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

Ishima, Rieko; Torchia, Dennis A.; Louis, John M.

doi:10.1074/jbc.m701304200

Cited by 52 publications

(111 citation statements)

References 35 publications

(40 reference statements)

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“…Almost all crystallized structures are for the dimeric mature protease, the final product of autoprocessing. In contrast, no structural information for the protease precursor is available except for a single monomer protease structure that has been reported using NMR analysis of a modified pseudo wild type protease containing a four-residue extension from the N-terminus and a four-residue deletion of the C-terminus [8,23]. Structural analyses of mature protease dimers alone cannot fully explain the autoprocessing mechanism or reveal the cause of drug resistance.…”

Section: Conclusion and Discussionmentioning

confidence: 99%

“…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. Consistent with this, a miniprecursor comprised of a slightly modified mature protease plus the upstream TFR has been utilized as a model system to study protease autoprocessing [3,23]. When expressed in E. coli , the miniprecursor is predominantly associated with inclusion bodies and is therefore purified under denaturing conditions and refolded in vitro .…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Conclusion and Discussionmentioning

confidence: 99%

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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“…Whereas adjacent p6* residues have been reported to facilitate the dimerization of a drugresistant and proteolytically inactive PR variant, p6* appears to have rather little influence on dimer formation of wild-type PR (10,42). While p6* seems to play a supportive or at least a neutral role in precursor dimerization, the amino-terminal extension of the PR by p6* residues has been demonstrated to affect the folding preferences within the PR domain, thereby destabilizing the folded PR structure, which results in a retarded PR maturation (6,17,23). This might explain the observation that Gag-PR precursors lacking the p6* domain are characterized by a faster autoactivation of the PR (26), whereas elongated or bulky p6*-GFP fusions might in return even prevent the formation of functional Pol dimers.…”

Section: Discussionmentioning

confidence: 99%

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication

Leiherer

Ludwig

Wagner

2009

J Virol

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Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6 gag sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3 end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6 gag reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function.

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“…Mature proteases were folded similarly or by the quench protocol as described previously (final pH 5.0) (24) in the absence or presence of the same molar excess of inhibitor. Thermal denaturation scans using a MicroCal VP-DSC microcalorimeter (GE Healthcare) and data handling were as described previously (5,13).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of autoprocessing of natural variants and multidrug resistant mutant precursors of HIV-1 protease by clinical inhibitors

Louis

Aniana

Weber

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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201

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Self-cleavage at the N terminus of HIV-1 protease from the Gag-Pol precursor (autoprocessing) is crucial for stabilizing the protease dimer required for onset of mature-like catalytic activity, viral maturation, and propagation. Among nine clinical protease inhibitors (PIs), darunavir and saquinavir were the most effective in inhibiting wild-type HIV-1 group M precursor autoprocessing, with an IC 50 value of 1-2 μM, 3-5 orders of magnitude higher than their binding affinities to the corresponding mature protease. Accordingly, both group M and N precursor-PI complexes exhibit T m s 17-21°C lower than those of the corresponding mature protease-PI complexes suggestive of markedly reduced stabilities of the precursor dimer-PI ensembles. Autoprocessing of group N (natural variant) and three group M precursors bearing 11-20 mutations associated with multidrug resistance was either weakly responsive or fully unresponsive to inhibitors at concentrations up to a practical limit of approximately 150 μM PI. This observation parallels decreases of up to 8 × 10 3 -fold (e.g., 5 pM to 40 nM) in the binding affinity of darunavir and saquinavir to mature multidrug resistant proteases relative to wild type, suggesting that inhibition of some of these mutant precursors will occur only in the high μM to mM range in extreme PI-resistance, which is an effect arising from coordinated multiple mutations. An extremely darunavir-resistant mutant precursor is more responsive to inhibition by saquinavir. These findings raise the questions whether clinical failure of PI therapy is related to lack of inhibition of autoprocessing and whether specific inhibitors can be designed with low-nM affinity to target autoprocessing.calorimetry | drug resistance | precursor processing | inhibitor binding | aspartic protease

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

Cited by 52 publications

References 35 publications

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

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

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication

Inhibition of autoprocessing of natural variants and multidrug resistant mutant precursors of HIV-1 protease by clinical inhibitors

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