance, suggesting that precursor autoprocessing is a critical step contributing to drug resistance. Taken together, this reported AlphaLISA platform will provide a useful tool for drug discovery targeting HIV-1 protease autoprocessing and for quantification of PI resistance.
HIV-1 protease autoprocessing is responsible for liberation of free mature protease (PR) from the Gag-Pol polyprotein precursor. A cell-based model system was previously developed to examine the autoprocessing mechanism of fusion precursors carrying the p6*-PR miniprecursor sandwiched between various proteins or epitopes. We here report that precursor autoprocessing is context-dependent as its activity and outcomes can be modulated by sequences upstream of p6*-PR. This was exemplified by the 26aa maltose binding protein (MBP) signal peptide (SigP) when placed at the N-terminus of a fusion precursor. The mature PRs released from SigP-carrying precursors are resistant to self-degradation whereas those released from SigP-lacking fusion precursors are prone to self-degradation. A H69D mutation in PR abolished autoprocessing of SigP-containing fusion precursors whereas it only partially suppressed autoprocessing of fusion precursors lacking SigP. An autoprocessing deficient GFP fusion precursor with SigP exhibited a subcellular distribution pattern distinct from the one without it in transfected HeLa cells. Furthermore, a SigP fusion precursor carrying a substitution at the P1 position released the mature PR and PR-containing fragments that were different from those released from the precursor carrying the same mutation but lacking SigP. We also examined autoprocessing outcomes in viral particles produced by a NL4-3 derived proviral construct and demonstrated the existence of several PR-containing fragments along with the mature PR. Some of these resembled the SigP precursor autoprocessing outcomes. This finding of context-dependent modulation reveals the complexity of precursor autoprocessing regulation that most likely accompanies sequence variation imposed by the evolution of the upstream Gag moiety.
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