Cell Type-Specific Anti-Human Immunodeficiency ... - Rega Institute

Vol. 36, No. 12

ANTIMIcROBLAL AGENTS AND CHEMOTHERAPY, Dec. 1992, p. 2628-2633

0066-4804/92/122628-06$02.00/0 Copyright © 1992, American Society for Microbiology

Cell Type-Specific Anti-Human Immunodeficiency Virus Type 1 Activity of the Transactivation Inhibitor Ro5-3335 MYRIAM WITVROUW,* RUDI PAUWELS, ANNE-MIEKE VANDAMME, DOMINIQUE SCHOLS, DIANE REYMEN, NAOHIKO YAMAMOTO, JAN DESMYTER, AND ERIK DE CLERCQ Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium Received 15 July 1992/Accepted 21 September 1992

The drug Ro5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-oneI inhibits human immunodeficiency virus type 1 (HIV-1) gene expression at the transcriptional level through interference with Tat-mediated transactivation (M.-C. Hsu, A. D. Schutt, M. Holly, L. W. Slice, M. I. Sherman, D. D. Richman, M. J. Potash, and D. J. Voisky, Science 254:1799-1802, 1991). We confirmed this specific inhibitory effect in a quantitative bioassay based on transactivation of a chimeric gene comprising the HlV-1 long terminal repeat promoter fused to the lacZ gene of Escherichua colt and transfected in a HeLa cell line expressing Tat. Ro5-3335 was found to inhibit HIV41 long terminal repeat-driven lIcZ gene expression at a 50%o inhibitory concentration of 0.5 ILM. The in vitro anti-HIV-1 activity of Ro5-3335 was highly dependent on the nature of the host cells. The highest selectivity index, 50, was found in phytohemagglutinin-stimulated peripheral blood lymphocytes. The selectivity index was between 1 and 10 in the CD4+ T-cell lines CEM, MOLT-4 (clone 8), and HUT-78. In MT-4 and MT-2 cells, Ro5-3335 had no inhibitory effect on HIV-i replication. The absence of anti-HIV-1 activity of Ro5-3335 in MT-4 cells was confirmed by using different parameters of virus replication and different multiplicities of infection. In persistently HIV-i-infected HUT-78/HIIILAI cells, Ro5-3335 failed to demonstrate any activity at subtoxic concentrations. The cytotoxicity of Ro5-3335 was significantly lower in peripheral blood lymphocytes than in the CD4+ T-cell lines. MATERIALS AND METHODS

Tat, one of the regulatory gene products encoded by human immunodeficiency virus (HIV), is a strong positive regulator essential for viral replication (1). The nuclear protein Tat increases gene expression directed by the HIV long terminal repeat (LTR) promoter and functions through interaction with the cis-acting Tat-responsive sequence (TAR) of the nascent RNA strain located immediately downstream of the transcription start site (5, 12, 14, 19, 21, 37, 44). Originally described as a transcriptional activator protein, Tat is now assumed to control gene expression at both the transcriptional and posttranscriptional levels (8, 9, 32, 34, 46, 47). Tat may be considered a suitable target for antiviral therapy of HIV infection, since an inhibitor of Tat may have the potential to keep the virus in its dormant state. Recently, the drug Ro5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one] was the first drug reported to inhibit transcriptional transactivation of HIV type 1 (HIV-1) by Tat (20). The compound did not inhibit the basal activity of the promoter. Ro5-3335 reduced the replication of HIV-1 in both acutely and chronically HIV-1-infected cells in vitro with a median inhibitory concentration (IC50) of 0.1 to 1 ,M. We used Ro5-3335 as a positive control in a transactivation assay, in which we could confirm its inhibitory effect on the transactivation of HIV-1. Furthermore, we evaluated the in vitro anti-HIV-1 and anti-HIV-2 activity and the cytotoxicity of Ro5-3335 in MT-4, MT-2, CEM, MOLT-4 (clone 8), and HUT-78 cells and phytohemagglutinin-stimulated peripheral blood lymphocytes (PBLs) as well as persistently HIV-1infected HUT-78 (HUT-78/IIIB/LAI) cells.

*

Compounds. Ro5-3335 (20) was synthesized by Wayne A. Spitzer and Frantz Victor at Lilly Research Laboratories, Indianapolis, Ind. Ro31-8959 {QC.AsnPhe[CH(OH)CH2N] DIQ.NHtBu, where QC is quinoline-2-carbonyl, DIQ is (4as, 8as)-decahydro-3(S)-isoquinolinecarbonyl, and tBu is tertbutyl} (35) was kindly provided by N. Roberts (Roche Products Limited, Welwyn Garden City, United Kingdom). Stock solutions (20 mg/ml) were prepared in dimethyl sulfoxide, divided into aliquots, and stored at -20°C under light-protected conditions. Cells. MT-4 cells (28), MT-2 cells (27), CEM cells (13), MOLT-4 cells (clone 8) (24), and HUT-78 cells (18) were grown and maintained in RPMI 1640 medium; and HLtat cells (10, 11, 41) were maintained in Dulbecco modified Eagle medium (4,500 mg/ml glucose). Both media were supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 0.1% sodium bicarbonate, and 20 ,ug of gentamicin per ml. MT-2 cells were cultured without sodium bicarbonate. HLtat cells contain stably integrated copies of

the HIV-1 LTR promoter linked to the synthetic first exon of the tat gene. This cell line was generated by cotransfection of HeLa cells with pSV2neo, which provides neomycin resistance, and pL3tat, which contains the HIV-1 LTR promoter, the synthetic first tat exon, and the simian virus 40 polyadenylation signal. HLtat cells were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Disease, and were contributed by Barbara K. Felber and George N. Pavlakis. Plasmid. The plasnid pHIVlacZ (25, 36) contains the lacZ gene driven by the HIV-1 LTR promoter. pHIVlacZ was obtained from Joseph J. Maio through the AIDS Research and Reference Reagent Program.

Corresponding author. 2628

VOL. 36, 1992

Viruses. The origins of the viruses were as described previously. HIV-1 strain IIIB/LAT (33) and HIV-2 strain ROD (7) were kindly provided by R. C. Gallo and L. Montagnier, respectively. Stocks were obtained from the culture supernatants of HIV-1- or HIV-2-infected cell lines (HUT-78/IIIB/LAI and MT-4/ROD, respectively) (31, 39). 13-Galactosidase (K-Gal) transactivation assay. (i) Transient transfectioi. protocol. HLtat cells were transfected with pHIVlacZ plasmid DNA by a modified calcium phosphate precipitation method. The calcium phosphate-DNA precipitate was prepared by using a Cellphect Transfection Kit (Pharmacia LKB, Uppsala, Sweden). To minimize the inherent variability of transfecting cells in monolayers, we transfected HLtat cells in bulk suspension before plating them in six-well plates at 5 x 105 cells per cup for drug testing. Exponentially growing HLtat cells were harvested by trypsinization. Cells were resuspended in medium containing serum, and aliquots that contained 107 cells were centrifuged at 800 x g for 5 min at 4°C. Each cell pellet was resuspended in 2 ml of calcium phosphate-DNA suspension containing 25 ,ug of DNA. After 15 min, the mixture was diluted with 10 ml of complete medium supplemented with 0.05 x Hanks balanced salt solution-6.25 mM CaCl2, and the cells were plated at 5 x 105 cells per cup in a six-well plate. The compound was added at various concentrations immediately after plating the cells. DNA uptake proceeded for 4 h, and then the cells were shocked by a 2.5-min glycerol treatment (15% in phosphate-buffered saline [PBS]) at room temperature. The cells were rinsed once with medium. Compound diluted in complete medium was added at various concentrations, and the cells were incubated for 48 h. (ii) Preparation of cell extracts. The medium was removed by gentle aspiration, and the monolayers were washed three times with PBS. Using a policeman and 1 ml of PBS, the cells were scraped into microcentrifuge tubes and were stored on ice until all the plates were processed. After recovering the cells by centrifugation at 12,000 x g for 10 s at room temperature, they were resuspended in 80 pl of 0.25 M Tris-HCl (pH 7.8) and disrupted by three cycles of freezing in dry ice-ethanol and thawing at 37°C. The cellular debris was precipitated at 12,000 x g for 5 min in a microcentrifuge and discarded, and the supematant containing the cell extracts was recovered. (iii) Assay for 1-Gal. The -Gal in the cell extracts was quantitated by a colorimetric assay as described by Sambrook et al. (38). Briefly, for each sample to be assayed, 30 ul of cell extract was mixed with 3.3 ,ullf a lOOx Mg solution (0.1 M MgCl2, 4.5 M 1-mercaptoethanol), 66.6 pl of a 4.5 x o-nitrophenyl-,B-galactopyranoside solution (4 mg/ml; dissolved in 0.1 M sodium phosphate [pH 7.5]), and 200 pl of 0.1 M sodium phosphate (pH 7.5). The reaction mixtures were incubated at 37°C for 2 h. The optical density was read at 420 nm. (iv) Protein assay. Protein concentrations in the cell extracts were determined by using an automated Bradford method (Bio-Rad). Antiviral assays. In MT4 and MT-2 cells, the anti-HIV-1 or anti-HIV-2 activities of the test compounds were determined by measuring the virus-induced cytopathogenicity (31). Briefly, MT-4 and MT-2 cells were suspended at 3 x 105 cells per ml and infected with HIV at 100, 50, and 10 times the 50% cell culture infective dose per ml. Immediately after infection, 100 pl of the cell suspension was placed into each well of flat-bottom 96-well microtiter trays containing various concentrations of the test compound. After 5 days of incubation at 37°C, the number of viable cells was deter-

ANTI-HIV-1 ACTIVITY OF Ro5-3335

2629

mined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method as described previously (30). In CEM, MOLT-4 (clone 8), HUT-78, and persistently HIV-1-infected HUT-78/IIIB/LAI cells cultured in the presence of various concentrations of the test compounds, anti-HIV activity was determined by monitoring viral antigen expression. All cells (2 x 105 cells per ml) were infected with HIV-1 and HIV-2 at 200 or 100 50% cell culture infective doses, respectively. Five days after infection of CEM and MOLT-4 (clone 8) cells with HIV-2 (strain ROD); 6 days after plating persistently HIV-1-infected HUT-78/ IIIB/LAI cells; 7 days after infection of CEM, MOLT-4 (clone 8), and MT-4 cells with strain IIIB/LAI;- 8 days after infection of PBLs with IIIB/LAI; and 12 days after infection of HUT-78 cells with strain ROD or strain IIIB/LAI, expression of HIV antigens was revealed by an indirect immunofluorescence procedure by using HIV type-specific polyclonal antibodies. The percentage of antigen-positive cells was determined by fluorescent-activated cell sorter (FACS) analysis (40, 48). The inhibitory effects of RoS-3335 on strain IIIWILAI replication in PBLs, MT-4, and persistently HIV-1-infected HUT-78/IIIB/LAI cells were monitored by the detection of HIV-1 p24 core antigens. PBLs and MT-4 cells were incubated with an excess 'of IIIB/LAI at 37°C for 45 min. All cells were washed three times with RPMI 1640 medium. Cells were plated at 5 x 105 cells per ml in the presence of various concentrations of the test compound. Eight days after plating PBLs (7 days for MT-4 and 6 days for HUT-78/IIIB/LAI cells), p24 antigens were detected by enzyme-linked immunosorbent assay (ELISA) (Du Pont). The supernatant of the HUT-78/IIIB/LAI cells incubated during 4 or 6 days in the presence of various concentrations of RoS-3335 was used to determine virus yield by titration on MT-4 cells. The cells from the same experiment were used to perform a giant cell assay in which MOLT-4 (clone 8) cells (106 cells per ml) were cultured with an equal number of HUT-78/IIIB/LAI cells in a 24-well plate. After a 24-h cocultivation period, the number of giant cells was recorded microscopically as described previously (2). Inhibition of DNA and RNA synthesis. CEM, MOLT-4 (clone 8), HUT-78, MT-4, and MT-2 cells were seeded in 96-well microtiter plates (100,000 cells per well). At that time, various concentrations of Ro5-3335 and 0.5 ,uCi of [methy1-3H]thymidine or [5-3H]uridine (25 to 30 Ci/mmol; Amersham) per well were added. After 24 h, the cells were lysed by adding an excess of water. The labeled DNA or RNA was precipitated with ice-cold 10% trichloroacetic acid and harvested with a semiautomated cell harvesting apparatus (GIBCO Life Technologies). Incorporation of labeled thymidine or uridine was measured by scintillation counting by using Ready Safe (Beckman) scintillation cocktail. RESULTS Inhibition of HIV transactivation. We established a quantitative bioassay (the 1-Gal transactivation assay) to visualize and quantify HIV-1 LTR transactivation by Tat in HLtat cells after transient transfection with pHIVlacZ. This bioassay is based on the transactivation by Tat of the lacZ gene through the HIV-1 LTR promoter. This Tat-dependent 13-Gal expression is measured by enzymatic staining of the cell extracts. RoS-3335, which was shown by Hsu et al. (20) to inhibit transactivation, displayed concentration-dependent activity in this assay (Fig. 1). 1-Gal expression was inhibited by 100% at RoS-3335 concentrations of 220 and 22 ,uM (50

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WITVROUW ET AL.

ANTIMICROB. AGENTS

CHEMOTHER.

TABLE 2. Anti-HIV-1 strain IIIW/LAI activity of Ro5-3335 in different host cells monitored by several viral parameters

_4.

0 -

C

0

u

IC50 (P.M) Cell line

0

Assay

*_l

Ro5-

Ro31-

3335

8959

Zod Zidovudie

a

4)

MT-4

MTP' Antigen expressionb p24c

HIJT-78/ IIIB/LM

Giant cell formationd >1

0 C0.

>1 >9 >9

0.003 ± 0.001 0.017 ± 0.006 0.026 ± 0.007

0

>44

Antigen expressionb p24C

>1 >44 >1 0.6 ± 0.3 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

4.

C 4P

a MTT, bromidebased viability assay. b Determined by FACS analysis. c Determined by ELISA. d Microscopic evaluation of syncytium (giant cell) formation with uninfected MOLT-4 (clone 8) cells.

CD

2 4., 0 C-

V-

4J

CD 4J

u

4i 0

tion was about 50-fold lower than the CC50 (Table 1). In CEM, MOLT-4 (clone 8), and HUT-78 cells, Ro5-3335 inhibited HIV-1 and HIV-2 replication at concentrations that were equal to or at most 10-fold less than the CC50 (Table 1). In MT-4 or MT-2 cells, no inhibition of HIV-induced cytopathicity could be found at subtoxic concentrations (CC50s, 1.3 and 0.9 FM, respectively). Also, no anti-HIV-1 or anti-HIV-2 activity was found in MT-4 cells when viral parameters other than viral cytopathogenicity were evaluated, i.e., antigen expression (FACS analysis) and p24 antigen production (ELISA) (Table 2). Even when the multiplicity of infection in MT-4 and MT-2 cells was lowered from 0.003 to 0.0003, no antiviral activity was observed microscopically at subtoxNc concentrations (data not shown). Up to concentrations that were toxic to the cells (55, 11, and 2.2 ,uM), Ro5-3335 failed to inhibit viral cytopathogenicity in MT-4 or MT-2 cells. When evaluated for its inhibitory effects on HIV-1 replication in persistently infected HUT-78/IIIILAI cells, Ro53335 at a subtoxic concentration of 0.35 ,uM reduced the HIV-1 progeny yield by no more than 1 log unit (Fig. 2). The protease inhibitor Ro31-8959 reduced the HIV-1 yield completely at 1.8 ,uM. No microscopically detectable cytotoxicity was observed at that concentration. A slight inhibitory effect in HIV-1-infected HUT-78 cells (20%) was achieved

0 0.22 2.2 22 220 Concentration of Ro5-3335 (yM) FIG. 1. Effect of Ro5-3335 on the expression of 1-Gal in the 13-Gal transactivation assay. Cytotoxicity was determined in the same cell cultures by measuring the amount of protein in an aliquot of the cell extracts. The bars represent the 3-Gal activities in cell extracts from transfected cells relative to the amount of surviving cells. Each datum point is the average of two separate experiments, each of which was performed in duplicate.

and 5 ,ug/ml, respectively) (Fig. 1). At 2.2 p,M, Ro5-3335 achieved 78% inhibition of transactivation. The IC50 of Ro5-3335 was 0.5 pM. The compound inhibited protein synthesis in transfected HLtat cells at concentrations down to 22 ,uM (50% cytotoxic concentration [CC50]; 105 pM). In vitro anti-HIV activity. Ro5-3335 was examined for its inhibitory effect on the cytopathogenicities of HIV-1 strain IIIB/LAI and HIV-2 strain ROD in acutely HIV-infected CD4+ cells, i.e., CEM, MOLT-4 (clone 8), HiUT-78, MT-4, and MT-2 cells and PBLs (Table 1). In PBLs, the IC50 of Ro5-3335 for HIV-1 replication was 2.6 ,uM. This concentra-

TABLE 1. Anti-HIV activity and cytotoxicity of Ro5-3335 in acutely infected CD4+ T-cell lines and PBLsa

IC50 (11MPb

IIIB/LAI

Cell line

Ro5-3335

CC50 (PLM) ROD

AZT

Ro5-3335

AZT

Ro5-3335

Zidovudine

0.4 ± 0.3 (6.5) CEM 0.003 ± 0.002 (28,000) 0.9 ± 0.1 (2.9) 0.002 ± 0.001 (37,000) 2.6 ± 1.0 67 ± 13 MOLT-4 (clone 8) 0.4 ± 0.2 (11) 0.0004 ± 0.0002 (260,000) 0.4 ± 0.2 (11) 0.0004 ± 0.0002 (260,000) 4.4 ± 2.1 104 ± 27 HUT-78 0.9 + 0.3 (1.4) 0.008 ± 0.003 1.3 ± 0.5 MT-4 >1.3 (1.3 (1.6 (