Development of a human immunodeficiency virus vector-based, single-cycle assay for evaluation of anti-integrase compounds

Abstract

Therapeutic strategies aimed at inhibiting human immunodeficiency virus type 1 (HIV-1) replication employ a combination of drugs targeted to two viral enzymes (reverse transcriptase and protease) and to the viral entry/fusion step. However, the high propensity of HIV-1 to develop resistance makes the development of novel compounds targeting different steps of the HIV-1 life cycle essential. Among these, integrase (IN) inhibitors have successfully passed the early phases of clinical development. By preventing integration, IN inhibitors preclude viral replication while allowing production of extrachromosomal forms of viral DNA (E-DNA). Here, we describe an improved and standardized assay aimed at evaluating IN inhibitors by taking advantage of the transcriptional activity of E-DNA produced by HIV-derived vectors in the absence of replication-competent virus. In this context, the use of the firefly luciferase gene as a reporter gene provides a rapid and quantitative measure of viral-vector infectivity, thus making it a safe and cost-effective assay for evaluating novel IN inhibitors.

FIG. 1.

Schematic representation of the vector used in this study. IN-competent (pCMVΔR8.2) and IN-defective (pCHelpIN⁻) packaging plasmids and a VSV.G envelope-coding plasmid (pMD.G) were used to produce recombinant viruses expressing luciferase (TY2-CMV-Luciferase) or GFP (TY2-CMV-GFP). The packaging signal (ψ), the primer binding site (PBS), the deleted packaging signal (ΔΨ), the major splice donor (SD) and acceptor (SA) sites, the bovine growth hormone polyadenylation signal (bGHpA), and the cPPT are indicated. X indicates nonfunctional envelope and/or integrase proteins in the packaging vectors.

FIG. 2.

Inhibition of p24 release in the supernatants of PHA-stimulated HIV-1_IIIB-infected PBMC using increasing amounts of known antiviral compounds. (a) Anti-IN L-731,988. (b and c) RT inhibitors AZT and ddI, respectively. At day 7 after infection, p24 antigen in the cell culture supernatants (bars) was evaluated with an enzyme-linked immunosorbent assay kit, and the percent inhibition (lines) was calculated, assuming 0% inhibition in the non-drug-treated PBMC. Each condition was tested in five replicates, and mean values with standard deviations are indicated.

FIG. 3.

Transduction of 293 cells with IN-competent (a) and IN-defective (b) GFP-expressing self-inactivating lentiviral vectors. The cells were infected in a 24-well plate format with 1 cpm/cell equivalent/well and treated with increasing amounts (range, 0.5 to 50 μM) of anti-IN drug (L-731,988). Three days postinfection, GFP expression was evaluated by fluorescence microscopy (a and b, top rows). Phase-contrast micrographs of the same fields are shown (bottom rows).

FIG. 4.

Luciferase activity in 293 cells infected with IN-competent (squares; left y axes) or IN-defective (diamonds; right y axes) luciferase-expressing self-inactivating lentiviral vectors. The cells were infected in a 96-well plate format with 1 cpm/cell equivalent/well and treated with increasing amounts of the anti-IN drug L-731,988 (range, 0.5 to 50 μM) (a) and the RT inhibitors AZT (range, 10 to 1,000 μM) (b) and ddI (range, 0,0005 to 10 μM) (c). Three days posttransduction, cell-associated luciferase activity was evaluated on a 96-well white plate with a clear bottom, using the Britelite Ultra-High Sensitivity Luminescence Reporter Gene Assay System. RLU, relative light units. Each condition was tested in three replicates, and mean values with standard deviations are shown.

FIG. 5.

Luciferase activities in 293 cells infected with IN-competent (squares; left y axes) or IN-defective (diamonds; right y axes) luciferase-expressing self-inactivating lentiviral vectors. The cells were infected in a 96-well plate format with 1 cpm/cell equivalent/well and treated with increasing amounts of the anti-IN drug L-731,988 (range, 0.5 to 50 μM) or the anti-IN DKA derivatives indicated (range, 1 to 50 μM). Three days posttransduction, cell-associated luciferase activity was evaluated on a 96-well white plate with a clear bottom, using the Britelite Ultra-High Sensitivity Luminescence Reporter Gene Assay System. RLU, relative light units. Each condition was tested in three replicates, and mean values with standard deviations are shown.