Human Immunodeficiency Virus Type 1 ... - Journal of Virology

JOURNAL OF VIROLOGY, Oct. 1994, p. 6598-6604

Vol. 68, No. 10

0022-538X/94/$04.00+0 Copyright C 1994, American Society for Microbiology

Synergistic Activation of Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Transcription by Retinoic Acid and Phorbol Ester through an NF-KB-Independent Mechanism JOSEPH W. MACIASZEK,"2 DAVID A. TALMAGE,3 AND GREGORY A. VIGLIANTIl24* Programs in Molecular Medicine' and Virology and Immunology2 and Department of Microbiology and Molecular Genetics,4 University of Massachusetts Medical Center, Worcester, Massachusetts 01605, and Institute of Human Nutrition, Columbia University, New York, New York 100323 Received 5 April 1994/Accepted 6 July 1994

The activation of human immunodeficiency virus type 1 (HIV-1) expression in latently infected cells by exogenous agents is believed to be important in the progression of AIDS. Most factors that are known to activate HIV-1 gene expression increase the binding of NF-KB or NF-KB-like transcription factors to the HIV-1 core enhancer region. In this report, we demonstrate that retinoic acid (RA) treatment of promonocytic U937 cells stimulates expression from the simian immunodeficiency virus (SIVmac) long terminal repeat (LTR). Furthermore, RA and phorbol 12-myristate 13-acetate (PMA) synergistically stimulated both SIVmac and HIV-1 LTRs to levels of expression comparable to that achieved by the viral transactivator Tat. The cis-acting elements required for a response to RA and PMA cotreatment are located between nucleotides -50 and +1 of SJVmac and between nucleotides -83 and +80 of HIV-1. Thus, the synergistic stimulation induced by RA and PMA is NF-KB independent. Analysis of deletion mutants of the SIVmac LTR demonstrates that RA and PMA stimulation cooperates with NF-KB and Spl. An SiVmac LTR-reporter gene construct [pLTR(-50/+466)CAT] lacking NF-KB and Spl binding sites was not activated by Tat in untreated cells but was activated in cells that were cotreated with RA and PMA. Furthermore, gel retardation assays demonstrated that RA treatment causes a change in the pattern of a cellular factor(s) which binds to the -50 through +1 region of the SIVmac LTR. These data suggest that RA induces a PMA-activatable cellular factor that cooperates with NF-KB, Spl, or Tat to stimulate LTR-directed transcription.

The pathogenesis of human and simian immunodeficiency viruses is thought to be controlled in part by complex interactions among viral regulatory proteins (such as Tat), cellular transcription factors (NF-KB, Spl, etc.), and proviral long terminal repeats (LTRs) (reviewed in references 32 and 34). Changes in the interplay among these factors conceivably have critical effects on the viral burdens of infected individuals. A number of stimuli that activate the transcription of virus in cells of both the myeloid and lymphoid lineages and that may be important in the transition from asymptomatic to symptomatic stages of disease have been identified. Retinoids, alone and in conjunction with various cytokines, are potent regulators of myeloid and lymphoid differentiation (28, 29, 51). This property is currently being utilized with dramatic results in retinoid-based differentiation therapy of acute promyelocytic and acute myelogenic leukemias (6, 22). Target cell responses to retinoids are largely mediated by two families of steroid and thyroid hormone-like nuclear receptors, the retinoic acid (RA) receptors and the retinoid X receptors (8). Although the critical primary target genes for transcriptional regulation by these receptors are largely unknown, there is ample evidence that retinoid-induced differentiation in many systems is accompanied by dramatic changes in the expression of transcription factors and in cellular responses to polypeptide hormones, growth factors, and cytokines whose signaling pathways lead to altered activity of transcription factors (15). The activation of human immunodeficiency virus type 1 *

(HIV-1) expression by exogenous agents is believed to be important in the progression of AIDS. Many factors (tumor necrosis factor alpha, phorbol esters, lipopolysaccharide, etc.) that are known to activate HIV-1 gene expression increase the binding of NF-KB to the HIV-1 core enhancer region (10, 13, 18, 26, 30, 33) (also reviewed in reference 32). Others (hexamethylene bisacetamide, adenovirus Ela, and herpesvirus ICPO) act through different elements in the HIV-1 LTR (23, 27, 50). The potential for retinoids to regulate the differentiation of cells of myeloid lineage led us to investigate the possible regulation of simian immunodeficiency virus from rhesus macaques (SIVmac) and HIV-1 expression in the U937 promonocytic leukemia cell line. In this report, we demonstrate that RA and phorbol 12-myristate 13-acetate (PMA) synergistically stimulate the transcription of SIVmac and HIV-1 LTRs independently of NF-KB binding. RA and PMA cotreatment induced LTR-directed expression to levels that were comparable to those achieved by the viral transactivator Tat. MATERIALS AND METHODS Cells and culture conditions. The promonocyte-like human histiocytic lymphoma cell line U937 (44) was obtained from the American Type Culture Collection. Cells were maintained in RPMI medium supplemented with 10% fetal calf serum at densities between 2 x 105 and 6 x 105 cells per ml. All-trans RA and retinol (dissolved in ethanol) were added daily as indicated below. For transfection experiments, cells not treated with RA were treated instead with ethanol. Therefore, control cells were grown in a parallel fashion to RA-treated cells. The ability of U937 cells to respond to RA was found to be serum

Corresponding author. Phone: (508) 856-3340. Fax: (508) 856-

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VOL. 68, 1994

lot dependent. Cells were treated with PMA (10 nM) for 18 to 20 h. Plasmids. PCR (25) was used to isolate a portion of SIVmac (isolate BK28) (20) including the entire 5' LTR and extending to the 5' splice site common to all subgenomic-length mRNAs (49) located at nucleotide position +466 (when + 1 is the start site of transcription). This fragment was inserted into the BglII site of pSVOCAT (12) to generate pLTR972CAT. An XbaIBamHI restriction fragment, including the SIVmac LTR and leader sequences as well as the chloramphenicol acetyltransferase (CAT) gene, simian virus 40 small t intron, and simian virus 40 polyadenylation sequences, was isolated from pLTR972CAT and inserted between the XbaI and BglII sites of pSP72 (Promega). The resultant clone, pLTR(-505/ +466)CAT, was used to generate a series of 5' deletion mutants beginning at the unique XbaI site adjacent to the 5' end of the SIVmaC LTR. Progressive 5' deletions were generated by unidirectional exonuclease III digestion. Exonuclease III-digested DNAs were blunt ended with nuclease Si and then ligated to Sacl linkers. The deletion endpoints were determined by DNA sequencing. PCR was used to isolate fragments of SIVmac including either nucleotides -50 through +466 or nucleotides -233 through +1. These fragments were inserted in place of the SIVmac LTR-leader sequences contained within a 5' deletion mutant clone, pLTR(-141/+466)CAT, to generate pLTR (-50/+466)CAT and pLTR(-233/+1)CAT. pCMVA31TAT contains the cytomegalovirus immediateearly promoter directing the expression of an SIVmac tat cDNA (49) that includes both coding exons. pHIVCAT contains a full-length wild-type HIV-1 LTR directing CAT expression (43). p(-83)CAT contains a portion of the HIV-1 LTR extending from nucleotides -83 through +80 and directing CAT expression (43). pCMVcTAT contains the cytomegalovirus immediate-early promoter directing the expression of the two-exon form of an HIV-1 tat cDNA (43). These plasmids were obtained from C. Southgate, University of Massachusetts Medical Center. Transfections and CAT assays. U937 cells were transfected by electroporation with a BRL Cell-Porator. Cells were suspended in serum-free RPMI (1 X 107 cells in 1 ml) containing 2 to 4 ,ug of plasmid DNA. Electroporation was performed at room temperature with settings of 800 ,uF and 300 V. Assays for CAT activity were performed as described previously (47) except that 0.2 mM phenylmethylsulfonyl fluoride was included in the buffer used to prepare cellular extracts. Spots on thin-layer chromatography plates corresponding to the different acetylated forms of chloramphenicol were cut out and quantified by liquid scintillation. Only values in the linear range of enzyme activity were used to determine the percentage of acetylated chloramphenicol. RNA isolation and analysis. Total cellular RNA was isolated as described previously (47). For RNA blot analysis, total RNAs were separated on 1% agarose-formaldehyde gels and transferred to nylon membranes. The filters were hybridized with a 32P-labeled probe homologous to the SIVmac LTR as described previously (48). Gel retardation assay. Cellular extracts for gel retardation assays were prepared as described previously (39) except that the extraction buffer contained 1 mM sodium orthovanadate and 1 mM ammonium molybdate as phosphatase inhibitors. Binding reactions were carried out for 30 min at 30°C under conditions shown to allow the detection of proteins bound to HIV-1 TATA element oligonucleotides (39). Binding reactions included 10 ,ug of cellular extract and 10,000 cpm of radioactive probe.

RA AND PMA ACTIVATION OF SIVmac AND HIV-1 Day P.T. L

Retinoic Acid (>M)

Retinol (pM)

6599

5 1

3

L-1 |-|-|ll-I -1- 1 h6L2t-i 1101

28S -w-

18S --

;

FIG. 1. Effects of retinoids on SIVmac mRNA accumulation in U937 cells. U937 promonocytic cells were transfected with a wild-type SIVnac provirus clone (pBK28) and then divided into four portions. Transfected cells were treated with the indicated concentrations of retinoids, and total RNA was prepared at the indicated times posttransfection (P.T.). RNAs were separated on formaldehyde-agarose gels, transferred to membranes, and hybridized with a probe homologous to the SIVma, LTR. Ethidium bromide staining of the gel (not shown) demonstrated that equivalent amounts of RNA were included in all lanes.

Radioactive probes were prepared from PCR-amplified DNAs corresponding to SIVmac LTR nucleotide positions -50 through + 1. The downstream end of the amplified fragment included an additional nine non-SIVmac nucleotides constituting a BglII restriction site. PCR-amplified DNAs were digested with BglII and then 32p labeled by using the Klenow fragment of Escherichia coli DNA polymerase I and [32P]dCTP. The labeled probe was purified on a 12% polyacrylamide gel. Competition assays were performed with either a 50-fold molar excess (relative to the probe) of plasmid DNAs or a 100-fold molar excess of oligonucleotides. The competitors were (i) a plasmid containing SIVmac sequences -505 through +466, including the entire LTR, and extending to the major splice donor site used for subgenomic-length mRNAs; (ii) a plasmid containing c-fos sequences -32 through +45, including the TATA element; (iii) a 22-bp oligonucleotide (5'GATCGATCGGGGCGGGGCGATC-3') with an Spl consensus binding site; and (iv) a 22-bp oligonucleotide (5'GATCGAGGGGACTIFCCCTAGC-3') with an NF-KB consensus binding site. RESULTS RA stimulates SIVmaC mRNA accumulation in U937 cells. The treatment of SIVmac-transfected U937 cells with 1 ,uM RA or 10 jiM retinol increased viral RNA levels (Fig. 1). Modest increases in viral transcripts were seen after 3 days (2- to 3-fold), with a significant (>10-fold) elevation maintained in5 days posttransfection. There was a time-dependent decrease virus-specific mRNA in transfected cells because of the inability of SIVmac (isolate BK28) to establish a productive infection in these cells. RA and phorbol ester synergistically activate SlVmac expression. RA does not induce terminal differentiation of U937 cells

6600

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MACIASZEK ET AL.

A

B [PMA (10 nM, 18 hr)]

*5

40

U

0

0

< 30 o

20

a)

,O) 10 cc

co a) 2

4

6

2

'*0

Days treament

6

4

8

10

Days treament

(1 icM retinoic acid)

(1 IcM retinoic acid)

FIG. 2. Time dependence of RA stimulation of SIVmac expression. U937 cells were treated with 1 ,uM RA for the indicated number of days and then transfected with an SIVmac LTR-CAT construct [pLTR(-505/+466)CAT]. Those cells that were pretreated with RA continued to receive RA after transfection. Cellular extracts were prepared 48 h after transfection, and the levels of CAT activity were determined. All CAT activities were normalized to the amount of protein contained within the reaction mixture. Only values in the linear range of CAT activity were used to calculate the relative CAT activities shown. (A) Cells pretreated with RA. (B) One-half of the transfected cells were treated with 10 nM PMA for 18 h prior to being harvested. as determined by the expression of cell surface markers and other characteristics associated with monocyte/macrophage phenotypes, including NF-KB activity (29, 45). It does, however, enhance the ability of U937 cells to differentiate in response to cyclic AMP and PMA (29, 51). Since the SIVmac LTR is known to be PMA responsive (35, 52), we sought to determine whether the RA-induced increase in viral mRNA accumulation described above was related to the enhanced PMA sensitivity of RA-pretreated U937 cells. U937 cells were pretreated with RA for various times and then transfected with an SIVmac LTR-CAT plasmid. We found that RA enhanced LTR-directed CAT activity three- to fourfold in cells that were pretreated for more than 4 days (Fig. 2). Maximal stimulation (- fourfold) occurred in cells pretreated for 7 days or longer. A similar time-dependent RA-induced

TatI Retinoic Acid PMA

F

1

I-I

-

-

20 hr

|

I-

+

I+

+

+

+_I +I --- +I+ X-+-l+

I1I

-I

+

increase in HIV-1 gene expression in THP-1 monocytes and primary adherent macrophages has been reported (46). To determine whether RA could potentiate the PMA responsiveness of the SIVmac LTR in U937 cells, one-half of each transfection was further treated with PMA (10 nM for 18 h). RA enhanced the PMA responsiveness of SIVmaC LTR expression with a time dependence similar to that observed for RA stimulation alone (Fig. 2). PMA stimulated LTR-directed CAT activity approximately sixfold in cells that were pretreated for less than 4 days. In contrast, PMA stimulated LTR-directed CAT activity approximately 20-fold in cells that were pretreated with RA for more than 4 days. Significantly, RA and PMA synergistically activated SIVmac LTR-directed expression. In a separate experiment (Fig. 3), treatment of U937 cells with RA for 7 days stimulated LTR expression 2-fold while PMA treatment of RA naive cells stimulated expression 15-fold. However, PMA treatment of cells that were pretreated with RA for 7 days stimulated expression 80-fold. This level of synergistic activation is comparable to the level induced by the viral activator Tat (100-fold in untreated cells). In control experiments, RA treatment had no effect on the transfection efficiency of U937 cells (as measured by Southern blot analysis of transfected DNA), indicating that the RAinduced differences in CAT activity resulted from changes in LTR-directed expression. RA and phorbol ester activation is NF-KB independent. The SIVmac LTR contains an imperfect direct repeat of the sequence GGTCA located between positions -172 and -185 (AGTCAggccTGTCA). This sequence is similar to hormone response elements that are bound by RA receptors (1). In addition, there are two known PMA response elements within the SIVmac LTR. These include SF-2 and NF-KB binding sites (Fig. 4) (35, 52). To determine whether the potential RARE or the PMA response elements are required for RA and PMA costimulation, we constructed a series of SIVmac LTR-CAT reporter constructs containing progressive 5' deletions (Fig. 4). These plasmids were transfected, either alone or with a

I

l

_

2 hr

FIG. 3. Synergistic stimulation of SIVmac LTR-directed expression by RA and PMA. Both untreated and RA-pretreated (7 days) U937 cells transfected with an SIVmaC LTR-CAT construct [pLTR(-505/+466)CAT] either alone or with a plasmid expressing SIVmac Tat. Cellular extracts were prepared, and CAT activities were determined as described in the legend to Fig. 2. One-half of the transfected cells were also treated for with 10 nM PMA for 18 h prior to being harvested. An autoradiograph of a representative CAT assay is shown. The lengths of CAT individual samples are shown at the bottom. The percent conversions of chloramphenicol to its acetylated forms for the various treatments shown are as follows: untreated, 1.3%; RA, 2.8%; PMA, 2.0%; RA and PMA, 10.9%; Tat, 13.7%; Tat and RA, 30.4%; Tat and PMA, 61.0%; Tat, RA, and PMA, >95% (in a reaction in the linear range of the assay, using less extract, a relative percent conversion of 213% was obtained). were

assays

RA AND PMA ACTIVATION OF SIVmac AND HIV-1

VOL. 68, 1994

RT?

RAFNE?

-505

SF-3 SF-2 11 SSF-11%SF

Nr-kB

-220 -200 -180-160 -140 -120 -100 -80 -60 -40 -20

+1 +466

FIG. 4. Transcription factor binding sites within the SIVmac LTR. A diagram of the SIVmac LTR and the positions of various transcription factor binding sites are shown. Arrows indicate the endpoints of various 5' deletion mutants. TBP, TATA-binding protein.

plasmid expressing SIVmac Tat, into U937 cells which had been pretreated with RA for 7 days. pLTR(-141/+466)CAT and pLTR(-120/+466)CAT lack both the putative RARE and a negative regulatory element (22a). In addition to the expected increase in basal activity, both of the constructs retained the same degree of RA responsiveness as the full-length LTR (Table 1). In fact, SIVmac LTR constructs lacking sequences upstream from nucleotide - 67 (when + 1 is defined as the start site of transcription) retained RA responsiveness (Table 1). The putative RARE is therefore not required for the RA responsiveness of the SIVmaC LTR. Since the deletion at nucleotide -67 also removes the binding sites for factors SF-1, SF-2, SF-3, and NF-KB (52), we can also conclude that these sites are not required for RA-induced stimulation. Deletions between nucleotides -120 and -78 remove the previously identified SF-2 and NF-KB binding sites implicated in the PMA responsiveness of the SIVmac LTR (52). As expected, only

6601

minor PMA enhancement (two- to threefold) was seen with either pLTR(-78/+466)CAT or pLTR(-67/+466)CAT. Surprisingly, we found that these two LTR constructs still responded to PMA in RA-treated cells. We suggest that the synergistic stimulation by RA and PMA resulted from RAinduced expression of a novel, PMA-activated cellular factor that is distinct from the previously identified PMA-responsive factors SF-2 and NF-KB. Although SIVmac LTR-reporter gene constructs which lacked NF-KB binding sites were synergistically stimulated by RA and PMA cotreatment, constructs which retained the NF-KB sites were stimulated to much higher levels (Table 1). For example, the SIVmac LTR-reporter gene construct lacking the NF-KB binding site [pLTR(-67/+466)CAT] was stimulated 13-fold by RA and PMA cotreatment compared with the over-100-fold stimulation of a reporter construct retaining the NF-KB binding site [pLTR(-95/+466)CAT]. pLTR(-95/ +466)CAT was stimulated 5- to 50-fold by PMA treatment alone, indicating that a functional PMA-stimulated NF-KB response was also present in these cells. These results are consistent with cooperation between a novel RA-induced, PMA-activated factor and PMA-activated NF-KB in stimulating LTR-directed expression. Costimulation by these two factors led to a level of expression comparable to the level achieved by the viral transactivator Tat (Table 1). The HIV-1 LTR is synergistically activated by RA and phorbol ester independently of NF-KB. The HIV-1 LTR was also synergistically activated by RA and PMA (Table 2). LTR-CAT constructs containing either a full-length HIV-1 LTR (pHIVCAT) or a deletion mutant [p(-83)CAT] lacking both NF-KB binding sites were transfected, alone or with a

TABLE 1. Synergistic activation of SIVmac LTR-CAT deletion mutants by RA and PMa Fold activation

pLTR(-505/+466)CAT

pLTR(-141/+466)CAT pLTR(-120/+466)CAT

pLTR(-95/+466)CAT pLTR(-78/+466)CAT pLTR(-67/+466)CAT

pLTR(-50/+466)CAT

With SIVmac Tat

Without Tat

Plasmid RA

PMA

2.2 1.7 2.6 1.8 2.1 1.8 3.0 2.1 1.3 1.5 3.8 1.6 2.4 1.6

15 43 13 28 ND 7.7 18 88 7.4 8.2 53

2.8 1.8 6.4 1.2 1.0 1.3 2.0 1.0 3.8

5.4 2.5

3.0 2.5 2.1 ND 2.8 1.0 1.0 2.0 3.7 3.3

RA and PMA

Untreated

RA

PMA

84

105 203 NDb 150 446 ND 142 351 ND 100 115 ND 100 102 ND 70 286 ND 2.5 1.7 2.5

233 291 ND 328 911 ND 372 515 ND 284 325 ND 232 221 ND 187 689 ND 3.0 5.0 6.5

469 758 ND 483 ND ND 1,037

333 57 222 ND 55 150 483 74 126 138 93 19 86 12 13 ND 14 3.5 5 7.5 16 29

2,309 ND 726 705 ND 195 300 ND 164 ND ND 10 3.7 4.0

RA and PMA

2,178 3,480 ND 2,743 ND ND

4,990 9,309 ND

3,496 4,225 ND

1,580 911 ND

1,020 ND ND 67 45 68 15 38

4.1 0.9 0.6 3.8 1.3 3.3 alone or with a plasmid either mutant a Both untreated and RA-treated (1 ,uM for 7 days) U937 cells were transfected with the indicated SIVmac LTR-CAT deletion

pLTR(-233/+1)CAT

expressing SIVmaC Tat. One-half of each transfection was also treated with 10 nM PMA for 18 h prior to being harvested. CAT activities were determined as described in Materials and Methods. The different rows contain data from separate experiments. The average conversions of CAT in untreated cells without Tat for the various LTR-CAT constructs were as follows: pLTR(-505/+466)CAT, 1.2%; pLTR(-141/+466)CAT, 3.9%; pLTR(-120/+466)CAT, 2.3%; pLTR(-95/+466)CAT, 1.1%; 0.7%; pLTR(-67/+466)CAT, 1.0%; pLTR(-50/+466)CAT, 0.25%; pLTR(-233/+1)CAT, 0.6%. pLTR(-78/+466)CAT, b ND, not done.

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TABLE 2. Synergistic activation of HIV-1 LTR-directed gene expression by RA and PMAa

COMPETITOR DNA X 0tL

Fold activation Without Tat

Plasmid

pHIVCAT

p(-83)CAT

RA

PMA

0.8 1.3 0.6 1.0 1.5

11 10 1.2 0.9 1.0

With HIV-1 Tat

RA and Untreated PMA 120 250 5.0 3.4 6.8

182 235 79 48 100

RA

PMA

and RAPMlA

176 707 79 154 512

670 550 238 70 95

2,941 784 800 1,305

1,622

a HIV-1 LTR-CAT reporter plasmids were analyzed as described in footnote a of Table 1. The average conversions of CAT in untreated cells without Tat were as follows: pHIVCAT, 4.9%; p(-83)CAT, 0.9%.

-F (flL Ot I-

B2-

BI-6'o A"'. '

p

46.0

40

40

q

FP -

plasmid expressing HIV-1 Tat, into U937 cells that had been pretreated with RA for 7 days. PMA stimulated expression of a full-length LTR about 10-fold in RA naive cells (Table 2). PMA stimulation of the HIV-1 LTR increased to approximately 190-fold in cells that were treated with RA. As seen with the SIVmac LTR, pLTR(-67/+466)CAT, an HIV-1 LTR lacking both NF-KB binding sites, retained a degree of PMA responsiveness in RA-treated cells (Table 2). We conclude that both the HIV-1 and SIVmaC LTRs can be synergistically stimulated in U937 cells by RA and PMA through an NF-KBindependent mechanism. RA and phorbol ester stimulation acts through promoterproximal cis-acting sequences and cooperates with Tat. The SIVmac and HIV-1 LTRs analyzed above included viral sequences extending downstream from the start of transcription. These sequences included the RNA TAR element, which mediates Tat stimulation of virus transcription and influences the translation of viral mRNAs (4, 7, 24, 31, 37, 38, 41, 42, 48). Since our data do not distinguish between increased transcriptional initiation and potential TAR-influenced translational events, we examined the effects of RA with and without PMA on pLTR(-233/+l)CAT, which lacks the SIVmac TAR element. This plasmid was synergistically activated by RA and PMA; therefore, the stimulation of viral LTR-regulated CAT expression did not require TAR or other downstream elements (Table 1). These results, together with those described above, are consistent with an RA- and PMA-responsive cis-acting element located between nucleotides -67 and +1, a region containing an Spl site, a TATA element, and part of a potential Inr element (9). We found that an LTR-CAT construct, pLTR(-50/ +466)CAT, lacking the single Spl site was poorly expressed and not significantly activated by either RA and PMA cotreatment or Tat (Table 1). Interestingly, pLTR(-50/+466)CAT became Tat responsive in RA- and PMA-treated cells (Table 1). RA induces changes in the binding of factors to the SIVmac promoter region. The analyses described above indicate that the sequences between nucleotides -50 and + 1 of the SIVmac LTR are required for responsiveness to RA and PMA activation. We therefore used a gel retardation assay (Fig. 5) to determine whether RA and PMA treatments influenced the binding of factors to this region of the SIVmac promoter. Binding conditions that have been shown to detect PMAinduced changes in factors bound to the HIV-1 TATA element were used (39). By using these conditions, two complexes were formed with extracts from untreated cells; the predominant complex was designated Bi, and the minor complex was

N"'Pill

PMA_l RA |l__

_

_

___

_l_l_l_

FIG. 5. Gel retardation assay with a 32P-labeled SIVmac promoter fragment. A PCR-generated fragment (nucleotides -50 through +1) of the SIVmac promoter was 32p labeled and incubated with cellular extracts prepared from U937 cells. Extracts from untreated cells formed two complexes, Bi and B2, with the probe. Extracts from RA-treated (0.5 ,uM for 7 days) U937 cells formed the same two complexes but in different proportions. Both complexes were specifically competed for by a plasmid containing the SIVmac LTR but not by a plasmid containing the c-fos promoter (nucleotides -32 through +45) and including the TATA element or by oligonucleotides containing either Spl or NF-KB binding sites. PMA treatment (10 nM for 1 h) of either RA naive or RA-treated cells had no effect on complex formation. FP, free probe.

designated B2. Significantly, the level of Bi decreased and the level of B2 increased when extracts from RA-treated cells were used in the assay. With these extracts, the levels of Bi and B2 were approximately equal. Both B1 and B2 were specifically competed for by SIVmaC LTR sequences but not by c-fos promoter sequences (including the TATA element) or oligonucleotides containing either Spl or NF-KB binding sites. PMA treatment did not affect the pattern of complexes formed by either RA naive or RA-treated cellular extracts. These results are consistent with RA causing a change in the factors binding to the SIVmac promoter. DISCUSSION We have demonstrated the presence of a retinoid-induced pathway in U937 cells that, activated by PMA, increases transcription from both SIVmac and HIV-1 LTRs. RA activation of viral LTRs required extended pretreatment of cells, did not require LTR sequences resembling defined RA receptor or retinoid X receptor response elements, and therefore appeared to result from a retinoid-induced change in U937 cells.

The induction of genes lacking classic RAREs by retinoids is a common event in cells that differentiate in response to retinoids (15). Although RA does not induce terminal differentiation of U937 cells into macrophages as determined by morphological criteria and expression of cell surface markers, it apparently primes cells for terminal differentiation induced by other agents, including phorbol esters and cyclic AMP (29, 45, 51). The synergistic interaction between RA and PMA on viral gene expression described above is consistent with an associa-

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tion between virus LTR activity and the state of differentiation of U937 cells. The viral DNA sequences required in cis for the RA and PMA response were localized between nucleotides -50 and + 1 of the SIVmac LTR and between nucleotides -83 and +80 of the HIV-1 LTR. An examination of these sequences revealed no obvious nucleic acid sequence elements, except for the TATA element, that are conserved between these two viruses. In addition, no factors other than the TATA-binding protein component of TFIID are known to bind this region of the SIVmaC LTR. Recently, NF-KB-independent activation of the HIV-1 LTR by PMA in megakaryocytes and T lymphocytes was related to the enhanced binding of factors to the TATA element (39). It is conceivable that NF-KB-independent, RAand PMA-activated expression from the HIV-1 and SIVmac LTRs in U937 cells results from increased binding of the same specific factor(s) to the viral TATA elements. Consistent with this idea, we found that RA induces a change in the binding of factors to an SIVmac promoter fragment which includes the TATA element. Although the RA- and PMA-responsive element lies between nucleotides -50 and + 1 of the SIVmac LTR and is capable of functioning in the absence of either Spl or TatTAR interactions, cooperation with one of these factors appeared to be necessary for significant RA- and PMA-enhanced transcription. When the Spl sites were deleted [pLTR(-50/ +466)CAT], significant activity was seen only in the presence of Tat, whereas in the presence of Spl sites [and other upstream sequences in pLTR(-233/+l)CAT], Tat expression was dispensable. Tat activation of the HIV-1 LTR requires functional interactions between Tat and other activating factors, such as Spl, and interactions between Tat and TFIID (3, 5, 16, 17, 43). In the latter case, substitution of other functional TATA elements for the naturally occurring HIV-1 TATA element reduces Tat activation, indicating that these interactions are TATA sequence specific (5). It is likely that Tat interacts not simply with TFIID but with a subset of TFIID that interacts preferentially with the HIV-1 TATA element. We found that pLTR(-50/+466)CAT lacking Spl binding sites was insensitive to Tat transactivation despite the presence of a normal TAR element. However, PMA stimulation of RA-pretreated U937 cells restored Tat responsiveness to this truncated LTR. These results are consistent with an RAinduced, PMA-activated factor that is capable of cooperating with Tat at the TATA element. Transcriptional regulation by both the simian virus 40 large T antigen and adenovirus Ela involves interactions among the viral regulators, TATA-binding protein, and upstream activators. In the case of large T antigen, these interactions are influenced by both the identity of the bound activator and the identity of the TATA element (11, 36). Although large T antigen directly interacts with TATA-binding protein (14), the dependence on the TATA element sequence for activation implies some degree of heterogeneity in TFIID complexes. Ela activity can be complemented by the cellular factor ElA-LA in embryonal carcinoma cells (21). EtA-LA is required for RA-regulated transcription of the RAR-,B 2 promoter, and its level is regulated by RA during the differentiation of these cells (2, 19, 21, 40). These results provide a precedent for regulated expression of a TFIID interacting factor (or component) by RA. Whether the effects of RA on SIVmac and HIV-1 expression in U937 cells is mediated by such a factor remains to be demonstrated. In this report, we have identified a novel cellular pathway for transcriptional transactivation of SIVmac and HIV-1. The level of LTR-directed gene expression that resulted from the acti-

RA AND PMA ACTIVATION OF

SIVmac

AND HIV-1

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vation of this pathway was comparable to that seen in response to Tat, the major viral transcriptional regulator. The agents used to induce and activate this cellular factor, RA and PMA, respectively, are known to mediate the in vitro differentiation of promonocytes into monocytes/macrophages. It is not inconceivable that combined retinoid and cytokine action in vivo induces the monocyte-to-macrophage transition in tissues and, in the case of persistently infected cells, activates SIVmaC and HIV-1 gene expression and replication. ACKNOWLEDGMENTS We thank C. Peterson and M. Somasundaran for comments on the manuscript and helpful discussions. We also thank M. Debatis for excellent technical assistance. This work was supported by grants to D.A.T. from the Public Health Service (HD26854) and the Lucille P. Markey Charitable Trust and by a grant to G.A.V. from the Public Health Service (AI31355). REFERENCES 1. Beato, M. 1989. Gene regulation by steroid hormones. Cell

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