Cysteine 95 and other residues influence the regulatory effects of Histidine 69 mutations on Human Immunodeficiency Virus Type 1 protease autoprocessing

Liangqun Huang,Alyssa Hall,Chaoping Chen

doi:10.1186/1742-4690-7-24

Abstract

BackgroundRegulated autoprocessing of HIV Gag-Pol precursor is required for the production of mature and fully active protease. We previously reported that H69E mutation in a pseudo wild type protease sequence significantly (>20-fold) impedes protease maturation in an in vitro autoprocessing assay and in transfected mammalian cells.ResultsInterestingly, H69E mutation in the context of a laboratory adapted NL4-3 protease showed only moderate inhibition (~4-fold) on protease maturation. There are six point mutations (Q7K, L33I, N37S, L63I, C67A, and C95A) between the NL4-3 and the pseudo wild type proteases suggesting that the H69E effect is influenced by other residues. Mutagenesis analyses identified C95 as the primary determinant that dampened the inhibitory effect of H69E. L63 and C67 also demonstrated rescue effect to a less extent. However, the rescue was completely abolished when H69 was replaced by aspartic acid in the NL4-3 backbone. Charge substitutions of surface residues (E21, D30, E34, E35, and F99) to neutral or positively charged amino acids failed to restore protease autoprocessing in the context of H69E mutation.ConclusionsTaken together, we suggest that residue 69 along with other amino acids such as C95 plus L63 and C67 to a less extent modulate precursor structures for the regulation of protease autoprocessing in the infected cell.

Highlights

Regulated autoprocessing of HIV Gag-Pol precursor is required for the production of mature and fully active protease
Ability to catalyze cleavages of a few sites in Gag and Gag-Pol polyproteins [8,9,10], but the full proteolytic activity is only associated with the mature protease after it is liberated from the precursor as a result of autoprocessing
Mutations Q7K, L33I, and L63I are known to minimize autoproteolysis; and C67A/C95A mutations prevent aggregation of E. coli expressed protease mediated by cysteine thiol oxidation

Summary

Introduction

Regulated autoprocessing of HIV Gag-Pol precursor is required for the production of mature and fully active protease. In the Gag-Pol precursor, HIV protease is flanked N-terminally by the transframe region (TFR) (Figure 1A) and C-terminally by the reverse transcriptase [5,7]. The embedded precursor protease has an intrinsic ability to catalyze cleavages of a few sites in Gag and Gag-Pol polyproteins [8,9,10], but the full proteolytic activity is only associated with the mature protease after it is liberated from the precursor as a result of autoprocessing. Mutations blocking the C-terminal cleavage have no significant influence on protease activity [13,14]

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Results

Conclusion