Azido-containing diketo acid derivatives inhibit human immunodeficiency virus type 1 integrase in vivo and influence the frequency of deletions at two-long-terminal-repeat-circle junctions

Abstract

We previously found that azido-containing beta-diketo acid derivatives (DKAs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase (IN) (X. Zhang et al., Bioorg. Med. Chem. Lett., 13:1215-1219, 2003). To characterize the intracellular mechanisms of action of DKAs, we analyzed the antiviral activities of two potent azido-containing DKAs with either a monosubstitution or a disubstitution of azido groups, using single- and multiple-replication-cycle assays. Both azido-containing DKAs significantly inhibited HIV-1 infection in 293T, CEM-SS, and H9 cells (50% inhibitory concentration = 2 to 13 micro M) and exhibited low cytotoxicity (50% cytotoxic concentration = 60 to 600 micro M). Inhibition of HIV-1 IN in vivo was demonstrated by the observation that previously described L-708,906 resistance mutations in HIV-1 IN (T66I and T66I/S153Y) also conferred resistance to the azido-group-containing DKAs. In vitro assays and in vivo analysis indicated that the DKAs did not significantly inhibit the 3' processing and selectively inhibited the strand transfer reaction. In addition, quantitative PCR indicated that two-long-terminal-repeat (2-LTR) circles were elevated in the presence of the azido-containing DKAs, confirming that HIV-1 IN was the intracellular target of viral inhibition. To gain insight into the mechanism by which the DKAs increased 2-LTR-circle formation of 3'-processed viral DNAs, we performed extensive DNA sequencing analysis of 2-LTR-circle junctions. The results indicated that the frequency of deletions at the circle junctions was elevated from 19% for the untreated controls to 32 to 41% in the presence of monosubstituted (but not disubstituted) DKAs. These results indicate that the structure of the DKAs can influence the extent of degradation of viral DNA ends by host nucleases and the frequency of deletions at the 2-LTR-circle junctions. Thus, sequencing analysis of 2-LTR-circle junctions can elucidate the intracellular mechanisms of action of HIV-1 IN inhibitors.

FIG. 1.

Structures of azido-containing DKAs and single-replication-cycle assay for screening inhibitors of HIV-1 replication. (A) Structures of DKAs shown are disubstituted azido-containing DKA (DA-DKA), disubstituted benzyloxy-containing DKA (L-708,906), monosubstituted azido-containing DKA (MA-DKA), and monosubstituted benzyloxy-containing DKA (MB-DKA). (B) Schematic representation of pNLuc vector and single-replication-cycle assay for testing antiviral activities of compounds. The pNLuc vectorcontains HIV-1 LTRs, all other cis-acting elements, and genes encoding all HIV-1 viral proteins (Gag, Pro, Pol, Vif, Vpr, Tat, Vpu, and Rev) except envelope (Env) and Nef. The vector also expresses the firefly luciferase gene (luc). Plasmid pHCMV-G encodes the G glycoprotein of the vesicular stomatitis virus envelope, which was expressed from the cytomegalovirus promoter. The shaded area represents treatment of target cells with DKAs. (C) Luciferase activity in 293T cells infected with pNLuc virus. Luciferase activity of infected cells in the absence of any drug (set to 100%) or in the presence of L-708,906 (25 μM) is shown. Luciferase activity in cells infected with pNLuc virus containing the D64E mutation in HIV-1 IN is also shown. The mean of three independent infections and standard error of the mean (error bars) are indicated. In these experiments, the average luminometer signal for wild-type (WT) virus infection in the absence of drug was 1,092 relative light units (RLU), whereas the negative control (uninfected cells) resulted in luminometer signals of <1 RLU.

FIG. 2.

Dose-dependent inhibition of replication-competent HIV-1 virus by MA-DKA and DA-DKA in H9 cells. Inhibition of virus replication was evaluated by measuring the amounts of p24 capsid antigen at days 3 to 14. MA-DKA and DA-DKA were tested at concentrations ranging between 0.6 and 40 μM as indicated.

FIG. 3.

Resistance to DKAs conferred by T66I and T66I/S153Y mutations in HIV-1 IN. Inhibition of integration was determined in the single-replication-cycle assay in the presence of 50 μM DKAs. Luciferase activities for control infections (No drug) were set at 100% for each experiment. The mean of three independent experiments and standard error of the mean (error bars) are indicated. To achieve similar luciferase activity in infected target cells, infections were performed at different dilutions for wild-type (WT) and mutant viruses (WT virus was diluted 100-fold; T66I virus was diluted 10-fold; T66I/S153Y mutant virus was diluted 2-fold). In these experiments, the average luminometer signal for WT virus infection in the absence of drug was 21,654 relative light units (RLU); the average luminometer signal for T66I virus infection in the absence of drug was 84,791 RLU; the average luminometer signal for T66I/S153Y virus infection in the absence of drug was 12,719 RLU. A negative control (uninfected cells) resulted in luminometer signals lower than 100 RLU.

FIG. 4.

Quantitative real-time PCR analysis of late RT product and 2-LTR-circle DNAs in the presence of DKAs. (A) Schematic representation of primer-probe sets that were used for real-time PCR. Primers ES531 and ES532 and probe LateRT-P were used to amplify late RT products (full-length viral DNA). Primers MH535 and MH536 and probe 2LTR-P were used to amplify the 2-LTR-circle form of viral DNA. (B) Full-length viral DNA copies detected by using primers andprobe for detecting late RT product at 12, 24, and 48 h postinfection in the absence or presence of DKAs (L-708,906, MA-DKA, and DA-DKA). Cell numbers were determined by quantification of the PBGD gene, which was present in a single copy in chromosomal DNA. (C) Copies of 2-LTR circles detected per cell at 12, 24, and 48 h postinfection in the absence or presence of DKAs and the cell numbers were determined by PBGD quantification. (D) 2-LTR circles presented as a percentage of full-length viral DNA transcripts.

FIG. 5.

In vivo analysis of 3′ processing in the absence or presence of DKAs. (A) Reverse transcription products that were detected by the RNA probe that hybridizes to the plus-strand of HIV-1 DNA (thick line) after HindIII digestion. The fragment of plus-strand strong-stop DNA that was detected by the probe was 122 nt in length (labeled +SSS). Unprocessed DNA generated a 105- or a 106-nt fragment (see text). The DNA ends that underwent the 3′-processing reaction carried out by HIV-1 IN generated a 103-nt product. RNA primers (PPT and tRNA) are shown as a dashed line. Minus-strand DNA is shown as a thin line. (B) Representative electrophoretic analysis of DNA products during HIV-1 infection. The lanes are labeled according to the drug treatment during infection. The sizes of the processed and unprocessed bands were determined by the migration of oligonucleotide markers of 103 and 105 nt length as described in Materials and Methods (results not shown). The band derived from plus-strand strong-stop DNA is labeled as +SSS, the unprocessed band is labeled as un, and the 3′-processed band is labeled as proc.

FIG. 6.

Deletions at 2-LTR-circle junctions that result from the host nuclease activity. A schematic representation of viral DNA is shown; the shaded GT dinucleotides were removed by HIV-1 IN during the 3′-processing reaction. Removal of the GT dinucleotide by HIV-1 IN through the 3′-processing reaction resulted in the generation of nonpalindromic ends. Deletion of additional sequences by host cell nuclease activity and ligation by host enzymes resulted in the formation of deletions at the 2-LTR-circle junctions. Absence of 3′ processing results in the generation of viral DNA with blunt ends, and ligation of the blunt-ended viral DNA with host enzymes results in the formation of wild-type circle junctions.

FIG.7.

Sequencing analysis of 2-LTR-circle junctions. The normal 2-LTR-circle junction is indicated as wild type (WT). GT and AC terminal nucleotides that were removed by IN during the 3′-processing reaction are shown in bold. Dashes represent deletions. All sequences containinga deletion or mutation are aligned to the WT sequence. All sequences containing an insertion are aligned to each other. PPT and PBS sequences are underlined. Insertions containing PBS sequences are adjacent to the 3′ LTR. Insertions containing PPT sequences are adjacent to the 5′ LTR. Large deletions or insertions are indicated by − or + followed by the numbers of total nucleotides deleted or inserted, respectively. The total number of each detected sequence is shown for wild-type virus in the absence of drug treatment (No drug), for D64E mutant of HIV-1 IN in the absence of drug treatment, and for wild-type virus in the presence of monosubstituted and disubstituted DKAs. The total number of sequences that were analyzed is also indicated in the bottom of the table.