Introduction

Despite its tremendous benefits, highly active antiretroviral therapy (HAART) is not able to eradicate human immunodeficiency virus (HIV) from treated patients (1). In this scenario, the continuously increasing number of viral strains that are resistant to therapy, as well as the adverse drug side effects that are frequently associated with long-term treatment, pose a serious threat to the current treatment success and require the development of new anti-HIV type 1 (HIV-1) compounds (2). In particular, the recent therapeutic success of T-20, a peptide inhibiting viral entry, has highlighted the general potential of HIV-1 entry/fusion inhibitors to complement current highly active antiretroviral therapy. While T-20 is an example of successful rational drug design, other compounds such as ALX 40-4C, a nonpeptide molecule inhibiting HIV-1 entry by targeting the HIV-1 coreceptor CXCR4, demonstrate the limitations of this approach: ALX 40-4C was initially designed to inhibit HIV-1 Tat activity, but in vitro experiments revealed that its mode of action was the inhibition of viral entry (3,4). Despite the tremendous advances rational drug design has brought to the field, such examples demonstrate the particular value of cell-based high-throughput screening (HTS) assays. For this purpose, a variety of cell lines and reporter assays that are suitable for HTS to various degrees have been established (5,6,7,8,9). Suitability is mostly defined by the costs of the actual screening and the time required for the assay. To justify the screening of an industrial-size compound library, which can hold several million compounds, the screening costs per compound need to be minimized. An ideal reporter cell line to screen for inhibitors of HIV-1 replication thus needs to fulfill several requirements: (i) the reporter cell line should be an immortalized T-cell line expressing both HIV-1 coreceptors, CXCR4 and CCR5, and (ii) the reporter cell line should provide a stable and quantifiable marker for HIV-1 expression, such as secreted alkaline phosphatase (SEAP), luciferase, or enhanced green fluorescent protein (EGFP) to avoid the costly and time-consuming direct detection and quantification of viral gene products [e.g., HIV-1 p24 enzyme-linked immuno-sorbent assay (ELISA) and reverse transcription assay]. Two reporter cell lines, MOCHA (10) and CEM. NKR-CCR5-Luc (11), which meet these criteria were recently published. The cell lines use SEAP or luciferase, respectively, under the control of an HIV-1 long terminal repeat (LTR) as the indicator of HIV-1 expression. However, during assay analysis, these cell lines still require time-consuming or relatively costly manipulation of the cell culture (cell lysis, supernatant transfer, and the addition of chemicals) to obtain the final readout.

Here we demonstrate that, based on a stably integrated LTR-EGFP reporter construct, HIV-1 expression in the JTRG-R5 reporter cell line can be directly detected and quantified in cell culture without additional manipulation. This not only significantly reduces the costs of any HTS effort but also provides the opportunity for the direct kinetic analysis of drug effects on HIV-1 expression over an extended time period. In addition, the cell line allows for single-cell analysis using flow cytometry. Using EGFP as a direct marker of HIV-1 infection, follow-up experiments that define the characteristics of an identified lead compound can then be performed with great convenience. If compound availability is low, cells from wells with positive drug hits identified during a drug screen can be removed from the plate and directly subjected to flow cytometry to account for potential cytotoxic or apoptotic effects of the tested compound. We demonstrate that JLTRG-R5 cells can be used in a 384-well plate-based format and quantitatively reflect previous findings obtained from peripheral blood mononuclear cell (PBMC) cultures. Thus, the cell line improves existing methodologies to identify novel anti-HIV-1 compounds.

Materials and Methods

Cell Culture and Reagents

The reporter T-cell lines JLTRG and JLTRG-R5 were maintained at an average cell density of 0.5 × 10⁶ cells/mL in RPMI 1640 (Mediatech, Herndon, VA, USA) supplemented with 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA) (12). The cell lines are identical with respect to CD4 and CXCR4 expression, but only JLTRG-R5 cells express CCR5. We found that CCR5 expression on JLTRG-R5 cells was relatively stable in long-term culture. Over a two-year culture period, only 30% of the cells lost CCR5 expression. Complete CCR5 expression on a population basis can be easily reestablished by enriching CCR5-positive cells using anti-CCR5 antibody-coated magnetic beads (Dynal Biotech, Lake Success, NY, USA) or by fluorescence-activated cell sorting techniques. Prior to the infection experiments, the cells were split to 1 × 10⁵ cells/mL and then grown to a density of 5 × 10⁵ cells/mL to assure optimal susceptibility to HIV-1 infection. JC53BL-13 cells (TZM-BL) were cultured and infected as previously described (13). Briefly, the cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Mediatech) supplemented with 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% heat-inactivated FBS.