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Antimicrobial Agents and Chemotherapy, September 2005, p. 3833-3841, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3833-3841.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

High-Throughput Human Immunodeficiency Virus Type 1 (HIV-1) Full Replication Assay That Includes HIV-1 Vif as an Antiviral Target

Joan Cao, Jason Isaacson, Amy K. Patick, and Wade S. Blair*

Department of Virology, Pfizer Global Research and Development, La Jolla Laboratories, 10777 Science Center Dr. (CB1), San Diego, California 92121

Received 3 March 2005/ Returned for modification 28 April 2005/ Accepted 26 May 2005


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ABSTRACT
 
Antiviral screens have proved useful for the identification of novel human immunodeficiency virus type 1 (HIV-1) inhibitors. In this study, we describe an HIV-1 full replication (HIV-1 Rep) assay that incorporates all of the targets required for replication in T-cell lines, including the HIV-1 Vif gene. The HIV-1 Rep assay was designed to exhibit optimal sensitivity to late-stage as well as early-stage inhibitors to maximize the likelihood of identification of novel target antiviral compounds in a screen. In addition, the flexibility of the HIV-1 Rep assay allows the rapid evaluation of antiviral compounds against different virus strains in different T-cell lines without significant modification of the assay format. We demonstrate that the HIV-1 Rep assay exhibits characteristics (e.g., a favorable Z' value) compatible with high-throughput screening in a 384-well format. The utility of the HIV-1 Rep assay was demonstrated in a high-throughput screen of >106 compounds. To our knowledge, this study represents the first example of an HIV-1 antiviral screen that includes Vif as a functional target and was executed on an industrial scale.


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INTRODUCTION
 
Current drugs approved for the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection fall into one of four therapeutic classes: the nucleoside reverse transcriptase inhibitors (NRTIs), the nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and fusion inhibitors. Although current therapies are clinically effective when they are used in combination, they are less than ideal due to drug-related side effects, inconvenient dosing requirements, and/or the emergence of drug-resistant virus (for a review, see reference 41). The issue of drug resistance is particularly problematic, given that viral variants resistant to one drug of a particular class often exhibit some level of cross-resistance to other drugs within the same class. Compounding the problem, recent studies show an increase in the transmission of resistant virus to drug-naïve patients (22). The identification of novel target inhibitors represents one approach that can be used to address the issue of drug resistance. Antiviral compounds that target new mechanisms, including CCR5 inhibitors (10), HIV-1 integrase inhibitors (INIs) (2, 30), a gp120/CD4 inhibitor (14), and a virion maturation inhibitor (40), are in development. However, the clinical utility of many of these agents remains to be determined. In addition, it is likely, if not a certainty, that drug resistance will be a recurrent problem in chronic antiviral therapy. Therefore, there still exists an urgent need for the identification and development of new HIV-1 inhibitors that exhibit improved safety and resistance profiles, and ideally, these inhibitors act against novel HIV-1 targets.

Cell-based antiviral screens have successfully been used to identify novel target inhibitors. Both BMS-488043 (3, 14, 21, 38) and PA-457 (11, 19), two novel target HIV-1 inhibitors that recently entered clinical development, were originally identified by using antiviral screens. Several different HIV-1 antiviral assay formats have been described that could be adapted for medium- to high-throughput screening (1, 5, 6, 7, 9, 20, 27, 29, 31, 32, 36), and many of these assays utilize either a reporter virus or a reporter cell to measure HIV-1 replication. In reporter virus assays, a reporter gene is introduced into the virus genome, usually in place of a viral gene not required for replication in the target cells of interest. The cells are then infected with the recombinant reporter virus, and virus replication is quantified by measuring the expression of the virus-encoded reporter gene (1, 5, 29, 31). In reporter cell assays, the target cells of interest are engineered to contain a reporter gene, which is activated upon viral infection. In this case, virus replication is measured by monitoring induction of the reporter gene in the infected target cells (9, 20, 27, 32, 36).

Cell-based screening methods offer the potential advantage of including antiviral targets that are not amenable to biochemical screenings methods. One potential target that has recently received a considerable amount of interest is the HIV-1 Vif protein. Vif (virion infectivity factor) is a late gene product that is encoded by most lentiviruses and that is required for HIV-1 replication in primary T cells and some T-cell lines and is presumably required for HIV-1 replication in vivo (for a review, see reference 28). Previous studies have shown that APOBEC3G (apolioprotein B editing complex protein 3G), formally known as CEM15, acts as part of a natural cellular defense mechanism to suppress HIV replication (15, 23, 24, 33, 43). Vif counteracts this defense mechanism by binding to APOBEC3G and targeting the complex for degradation through the proteosome pathway (8, 18, 24, 25, 26, 34, 37, 42). Unfortunately, biochemical assays that fully recapitulate the function of Vif are not available. Therefore, cell-based screening methods are currently the most attractive option for use in the exploration of Vif as a potential target.

Although current HIV-1 reporter virus-cell approaches are more convenient for antiviral screening than traditional assays, they have limitations as well. Labor-intensive reagent construction is usually required to establish new assays, and often, HIV-1 reporter assays do not maximize the number of HIV-1 targets adequately represented in an antiviral screen. The HIV-1 full replication (HIV-1 Rep) assay described here represents a simple yet unique approach that was designed to avoid many of the issues associated with current HIV-1 reporter assays. We show that a functional HIV-1 Vif gene is required for maximal signal in the HIV-1 Rep assay and that the assay is sensitive to known HIV-1 inhibitors of different classes that target both the early and the late steps in the viral replication cycle. In addition, we show that the HIV-1 Rep assay exhibits properties favorable for the establishment of a 384-well high-throughput screen (HTS) and demonstrate the utility of the assay by executing a screen of >106 compounds.


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MATERIALS AND METHODS
 
Cells. HeLa CD4 LTR/beta-Gal (catalog no. 1470), MT-2 (catalog no. 237), PM1 (catalog no. 3038), H9 (catalog no. 87), 174XCEM (catalog no. 272), CEM-SS (catalog no. 776), C8166 (catalog no. 404), CEM-T4 (catalog no. 117), SupT1 (catalog no. 100), and HEK 293 (catalog no. 103) cells were obtained through the AIDS Research and Reference Reagent Program, National Institutes of Health, Bethesda, MD. HeLa CD4 LTR/beta-Gal cells and HEK 293 cells were propagated in Dulbeccos's modified Eagle medium (DMEM; Life Technologies, Gaithersburg MD) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT). MT-2, PM1 H9, 174XCEM, CEM-SS, C8166, CEM-T4, and SupT1 cells were propagated in RPMI 1640 medium (RPMI; Life Technologies, Gaithersburg MD) containing 10% FBS (HyClone).

Plasmids and virus. HIV-1 strain RF (catalog no. 2803), strain IIIB (catalog no. 398), and strain 92HT599 (catalog no. 3301) and pNL4-3 (catalog no. 114; GenBank accession no. AF324493) infectious HIV-1 cDNAs were obtained through the AIDS Research and Reference Reagent Program, National Institutes of Health. To construct the NL4-3 Vif mutant virus (HIV-1 NL4-3/deltaVif), a 169-nucleotide deletion was introduced in the Vif-coding region of pNL4-3 (nucleotide positions 5151 to 5320) by PCR-based mutagenesis (16). The resulting pNL4-3/deltaVif cDNA encoded a 56-amino-acid deletion in Vif sequences. To generate infectious virus, pNL4-3 or pHIV-1 NL4-3/deltaVif was transfected into HEK 293 cells by using the LipofectAMINE Plus transfection kit, according to the manufacturer's protocol (Life Technologies). Seventy-two hours after transfection, infectious wild-type (wt) NL4-3 virus or NL4-3/deltaVif mutant virus was harvested from the supernatants of the transfected cells and clarified by centrifugation (500 x g). The titers (i.e., the 50% tissue culture infective dose [TCID50] per ml) of the resulting viral stocks were determined after infection of HeLa CD4 LTR/beta-Gal target cell lines with serial dilutions of the viral stocks (17) and measurement of the beta-galactosidase (beta-Gal) activity in the HeLa CD4 LTR/beta-Gal cells 72 h after infection by using the Dual-Light reporter gene assay kit (Applied Biosystems, Bedford, MA).

Compounds. Nelfinavir (NFV) and flavorpiridol were synthesized at Pfizer (formerly Agouron Pharmaceuticals Inc., San Diego, CA. Nevirapine (NVP; catalog no. 4666), saquinavir (SQV; catalog no. 4658), efavirenz (EFV; catalog no. 4624), and indinavir (IDV; catalog no. 8145) were obtained through the AIDS Research and Reference Reagent Program, National Institutes of Health. Delavirdine (DLV), lamivudine (3TC), and stavudine (d4T) were kindly provided by Pharmacia and Upjohn (Kalamazoo, MI), Glaxo Wellcome (Research Triangle Park, NC), and Bristol-Myers Squibb (Wallingford, CT), respectively. 3'-Azido-3'-deoxythymidine (AZT), aurintricarboxylic acid (ATA), and didanosine (ddI) were purchased from Sigma-Aldrich (St Louis, MO).

HIV-1 p24 assay. MT-2 cells or PM1 cells were infected with NL4-3 wt virus or NL4-3/deltaVif virus using equivalent multiplicities of infection (MOI; 0.16 TCID50 per cell) for 2 h. The infected cultures were then washed with RPMI resuspended in 5 ml of RPMI at final cell densities of 2 x 105 cells/ml and incubated at 37°C in a 5% CO2 atmosphere. Virus replication was measured at various times after infection by quantifying the HIV-1 p24 antigen present in 1 ml of cell-free supernatants of infected cell cultures by using the COULTER HIV-1 p24 antigen assay kit (Beckman Coulter, Miami, FL), according to the manufacturer's protocol. Data are presented as the ng/ml of p24 measured versus days in culture.

HIV-1 Rep infection assays. HeLa CD4 LTR/beta-Gal indicator cells were added to 96-well microtiter plates at cell densities of 1 x 104 cells/well in DMEM or RPMI (Life Technologies) containing 10% FBS (HyClone). MT-2 or PM-1 cells were infected with NL4-3 wt or NL4-3/deltaVif by using an MOI of 0.08 TCID50 per cell. Two hours after infection, infected MT-2 or PM1 cells were washed with RPMI (Life Technologies) and added to the 96-well microtiter plates containing the HeLa CD4 LTR/beta-Gal indicator cells at final infected T-cell densities of 1.6 x 104 cells/well (MT-2) or 2 x 104 cells/well (PM1). Alternatively, HeLa CD4 LTR/beta-Gal indicator cells were infected directly with NL4-3 wt or NL4-3/deltaVif by using an MOI of 0.08 (TCID50 per cell) at a final cell density of 1 x 104 cells. NL4-3 wt infections were performed either in the absence of compound or in the presence of NFV or EFV at final concentrations of 1 µM and 0.1 µM, respectively. Virus replication was measured 4 days after infection by quantifying HIV-1 Tat-induced beta-Gal activity in the HeLa CD4 LTR/beta-Gal indicator cells by using the Dual-Light system (Applied Biosystems) and a Microbeta luminometer (Perkin-Elmer), according to the manufacturers' protocols. Data were expressed as the fold induction of beta-Gal reporter activity in infected wells relative to the background reporter signal measured in the absence of virus. All experiments were performed with three or more replicates, and P values were determined by using a Student's t test. The percent inhibition values derived from the viral infection experiments in the presence of a single concentration of compound were calculated based on the following equation: 100 x {1 – [(mean reporter signal in the presence of compound-mean reporter signal in the absence of virus)/(mean reporter signal in the absence of compound-mean reporter signal in the absence of virus)]}.

For the HIV-1 Rep screens, compound was added to the test wells at a final concentration of 10 µM with dimethyl sulfoxide (DMSO) at a final concentration of 1% or DMSO alone was added at a final concentration of 1% (no-compound control wells). Antiviral maximum signals (AV max) and antiviral minimum signals (AV min) were calculated from the no-compound control wells. Z' coefficients were calculated from the no-compound control wells by using the equation 1 – {[(3 x SD AV max)-(3 x SD AV min)]/(AV max-AV min)}, where SD is the standard deviation (44). Wells that exhibited ≥50% or ≥60% inhibition of the reporter gene signal relative to that in the no-compound control wells (AV max) were scored as hits in the mock screen and HTS, respectively. Hits from the HTS were evaluated again in the HIV-1 Rep assay, and compounds showing ≥50% inhibition were designated confirmed hits. In addition, the cytotoxicity of compounds that scored as confirmed hits in the primary screen was measured by incubating the compounds with MT-2 cells at a final concentration of 10 µM for 4 days and determining cell viability by the CellTiter-Glo assay (Promega). Compounds showing ≥50% inhibition of the luminescent end point in the CellTiter-Glo assay were designated cytotoxic and subtracted from the confirmed hit list to generate the number of specific antiviral confirmed hits.

Susceptibility assays. Half-log dilutions of the test compounds were added to HeLa CD4 LTR/beta-Gal indicator cells seeded in 96-well microtiter plates at a cell density of 1 x 104 cells per well in DMEM or RPMI (Life Technologies) containing 10% FBS (HyClone). MT-2 or PM1 cells were infected with NL4-3 wt virus at an MOI of 0.08 TCID50 per cell. Two hours after infection, infected cells were washed with RPMI, resuspended in RPMI, and then added to the 96-well microtiter plates containing compound-treated or compound-free HeLa CD4 LTR/beta-Gal indicator cells. Virus replication was measured 3 or 4 days after infection by quantifying HIV-1 Tat-induced beta-Gal activity in the HeLa CD4 LTR/beta-Gal indicator cells as described above. Data from the reporter gene measurements were expressed as the percentage of reporter gene activity in infected compound-treated cells relative to that in infected, compound-free cells. The 50% effective concentration (EC50) was calculated as the concentration of compound that effected a decrease in the percentage of the virus-encoded reporter gene activity in infected, compound-treated cells to 50% of that produced in infected, compound-free cells. In addition, the EC90 was calculated as the concentration of compound that effected a decrease in the percentage of the virus-encoded reporter gene activity in infected, compound-treated cells to 90% of that produced in infected, compound-free cells. In experiments where 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) dye reduction (39) was used as the assay end point (cell protection assays), subject cells were infected with HIV-1 RF virus at an MOI of 0.025 to 0.819 TCID50 per cell or mock infected with medium only and were added at 2 x 104 cells per well into 96-well microtiter plates containing half-log dilutions of test compounds. Six days later, 50 µl of XTT (1 mg/ml XTT, 0.02 nM phenazine methosulfate) was added to the wells and the plate was reincubated for 4 h. Viability, as determined by the amount of XTT formazan produced, was quantified spectrophotometrically by determination of the absorbance at 450 nm. Data from the cell protection assays were expressed as the percentage of formazan produced in compound-treated cells compared to the amount of formazan produced in the wells with uninfected, compound-free cells. The EC50 was calculated as the concentration of compound that effected an increase in the percentage of formazan production in infected, compound-treated cells to 50% of that produced by uninfected, compound-free cells. The 50% cytotoxic concentration was calculated as the concentration of compound that decreased the percentage of formazan produced in uninfected, compound-treated cells to 50% of that produced in uninfected, compound-free cells.


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RESULTS
 
The HIV-1 Rep assay includes Vif as a functional target. We designed an HIV-1 Rep assay for the purpose of executing a high-throughput antiviral screen that incorporates all of the HIV-1 targets required for replication in culture, including the HIV-1 Vif gene. In the HIV-1 Rep assay (Fig. 1), T-cell lines that support high levels of virus replication (e.g., MT-2 cells) are infected for 2 h with HIV-1 at a relatively low MOI. The infected T cells are then added to microtiter plates containing HeLa CD4 LTR/beta-Gal indictor cell lines in the presence or absence of compound. Virus replication occurs rapidly in the highly permissive T-cell lines, resulting in the amplification of infectious virus in the wells of the microtiter plate, infection of the HeLa CD4 LTR/beta-Gal indictor cells, and significant levels of beta-Gal reporter gene induction in the indicator cells. The HIV-1 Rep assay design ensures that significant reporter gene induction is dependent on multiple rounds of virus replication (see below).



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  		FIG. 1. HIV-1 Rep assay design. T cells (TC) are infected with virus and then added to microtiter plates containing a separate indicator cell line (IC) (e.g., HeLa CD4/LTR beta-Gal cell lines) in the presence or absence of compound, as described in Materials and Methods. Four days after infection, virus replication in the infected (Inf.) T cells (ITC) and indicator cells is measured by quantifying Tat-mediated reporter gene induction in the indicator cell line.

To identify the optimal T-cell lines for the HIV-1 Rep assay format, we evaluated eight different T-cell lines (MT-2, PM1, H9, 174XCEM, CEM-SS, C8166, CEM-T4, and SupT1) and determined that MT-2 and PM1 cells both exhibited a Vif nonpermissive phenotype and supported high levels of virus replication. To achieve this, we compared the replication kinetics of HIV-1 NL4-3 wt to that of an HIV-1 NL4-3 mutant virus, which contains a deletion in Vif-coding sequences (NL4-3/deltaVif). MT-2 (Fig. 2A) or PM1 (Fig. 2B) cells were infected with NL4-3 wt or NL4-3/deltaVif with equivalent MOIs, and the levels of p24 in the supernatants of infected cells were measured either immediately after infection (day 0) or on 3, 6, or 10 days after infection, as described in Materials and Methods. As shown in Fig. 2, significantly higher levels of p24 were produced in the supernatants of MT-2 or PM1 cells infected by NL4-3 wt than in the supernatants of the same cells infected with comparable amounts of NL4-3/deltaVif. In MT-2 cells (Fig. 2A), sixfold higher levels of p24 were measured in cells infected with NL4-3 wt than in the same cells infected with NL4-3/deltaVif 10 days after infection (i.e., an 83% reduction in NL4-3/deltaVif virus replication). In PM1 cells (Fig. 2B), 21-fold higher levels of p24 were measured in cells infected with NL4-3 wt than in the same cells infected with NL4-3/deltaVif 10 days after infection (i.e., a 95% reduction in NL4-3/deltaVif virus replication). Consistent with reports in the literature, H9 cells exhibited a Vif nonpermissive phenotype as well (12); however, MT-2 and PM1 cells supported significantly higher levels of virus replication than H9 cells (data not shown). The remaining cell lines tested (174XCEM, CEM-SS, C8166, CEM-T4, and SupT1) exhibited a Vif permissive phenotype (data not shown). These data demonstrate that both MT-2 cells and PM1 cells exhibit a Vif nonpermissive phenotype and support high levels of wt virus replication.




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  		FIG. 2. Replication of NL4-3 wt or HIV-1 NL4-3/deltaVif in MT-2 or PM1 cells. (A) MT-2 or (B) PM1 cells were infected with wild-type HIV-1 NL4-3 (NL4-3 wt) or HIV-1 NL4-3/deltaVif using equivalent MOIs (0.16). Virus replication was then measured by quantifying p24 production (ng/ml) in the supernatants of infected cells either immediately after infection (day 0) or 3, 6, or 10 days after infection, as described in Materials and Methods.

To determine whether MT-2 and/or PM1 cells could be utilized to establish an antiviral HTS, such cells were infected with NL4-3 wt and evaluated in the HIV-1 Rep assay format, as described in Materials and Methods. For comparison, HeLa CD4/LTR beta-Gal indicator cells were infected directly with NL4-3 wt. Virus replication was measured 4 days after infection by quantifying HIV-1 Tat-induced beta-Gal activity in the HeLa CD4 LTR/beta-Gal indicator cells. The results showed a 71-fold induction of beta-Gal activity after infection of MT-2 cells in the HIV-1 Rep assay format (Fig. 3). A 34-fold induction of beta-Gal reporter activity was measured after infection of PM1 cells in the HIV-1 Rep assay, and a 49-fold induction of reporter activity was observed after direct infection of the HeLa CD4 LTR/beta-Gal indicator cells (Fig. 3). To demonstrate that the reporter gene signal was dependent on virus replication, infections were performed in the presence of EFV, an NNRTI, or NFV, an HIV-1 PI, at concentrations that were ≥10-fold higher than the EC90 of each compound. The results showed a ≥99% inhibition of the reporter signal when MT-2, PM1, or HeLa CD4 LTR/beta-Gal cells were infected in the presence of EFV (0.1 µM) (Fig. 3). When MT-2 or PM1 cells were infected in the presence of NFV (1 µM), >97% reductions in the reporter signal were observed for both cell types. Alternatively, a more modest 47% reduction in reporter activity was observed after direct infection of the HeLa CD4 LTR/beta-Gal cells in the presence of NFV compared to that for the no-compound control. These results demonstrate that the reporter signals in the MT-2 and PM1 cell-based HIV-1 Rep assays are dependent on multiple rounds of virus replication and suggest that both HIV-1 Rep assay formats would be fully sensitive to inhibitors that target any step in the HIV-1 replication cycle. In contrast, a significant fraction of the signal observed after direct infection of HeLa CD4 LTR/beta-Gal indicator cells with HIV-1 appeared to result from the initial round of infection rather than from multiple rounds of virus replication. As such, this fraction of the reporter signal (53%) was not suppressed by an HIV-1 PI (NFV), which acts during the later stages of the replication cycle (Fig. 3).



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  		FIG. 3. Replication of NL4-3 wt and NL4-3/deltaVif in the HIV-1 Rep assay. MT-2 cells or PM1 cells were infected with NL4-3 wt or NL4-3/deltaVif for 2 h and then added to microtiter plates containing HeLa CD4/LTR beta-Gal cells. Virus replication was then measured by monitoring beta-Gal induction 4 days after infection, as described in Materials and Methods. Alternatively, HeLa CD4/LTR beta-Gal cells were infected directly with NL4-3 wt or NL4-3/deltaVif virus, and virus replication was measured 4 days after infection, as described in Materials and Methods. Infections with NL4-3 wt were performed in the presence or absence of the HIV-1 nonnucleoside reverse transcriptase inhibitor EFV or the protease inhibitor NFV at ≥10 times the EC90s. The results are presented as the mean fold induction of reporter gene activity over that of the background (i.e., in the absence of virus) ± standard deviation. Mean values were determined from three or more experiments.

As stated above, our goal was to establish a high-throughput antiviral screen that includes the HIV-1 Vif gene as a functional target. To demonstrate that HIV-1 Rep assays with MT-2 or PM1 cells are Vif dependent, we compared the reporter signal generated after infection with NL4-3 wt with that generated after infection with the NL4-3/deltaVif mutant. In the MT-2 cell-based HIV-1 Rep assay, a 79% reduction in virus replication was observed for the NL4-3/deltaVif mutant virus compared to the level for the wt (P = 0.004), while a 93% reduction in NL4-3/deltaVif mutant virus replication was observed in the PM1 cell-based HIV-1 Rep assay (P = 0.0003). These results are consistent with those observed when NL4-3 wt and NL4-3/deltaVif virus replication was compared in MT-2 cells or PM1 cells by using a p24 end point (Fig. 2A and B). It should be noted that the differences between wt and NL4-3/deltaVif virus replication were maximal in HIV-1 Rep assays of ≥4 days in duration (data not shown). As expected, similar levels of virus replication were measured after direct infection of the HeLa CD4 LTR/beta-Gal cells (Vif permissive cells) with NL4-3 wt or the NL4-3/deltaVif mutant virus. These results confirm that maximum levels of virus replication in the MT-2 or PM1 cell-based HIV-1 Rep assays depend on a functional Vif gene. Therefore, the MT-2 and PM1 cell-based HIV-1 Rep assays represent novel high-throughput assays that could be used to screen for inhibitors of HIV-1 Vif function.

HIV-1 inhibitors exhibit similar activities in HIV-1 Rep and HIV-1 cell protection assays. To demonstrate that the activities of the HIV-1 inhibitors measured in the HIV-1 Rep assay are consistent with the activities measured in conventional HIV-1 assays, EC50 and EC90 values were determined for HIV-1 NNRTIs, PIs, NRTIs, an entry inhibitor, and a Tat inhibitor, as described in Materials and Methods. As shown in Table 1, the majority of the EC50 and the EC90 values measured for inhibitors tested in HIV-1 Rep assays with MT-2 or PM1 cells were comparable to those values measured in a more conventional HIV-1 RF cell protection assay. In addition to the inhibitors shown in Table 1, EC50 and EC90 values were determined for two HIV-1 integrase inhibitors in the HIV-1 Rep and HIV-1 RF cell protection assays, and again, similar results were observed (data not shown). Therefore, these data demonstrate that the MT-2 and PM1 cell-based HIV-1 Rep assays are valid for measurement of the antiviral activities of HIV-1 inhibitors.


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  		TABLE 1. Antiviral activities of HIV-1 inhibitors in the HIV-1 Rep and HIV-1 RF cell protection assays

To demonstrate the flexibility of the assay format, HIV-1 Rep assays were established using four other T-cell lines (CEM-SS, C8166, CEM-T4, and 174XCEM) and two additional HIV-1 strains (HIV-1 IIIB and HIV-1 92HT599), and the activities of HIV-1 inhibitors were evaluated. As expected, known HIV-1 inhibitors exhibited activity in such assays within the expected concentration ranges (data not shown).

The MT-2 cell-based HIV-1 Rep assay can be formatted as an HTS. The MT-2 cell-based HIV-1 Rep assay showed better signal-to-background ratios and lower interassay variability than the PM1 cell-based assay (data not shown); therefore, the MT-2 cell-based assay was formatted for HTS. To demonstrate that the MT-2 cell-based HIV-1 Rep assay is suitable for HTS, the assay was automated and 11 96-well mock screening plates were evaluated initially. Five known HIV-1 inhibitors representing different classes (two NNRTIs, one NRTI, one INI, and one PI) were distributed randomly on the 11 microtiter plates at their respective EC50, EC90, or 2x EC90. Each inhibitor was distributed on at least three different plates at each concentration (EC50, EC90, or 2x EC90). As described in Materials and Methods, wells that exhibited a ≥50% inhibition of the reporter gene activity compared to that in the no-compound control wells were scored as hits, and the number of hits identified in the mock screen were then plotted relative to the number of total inhibitors present (Fig. 4). The results showed that 79% (26 of 33) of the inhibitors present at their respective EC50s were identified as antiviral hits in the MT-2 cell-based mock screen and 100% of the compounds present at their respective EC90 or 2x EC90 were identified as hits (Fig. 4). These data demonstrate that the MT-2 cell-based HIV-1 Rep assay is sufficiently sensitive to reproducibly identify diverse classes of HIV-1 inhibitors in an HTS format and suggest low rates of false-negative results for an HIV-1 Rep screen.



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  		FIG. 4. Known HIV-1 inhibitors are identified as hits in a mock screen using the MT-2 cell-based HIV-1 Rep assay. Eleven 96-well mock screening plates that contained five different HIV-1 inhibitors (two NNRTIs, one NRTI, one INI, and one PI) distributed randomly at their respective EC50, EC90, or 2x EC90 were evaluated in the HIV-1 Rep assay, as described in Materials and Methods. Wells exhibiting ≥50% inhibition of the reporter gene activity compared to that in the no-compound control wells were scored as hits and compared to the total number of inhibitors present.

Prior to the initiation of an antiviral HTS, the HIV-1 Rep assay was converted to a 384-well plate format and was validated for screening. Miniaturization of the HIV-1 Rep assay from the 96-well format to the 384-well format allowed for an increase in throughput and a reduction in screen cost, while similar assay parameters were maintained. To illustrate this, signal-to-background ratios and Z' values were calculated from the control wells of the 96-well or 384-well screen plates, as described in Materials and Methods. The Z' value is reflective of the dynamic range as well as the variation of the assay and is a useful tool for assay comparisons and assay quality determinations (44). Typically, a Z' value >0.5 is considered favorable for high-throughput screening. The HIV-1 Rep assay exhibited signal-to-background ratios of 25 and 41 in the 96-well and the 384-well formats, respectively, and Z' values of 0.82 and 0.62 in the 96-well and 384-well formats, respectively. These data demonstrate that the HIV-1 Rep assay exhibits similar screening parameters in the 96- and 384-well formats and strongly suggest that the MT-2 cell-based HIV-1 Rep assay is suitable for HTS in a 96- or 384-well format.

To demonstrate the utility of the HIV-1 Rep assay for the execution of industrial scale high-throughput screens, >106 compounds were screened at a throughput of up to 72,000 wells per week (Table 2). Hits were defined as those compounds showing percent inhibition values ≥60% compared to that for the no-compound controls. A total of 21,077 primary hits were identified from the 1,251,148 wells screened, resulting in a hit rate of 1.7%. Although such a hit rate may be considered high by conventional HTS standards, we expected hit rates greater than 1% based on the large number of potential targets in the HIV-1 replication cycle and previous antiviral screening experience (4). The primary hits were tested again in the HIV-1 Rep assay, and 10,445 of the 21,077 hits exhibited reproducible activity, resulting in a confirmation rate of 50% (Table 2). Based on our previous screening experience (4), we predicted that a significant portion of the confirmed hits would be cytotoxic or nonspecific inhibitors (i.e., false positives). Therefore, confirmed hits were evaluated in a separate cytotoxicity assay with MT-2 cells and an ATP-dependent luciferase end point (CellTiter-Glo; Promega), and 4,878 compounds (47%) were identified as cytotoxic false positives. The remaining 5,567 compounds (26% of the primary hits) represented specific antiviral hits (Table 2) and will be characterized further in antiviral and mechanism-of-action assays.


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  		TABLE 2. Hit confirmation rates in a high-throughput HIV-1 Rep screen


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DISCUSSION
 
We describe an HIV-1 full replication assay and demonstrate the utility of the assay for executing an antiviral HTS. We show that a significant reporter signal is induced in the HIV-1 Rep assay 4 days after infection and that the reporter signal is fully suppressed (>97%) by known inhibitors that target both early (NNRTI) and late (PI) stages of the HIV-1 replication cycle. These results suggest that the HIV-1 Rep assay will be sensitive to inhibitors targeting any step of the replication cycle. We provide additional data in support of this by showing that HIV-1 NNRTIs, NRTIs, PIs, an entry inhibitor, and a Tat inhibitor exhibit EC50 and EC90 values in the expected ranges compared to those provided by a more conventional HIV-1 antiviral assay format (HIV-1 RF cell protection assay). However, it should be noted that HIV-1 NL4-3 is an X4-tropic virus and MT-2 and HeLa LTR/beta-Gal cells do not express CCR5; therefore, CCR5 is not a target in the current form of the HIV-1 Rep assay. In contrast to that observed for the HIV-1 Rep assay, a significant portion of the reporter signal induced after direct infection of the HeLa LTR/beta-Gal indicator cells resulted from the initial round of infection and could not be inhibited by high concentrations of a PI. Based on these observations, we would expect that an antiviral screen by a direct infection (reporter cell) assay may be less sensitive to late-stage inhibitors than the HIV-1 Rep assay and that EC90 values would be difficult to measure for such inhibitors in the direct infection reporter cell assay. A fundamental advantage of the HIV-1 Rep assay design is that at least one round of HIV replication in the target cells is required in the HIV-1 Rep assay to generate a detectable signal in the indicator cells and multiple rounds of replication in the target cells are required for maximal reporter gene signals. As such, the HIV-1 Rep assay design allows for the development of an antiviral screen with optimal sensitivity to late-stage inhibitors.

The HIV-1 Rep assay design also allows the inclusion of Vif as a target in an antiviral screen. We identified two T-cell lines (MT-2 and PM1) that both exhibited a Vif nonpermissive phenotype and supported high levels of wt HIV-1 replication, and these cell lines were each utilized in the HIV-1 Rep assay format. We demonstrate that a functional Vif gene is required for maximal reporter signals in the HIV-1 Rep assay format. In fact, Vif mutant virus replication was attenuated to similar extents in the HIV-1 Rep assay and in p24 assays with MT-2 or PM1 cells. Given that the HeLa CD4 LTR/beta-Gal indicator cells used in the HIV-1 Rep assay are Vif permissive, these results demonstrate that virus replication is primarily driven by the T-cell component of the assay. Other HIV assays that are dependent on a functional Vif gene have been described (12, 24, 25, 33, 34, 35, 37); however, previous assay methods rely either on end points (e.g., measurement of the production of p24 or RT activity in the supernatants of infected cells) or on transient transfection protocols that cannot be formatted for HTS. The HIV-1 Rep assay is unique in that it includes Vif as an antiviral target and is compatible with high-throughput screening. In the event that inhibitors of Vif function are identified from the HIV-1 Rep screen, previously described transfection assays for Vif function (24, 25, 34, 37) or antiviral assays with Vif permissive cell lines (12, 33, 35) may be utilized for target validation.

Another advantage of the HIV-1 Rep assay is that it allows for the rapid evaluation of different target cells (e.g., multiple T-cell lines or primary T cells) or different virus strains (e.g., different HIV-1 laboratory strains or primary isolates) without having to generate new stable reporter cell lines or new reporter virus constructs. In principle, the HIV-1 Rep assay allows the analysis of any target cell that supports HIV-1 replication and that can be propagated for short periods of time, including primary cells. Thus far, we have established HIV-1 Rep assays using six different T-cell lines and three different HIV-1 strains, including one clinical isolate (HIV-1 92HT599). This has afforded us the opportunity to rapidly evaluate the activities of HIV-1 inhibitors against different virus strains in different T-cell lines in a high-throughput manner without having to generate new reagents or modify the assay format. This is not the case with typical reporter cell assays, which depend on the engineering of individual stable cell lines, or reporter virus assays, which require the generation of new reporter viruses with each HIV-1 variant being analyzed.

Several different antiviral assay formats that could potentially be used for antiviral high-throughput screening have been proposed in the literature (1, 5, 6, 7, 9, 20, 27, 29, 31, 32, 36). Despite this, the execution of an antiviral HTS is not trivial, and few have been successfully achieved on an industrial scale (>106 compounds). HIV-1 cell protection assays similar to that described in Table 1 have been used in random compound library screens (6, 13, 39). In cell protection assays, virus replication is measured indirectly by monitoring cell viability by using a dye reduction method (e.g., XTT). Although Vif is not required for HVI-1 RF replication in the CEM-SS cell-based assay described in Table 1, a cell protection assay with HIV-1 NL4-3 and MT-2 cells might be used to establish an antiviral screen that would incorporate Vif as a functional target. However, in our experience HIV-1 cell protection assays with HIV-1 NL4-3 and MT-2 cells exhibit higher interassay variability than the HIV-1 Rep assay. In addition, the HIV-1 Rep assay is not affected by colored or fluorescent compounds, which may significantly affect the rates of false-positive or false-negative results in an HTS using traditional cell protection assay formats. Furthermore, the flexibility of the HIV-1 Rep assay design allows the utilization of a large number of the cell type-virus strain combinations in an antiviral screen. Alternatively, cell protection assays are limited to a smaller set of virus strain-cell type combinations that result in highly cytopathic infections.

In this study, we describe an antiviral screening method that is a simple yet unique adaptation of existing assay methods that use reagents that can be easily acquired. The HIV-1 Rep assay offers several advantages over current antiviral assays, the most notable of which are compatibility with high-throughput screening and the inclusion of Vif as an antiviral target. To our knowledge, the HIV-1 Rep assay represents the first HIV-1 full-replication HTS to be executed on an industrial scale and has the potential to identify novel target inhibitors.


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ACKNOWLEDGMENTS
 
We thank the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, for providing the reagents listed in the Materials and Methods section.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Virology, Pfizer Global Research and Development, La Jolla Laboratories, 10777 Science Center Dr. (CB1), San Diego, CA 92121-1111. Phone: (858) 622-7692. Fax: (858) 526-4451. E-mail: wade.blair{at}pfizer.com. Back


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Antimicrobial Agents and Chemotherapy, September 2005, p. 3833-3841, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3833-3841.2005
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