Satish K. Sharma

Human immunodeficiency virus type 1 (HIV-1) recombinant reverse transcriptase: Asymmetry in p66 subunits of the p66/p66 homodimer

A recombinant p66 form of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) can be obtained [(1991) Biotechnol. Appl. Biochem. 14, 69‐81] from crude Escherichia coli extracts by immobilized metal affinity chromatography (IMAC). We have analyzed the p66 HIV‐1 RT, isolated in the presence of 0.3 M imidazole, by gel permeation HPLC on Superose 12. The results show that it contains two major distinct p66 forms (24.1 min and 28.3 min peaks) which are distinguishable from the purified homodimeric (p66/p66) HIV‐1 RT (22.2 min peak). Protein peak 1 (24.1 min) is converted to a 22.3 min peak upon storage for 20 h at 4°C. Under identical conditions, the isolated peak 2 (28.3 min) appeared as a conformationally heterogeneous mixture elaborated by peaks at 22.3 min and 25.9 min. The protein species thus obtained were active in the RNA‐dependent DNA polymerase and RNase H activity assays and produced heterodimeric HIV‐1 RT upon incubation with the HIV‐1 protease. When the IMAC‐purified, imidazole‐free homodimeric (p66/p66) form of the enzyme was incubated with 0.3 M imidazole for 16 h at 4°C, protein peaks at 28.3 min (peak A) and 30.5 min (peak B) were isolated by gel permeation HPLC. While both of these p66‐containing species were stable and displayed identical RNA‐dependent DNA polymerase activities, the protein in peak B was only 50% active in RNase H function compared with the protein from peak A. These imidazole‐mediated dissociation studies support the hypothesis of partial unfolding of one of the RNase H domains of the p66/p66 homodimer, suggesting that the p66 subunits are asymmetric in the native enzyme.

Mechanism of resistance to U-90152S and sensitization to L-697,661 by a proline to leucine change at residue 236 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase

Bisheteroarylpiperazines (BHAPs) are highly specific inhibitors of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT). BHAP‐resistant HIV‐1 is sensitized to other classes of nonnucleoside RT inhibitors and this has been primarily attributed to a proline‐to‐leucine substitution at amino acid 236 (P236L) of HIV‐1 RT. To understand the basis for the in vitro sensitization‐resistance phenomenon, single base pair mutations at amino acid P236 in HIV‐1 RT were introduced to obtain P236L, P236T, P236H, P236R, and P236A HIV‐1 RT mutants. Active HIV‐1 RT mutants H235W, D237T, and H235W/D237T/T240K, containing substitutions from HIV‐2 RT, were also cloned, expressed, and purified. Three BHAPs (U‐88204E, U‐87201E, and U‐90125S) and the pyridinone L‐697,661 were selected to quantitatively assess the effects of these amino acid substitutions on sensitization to L‐697,661 and resistance to the BHAPs. The HIV‐1 RT mutants bearing single (H235W; D237T) or multiple (H235W/D237T/T240K) HIV‐2 RT substitutions around the conserved P236 conferred little resistance or sensitization to these RT inhibitors. The inhibition profiles of the P236 HIV‐1 RT mutants demonstrated a direct correlation between sensitization to L‐697,661 and resistance to the BHAPs. These results suggest alterations in the shape of the binding pocket as the mechanism by which the P236L mutation confers resistance to the BHAPs and sensitization to L‐697,661.

Simultaneous mutations at Tyr-181 and Tyr-188 in HIV-1 reverse transcriptase prevents inhibition of RNA-dependent DNA polymerase activity by the bisheteroarylpiperazine (BHAP) U-90152s

The replacement of either Tyr‐181 or Tyr‐188 of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) by the corresponding HIV‐2 RT amino acids Ile‐181 or Leu‐188 is known to result in active mutant enzymes (Y181I; Y188L) with virtual loss of sensitivity towards three structural classes of nonnucleoside RT inhibitors; L‐697,661, nevirapine, and TIBO R82913. The bisheteroarylpiperazine (BHAP) U‐90152S, a highly specific inhibitor (IC50, 0.29 ± 0.01 μM) of HIV‐1 RT, inhibited the recombinant Y181I and Y188L HIV‐1 RT mutants with IC50 values of 3.6 ± 0.15 μM and 0.71 ± 0.02 μM, respectively. Construction and in vitro analysis of double mutants Y181I/Y188L and Y181C/Y188L of HIV‐1 RT showed > 150‐fold resistance to U‐90152S. An HIV‐2 mutant containing amino acids 176–190 from HIV‐1 RT acquired full sensitivity to U‐90152S (IC50, 0.29 ± 0.01 μM). It is concluded tha simultaneous mutations at Tyr‐181 and Tyr‐188 of HIV‐1 RT promotes resistance to U‐90152S.

Development of Activity Assays for High-Volume Evaluation of Human Immunodeficiency Virus (HIV) Protease Inhibitors in Rat Serum: Results with Ditekiren

We showed previously that a commercially available synthetic tetradecapeptide, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, produces authentic angiotensin I (Ang I) upon incubation with the HIV-1 protease (S. K. Sharma et al., Anal. Biochem. 198:363, 1991). Therefore, we developed an Ang-I based activity assay for HIV protease inhibitors based on the technology developed earlier (M. J. Ruwart et al., Pharm. Res. 7:407, 1990; S. K. Sharma et al., Anal. Biochem. 186:24, 1990) for tracking renin inhibitors in rat sera. Ditekiren was either extracted from sera with ethyl acetate or assayed after the interfering substances in sera were precipitated with acetonitrile. Purified recombinant HIV-1 protease was added to extracted rat serum and the enzymatic reaction was initiated in the presence of the tetradecapeptide substrate. The inhibition of Ang I production was measured by a commercially available RIA kit. The cleanup methodology also enabled a commercially available Proteinase Scintillation Proximity Assay (SPA, Amersham) to quantify ditekiren in rat serum through the addition of recombinant HIV-1 protease and cleavage of substrate from SPA beads. Results were confirmed by HPLC or by the renin assay for ditekiren, which inhibits both aspartyl proteases. These technologies should prove useful for assessing serum levels of HIV protease inhibitors in rat.