Actinomycin D (ActD) is generally used as an inhibitor of transcription. The agent is thought to intercalate DNA, which thereby prevents the progression of RNA polymerase.1,2 The agent is currently used to treat certain forms of cancer.3-5 Several studies have reported that ActD inhibits HIV-1 reverse transcription, and it has been proposed that ActD might inhibit HIV-1 replication as a potent anti-HIV-1 agent.6-9
HIV-1 reverse transcription is composed of multiple steps in the process.10 The initial step of HIV-1 reverse transcription involves annealing of host tRNALys to a primer-binding site near the 5′ end of HIV-1 genomic RNA. HIV-1 reverse transcriptase (RT) then catalyzes the synthesis of a short length of complementary DNA (cDNA) from the annealing site to the 5′ end of the viral genome. This short length of cDNA (minus strand) is translocated to the 3′ terminus of the genomic RNA, and then HIV-1 RT catalyzes the synthesis of a full-length cDNA of the HIV-1 genome. It is reported that ActD inhibits the process of the minus strand transfer and then prevents the RT reaction. The median inhibitory concentration (IC50) of ActD for the inhibition of the minus strand transfer was 2 to 100 μM.6-9
In our previous work, we evaluated the inhibitory effect of ActD on HIV-1 replication using several HIV-1-infected cells.11 HIV-1-infected cells were cultured for 7 days in the presence of different concentrations of ActD. High concentrations of ActD (>100 nM) showed a strong cytotoxic effect in all cells tested. Of interest and rather unexpectedly, lower concentrations of ActD (1-10 nM) enhanced HIV-1 replication 8- to 10-fold in MT-2 cells, a human T-cell leukemia virus type 1 (HTLV-1)-infected cell line. HIV-1 replication kinetic studies showed that virus replication was prolonged for 7 days and that ActD downregulated the expression of the HIV-1 receptors CD4 and CXCR4 on MT-2 cells. This increase in HIV-1 replication was not seen in MT-4 cells, another HTLV-1-infected cell line, or in non-HTLV-1-infected cells such as Jurkat cells and peripheral blood mononuclear cells (PBMCs). HTLV-1-infected cells produce infectious HTLV-1 virions.12,13 It is reported that HTLV-1 virions enhance HIV-1 replication in cell culture,14-18 and MT-2 cells spontaneously produce a higher level of infectious HTLV-1 virions in culture supernatant compared with MT-4 cells.13 Thus, it was reasonable to postulate that the action of ActD correlated with differences in production of HTLV-1 or HTLV-1-related proteins. A quantitative analysis showed that ActD treatment inhibited HTLV-1 production in culture supernatants of MT-2 cells; thus, the mechanism by which ActD enhances HIV-1 replication in MT-2 cells remains unclear.
HTLV-1-infected cells spontaneously produce multiple cytokines in tissue culture supernatants.19,20 Several proteins such as tumor necrosis factor-β (TNFβ; lymphotoxin), HTLV-1 Tax protein, and chemokines have been identified as HIV-inducing or-suppressing factors in HTLV-1 supernatants.21-23 The purpose of the present study was to attempt to identify which, if any, of the factors produced by MT-2 cells were responsible for the increase in HIV replication induced by ActD.
MATERIALS AND METHODS
Cells and Reagents
The MT-2,24,25 U1,26 ACH-2,27,28 and H-9/HTLV-IIIMN (H-9MN)29,30 cell lines and anti-Tax serum31,32 were obtained from the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases (Rockville, MD). MT-2 was obtained from Douglas Richman, U1 and ACH-2 were from Thomas Folks, H-9MN was from Robert Gallo, and anti-Tax serum was from Kuan-Teh Jeang. All cell lines were maintained in RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT), 10 mM of L-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Quality Biologic, Gaithersburg, MD) (RPMI-10) in T175 cm2 flasks (Sarstedt, Newton, NC) at 0.2 × 106 cells/mL for MT-2 cells and 0.1 × 106 cells/mL for U1, ACH-2, and H-9MN cells. PBMCs were isolated from heparinized whole blood from healthy donors using lymphocyte separation medium (ICN Biomedical, Aurora, OH) as described previously.33
ActD was purchased from Sigma (St. Louis, MO). Goat anti-IL-6, goat anti-TNFβ, goat anti-tissue inhibitor of metalloproteinase-1 (TIMP-1) IgG, goat normal IgG, recombinant interleukin (IL)-6, recombinant TNFβ, and recombinant TIMP-1 were purchased from R&D Systems (Minneapolis, MN). Goat anti-Cystatin C IgG was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Generation of Conditioned Medium and Evaluation of HIV-Inducing Activity
MT-2 cells (4 × 106 cells/mL) were pretreated with 0 or 200 nM of ActD for 4 hours at 37°C. To remove unincorporated ActD, the treated cells were washed 3 times with warmed RPMI-10. The pretreated MT-2 cells were then cultured for 72 hours at 37°C. Crude supernatants were clarified by low-speed centrifugation (500 g for 5 minutes) and filtrated through a Millex-GV 0.22-μm PVDF membrane (Millipore, Billerica, MA) to remove cellular debris. To pellet HTLV-1 virions in the conditioned medium, ultracentrifugation (24,000 rpm for 2 hours at 4°C) was performed using 20% sucrose (wt/vol) in 150 mM of sodium chloride (NaCl) and 50 mM of HEPES, pH 7.4.11
Evaluation of HIV-inducing activity in the culture supernatants was determined as follows. Fresh MT-2 cells and phytohemagglutinin (PHA)-stimulated PBMCs were infected with recombinant HIV-1NL4.3 as previously described.11 The infected MT-2 cells, infected PBMCs, U1 cells, ACH-2 cells, and H-9MN cells were cultured in the presence of 1:4 diluted supernatants derived from untreated MT-2 cells (Sup0) or MT-2 cells treated with 200 nM of ActD (Sup200) for 7 days at 37°C. MT-2 cells and PBMCs were cultured at 0.2 × 106 cells/mL and 1 × 106 cells/mL, respectively. U1, ACH-2, and H-9MN cells were cultured at 0.1 × 106 cells/mL. HIV-1 induction was determined by measuring the amount of HIV-1 core protein, p24, in culture supernatants. Levels of p24 were monitored by a p24 antigen capture assay kit (Beckman-Coulter, Miami, FL).
Isolation of HIV-Inducing Factors From Culture Supernatants
Isolation of HIV-inducing factors from Sup200 was performed as follows. A total of 1.4 × 109 MT-2 cells were pretreated with 200 nM of ActD in RPMI-10 at 4 × 106 cells/mL at 37°C for 4 hours. After washing the cells with RPMI-1640 containing 0.1% bovine serum albumin (RPMI-BSA; Sigma), the pretreated cells were cultured for 72 hours at 0.4 × 106 cells/mL in RPMI-BSA with 10 mM of L-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. A bulk scale (total of 3.5 L) of Sup200 was centrifuged at 500 g for 5 minutes at 4°C, followed by filtration through 0.2-μm pore membranes (Corning, Park Ridge, IL). To concentrate Sup200, the pH of the culture supernatants was adjusted to 4.5 using 1 N of hydrochloric acid (HCl) and diluted by 2-fold with buffer A (50 mM of sodium acetate [NaOAc] buffer, pH 4.5) with a protease inhibitor cocktail for use with mammalian cells (Sigma). All purification procedures (Fig. 1) were carried out at 4°C. Chromatography was performed using the BioLogic LP system (Bio-Rad, Hercules, CA). The diluted Sup200 was applied on a 35-mL SP-Sepharose Fast Flow column (Amersham Pharmacia, Piscataway, NJ) equilibrated with buffer A containing 75 mM of NaCl at a flow rate of 3 mL/min. After the column was washed with buffer A containing 75 mM of NaCl, the bound proteins were eluted with buffer A containing 600 mM of NaCl. The eluted proteins were immediately dialyzed against buffer B (20 mM of Tris-HCL, pH 8.0, and 1 mM of EDTA). After dialysis, the pooled fractions were loaded onto a 35-mL Q-Sepharose Fast Flow column (Amersham Pharmacia) equilibrated with buffer B. The unbound (flow-through) fraction was collected (pool 1, see Fig. 1) and stored at −20°C. The column was then washed with buffer B, and bound proteins were eluted by a linear gradient increase of NaCl concentration from 0 to 200 mM in 300 mL of buffer B at a flow rate of 1.5 mL/min. Fractions of 3 mL were collected, and every other fraction (100 μL) was then diluted 1:4 with RPMI-10 and filter-sterilized using a Millex-GV filter (Millipore). The HIV-inducing activity in a 1:8 dilution of the fractions was analyzed by culturing with U1 cells in 96-well flat-bottom plates. After 3 days of incubation, the HIV-inducing activity was determined by measuring the concentration of p24 in the culture supernatants. The fractions containing HIV-inducing activity were pooled (pool 2, see Fig. 1). The pH of pool 2 was adjusted to 5.0, diluted with 10 vol of buffer C (50 mM of NaOAc buffer, pH 5.0), and directly applied to a HiTrap SP-HP column (1 mL; Amersham Pharmacia) equilibrated with buffer C containing 75 mM of NaCl at a flow rate of 1 mL/min. The flow-through fractions were collected, the pH of the collected flow-through was adjusted to 4.5 with 1 N of HCl, and the collected flow-through was then applied to a HiTrap SP-HP column (1 mL) equilibrated with buffer A containing 75 mM of NaCl. Bound proteins were eluted in a 0.5-mL fraction by a linear gradient increase of the NaCl concentration from 75 to 600 mM in buffer A in 20 mL at a flow rate of 0.5 mL/min. Fractions containing HIV-inducing activity from the separation were pooled and immediately dialyzed against buffer D (20 mM of Tris-HCl, pH 7.0, 10 mM of NaCl, and 1 mM of EDTA). After dialysis, the pooled fraction was loaded to a 1-mL UNO-Q1 chromatography column (Bio-Rad) using the BioLogic DuoFlow Pathfinder Chromatography system (Bio-Rad). Bound proteins were eluted by a linear gradient increase of the NaCl concentration from 0 to 200 mM in buffer D at 3.5 mL/min. The active fractions were pooled and stored at −80°C.
The pool 1 fraction was applied to a 1-mL HiTrap SP-HP column in buffer B, and the bound protein was eluted using a linear gradient increase of the NaCl concentration from 0 to 600 mM in buffer B. The fractions containing HIV-inducing activity were diluted with 10 vol of buffer B and then applied on a 1-mL UNO-S1 chromatography column (Bio-Rad) using the BioLogic DuoFlow Pathfinder Chromatography system. Bound proteins were eluted by a linear gradient increase of NaCl concentration from 0 to 200 mM in buffer D at 3.5 mL/min. The active fractions were pooled and stored at −80°C. The amounts of protein in the pooled fractions were determined using the bicinchoninic acid (BCA) protein assay kit (Pierces Biotechnology, Rockford, IL).
Protein Identification by Microcapillary Chromatography and Ion Trap Mass Spectrometry
Fractions from the UNO-S1 or UNO-Q1 chromatography column were separated by 12% Bis-Tris polyacrylamide gel electrophoresis (PAGE; Invitrogen) in 1 × 3-(N-morpholino) propanesulfonic acid buffer (Invitrogen). Proteins were stained with Coomassie Brilliant blue R250 (Bio-Rad). Protein bands were digested with trypsin, and the peptides were extracted as previously described.34 The samples were desalted with C18 Zip Tips (Millipore) before analysis by microcapillary reverse-phase liquid chromatography (μRPLC) using an Agilent 1100 capillary liquid chromatography system (Agilent Technologies, Palo Alto, CA) coupled on-line to an ion trap (IT) mass spectrometer (LCQ Deca XP; Thermo Electron, San Jose, CA). Reverse-phase separations were performed using 75 m id Å × 360 m od Å × 10-cm-long capillary columns (Polymicro Technologies, Phoenix, AZ) that were slurry-packed in-house with 3 μm, 300 Å of pore size C-18 silica-bonded stationary phase (VYDAC/The Separations Group). After being injected with 7 nL of sample, the column was washed for 20 minutes with 98% solvent E (0.1% formic acid in water, vol/vol) and peptides were eluted using a linear gradient of 2% solvent F (0.1% formic acid in 100% acetonitrile, vol/vol) to 85% solvent F for 160 minutes at a constant flow rate of 500 nL/min.
The IT mass spectrometry (MS) was operated in a data-dependent mode in which each full MS scan was followed by 3 tandem MS (MS/MS) scans, where the 3 most abundant peptide molecular ions were dynamically selected from the prior scan for collision-induced dissociation (CID) using a normalized collision energy of 8%. Dynamic exclusion was used to prevent redundant acquisition of peptides previously selected for MS/MS. The temperatures of the heated capillary and electrospray voltage were 160°C and 1.5 kV, respectively.
Tandem MS spectra were searched against the Expert Protein Analysis System's human proteomic database (http://www.expasy.org) with SEQUEST operating on an 18-node Beowulf cluster (Thermo Electron).
Western Blot Analysis
Western blotting was performed as previously described.11 Samples containing a total of 1 μg of protein were concentrated using the StrataClean Resin (Stratagene, La Jolla CA), fractionated by 10% sodium dodecyl sulfate (SDS) Bis-Tris Gels (Invitrogen) and then transferred onto nitrocellulose membranes. Signals on the membranes were detected using the ECL-plus kit (Amersham Pharmacia).
Preparation of Immunodepleted Conditioned Medium (CM) and Evaluation of HIV-Inducing Activity
Cytokines in crude supernatants were depleted as follows. A total of 0.8 mL of supernatants was incubated with normal IgG or anticytokine IgG at 4°C for 2 hours with rocking, followed by immunoprecipitation (IP) using protein G plus agarose (Santa Cruz Biotechnology) at 4°C for 2 hours. The suspension was centrifuged at 300 g at 4°C for 1 minute to pellet the IP complexes containing protein G agarose. The resulted supernatants were added to cell cultures at a final dilution of 1:8, and the HIV-inducing activity was measured using U1 cells.
Flow Cytometric Analysis
Flow cytometric analyses were performed as described previously.11 Using fluorescein isothiocyanate (FITC)-conjugated anti-CD4 and phycoerythrin (PE)-conjugated anti-CXCR4, antibodies were obtained from BD Biosciences (San Diego, CA).
Enzyme-Linked Immunosorbent Assay
Levels of IL-6 and TNFβ production in culture supernatants were quantified using enzyme-linked immunosorbent assay (ELISA) kits for human IL-6 and human TNFβ (R&D Systems). HTLV-1 production was determined by measurement of p19 antigen using a p19 antigen ELISA kit (Zeptometix Corporation, Buffalo, NY).
Northern Blot Analysis
Total cellular RNA was extracted from 10 × 106 cells using TRIzol Reagent (Invitrogen). A total of 20 μg of RNA was loaded per lane and resolved in 1.2% agarose/formaldehyde gel; using the Turbo Blotter Systems (Schleicher & Schuell BioScience, Keene, NH), the product was transferred to a Nytran SuPerCharge Nylon membrane. After transfer, the RNA was cross-linked onto the membranes by ultraviolet irradiation. Using the Prime-It RmT Random Primer labeling kit (Stratagene), gene-specific cDNA fragments were labeled with [α-32P] deoxycytidine triphosphate (dCTP; Amersham Pharmacia). Probe sizes of IL-6 and TNFβ were 421 base pairs (bp) and 442 bp, respectively. Membranes were prehybridized for 1 hour and then hybridized at 56°C overnight with the probes in the QuikHyb hybridization solution (Stratagene). After hybridization, the membranes were washed at 56°C for 30 minutes under a low-stringency condition (2 × SSC, 0.1% SDS), followed by a high-stringency condition (0.1 × SSC, 0.1% SDS); they were then exposed to Kodak BioMax MS-1 film (Eastman Kodak Company, Rochester, NY) at −80°C overnight. The intensity of the bands was quantified using a bioimaging analyzer (Bas 1000; Fuji Film Medical Systems USA, Stanford, CT).
Differences in cytokine production between the untreated and the ActD-treated MT-2 cells were determined by using the paired t test of the StatView program (Abacus Concepts, Berkeley, CA).
Evaluation of HIV-Inducing Activity in Culture Supernatants From ActD-Treated MT-2 Cells
We have reported that low concentrations of ActD (1-10 nM) increased HIV-1 replication by 8- to 10-fold in MT-2 cells but not in PBMCs or Jurkat cells.11 In this study, to characterize the effect of ActD on HIV-1 replication further, PBMCs as well as MT-2 and Jurkat cells were pulse-treated with 0, 1, or 200 nM of ActD for 4 hours and then infected with HIV-1NL4.3, followed by incubation for 7 days. The pulse treatment with 200 nM (but not 1 nM) of ActD enhanced HIV-1NL4.3 replication by 8-fold in MT-2 cells compared with that seen in untreated MT-2 cells (Fig. 2A). This enhancement was not seen in PBMCs or Jurkat cells (data not shown). In contrast, another transcriptional inhibitor, α-Amanitin, did not show the enhancement of HIV in MT-2 cells (data not shown).
It is known that HTLV-1-infected cells produce soluble proteins, including cytokines and Tax protein in culture supernatants.21-23,35,36 To define whether changes in any of these soluble proteins were responsible for HIV induction in the pulse-treated MT-2 cells, culture supernatants from untreated and ActD-treated MT-2 cells were generated as follows. MT-2 cells were pulse-treated with 0 or 200 nM of ActD for 4 hours and then washed to remove unincorporated ActD. The cells were then cultured for 3 days, and the cell-free CM was collected. No significant cytotoxicity was seen between the untreated and pulse-treated MT-2 cells during the incubation; the viability of untreated and treated cells was 94% ± 1.4% and 92% ± 1.8%, respectively. To evaluate the impact of CM on HIV-1 replication, fresh MT-2 cells were infected with HIV-1NL4.3 and then cultured in the presence of 1:4 diluted CM. The CM from the ActD-treated MT-2 cells (Sup200) showed a 7-fold upregulation of HIV-1 replication in MT-2 cells (see Fig. 2B); in contrast, the CM derived from MT-2 cells (Sup0) had no impact on virus replication. Because CM from untreated MT-2 cells increased HIV-1 replication in HIV-1-infected PBMCs,22,23 we also evaluated the impact of Sup0 and Sup200 on HIV-1 replication in HIV-1NL4.3-infected PBMCs. Sup0 and Sup200 showed partial inhibition rather than induction of HIV-1 replication. To evaluate the HIV-inducing activity in Sup0 and Sup200 further, chronically infected cell lines and U1, ACH-2, and H-9MN cells were also tested. U1 and ACH-2 cells are latently infected monocyte and T-cell lines, respectively. U1 and ACH-2 cells both produce a high level of HIV-1 replication on stimulation with TNFα or phorbol myristate acetate (PMA).26-28 H-9MN cells are a cell line that constitutively produces HIV-1 in culture without stimulation.29,30 Of note, Sup0 induced virus replication by 150-fold in U1 cells, and Sup200 further increased HIV-1 replication by 1200-fold (see Fig. 2B). In the case of ACH-2 cells, Sup0 enhanced HIV-1 replication by 24-fold, but Sup200 increased it only by 5-fold. Meanwhile, no changes were seen in HIV-1 replication in H-9MN cells in the presence of Sup0 or Sup200 (see Fig. 2B).
To assess differences in HIV-1 induction between Sup0 and Sup200, U1 cells were cultured for 3 or 7 days in the presence of Sup0 or Sup200 diluted to 1:4. Sup0 induced maximum HIV-1 replication after 3 days of incubation, but the replication was not further increased at 7 days (see Fig. 2C). In contrast, Sup200 constitutively induced HIV-1 replication for 7 days.
It has been reported that HTLV-1 virus activates HIV-1 replication.14-18 Thus, we also evaluated the impact of ActD treatment on HTLV-1 production in MT-2 cells. The concentrations of HTLV-1 p19 antigen in Sup0 and Sup 200 were 60 ± 9.3 ng/mL and 10 ± 2.7 ng/mL, respectively; therefore, ActD treatment significantly inhibited HTLV-1 production from MT-2 cells (P = 0.006). In addition, the depletion of HTLV-1 virions using ultracentrifugation had no effect on HIV-1 induction by Sup200 (data not shown). Thus, the HIV-inducing activity in Sup200 does not seem to be associated with HTLV-1. To elucidate the impact of HTLV-1 on ActD-induced HIV-1 production further, Tax protein in Sup200 was immunoabsorbed through the use of anti-Tax serum. The immunoabsorption had no effect on HIV-1 induction in U1 and ACH-2 cells (data not shown).
A recent report described the activation of the HIV promoter long terminal repeat (LTR) by ActD.37 To define whether residual ActD in the Sup200 was sufficient to induce HIV-1 replication, U1 or ACH-2 cells were cultured in the presence of different concentrations (0-100 nM) of ActD for 7 days. Untreated U1 cells produced low concentration of p24 (0. 33 ± 0.04 ng/mL) in culture. Of interest, 1 nM of ActD increased HIV-1 replication by 33-fold (10.9 ± 2.0 ng/mL of p24 production; see Fig. 2D). Further induction was not seen with increases in the concentration of ActD, however. One hundred nanometers of ActD showed a strong cytotoxic effect and decreased HIV-1 production in U1 cells. Similarly, ACH-2 cells were cultured in the presence of ActD. A low concentration of ActD (3 nM) induced virus replication by 5-fold (see Fig. 2D). Therefore, low concentrations of ActD by themselves enhanced HIV-1 replication in latently infected monocyte and T-cell lines. The enhancement was not comparable to the level of induction by Sup200, however. These results suggested that most HIV-inducing activity in Sup200 was not caused by the remaining small amounts of ActD in Sup200. Thus, Sup200 seems to contain factor(s) associated with the high level of HIV-1 replication in U1 cells.
Identification of HIV-Inducing Factors in Sup200
The approximate molecular size of the HIV-inducing factor(s) in Sup200 was first determined by means of gel filtration with S300 HR chromatography (Amersham Pharmacia). Activity was observed in fractions between 25 and 75 kd (data not shown). Heating of Sup200 at 95°C for 30 minutes diminished the HIV-inducing activity (data not shown). These results suggested that the factor(s) might be protein rather than peptide in nature.
To isolate the HIV-inducing factor(s) in Sup200, we used a sequential chromatographic approach that involved SP-Sepharose chromatography (cationic exchange), Q-Sepharose chromatography (anion exchange), and HPLC (see Fig. 1). Table 1 summarizes recoveries and yields of each purification step. After each separation, the HIV-inducing activity in each of the pooled fractions was measured by p24 antigen capture assays using U1 cells.
A total of 3.5 L of Sup200 was concentrated using SP-Sepharose chromatography at pH 4.5. The concentrated Sup200 was then subjected to Q-Sepharose chromatography at pH 8.0. HIV-inducing activity in the concentrated Sup200 was separated into 2 fractions: the unbound fraction (flow-through, pool 1) and the bound fraction eluted at 50 to 150 mM of NaCl (pool 2) (see Fig. 1). Pool 1 was applied to a HiTrap SP-HP Sepharose column equilibrated at pH 8.0. Active fractions from the HiTrap SP-HP Sepharose were pooled, and loaded onto a UNO-S1 HPLC column equilibrated at pH 8.0. The HIV-inducing activity predominantly eluted at 100 to 135 mM of NaCl (Fig. 3A). Pool 2 was applied to a HiTrap SP-HP column equilibrated at pH 5.0. The HIV-inducing activity was found in the flow-through fraction. The pH of the flow-through fraction was adjusted to 4.5, and the flow-through fraction was applied to a HiTrap SP-HP column equilibrated at pH 4.5. Bound proteins were eluted from the column, and fractions containing HIV-inducing activity were subjected to UNO-Q1 HPLC at pH 7.0. The HIV-inducing activity was eluted from the UNO-Q1 HPLC as a single peak at 100 to 120 mM of NaCl (see Fig. 3C). Fraction 33 of the UNO-S1 HPLC (see Fig. 3A) and fraction 29 of the UNO-Q1 HPLC (see Fig. 3C) were then subjected to 12% SDS-PAGE, followed by protein detection with Coomassie Brilliant blue staining (see Figs. 3B, D). Individual protein bands were excised from the stained gels and digested with trypsin. The resulting tryptic peptides were analyzed by μRPLC and MS/MS. Tryptic peptides from bands 1, 2, 3, and 4 in fraction 33 were unambiguously identified as arising from BSA, TIMP-1, TNFβ, and Cystatin C, respectively (see Fig. 3B). Band 5 in fraction 29 was identified as IL-6 (see Fig. 3D). To confirm the presence of TIMP-1, TNFβ, Cystatin C, and IL-6 in the pooled fractions from UNO-S1 and UNO-Q1 HPLC, Western blot analyses were performed. The Western blot analyses showed a single band of TIMP-1 and Cystatin C at 30 kd and 13 kd (data not shown), respectively, and a single band of TNFβ at 25 kd in the pooled fractions from UNO-S1 HPLC (Fig. 4A). Using an antibody to IL-6, the pooled fractions of UNO-Q1 HPLC showed 2 bands at 30 kd and 28 kd (see Fig. 4B).
Impact of Tumor Necrosis Factor-β and Interleukin-6 on HIV-1 Replication
To determine the relative roles of TNFβ, IL-6, TIMP-1, and Cystatin C in culture supernatants on HIV-1 replication in U1 cells, each protein was immunodepleted using anti-TNFβ, anti-IL-6, anti-TIMP-1, or anti-Cystatin C antibodies. As a control for immunodepletion, each Sup0 and Sup200 was treated with normal IgG. Sup0 treated with normal IgG induced 38.3 ± 4.3 ng/mL of HIV-1 replication in U1 cells (Fig. 5A). This induction was immunoabsorbed 97% by anti-TNFβ but not by anti-IL-6 (see Fig. 5A). In contrast, Sup200 treated with normal IgG induced 182 ± 25 ng/mL of HIV-1 induction in U1 cells (see Fig. 5B). Immunodepletion of TNFβ or IL-6 from Sup200 inhibited the induction by 90% or 62%, respectively. Immunodepletion of TNFβ and IL-6 showed further inhibition of HIV induction by 98%. Neither anti-TIMP-1 nor anti-Cystatin C antibodies absorbed HIV-inducing activity in Sup 0 and Sup200 (data not shown). These results indicated that the HIV-inducing factor in Sup0 was TNFβ, whereas the factors in Sup200 were TNFβ and IL-6.
To assess the impact of ActD treatment on the production of TNFβ and IL-6 from MT-2 cells, an ELISA was performed, indicating that the concentration of IL-6 in Sup200 was increased by 8-fold compared with that in Sup0 (Table 2). In contrast, TNFβ in Sup200 was 80% lower than that in Sup0. These results suggest that ActD treatment increases IL-6 production, although decreasing the production of TNFβ in MT-2 cells.
To determine whether a combination of TNFβ and IL-6 induces HIV-inducing activity comparable to that of Sup200, a cytokine cocktail was constituted using recombinant TNFβ and IL-6. Sup 200 diluted at 1:4 was used as a positive control. Because the diluted Sup200 contained 5 ng/mL of TNFβ and 1 ng/mL of IL-6, the cytokine cocktail was constituted using the same concentration of each recombinant cytokine. The cytokine cocktail showed an effect comparable to that of Sup200 on HIV-1 induction from U1 cells. Either cytokine by itself did not induce similar levels of HIV-1 replication to those induced by Sup200, however (see Fig. 5E). Therefore, TNFβ and IL-6 were the HIV-inducing factors in Sup200, and a combination of both cytokines synergistically induced HIV-1 replication in U1 cells.
Immunodepletion of TNFβ from Sup0 and Sup200 abolished the ability to induce HIV-1 replication in ACH-2 cells, but immunodepletion of IL-6 did not change the induction in Sup0 or Sup200 (see Figs. 5C, D). Because recombinant TNFβ but not recombinant IL-6 induced HIV-1 replication in ACH-2 cells (see Fig. 5E) and no detection of IL-6 receptor38 on ACH-2 cells was observed by fluorescence-activated cell sorting (FACS) analysis (data not shown), it seemed that only TNFβ in Sup0 and Sup200 induced HIV-1 replication in ACH-2 cells.
Actinomycin D Activates Interleukin-6 Gene but Not Tumor Necrosis Factor-β in MT-2 Cells
Although ActD is generally used as a transcriptional inhibitor, the protein levels of IL-6 in Sup200 were 7-fold higher than those in Sup0 (see Table 2). To define the impact of ActD on IL-6 and TNFβ gene expression in MT-2 cells, Northern blot analysis was performed. Total cellular RNA was isolated from untreated and ActD-treated MT-2 cells that had been cultured for 24, 48, or 72 hours. Northern blot analysis showed constitutive expression of TNFβ and IL-6 messenger RNA (mRNA) in MT-2 cells (Fig. 6A). No significant change was seen in the expression of the TNFβ gene between untreated and ActD-treated MT-2 cells during 72 hours of incubation. In contrast, the expression of the IL-6 gene in ActD-treated cells was seen to be upregulated by 7-fold after 24 hours of incubation, which was maintained by 48 hours. The expression level was decreased by 72 hours. Because the concentration of extracellular TNFβ protein was 80% lower in ActD-treated cells than in controls (see Table 2), without changes in TNFβ transcripts, it was postulated that ActD might suppress at a posttranscriptional level. To elucidate whether ActD treatment decreased the secretion or the translation of proteins, the concentration of intracellular cytokines was compared between untreated and ActD-treated MT-2 cells (see Figs. 6B, C). As shown, the ActD treatment of MT-2 cells did not change intracellular levels of TNFβ but did significantly decrease extracellular levels of TNFβ (see Fig. 6B). In contrast, the ActD treatment increased intracellular and extracellular levels of IL-6 production (see Fig. 6C). Thus, the increase of HIV-1 replication in U1 cells by Sup200 seems to be attributable to a combination of an increase in the expression of IL-6 at the RNA and protein level and a decrease in the secretion of TNFβ from MT-2 cells.
In the present study, we have demonstrated that the ActD-mediated induction of HIV replication seems to be secondary to activation of the IL-6 gene. Given that ActD has generally been used as an inhibitor of transcription, this finding was unanticipated. HTLV-1-infected cell lines such as MT-2 cells spontaneously release multiple cytokines in tissue culture.19,20 It has been reported that MT-2 supernatants can enhance or suppress HIV-1 replication in T cells.21-23 In this study, we evaluated the impact of ActD on the production of cytokines and soluble proteins associated with HIV-1 replication. IL-6 and TNFβ were identified as soluble factors responsible for the induction of HIV-1 by supernatants of MT-2 cells treated with ActD. ActD treatment increased production of IL-6 from MT-2 cells in association with an increase in the transcription of IL-6 mRNA. In contrast, ActD treatment decreased production of TNFβ from MT-2 cells without changes in gene expression as determined by Northern blot analysis or in intracellular concentrations of TNFβ protein as determined by ELISA. To our knowledge, this is the first report that ActD could suppress secretion of a cytokine without causing changes in gene expression.
We have shown that TNFβ and IL-6 synergistically induce a high level of HIV-1 replication in U1 cells but not in ACH-2 cells. These findings complement earlier data on the synergistic effect between TNFα and IL-6 on HIV-1 replication in U1 cells.39 This latter effect involves a posttranslational modulation in HIV-1 gene activation. TNFα and TNFβ belong to the TNF superfamily and share common receptors (TNFR-I and TNFR-II).40,41 Thus, the mechanism(s) of HIV-1 induction by TNFβ and IL-6 may be similar to that seen with TNFα and IL-6. FACS analysis revealed high levels of expression of the IL-6 receptor38 on U1 cells but not on ACH-2 cells, thus offering an explanation as to why the synergistic effect between TNFβ and IL-6 was not seen in ACH-2 cells.
It is reported that the IL-6 gene can be activated via a Tax-dependent pathway.42,43 In this study, we showed that ActD treatment increased the expression of IL-6 genes but decreased the production of HTLV-1. Thus, it is unlikely that HTLV-1 plays an important role in the induction of HIV by ActD. Given the well-described character of ActD to interfere with gene transcription, it is possible that ActD works in the setting to block production of a repressor of IL-6 transcription. Studies are underway to address this possibility. Such studies could help to identify novel regulatory pathways of cytokine gene activation.
In this study, we showed that not only culture supernatants from MT-2 but low concentrations of ActD by themselves enhanced HIV-1 replication in U1 and ACH-2 cells. Given that U1 cells produce noninfectious HIV-1 because of mutations in the HIV-1 trans-activator protein (Tat),44 it is unlikely that the enhanced HIV-1 replication by Sup200 was attributable to an increased rate of reinfection. Although these same phenomena were not seen with PBMCs, the data generated here provide insight as to the regulation of HIV-1 replication.
ActD is generally used as a transcriptional inhibitor. The agent is currently used in the treatment of certain forms of cancer.3-5 In the present study, we have shown that the transcriptional inhibitor enhanced IL-6 transcription and subsequently increased IL-6 production. Although no change was seen in TNFβ transcription, ActD treatment suppressed TNFβ secretion. This change in cytokine profile resulted in an increase in HIV-1 replication from latently infected cells. We have also shown that low concentrations of ActD by themselves induce HIV-1 replication in latently infected cells. Elucidating the mechanism of activation may help to define a strategy to deplete the HIV-1 reservoir (latently infected cells).45-47
The authors thank A. Fauci for guidance and support. They are indebted to C. Watkins, C. Yeager, D. Peiot, M. Murphy, G. DeGray, and T. Brann for technical support. They are grateful to S. Nagasawa for guidance in isolating proteins.
1. Perry RP, Kelley DE. Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species. J Cell Physiol
2. Sobell HM. Actinomycin and DNA transcription. Proc Natl Acad Sci USA
3. Craft AW, Cotterill SJ, Bullimore JA, et al. Long-term results from the first UKCCSG Ewing's Tumour Study (ET-1). United Kingdom Children's Cancer Study Group (UKCCSG) and the Medical Research Council Bone Sarcoma Working Party. Eur J Cancer
4. Green DM, Breslow NE, Beckwith JB, et al. Comparison between single-dose and divided-dose administration of dactinomycin and doxorubicin for patients with Wilms' tumor: a report from the National Wilms' Tumor Study Group. J Clin Oncol
5. Estlin EJ, Veal GJ. Clinical and cellular pharmacology in relation to solid tumours of childhood. Cancer Treat Rev
6. Davis WR, Gabbara S, Hupe D, et al. Actinomycin D inhibition of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase and nucleocapsid protein. Biochemistry
7. Guo J, Wu T, Bess J, et al. Actinomycin D inhibits human immunodeficiency virus type 1 minus-strand transfer in in vitro and endogenous reverse transcriptase assays. J Virol
8. Jeeninga RE, Huthoff HT, Gultyaev AP, et al. The mechanism of actinomycin D-mediated inhibition of HIV-1 reverse transcription. Nucleic Acids Res
9. Gabbara S, Davis WR, Hupe L, et al. Inhibitors of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Biochemistry
10. Telesnitsky A, Goff SP. Reverse transcriptase and the generation of retroviral DNA. In: Coffin JM, Hughes SH, Varmus HE, eds. Retroviruses
. New York: Cold Spring Harbor Laboratory Press; 1977:121-160.
11. Imamichi T, Murphy MA, Joseph WA, et al. Actinomycin D induces high-level resistance to thymidine analogs in replication of human immunodeficiency virus type 1 by interfering with host cell thymidine kinase expression. J Virol
12. Koyanagi Y, Hinuma Y, Schneider J, et al. Expression of HTLV-specific polypeptides in various human T-cell lines. Med Microbiol Immunol (Berl)
13. Dhib-Jalbut S, Hoffman PM, Yamabe T, et al. Extracellular human T-cell lymphotropic virus type I Tax protein induces cytokine production in adult human microglial cells. Ann Neurol
14. Boehnlein E, Siekevitz M, Ballard DW, et al. Stimulation of the HIV-1 enhancer by the HTLV-I Tax
gene product involves the action of inducible cellular proteins. J Virol
15. Cheng H, Tarnok J, Parks W. Human immunodeficiency virus type 1 genome activation induced by human T-cell leukemia virus type 1 Tax protein is through cooperation of NF-κB and Tat. J Virol
16. Zack JA, Cann AJ, Lugo JP, et al. HIV-1 production from infected peripheral blood T cells after HTLV-I induced mitogenic stimulation. Science
17. Lindholm PF, Marriott SJ, Gitlin SD, et al. Induction of nuclear NF-κB DNA binding activity after exposure of lymphoid cells to soluble Tax1 protein. New Biol
18. Marriott SJ, Lindholm PF, Reid RL, et al. Soluble HTLV-I Tax1 protein stimulates proliferation of human peripheral blood lymphocytes. New Biol
19. Hollsberg P, Hafler DA. Pathogenesis of diseases induced by human lymphotropic virus type I infection. N Engl J Med
20. Matsuoka M. Human T-cell leukemia virus type I and adult T-cell leukemia. Oncogene
21. Matsuyama T, Hamamoto Y, Yoshida T, et al. Effect of culture supernatant of MT-2 cells on human immunodeficiency virus-producing cells, MOLT-4/HIVHTLV-IIIB cells. Jpn J Cancer Res
22. Moriuchi H, Moriuchi M, Fauci AS. Factors secreted by human T lymphotropic virus type I (HTLV-I)-infected cells can enhance or inhibit replication of HIV-1 in HTLV-I-uninfected cells: implications for in vivo coinfection with HTLV-I and HIV-1. J Exp Med
23. Moriuchi H, Moriuchi M. In vitro induction of HIV-1 replication in resting CD4(+) T cells derived from individuals with undetectable plasma viremia upon stimulation with human T-cell leukemia virus type I. Virology
24. Harada S, Koyanagi Y, Yamamoto N. Infection of HTLV-III/LAV in HTLV-carrying cells MT-2 and MT-4 and application in a plaque assay. Science
25. Haertle T, Carrera CJ, Wasson DB, et al. Metabolism and anti-human immunodeficiency virus-1 activity of 2-halo-29, 39-dideoxyadenosine derivatives. J Biol Chem
26. Folks TM, Justement J, Kinter A, et al. Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line. Science
27. Clouse KA, Powell D, Washington I, et al. Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J Immunol
28. Folks TM, Clouse KA, Justement J, et al. Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc Natl Acad Sci USA
29. Gallo RC, Salahuddin SZ, Popovic M, et al. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science
30. Shaw GM, Hahn BH, Arya SK, et al. Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome. Science
31. Jeang KT, Giam CZ, Nerenberg M, et al. Abundant synthesis of functional human T-cell leukemia virus type I p40x protein in eucaryotic cells by using a baculovirus expression vector. J Virol
32. Jeang KT, Widen SG, Semmes OJ, et al. HTLV-I trans-activator protein, Tax, is a trans-repressor of the human beta-polymerase gene. Science
33. Imamichi T, Berg SC, Imamichi H, et al. Relative replication fitness of a high-level 3′Azido-3′-deoxythymidine resistant variant of human immunodeficiency virus type-1 possessing an amino acid deletion at codon 67 and a novel substitution (Thr to Gly) at codon 69. J Virol
34. Wilm M, Shevchenko A, Houthaeve T, et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature
35. Johnson JM, Harrod R, Franchini G. Molecular biology and pathogenesis of the human T-cell leukaemia/lymphotropic virus type-1 (HTLV-1). Int J Exp Pathol
36. Albrecht B, Lairmore MD. Critical role of human T-lymphotropic virus type 1 accessory proteins in viral replication and pathogenesis. Microbiol Mol Biol Rev
37. Cassé C, Giannoni F, Nguyen VT, et al. The transcriptional inhibitors, actinomycin D and α-amanitin activate the HIV-1 promoter and favor phosphorylation of the RNA polymerase II C-terminal domain. J Biol Chem
38. Kishimoto T, Akira S, Taga T. Interleukin-6 and its receptor: a paradigm for cytokines
39. Poli G, Bressler P, Kinter A, et al. Interleukin 6 induces human immunodeficiency virus expression in infected monocytic cells alone and in synergy with tumor necrosis factor alpha by transcriptional and post-transcriptional mechanisms. J Exp Med
40. Wallach D, Varfolomeev EE, Malinin NL, et al. Tumor necrosis factor receptor and Fas signaling mechanisms. In: Paul WE, Fathman CG, Mezger H, eds. Annual Review of Immunology, vol. 17
. Palo Alto: Annual Review; 1999:331-367.
41. Aggarwal BB. Signaling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol
42. Yamashita I, Katamine S, Moriuchi R, et al. Transactivation of the human interleukin-6 gene by human T-lymphotropic virus type 1 Tax protein. Blood
43. Lal RB, Rudolph D, Buckner C, et al. Infection with human T-lymphotropic viruses leads to constitutive expression of leukemia inhibitory factor and interleukin-6. Blood
44. Emiliani S, Fischle W, Ott M, et al. Mutations in the tat gene are responsible for human immunodeficiency virus type 1 postintegration latency in the U1 cell line. J Virol
45. Butera ST. Therapeutic targeting of human immunodeficiency virus type-1 latency: current clinical realities and future scientific possibilities. Antiviral Res
46. Blankson JN, Persaud D, Siliciano RF. The challenge of viral reservoirs in HIV-1 infection. Annu Rev Med
47. Pomerantz RJ. Reservoirs of human immunodeficiency virus type 1: the main obstacles to viral eradication. Clin Infect Dis