Toward the development of a virus-cell-based assay for the discovery of novel compounds against human immunodeficiency virus type 1

Abstract

The emergence of human immunodeficiency virus type 1 (HIV-1) strains resistant to highly active antiretroviral therapy necessitates continued drug discovery for the treatment of HIV-1 infection. Most current drug discovery strategies focus upon a single aspect of HIV-1 replication. A virus-cell-based assay, which can be adapted to high-throughput screening, would allow the screening of multiple targets simultaneously. HIV-1-based vector systems mimic the HIV-1 life cycle without yielding replication-competent virus, making them potentially important tools for the development of safe, wide-ranging, rapid, and cost-effective assays amenable to high-throughput screening. Since replication of vector virus is typically restricted to a single cycle, a crucial question is whether such an assay provides the needed sensitivity to detect potential HIV-1 inhibitors. With a stable, inducible vector virus-producing cell line, the inhibitory effects of four reverse transcriptase inhibitors (zidovudine, stavudine, lamivudine, and didanosine) and one protease inhibitor (indinavir) were assessed. It was found that HIV-1 vector virus titer was inhibited in a single cycle of replication up to 300-fold without affecting cell viability, indicating that the assay provides the necessary sensitivity for identifying antiviral molecules. Thus, it seems likely that HIV-1-derived vector systems can be utilized in a novel fashion to facilitate the development of a safe, efficient method for screening compound libraries for anti-HIV-1 activity.

FIG. 1.

Schematic diagrams of expression plasmids and retroviral transducing vector used to establish the #6 producer cell line. The shaded boxes represent nonfunctional HIV-1 genes. Δψ represents a 33-bp deletion within the encapsidation signal downstream of the splice donor site. P_RSV, Rous sarcoma virus promoter; P_CMV, immediate-early promoter from human cytomegalovirus; P_EM-7, synthetic Escherichia coli T7 promoter; P_Ec, ecdysone promoter; P_SNV, spleen necrosis virus U3 promoter; IRES, internal ribosomal entry site from encephalomyocarditis virus (EMCV); gfp, sequence encoding the enhanced green fluorescent protein; P_SV40, simian virus 40 early gene promoter; hygro, hygromycin resistance gene; SV40 pA, simian virus 40 late polyadenylation sequence; BGH pA, bovine growth hormone polyadenylation sequence; puro, puromycin resistance gene; rre, cis-acting Rev response element; SD, splice donor site; SA, splice acceptor site; pbs, primer binding site; ppt, polypurine tract; LTR, long terminal repeat.

FIG. 2.

Biochemical characterization of #6 producer cell line. (A) Upregulation of reverse transcriptase activity. Supernatant samples from the #6 HIV-1 producer cell line were collected prior to induction and on days 1 through 6 postinduction. RT activity was assayed as described in Materials and Methods. Arrows indicate times at which the medium was replaced with fresh 10% MEM containing 10 μM ponasterone A. The inset depicts the actual phosphorimager induction profile of RT. (B) Induction of p24^gag expression. Supernatant was harvested from uninduced and ponasterone A-induced #6 HIV-1 producer cells from day 1 through day 6. The quantity of p24^gag was determined by enzyme-linked immunosorbent assay. (C) Western blot analysis of inducible HIV-1 envelope expression. Lane +, cell lysate from wild-type HIV-1-infected HeLaT4 cells; lane −, uninfected HeLaT4 cell lysates.

FIG. 3.

Outline of drug-screening assay system. Vector virus undergoes one round of replication when transmitted from producer cells to target cells. Inhibitory effects of drugs can be evaluated on both producer cells and target cells to distinguish between compounds that act during each stage of replication. See Fig. 1 for an enlargement of the recombinants integrated within the #6 producer cell line. The protease and RT inhibitors are listed in boldface at their stage of action. AZT, zidovudine; ddI, didanosine; 3TC, lamivudine; d4T, stavudine; for other abbreviations, see the legend to Fig. 1.

FIG. 4.

Effect of inhibitors on HIV-1 vector virus. RT inhibitors and indinavir were incubated with producer cells (at the time of induction with 10 μM ponasterone A until harvest of the supernatant on day 4) or target cells (added at the time of infection along with 8 μg of Polybrene per ml and maintained through the addition of 1 μg of puromycin per ml with 10% MEM during the selection process). Results shown are the means (± standard deviations) of quadruplicate titer determinations. The control consisted of producer or target cells not treated with protease and RT inhibitors. AZT, zidovudine; ddI, didanosine; 3TC, lamivudine; d4T, stavudine.

FIG. 5.

Effects of increasing concentrations of RT inhibitors and indinavir on viral vector titer. (A) The indicated concentrations of RT inhibitors (zidovudine, didanosine, lamivudine, and stavudine) were added at the time of infection with 8 μg of Polybrene per ml and maintained through the initiation of puromycin selection. AZT, zidovudine; ddI, didanosine; 3TC, lamivudine; d4T, stavudine. (B) Indinavir was added at the time of induction with ponasterone A and maintained through the harvesting of viral supernatant for target cell transduction. Any indinavir remaining from the induction phase would be diluted 10- to 1,000-fold during the serial dilutions to calculate titer. For both panels, the results shown are the means (± standard deviations) of quadruplicate determinations and are representative of three independent experiments.

FIG. 6.

Drug cytotoxicity assays. HeLaT4 cells were assayed for cytotoxicity in the presence of the RT inhibitors zidovudine, didanosine, lamivudine, and stavudine at the indicated concentrations for 72 h (A). AZT, zidovudine; ddI, didanosine; 3TC, lamivudine; d4T, stavudine. Viability of treated cells was assessed with the tetrazolium salt WST-1 reagent as per the manufacturer's instructions (Roche). The cytotoxicity of indinavir was evaluated on the #6 producer cell line (B). Results shown are the means (± standard deviations) of triplicate readings. The shaded region in panel A depicts the effective range of drug inhibitory concentrations towards supernatant harvested from induced #6 HIV-1 producer cells as displayed in Fig. 5. A shaded area is not shown in panel B because it would be too narrow to be seen with the range of coordinates depicted on the abscissa.