A Transient Precursor of the HIV-1 Protease

Wondrak, Ewald M.; Nashed, Nashaat T.; Haber, Martin T.; Jerina, Donald M.; Louis, John M.

doi:10.1074/jbc.271.8.4477

Cited by 60 publications

(59 citation statements)

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“…The first step involves an intramolecular cleavage of the N terminus that is followed by intermolecular cleavage of the C terminus (19,21). A relatively low K m for peptide substrates representing the p6*-PR (where p6* is TFPϩp6 pol ) cleavage site, compared with that for oligopeptides corresponding to other Gag or Pol cleavage sites (23) supports the view that the Nterminal cleavage is an early event in the proteolytic cascade.…”

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confidence: 65%

“…Earlier studies have shown that premature activation or partial inhibition of the protease leads to retarded viral maturation (15)(16)(17)(18). Hence understanding the exact sequence of protease maturation from the Gag-Pol precursor has gained importance in recent years because of its intrinsic importance in viral maturation and as a target for drugs against AIDS (19).…”

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confidence: 99%

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Folding Regulates Autoprocessing of HIV-1 Protease Precursor

Chatterjee

Mridula

Mishra

et al. 2005

Journal of Biological Chemistry

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Autoprocessing of HIV-1 protease (PR) precursors is a crucial step in the generation of the mature protease. Very little is known regarding the molecular mechanism and regulation of this important process in the viral life cycle. In this context we report here the first and complete residue level investigations on the structural and folding characteristics of the 17-kDa precursor TFR-PR-C nn (161 residues) of HIV-1 protease. The precursor shows autoprocessing activity indicating that the solution has a certain population of the folded active dimer. Removal of the 5-residue extension, C nn at the C-terminal of PR enhanced the activity to some extent. However, NMR structural characterization of the precursor containing a mutation, D25N in the PR at pH 5.2 and 32°C under different conditions of partial and complete denaturation by urea, indicate that the precursor has a high tendency to be unfolded. The major population in the ensemble displays some weak folding propensities in both the TFR and the PR regions, and many of these in the PR region are the non-native type. As both D25N mutant and wild-type PR are known to fold efficiently to the same native dimeric form, we infer that TFR cleavage enables removal of the non-native type of preferences in the PR domain to cause constructive folding of the protein. These results indicate that intrinsic structural and folding preferences in the precursor would have important regulatory roles in the autoprocessing reaction and generation of the mature enzyme.

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confidence: 65%

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confidence: 99%

Folding Regulates Autoprocessing of HIV-1 Protease Precursor

Chatterjee

Mridula

Mishra

et al. 2005

Journal of Biological Chemistry

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“…Structural analyses of mature protease dimers alone cannot fully explain the autoprocessing mechanism or reveal the cause of drug resistance. Proviral DNA mutagenesis on the other hand has provided insightful information regarding protease autoprocessing mechanism [14,19-22,24,28], however, sensitive and direct detection of the mature protease, along with its precursor and processing intermediates, has been restrained due to lack of highly specific and sensitive antibodies. Consequently, most information is indirectly derived from analysis of Gag processing efficiency and/or p24 production [14,24].…”

Section: Conclusion and Discussionmentioning

confidence: 99%

“…Between the two cleavage sites that lead to liberation of the mature protease, the C-terminal cleavage seems to have less of an impact on the regulation of autoprocessing as mutations blocking this cleavage have no significant influence on protease activity or Gag processing in transfected mammalian cells [19,20]. In contrast, mutations blocking N-terminal cleavage abolish Gag processing and lead to loss of viral infectivity [21,22], suggesting that N-terminal cleavage plays an important role in regulating autoprocessing.…”

Section: Introductionmentioning

confidence: 99%

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

Huang

Chen

2010

AIDS Res Ther

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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.

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“…Unlike the flanking C-terminal residues (reverse transcriptase region), which do not appear to affect the catalytic activity or the K d in fusion with the protease (9,22,23), the native transframe region, which flanks the N terminus of the protease in the Gag-Pol precursor, drastically influences the dimer stability of the protease indicated by the very low catalytic activity of the protease prior to its maturation at its N terminus ( Fig. 1) (10, 11).…”

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Mutational and Structural Studies Aimed at Characterizing the Monomer of HIV-1 Protease and Its Precursor

Ishima

Torchia

Louis

2007

Journal of Biological Chemistry

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An experimental protocol for folding the mature human immunodeficiency virus-1 (HIV-1) protease is presented that facilitates NMR studies at a low protein concentration of ϳ20 M. Under these conditions, NMR spectra show that the mature protease lacking its terminal ␤-sheet residues 1-4 and 96 -99 (PR 5-95 ) exhibits a stable monomer fold spanning the region 10 -90 that is similar to that of the single subunit of the wildtype dimer and the dimer bearing a D25N mutation (PR D25N ). Urea-induced unfolding monitored both by changes in 1 H-15 N heteronuclear single quantum correlation spectra and by protein fluorescence indicates that although PR 5-95 monomer displays a transition profile similar to that of the PR D25N dimer (50% unfolded (U 50 ) ‫؍‬ ϳ1.9 M), extending the protease with 4 residues (SFNF) of its N-terminally flanking sequence in the Gag-Pol precursor ( SFNF PR D25N ) decreases the stability of the fold (U 50 ‫؍‬ ϳ1.5 M). Assigned backbone chemical shifts were used to elucidate differences in the stability of the PR T26A (U 50 ‫؍‬ 2.5 M) and SFNF PR D25N monomers and compared with PR D25N/T26A monomer. Discernible differences in the backbone chemical shifts were observed for N-terminal protease residues 3-6 of SFNF PR D25N that may relate to the increase in the equilibrium dissociation constant (K d ) and the very low catalytic activity of the protease prior to its autoprocessing at its N terminus from the Gag-Pol precursor.

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A Transient Precursor of the HIV-1 Protease

Cited by 60 publications

References 20 publications

Folding Regulates Autoprocessing of HIV-1 Protease Precursor

Folding Regulates Autoprocessing of HIV-1 Protease Precursor

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

Mutational and Structural Studies Aimed at Characterizing the Monomer of HIV-1 Protease and Its Precursor

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