AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 2, 1992 Mary Ann Liebert, Inc., Publishers

Binding of NF-kB

to the HIV-1 LTR Is Not Sufficient to

HIV-1 LTR

Induce

Activity

CLEMENS DOPPLER, GUNNAR SCHALASTA, EBERHARD AMTMANN, and GERHARD SAUER

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) spends a significant part of its life cycle as latent provirus in nonactivated cells. Its induction requires mitogen stimulation. TPA treatment induces HIV-1 transcription by protein kinase C (PKC) -mediated activation of the cellular transcription factor NF-kB. PKC activation induces the dissociation of NF-kB from its inhibitor protein (IkB). The liberated NF-kB then binds to its proviral recognition sequence in the HIV-1 long terminal repeat (LTR) sequence. This step, however, is not sufficient to augment transcription. We demonstrate that NF-kB-mediated HIV-1 LTR activation is regulated by an additional event that is not dependent on IkB. A further phosphorylation event is proposed, since this step could be blocked by an inhibitor of a phospholipase C (PLC) type reaction. This inhibitor precludes the formation of diacylglycerols, which are required for activation of PKC isoenzymes. As an alternative pathway that is not dependent on PLC reactions, high-level transcription from the HIV-1 LTR is shown to require binding of both NF-kB and TAT.

INTRODUCTION

strategies against HIV-1 that either viral the rationale mainly by guided gene products or virus-coded regulatory gene functions might be the appropriate targets. A major problem is posed, however, by the latency of this virus during which its genome remains unexpressed, thus precluding any access to virus-coded pro-

The

development of antiviral

has been

teins. The process of activation that terminates the period of dormancy and leads to virus propagation is mediated, as shown by Nabel and Baltimore,' by an inducible host cell transcription factor. This nuclear factor (NF-kB) responds to mitogenic signals that can be exerted by a number of biomodulators, TPA, and physical agents such as ultraviolet (UV) light.2'3 In non-B cells, NF-kB is maintained in an inactive, cytoplasmatic state complexed with a 37 kD inhibitor protein (IkB).4-6 The stimulation of cells with TPA induces PKC, which leads to the phosphorylation of IkB,5 rendering the dissociation of NF-kB from IkB possible. Subsequently, NF-kB can be trans-

German Cancer Research Center, Institute for Virus Research, 6900

located into the nucleus, where it binds to its recognition The latter functions as enhancer in the HIV-1 sequence.7'8 1.9.10 LTR The NF-kB-mediated HIV-1 LTR induction augments a low level of transcription, leading to synthesis of the viral regulatory TAT, NEF, and REF proteins."12 TAT, in turn, enhances viral transcription by several orders of magnitude,'3_l6 in such a way that ultimately the process of virus maturation can take place. A key role in the early events that initiate viral transcription falls to the PKC-mediated phosphorylation of IkB. We have focused our interest particularly on such early phosphorylation events, since our antiviral work during recent years has been concerned with an inhibitory compound, tricyclodecan-9-ylxanthogenate (D609), that permits the identification of regulatory events involving different members of the PKC isoenzyme family. This compound has been shown to display a broad range of antiviral properties. DNA and RNA viruses, including HIV-1, are inhibited by D609.17-'9 D609 interacts indirectly with PKC by precluding the formation of phospholipase C-generated diacylglycerols that serve as second messengers for the

Heidelberg, Germany. 245

246

DOPPLER ET AL.

activation of PKC isoenzymes. Furthermore, in contrast to H7,20 a direct interaction of D609 with PKC has been excluded.21 As a consequence, in D609-treated cells PKC-mediated phosphorylation of target proteins, such as frani-acting transcription factors can be selectively inhibited.22'23 We have employed D609 and the protein kinase inhibitor H7, which leads to inactivation of PKC by direct binding although with poor selectivity. This approach has revealed that binding of NF-kB to the HIV-1 LTR per se is not sufficient to augment transcription. We show in this report that NF-kB-mediated HIV-1 activation is accomplished by an additional event not dependent on IkB, which requires the activation of a putative PkC isoenzyme through PLC-generated diacylglycerols.

MATERIALS AND METHODS

were left mitotically active with the same concentration. The cells were washed 22 h after treatment with icecold phosphatebuffered saline and collected in 40 mM Tris, pH 7.8, 1 mM EDTA, 150 mM NaCl, 4°C, by scraping. Cell pellets (30 s, centrifugation in an Eppendorf fuge) were resuspended in 250 mM Tris, pH 7.8, and disrupted by four cycles of freezing and thawing. The total protein amount of the cleared supernatant (10 min, 15,000 x g, 4°C) was determined by Bradford assay (Biorad, Munich, Germany). For each assay three parallel cultures were used.

Quantitation of virus progeny After low-speed centrifugation of tissue culture medium, the virus concentration was determined with the aid of an anti-p24 elisa (Organon, Eppelheim, Germany).

Cell culture HeLa cells and HeLa tat-III cells (described by A. Rice)2426 were grown in basal medium Eagle (BME) supplemented with 5% fetal bovine serum (FBS). Cells (0.8 x 106) were seeded in 6 cm dishes 14-18 h before transfection. Jurkat (2 x 105 cells/ml) were cultured in RPMI-1640 supplemented with 10% inactivated FBS. For gel retardation assays, cells were washed three times and cultured for 24 h prior to stimulation in BME supplemented with 5% FBS that had been extensively dialysed against 0.9% NaCl.

Plasmids The HIV-1 LTR-CAT vectors lacking a TPA responsive element of the basic cat construct have been described previously.10 In the plasmid m-633/+80 LTR-CAT the enhancer sequence (- 105/-80) was point-mutated in both NF-kB binding regions (CTC instead of GGG10) abolishing NF-kB binding.

CAT assays

Assays using chloramphenicol acetyltransferase (CAT) were performed as described elsewhere.27 The measurements were made in the linear range of the assay; radioactivity was measured with a thin layer Chromatographie (TLC) radio analyzer (Berthold, Wildbad, Germany) and the conversion of CAT into acetylated isoforms was quantitated. The CAT turnover was calculated as picomoles per milligram of protein used per minute (pmol/mg x min). AH transfection assay experiments were repeated six times. The standard deviation in parallel assays was found to range between 2% and 15%, as indicated in the figure legends. Gel retardation assay

Quiescent HeLa cells were cultivated in BME supplemented dialysed FBS as described. The nuclear protein

with 5.0%

Transfection

and

treatment

of cells

Cells were transfected after overnight culture using DOTAP liposomes (for details, see below under "chemicals"). DOTAP (6 p,g DOTAP per 1 pg DNA) and DNA (5 u-g vector DNA) were diluted separately in 20 mM Hepes pH 7.0 (cell culture grade). DOTAP was diluted to a concentration of 0.3 p.g/u,l in an Eppendorf tube. DNA (diluted to a concentration of 0.2 pg/pl) was

added and the mixture

was

incubated for 15 min to allow

DNA/liposome complex formation at room temperature. The dish then was gently agitated while the complex was added to fresh BME supplemented with 5% dialysed FBS and 1 % penicillin (pen.), 1 % streptomycin (strep.). The transfection was stopped 6 h later by discarding the transfection medium and adding fresh BME, pH 7.0 (5% dialysed FBS, 1% pen., 1% strep.). Cells were treated with D609 (15 p-g/ml) or H7 (27.5 p.M) 15 min before stimulation with TPA (10~7 M). In the case of Jurkat cells, transfection was carried out in 5 ml tissue culture medium (4 x 105 cells/ml). For TPA stimulation (10~8 M together with 20 p-g/ml Concanavalin A) and D609 (30 p.g/ml) or H7 (27.5 pJVl) treatment the cells were refed with RPMI-1640 pH 7.0, 10% inactivated FBS, 1% penicillin 1% streptomycin. The dose of D609 was chosen because it was found to be high enough to inhibit virus replication17'19'37 while uninfected cells

prepared28 from HeLa cells 20 h after stimulation. The content of active enhancer binding protein was determined by gel retardation analysis29 with slight modifications: 5 u,g extract protein was incubated with 0.5 p,g poly dl-dC and the 32P end-labeled oligonucleotide (5-10 x 103 cpm) for 30 min followed by electrophoresis in native polyacrylamide gels. After drying, x-ray films were exposed to the gels, using intensifying screens. In order to determine NF-kB specific binding we used the double-stranded synthetic oligonucleotide 5'-CCTextracts were

TATGGGGACTTTCCGTCTGCAGCA-3'

as a

probe.30

Chemicals was provided by Merz & Co., Frankfurt, Germany. It synthesized according to published methods.31 A'-[l-(2,3-dioIeoyloxy)propylJ-N,N,N-trimethylammoniumethyl-sulfate (DOTAP), acetyl-coenzyme A (Li-salt), and Hepes were provided by Boehringer Mannheim, Germany. lf(5-Isoquinolinsulfonyl)]-2-methylpiperazine dihydrochloride (H-7) was obtained from Calbiochem (Frankfurt, Germany). 12-0-Tetradecanoyl-phorbol-13-acetate (TPA) was kindly provided by Dr. Hecker, German Cancer Research Center. All other chemicals were obtained from Sigma (Munich, Germany).

D609

was

NF-kB BINDING TO HIV-1 LTR

247

RESULTS D609 inhibits the induction

of provirus by

TPA

It has been shown recently that an early event in the replication cycle of HIV-1 is interfered with by the antiviral xanthate compound D609.19 In the case of other viruses (BPV-1, herpes, SV-40, and VSV) transcriptional processes were found to be affected.17'21 '37'47 D609 is an indirect PKC inhibitor capable of blocking PKC-mediated phosphorylation steps36 that were found to be required for the activation of trans-acting factors such as serum response factor (SRF) and NF-kB.5'23 Since HIV-1 virus production can be induced by TPA' which involves PKC-dependent phosphorylation steps, we have studied the effect of D609 on this event. We used cloned HIV-1 proviral DNA (plasmid pNL4-3);40'45 after transfection in quiescent HeLa cells as a pseudo-latent model system which produces HIV-1 virions at a low level. After a 40-h treatment with TPA the virus production was stimulated by 4-5-fold (Fig. 1) which could be inhibited by simultaneous exposure to D609. In the latter case virus production was found to be even lower than in the unstimulated control cells. This effect can be attributed to the fact that the growth of HeLa cells cannot be completely arrested and therefore a residual PKC activity, which can be quenched, however, by D609, is encountered. After removal of D609 from the tissue culture medium the inhibitory effect is completely reversed and undiminuished virus production is resumed (data not shown).

induction of latent HIV-1 provirus.1'910 The major HIV-1 LTR enhancer element (— 105/—80) functions as the binding region for NF-kB.1 TPA treatment of cells was shown to lead to migration of liberated NF-kB to the nucleus recognizing its cognate binding sequences.6'30'32 PKC is implicated in the phosphorylation of the NF-kB inhibitor protein (IkB), which liberates NF-kB. The direct PKC inhibitor H7 is capable of inhibiting the activation of NF-kB.5 We performed mobility shift experiments (Fig. 2) to find whether the liberation of NF-kB after TPA stimulation is influenced by treatment either with the PLC-reaction inhibitor D609 or H7. In nuclear extracts from quiescent, serum-starved HeLa cells, a low level of binding activity is detectable (Fig. 2, lane 1). In nuclear extracts from TPA-stimulated cells (lane 2) the binding activity was increased about 15-fold. This binding is specific to the NF-kB recognition sequence, since competition with unlabeled authentic oligonucleotide prevents formation of the complex (lanes 6, 7). The synthetic oligonucleotide comprising the SRF binding site23 fails to compete for binding (lane 5). Nuclear extracts from cells treated simultaneously with H7 and TPA (lane 4) revealed a binding activity comparable to that of extracts from unstimulated cells (lane 1). In contrast, stimulation with TPA in combination with D609 (lane 3) led to a binding activity corresponding to the level in TPA stimulated cells (lane 2).

Effect ofD609 on TPA-induced NF-kB liberation The cellular transcription factor NF-kB mammalian cells tested.7 This factor plays

detected in all crucial role in the

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FIG. 1. Inhibition of provirus induction by D609. HeLa cells were treated with TPA (filled boxes) 6 h after transfection with provirus DNA (plasmid pNL4-3) or remained untreated (filled circles). To one TPA-treated culture 15 pg/ml D609 was added 15 minutes prior to TPA stimulation (open boxes). The virus yield was determined as p24 concentration in tissue culture medium

by an elisa.

FIG. 2. NF-kB binding was assayed in mobility shift assays using the specific NF-kB sequence described in Materials and Methods. Nuclear extracts were prepared from quiescent Hela cells (lane 1 ), from cells 20 h after treatment with TPA (10~7 M; lanes 2-7) or from cells additionally treated with D609 (lane 3) or with H7 (lane 4). In addition to radioactive-labeled oligonucleotide NF-kB, specific nonlabeled oligonucleotide was added to the sample in lane 6 (5 ng) and in lane 7 (20 ng). To the sample in lane 5. 20 ng of a nonlabeled SRF-specific oligonucleotide was added.

248 TPA-induced liberation of NF-kB was not affected by D609 treatment, which suggests that PLC-dependent reactions are not involved in TPA-induced dissociation of the NF-kB/IkB com-

plex. TPA induction of HIV-1 LTR-controlled CAT expression is inhibited by both HI and D609 In order to assess the influence of PKC on the NF-kBmediated HIV-1 LTR activation various HIV-1 LTR CAT expression vectors bearing mutations in the NF-kB-binding site10 were employed. The position of the mutations and the designation of the expression vectors are indicated in Table 1. Before the results are discussed, a few remarks are necessary about the problems arising from the impact of transfection techniques in conjunction with the subsequent treatment with inhibitory compounds on the viability of the recipient cells. This situation demanded the development of a particular experimental design. First of all, the aggressive method of DEAE-dextran treatment or electroporation had to be replaced by the gentler method of liposome-mediated transfection of the plasmids. In this way toxic effects were avoided. In addition, prestarvation of the HeLa cells by serum deprivation (in order to prepare quiescent cells) prior to the transfection step resulted in an unacceptably low transfection efficiency. Therefore, we had to omit the serum deprivation, and the cells had to be maintained in the presence of 5% serum up to the application of agonists and inhibitory compounds. The presence of serum, although it was dialysed (which at least partially removes growth factors), however, led to increased dissociation of NF-kB from the complex, to the extent that only marginal stimulation of binding to the NF-kB binding sequence ensued after TPA treatment (less than 15-fold). These restrictions must be borne in mind when the data below are considered. The LTR of HIV-1 contains four major functional CM-acting elements, which are indicated in the collection of plasmids displayed in Table 1. To assess whether the NF-kB binding site is the crucial element for TPA induction of transcription in our HeLa cell system various mutants were used. In the sections above it has been shown that the TPA induced dissociation of NF-kB and IkB can be inhibited by H7 while D609 fails to inhibit this. The next question was whether H7 and D609 influenced the TPA-mediated and NF-kB-controlled activation of HIV-1 LTR transcription. We used CAT-expression vectors carrying either the entire HIV-1 LTR or various mutants of the LTR (Table 1 ). The induction of CAT activity by TPA was measured in the presence and absence of the inhibitory compounds. In case of the plasmid carrying the entire LTR sequences (plasmid: —633/+80 LTR-CAT), the stimulation by TPA (factor 2.4) was inhibited by both substances, H7 (0.1-fold induction) and D609 (0.3-fold induction Table 1). The CAT expression level was even lower than in the case of uninduced cultures. As mentioned above, these experiments were carried out in tissue culture medium containing dialysed serum. This medium contains at least low amounts of growth factors, which probably induce PKC and, as a consequence, LTR-CAT activity (data not shown). As this induction can be inhibited by H7 and D609, lower CAT expression was obtained. Transfection of the vector containing the undeleted LTR sequence (— 121/+232 LTR-CAT) and subsequent TPA stimu-

DOPPLER ET AL.

lation resulted in an induction of about 2.5-fold CAT expression (Table 1). This induction could be inhibited by the treatment with either D609 (0.3-fold induction) or H7 (0.2-fold induction). Transfection of the enhancer-bearing vector from which the SP1 and TAR sites were deleted (enhancer LTR-CAT), TPA stimulation (1.9-fold induction) was inhibited by both D609 (0.6-fold induction) and H7 (0.4-fold induction). In cells transfected with the construct containing the point mutated enhancer site (m-633/+80 LTR-CAT) no significant TPA stimulation of the CAT activity was observed (1.0-fold induction), and additional treatment with D609 (1.1 -fold induction) or H7 treatment (1.0-fold induction) had, as expected, no effect. The NF-kB deletion vector —76/+232 LTR-CAT reacted in a similar way in that TPA (0.9-fold induction) failed to stimulate CAT expression. Subsequent D609 or H7 treatment had no influence on CAT expression (Table 1). The TAT-responsive site TAR ( 121 /+5 LTR-CAT) was not sufficient to inhibit CAT expression by D609 or H7 (Table 1 ). The inhibitory effect of both D609 and H7 is confined exclusively to NF-kB-mediated CAT expression. Deletion of the TAR site, on the other hand (—121/+5 LTR-CAT; enhancer LTR-CAT) was immaterial to both the augmentation of the CAT activity of TPA and subsequent inhibition by D609 and H7 (Table 1). When similar experiments were carried out in Jurkat cells, in which the original observation, namely stimulation of the HIV-1 enhancer by TPA was described, ' essentially similar results as in HeLa cells were observed. The CAT activity was found in this cell line to be stimulated by TPA/lectin by a factor of even 22-fold. Both D609 and H7 inhibited this stimulation com-

pletely (Table 1).

Cooperative interaction between NF-kB influenced by D609

and TAT is

not

One of the early HIV-1 transcripts codes for the viral protein TAT. This protein acts as an effective fra«s-activator of HIV-1 LTR (for review, see 33) by binding to the TAR element (for review, see 34) and interacting with the proviral LTR. 13~16 We compared the CAT activity in cells either containing or lacking TAT protein after transfection with the CAT plasmids specified above. TAT was provided by HeLa tat-III cells,24'25 which constitutively express the protein. Provided a functional TAR site was available in TAT-containing cells, a 20-30-fold increase of CAT activity was obtained compared with that in cells lacking TAT (-TAT; Table 2). The TAR-deleted plasmid (—121/+5 LTR-cat) displayed similar CAT activity in all three cell systems (Table 2). It also is shown that the presence of a functional NF-kB site in TAT-expressing cells leads cat activity to increase by 80-90-fold compared with cells lacking a functional NF-kB site (-633/+80 LTR-CAT versus m-633/+80 LTR-CAT; Table 2). The interaction between NF-kB and TAT is not affected by the treatment with D609 (Table 2). Regardless of the presence of D609 in TAT-expressing cells the CAT activity was found to be 16-34-fold higher (Table 2; factor of inhibition: 0.8-1.2) than in cells lacking TAT. In contrast to D609, H7 was shown to inhibit TAT-mediated /rafw-activation (data not shown). The TATmediated LTR stimulation was shown to be dependent on the presence of the TAR site and did not depend on the presence of a functional enhancer site; however, maximal transcription

NF-kB BINDING TO HIV-1 LTR

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Binding of NF-kB to the HIV-1 LTR is not sufficient to induce HIV-1 LTR activity.

Human immunodeficiency virus type 1 (HIV-1) spends a significant part of its life cycle as latent provirus in nonactivated cells. It induction require...
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