Abstract
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.
Highlights
The Human Immunodeficiency Virus type 1 (HIV-1) protease (PR) is one of the three indispensable enzymes encoded by the viral genome with the other two being reverse transcriptase (RT) and integrase (IN)
We have reported that precursor autoprocessing is more resistant than mature PR to suppression by known PR inhibitors [7, 8], which is consistent with previous observations made with purified recombinant miniprecursor [10,11,12] or in vitro translated Gag-Pol polyprotein precursor [13]
We recently described point mutations that emerged in the HIV-1 viruses isolated from a subpopulation of Women’s Interagency HIV Study (WIHS) participants where anti-retroviral agents, including indinavir (IDV), had failed to suppress the viral load [28, 29]
Summary
The Human Immunodeficiency Virus type 1 (HIV-1) protease (PR) is one of the three indispensable enzymes encoded by the viral genome with the other two being reverse transcriptase (RT) and integrase (IN). In the HIV-infected cell, these enzymes are initially synthesized as part of the Gag-Pol polyprotein precursor which shares the same N-terminus with the Gag structural precursor polyprotein. Within the Gag-Pol precursor, PR is flanked by an upstream peptide sequence and by the downstream RT. The upstream peptide is named transframe region (TFR) or p6Ã [1, 2] as its coding sequence overlaps with the p6 region of the Gag reading frame. During the late stage of virus replication, Gag and Gag-Pol co-assemble into virus particles that subsequently bud off from the infected cell. Upon or shortly after virion release, the Gag-Pol polyprotein undergoes autoproteolysis and liberates free mature PR–a process generally referred to as PR autoprocessing
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