Abstract

BackgroundThe HIV-1 protease is initially synthesized as part of the Gag-Pol polyprotein in the infected cell. Protease autoprocessing, by which the protease domain embedded in the precursor catalyzes essential cleavage reactions, leads to liberation of the free mature protease at the late stage of the replication cycle. To examine autoprocessing reactions in transfected mammalian cells, we previously described an assay using a fusion precursor consisting of the mature protease (PR) along with its upstream transframe region (p6*) sandwiched between GST and a small peptide epitope.ResultsIn this report, we studied two autoprocessing cleavage reactions, one between p6* and PR (the proximal site) and the other in the N-terminal region of p6* (the distal site) catalyzed by the embedded protease, using our cell-based assay. A fusion precursor carrying the NL4-3 derived protease cleaved both sites, whereas a precursor with a pseudo wild type protease preferentially autoprocessed the proximal site. Mutagenesis analysis demonstrated that several residues outside the active site (Q7, L33, N37, L63, C67 and H69) contributed to the differential substrate specificity. Furthermore, the cleavage reaction at the proximal site mediated by the embedded protease in precursors carrying different protease sequences or C-terminal fusion peptides displayed varied sensitivity to inhibition by darunavir, a catalytic site inhibitor. On the other hand, polypeptides such as a GCN4 motif, GFP, or hsp70 fused to the N-terminus of p6* had a minimal effect on darunavir inhibition of either cleavage reaction.ConclusionsTaken together, our data suggest that several non-active site residues and the C-terminal flanking peptides regulate embedded protease activity through modulation of the catalytic site conformation. The cell-based assay provides a sensitive tool to study protease autoprocessing reactions in mammalian cells.

Highlights

  • The HIV-1 protease is initially synthesized as part of the Gag-Pol polyprotein in the infected cell

  • In the HIV-1 infected cell, the protease is initially synthesized as part of the Gag-Pol polyprotein precursor, within which the HIV-1 protease is flanked at the N-terminus by a transframe region named TFR or p6*, and at the C-terminus by the reverse transcriptase (RT) [2,5,6]

  • In this study, we studied two proteolysis reactions involved in HIV-1 protease autoprocessing in transfected cells expressing engineered fusion precursors

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Summary

Introduction

The HIV-1 protease is initially synthesized as part of the Gag-Pol polyprotein in the infected cell. In the HIV-1 infected cell, the protease is initially synthesized as part of the Gag-Pol polyprotein precursor, within which the HIV-1 protease is flanked at the N-terminus by a transframe region named TFR or p6*, and at the C-terminus by the reverse transcriptase (RT) [2,5,6]. The regulated cleavage reactions, in which the Gag-Pol precursor is both the enzyme and substrate, lead to liberation of the free mature HIV-1 PR. This process is generally referred to as protease autoprocessing

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