Human Immunodeficiency Virus Type 1 Reverse Transcriptase Dimer Destabilization by 1-{Spiro[4‘ ‘-amino-2‘ ‘,2‘ ‘-dioxo-1‘ ‘,2‘ ‘-oxathiole-5‘ ‘,3‘- [2‘,5‘-bis-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]]}-3-ethylthymine

Abstract

The nonnucleoside inhibitor binding pocket is a well-defined region in the p66 palm domain of the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). This binding pocket opens toward the interface of the p66/p51 heterodimer and we have investigated whether ligand binding at or near this site induces structural changes that have an impact on the dimeric structure of HIV-1 RT. 1-[2',5'-bis-O-(tert-butyldimethylsilyl]-3'-spiro-5' '-(4' '-amino-1' ',2' '-oxathiole-2' ',2' '-dioxide)-3-ethylthymine (TSAOe(3)T) was found to destabilize the subunit interactions of both the p66/p51 heterodimer and p66/p66 homodimer enzymes. The Gibbs free energy of dimer dissociation (DeltaG(D)(H)2(O)) is decreased with increasing concentrations of TSAOe(3)T, resulting in a loss in dimer stability of 4.0 and 3.2 kcal/mol for the p66/p51 and p66/p66 HIV-1 RT enzymes, respectively. This loss of energy is not sufficient to induce the dissociation of the subunits in the absence of denaturant. This destabilizing effect seems to be unique for TSAOe(3)T, since neither the tight-binding inhibitor UC781 nor nevirapine showed any effects on the stability of HIV-1 RT dimers. TSAOe(3)T was unable to destabilize the subunit interactions of the E138K mutant enzyme, which exhibits significant resistance to TSAOe(3)T inhibition. Molecular modeling of TSAOm(3)T into the nonnucleoside inhibitor binding pocket of wild-type RT suggests that it makes significant interactions with the p51 subunit of the enzyme, a feature that has not been observed with other types of nonnucleoside inhibitors. The observed destabilization of the dimeric HIV-1 RT may result from structural/conformational perturbations at the reverse transcriptase subunit interface.

Functional Characterization of Chimeric Reverse Transcriptases with Polypeptide Subunits of Highly Divergent HIV-1 Group M and O Strains

“…Analysis of RT Activity in Crude Bacterial Lysates-DNA polymerization assays were carried out by mixing 20 l of the obtained lysate and 80 l of 62.5 mM Tris-HCl (pH 8.0) containing 25 mM NaCl, 12.5 mM MgCl 2 , 2 mM DTT, 0.5 g/ml poly(rC)/oligo(dG) [12][13][14][15][16][17][18] , and 10 M [␣-…”

“…RT concentrations were determined using the Bio-Rad protein assay. The specific activity of the purified enzyme was estimated in DNA polymerase assays carried out in 50 l of 50 mM Tris-HCl (pH 8.0) containing 20 mM NaCl, 10 mM MgCl 2 , 8 mM DTT, 5 M dTTP (3-5 Ci/ml [ 3 H]dTTP), and 0.5 M poly(rA)/oligo(dT) [12][13][14][15][16][17][18] (concentration expressed as 3Ј-hydroxyl primer termini) as previously described (22,27). One activity unit is the amount of enzyme that incorporates 1 nmol of [ 3 H]dTMP into acid-insoluble products in 10 min at 37°C.…”

“…RT inhibition assays were carried out with poly(rA)/oligo(dT) [12][13][14][15][16][17][18] or D2-47/PG5-25 under the conditions described for kinetic studies. In assays using poly(rA)/oligo(dT) [12][13][14][15][16][17][18] , incorporation of radioactive dTMP was measured in the presence of increasing amounts of nevirapine. In singlenucleotide extension assays, the RT was preincubated with the inhibitor at 37°C for 10 min before the addition of the D2-47/PG5-25 template-primer.…”

“…1). RNA-dependent DNA polymerase activity was determined using poly(rC)/oligo(dG) [12][13][14][15][16][17][18] and [␣-32 P]dGTP as substrates. All five RTs from this ESP2 sample had nearly identical amino acid sequence in the polymerase domain, and we selected clone 46 as the group O RT prototype in the biochemical studies described in this work.…”

“…Peptides mimicking the dimer interface of p66 and p51 have been used to prevent heterodimer formation (14,15). In addition, certain non-nucleoside RT inhibitors (NNRTIs), such as [1-[2Ј,5Ј-bis-O-(tert-butyldimethylsilyl)-␤-D-ribofuranosyl]-N 3 ethylthymine]-3Ј-spiro-5Љ-(4Љ-amino-1Љ,2Љ-oxathiole-2Љ,2Љ-dioxide), are known to produce a modest, but significant destabilizing effect on the heterodimer (16). However, the molecular determinants of heterodimer stability are not known.…”