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.