Dissociation and association of the HIV-1 protease dimer subunits: Equilibria and rates

Darke, Paul L.; Jordan, Susan P.; Hall, Dawn L.; Zugay, Joan A.; Shafer, Jules A.; Kuo, Lawrence C.

doi:10.1021/bi00167a013

Cited by 64 publications

(42 citation statements)

References 22 publications

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“…Our results show, however, that HIV-1 PR dissociation is rather slow at pH 5.0 and 25°C. This observation is in perfect agreement with data from Darke and colleagues (44), who applied subunit exchange to investigate the interconversion between the active dimer of HIV-1 PR and its inactive monomers . The Asp 25 3 Asn PR subunit used in this study may be regarded as the ideal dimerization inhibitor, since it is, except for the Asp 25 3 Asn substitution, indistinguishable from the native subunit.…”

Section: Discussionsupporting

confidence: 90%

Competitive Inhibition of Human Immunodeficiency Virus Type-1 Protease by the Gag-Pol Transframe Protein

Paulus¹,

Hellebrand²,

Tessmer³

et al. 1999

Journal of Biological Chemistry

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The human immunodeficiency virus type-1 (HIV-1) transframe protein p6* is located between the structural and enzymatic domains of the Gag-Pol polyprotein, flanked by the nucleocapsid (NC) and the protease (PR) domain at its amino and carboxyl termini, respectively. Here, we report that recombinant highly purified HIV-1 p6* specifically inhibits mature HIV-1 PR activity. Kinetic analyses and cross-linking experiments revealed a competitive mechanism for PR inhibition by p6*. We further demonstrate that the four carboxyl-terminal residues of p6* are essential but not sufficient for p6*-mediated inhibition of PR activity. Based on these results, we suggest a role of the transframe protein p6* in regulating HIV-1 PR activity during viral replication.

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Section: Discussionsupporting

confidence: 90%

Competitive Inhibition of Human Immunodeficiency Virus Type-1 Protease by the Gag-Pol Transframe Protein

Paulus¹,

Hellebrand²,

Tessmer³

et al. 1999

Journal of Biological Chemistry

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“…The HTLV-1 Cterminal peptide (YLPEAKRPPVIL-OH) was a more effective inhibitor than the C-terminal HIV-1 peptides, although the K s of 5'7 gU suggests that the peptides either fit poorly, or the monomeric form is not accessible for binding to free peptides. Similar experiments with HIV-1 protease indicate that the dissociation of the dimeric enzyme is slow (Darke et al, 1994) and that inhibition can be increased by preincubation of the protease with peptide at 37 °C for 2 to 3 h (Schramm et al, 1992). However long preincubation of PR14 at 37 °C resulted in the inactivation of the enzyme in the absence of N-and C-terminal peptides and hence this could not be tested (data not shown).…”

Section: Discussionmentioning

confidence: 91%

Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preferences and specificity of binding

Daenke

Schramm

Bangham

1994

Journal of General Virology

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Human T cell leukaemia virus type 1 (HTLV-1) protease (PR14) was expressed in bacteria and purified by gel filtration. A continuous spectrophotometric assay was used to measure the kinetic parameters of substrate hydrolysis by PR14. Several peptide substrates containing HTLV-1 sequences known to be cleaved by PR14 were used. Cleavage analysis showed that the affinity with which PR14 binds these substrates is higher than that previously reported for HTLV-1 Gag peptides. Also, the affinities of peptides containing the sites involved in autocleavage of protease from its precursor are higher than for the peptides containing sites required for structural protein maturation. This suggests that the autocatalysis of protease from its own precursor has priority over other cleavage reactions and supports similar observations of an ordered hierarchy of processing events by retroviral proteases. As the N-and Cterminal regions of retroviral aspartic proteases are known to contribute to stability of the dimer by forming antiparallel fl-strands, short peptides corresponding to these terminal sequences of HTLV-1 protease were tested for their ability to inhibit cleavage of substrates by PRI4. Inhibition was seen with a C-terminal peptide corresponding exactly to the C-terminal 11 amino acids of the processed PR14, whereas a peptide containing a sequence situated further from the C terminus was less effective. An inhibitor of the protease of human immunodeficiency virus type 1, Ro 31-8959, was found to be a poor inhibitor of PR14.

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“…Heterodimer formation and subunit exchange in vitro between wt HIV PR and defective HIV PR variants, as well as between nonidentical subunits of HIV PR, have been reported (26,27). In these cases, heterodimer formation leads to HIV PR inactivation.…”

Section: Discussionmentioning

confidence: 99%

Macromolecular Inhibitors of HIV-1 Protease

Rozzelle¹,

Dauber²,

Todd³

et al. 2000

Journal of Biological Chemistry

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The mechanism of action of these dominant-negative inhibitors was established using recombinantly expressed defective monomers. The defective monomers were refolded in vitro in the presence of wt HIV PR and showed dose-dependent inhibition of proteolytic activity. This inhibition was shown to result from the formation of inactive heterodimers between defective and wt HIV PR monomers. Heterodimer formation was detected by (i) isolating refolded, inactive heterodimers using histidine-tagged defective monomers and (ii) isolating heterodimers from bacteria coexpressing both wt and defective variants of HIV PR. Single-chain variants of HIV PR, in which the C terminus of the wt HIV PR monomer was covalently tethered to the N terminus of the defective monomer, were also expressed and analyzed. Thermal denaturation of these single-chain heterodimers using differential scanning calorimetry revealed a 1.5-7.2°C greater thermal stability than single-chain wt HIV PR. The thermodynamic trend shown by these three variants mirrors their relative inhibition in provirus transfection assays. These data support the model that the effects seen both in tissue culture and in vitro arise from an increase in stability conferred on these heterodimers by interface mutations and identifies heterodimer formation as their mechanism of inhibition.

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Dissociation and association of the HIV-1 protease dimer subunits: Equilibria and rates

Cited by 64 publications

References 22 publications

Competitive Inhibition of Human Immunodeficiency Virus Type-1 Protease by the Gag-Pol Transframe Protein

Competitive Inhibition of Human Immunodeficiency Virus Type-1 Protease by the Gag-Pol Transframe Protein

Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preferences and specificity of binding

Macromolecular Inhibitors of HIV-1 Protease

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