INTRODUCTION

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Primate retroviruses belonging to the foamy virus, or spumavirus, subfamily encode not only the structural proteins Gag, Pol, and Env but also a potent transcriptional transactivator and at least two auxiliary proteins of currently unknown function (7, 11, 26, 29). The transcriptional transactivator, which is termed Bel-1 in the case of human foamy virus (HFV) and Taf or Tas in the case of simian foamy viruses (SFV), has been shown to be essential for foamy virus replication in culture (1, 24). Foamy viruses contain at least two promoter elements that are highly responsive to the Bel-1/Tas protein. The first is the long terminal repeat (LTR) promoter, which may contain as many as three Bel-1/Tas DNA target sites and which is responsible for transcription of genome-length viral transcripts (8, 18, 20, 28, 30, 33). A second, internal promoter element is located towards the 3' end of the viral envelope gene and directs transcription of mRNAs encoding the viral auxiliary proteins, including Bel-1/Tas (5, 22, 25). The internal promoter element is thought to activate expression of these auxiliary proteins early in the viral life cycle and is clearly critical for their efficient expression (21, 23, 25). Therefore, the internal promoter element is required for effective virus replication in culture.

Research into the mechanism of action of the HFV Bel-1 protein has identified an acidic transcription activation domain located within the carboxy-terminal ~40 amino acids (aa) of this 300-aa viral regulatory protein and has also defined a DNA targeting domain occupying ~120 aa in the core of Bel-1 (3, 12, 16, 32). While the domain organization of the related SFV type 1 (SFV-1) Tas protein appears to be very similar to that observed in Bel-1 (27), Tas and Bel-1 both fail to activate transcription directed by promoters containing functional DNA target sites specific for the other protein (5, 12).

Although several DNA target sites for Bel-1 have been mutationally defined, these have little evident sequence homology (8, 17, 18, 20, 22, 33). Nevertheless, it has been demonstrated that Bel-1 can directly and specifically bind to the major, cap-proximal Bel-1 response element (BRE) located in the viral LTR promoter and also to sequences present in the HFV internal promoter element (15). Similarly, specific Tas binding to the SFV-1 internal promoter, and to a proposed Tas-dependent enhancer element located in the SFV-1 gag gene, has also been reported (4, 34). Surprisingly, for both Tas and Bel-1, DNA sequences that are sufficient for DNA binding in vitro were found to be necessary but not sufficient for Tas or Bel-1 function in vivo (15, 34). This observation raises the possibility that other, cellular DNA binding proteins may play a critical role in mediating Bel-1 and Tas function in vivo.

Although target sequences for the Bel-1 protein have been loosely defined both based on functional criteria and by in vitro DNA binding (8, 15, 17, 18, 20, 22, 33), the actual DNA target specificity of Bel-1 remains far from clear. In this study, we attempted to shed further light on the DNA binding specificity of Bel-1 by comparing the interactions of Bel-1 with the HFV internal and LTR promoter elements. These experiments demonstrate that Bel-1 binds to the BRE present in the internal promoter with significantly higher affinity than to the major LTR promoter BRE. Using modification interference, we have identified individual bases within both the internal and LTR promoters that are critical for Bel-1 DNA binding in vitro. This analysis has permitted the definition of a minimal, 25-bp DNA sequence that is sufficient to mediate Bel-1 binding both in vitro and in vivo.

	MATERIALS AND METHODS

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Construction of molecular clones. The full-length HFV bel-1 gene was amplified by PCR with primers that introduced flanking XbaI sites and then ligated into the XbaI-digested plasmid pYCplacIII (13). pYCplacIII is a single-copy, ARS-CEN based yeast (Saccharomyces cerevisiae) expression plasmid that contains ADH1 promoter and terminator sequences. Similarly, Tas coding sequences from SFV-1 were amplified and cloned between the XbaI and EcoRI sites of pYCplacIII. We have previously described (15) a high-copy-number yeast expression plasmid for Bel-1, based on the 2µm origin of replication, in which Bel-1 expression is directed by the potent phosphoglycerate kinase promoter.

Gel retardation analysis. A fusion protein consisting of glutathione S-transferase (GST) linked to residues 1 to 228 of Bel-1 was expressed in the protease-deficient XA90 strain of Escherichia coli by using the pGST/Bel-1(1-228) plasmid described previously (15). The fusion protein was purified to homogeneity by glutathione affinity chromatography followed by chromatography with a Bio-Rex 70 anion-exchange column (Bio-Rad).

Methylation interference analysis. The LTR probe was uniquely end labeled on the coding strand by filling in the recessed 3' end at the introduced BamHI site with [-³²P]GTP by using avian myeloblastosis virus reverse transcriptase. The internal promoter probe was similarly labeled on the coding strand at an XbaI site with [-³²P]CTP. About 10⁶ cpm of each probe was methylated with 1 µl of dimethylsulfide for 1 min at room temperature in 200 µl of methylation buffer (50 mM sodium cacodylate, pH 8.0; 1 mM EDTA, pH 8.0) (31). The methylation reaction was stopped by adding 40 µl of stop buffer (1.5 M sodium acetate, pH 7.0; 1 M 2-mercaptoethanol), and the probe was isolated by ethanol precipitation. The probe was then incubated with 200 ng of the GST-Bel-1(1-228) fusion protein in 200 µl of binding buffer for 20 min at 4°C, and the unbound and bound DNA fractions were resolved by native polyacrylamide gel electrophoresis, located by autoradiography, excised, and eluted. The purified DNA was dissolved in 30 µl of 10 mM sodium phosphate (pH 6.8)-1 mM EDTA and incubated for 10 min at 92°C, and then 3 µl of 1 M NaOH was added to the solution and the incubation was continued for 30 min at 92°C. The DNA was precipitated and dissolved in sequencing buffer. The resultant end-labeled DNA fragments were resolved on an 8% sequencing gel.

Yeast transformation and analysis. The Bel-1 and Tas yeast expression plasmids and pJLB-derived lacZ indicator plasmids were cotransformed into the yeast strain PSY 316 (2). After 3 days of growth selection in media lacking uracil and leucine, yeast cell extracts were prepared and assayed for -galactosidase (-Gal) activity as described previously (3). Yeast plasmids expressing fusion proteins consisting of the GAL4 DNA binding domain and the various viral activation domains were transformed into the yeast strain Y190, which harbors an integrated lacZ reporter construct (14). After 3 days of growth selection in medium lacking leucine, -Gal assays were carried out as described previously (3).

	RESULTS

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Using DNase I protection analysis, we have previously mapped DNA binding sites for the HFV Bel-1 protein to between 84 and 32 relative to the LTR promoter cap site and between 171 and 135 relative to the internal promoter cap site (15). The ability of these HFV DNA sequences to bind the Bel-1 protein specifically in vitro was confirmed in the electrophoretic mobility shift assay (EMSA) shown in Fig. 1, which shows that a recombinant GST-Bel-1 fusion protein is able to interact with both an LTR DNA probe (LTR sequences 108 to 35) (lanes 3 to 5) and an internal promoter DNA probe (residues 191 to 105) (lanes 9 to 11). Of interest, under identical assay conditions, and with similar levels of LTR and internal promoter probes labeled to closely comparable specific activities, a significantly higher percentage of the internal promoter probe than of the LTR probe was bound by GST-Bel-1 (compare lanes 5 and 11). The observed probe shifts were due to the Bel-1 protein, in that GST itself failed to bind either probe (lanes 2 and 8). All three observed protein-DNA complexes were supershifted by addition of an anti-GST monoclonal antibody (Fig. 1, lanes 6 and 12), thus demonstrating that these were due to the GST-Bel-1 fusion protein and not to a contaminating DNA binding activity. The fact that at least three distinct protein-DNA complexes, labeled C1, C2, and C3, were observed in this EMSA is of interest, given the previous report that Bel-1 is able to form multimers in vivo (6). However, it should be noted that the linked GST protein can also form dimers.

View larger version (89K): [in this window] [in a new window]   FIG. 1.   Bel-1 binds to target DNA sites located in both the LTR and internal promoters of HFV. EMSA showing binding of increasing levels of a purified, recombinant GST-Bel-1(1-228) fusion protein to an HFV LTR and internal promoter (Int. Pr.) probe. Numbers above the lanes indicate the amounts, in nanograms, of fusion protein used. Controls include no added protein (Neg.) or addition of 200 ng of purified GST. Lanes 6 and 12 show a supershift obtained by addition of an anti-GST monoclonal antibody (~50 ng). C1, C2, and C3 are retarded complexes displaying different electrophoretic mobilities.

We next sought to confirm that the observed Bel-1-DNA interactions are specific. As shown in Fig. 2, the ability of GST-Bel-1 to bind to either an LTR DNA probe (Fig. 2A) or an internal promoter probe (Fig. 2B) was efficiently competed by an excess of either the unlabeled internal promoter probe (lanes 3 and 4) or the LTR probe (lanes 5 and 6) but was essentially unaffected by the same level of a nonspecific DNA competitor (lanes 7 and 8). Therefore, this DNA binding event is clearly specific and the interactions of Bel-1 with the DNA target sequences are likely to be mechanistically similar.

View larger version (69K): [in this window] [in a new window]   FIG. 2.   DNA binding by HFV Bel-1 is specific. The direct interaction of GST-Bel-1 (25 ng) with an HFV LTR (A) or internal promoter (Int. Pr.) DNA probe (B) was specifically blocked by preincubation with an 80- or 320-fold molar excess of unlabeled forms of these same two DNA probes but was not blocked by preincubation with a similar excess of a nonspecific (N.S.) DNA competitor.

Bel-1 binds the internal promoter more effectively than the LTR promoter. As noted above, the GST-Bel-1 fusion protein used in these assays bound to a higher percentage of the internal promoter probe than of the LTR promoter probe when incubated under the same assay conditions (Fig. 1). This observation suggests that the internal promoter may contain a higher-affinity binding site for Bel-1 than the one present in the LTR. To address this issue in more detail, we performed a quantitative EMSA with the internal promoter probe and several different levels of unlabeled internal promoter, LTR, or nonspecific competitor DNAs. As shown in Fig. 3, the internal promoter-derived competitor DNA (lanes 3 to 6) proved a far more effective competitor of the internal promoter probe-Bel-1 binding reaction than did the LTR-derived competitor (lanes 7 to 10). Therefore, it is clear that this internal promoter target sequence is a significantly higher-affinity in vitro DNA binding site for Bel-1 than is the tested HFV LTR target sequence.

View larger version (69K): [in this window] [in a new window]   FIG. 3.   Bel-1 binds the internal promoter DNA target more effectively than the LTR DNA target. This EMSA measures the degree of inhibition of GST-Bel-1 protein (25 ng) binding to an internal promoter (Int. Pr.) DNA probe observed upon preincubation with a 5-, 10-, 20-, or 40-fold molar excess of unlabeled forms of the internal promoter or LTR Bel-1 binding site or a nonspecific (N.S.) DNA competitor. The degree of inhibition seen with each competitor was measured with a phosphorimager and is given at bottom as percent residual binding, with binding in the absence of competitor (lane 2) set at 100%. As can be seen, the internal promoter probe competes more effectively for Bel-1 binding than does the LTR DNA probe. Little competition is observed with similar levels of the nonspecific DNA competitor. Neg., no added protein (control).

Modification interference analysis of Bel-1 DNA binding. We next wished to identify specific residues within the internal promoter and LTR DNA probes required for Bel-1 binding by modification interference analysis. For this purpose, the 108 to 35 HFV LTR probe and the 191 to 105 internal promoter probe were each uniquely end labeled and then incubated with dimethylsulfide, which specifically methylates G and A residues (31). The modified probes were then used for EMSA, and the C1 and C2 complexes (Fig. 1) observed with the internal promoter probe and the C1 complex observed with the LTR probe were excised, purified, and chemically cleaved. The pattern of DNA modification observed in these shifted DNA complexes was then compared to the pattern seen in the free-DNA probes.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Identification of critical residues for Bel-1 binding in the internal and LTR promoters. Modification interference analysis was used to identify purine residues present in the internal promoter (Int. Pr.) and LTR promoter that are critical for in vitro binding by the GST-Bel-1 fusion protein. Two DNA-protein complexes of different electrophoretic mobilities that form on the internal promoter probe, labeled C1 and C2 (Fig. 1), were analyzed independently, with comparable results. Residues showing marked interference are indicated by asterisks.

View larger version (13K): [in this window] [in a new window]   FIG. 5.   Alignment of possible Bel-1 and Tas minimal binding sites. Candidate minimal binding sites for HFV Bel-1 located in the LTR and internal promoter (Int. Pr.) elements are indicated and aligned. Purine residues that gave rise to detectable inhibition of Bel-1 binding after methylation are indicated. A candidate minimal SFV-1 Tas DNA binding site, based on the DNase I footprinting studies of Zou and Luciw (34), is also given, although the alignment used is largely arbitrary and does not show significant conservation of purine residues shown to be critical for Bel-1 binding. DNA sequences used for subsequent analyses are in capital letters. Boxes define residues whose mutation blocks Bel-1 binding in vitro and function in vivo (15).

Identification of a minimal Bel-1 DNA binding site. We next wished to determine if the minimal, ~25-bp internal promoter Bel-1 binding site delineated in Fig. 5 is indeed a complete, functional Bel-1 binding site. For this purpose, we prepared synthetic, double-stranded DNA oligonucleotides containing the wild-type 25-bp internal promoter sequence shown in Fig. 4 or containing the same sequence bearing a 6-bp mutation between residues 144 and 149. As shown in Fig. 6, the wild-type oligonucleotide proved able to effectively compete for Bel-1 binding to the full-length (191 to 105) internal promoter probe (lanes 3 to 5). However, introduction of a mutation into this minimal DNA binding site blocked this competition (lanes 6 to 8).

View larger version (84K): [in this window] [in a new window]   FIG. 6.   A 25-bp internal promoter sequence competes for specific Bel-1 DNA binding. A double-stranded, synthetic oligonucleotide consisting of the 25-bp internal promoter (Int. Pr.) sequence (164 to 140) shown in Fig. 5 specifically competed Bel-1 binding to the full-length (191 to 105) internal promoter probe (lanes 3 to 5). In contrast, a similar internal promoter-derived oligonucleotide containing mutations at residues 149 to 144 (Fig. 5) failed to compete (lanes 6 to 8). This competition experiment used 4-, 20-, and 80-fold molar excesses of each competitor oligonucleotide. WT, wild type; oligo, oligonucleotide.

Comparison of the activation domains of Bel-1 and Tas. A surprising result reported in Table 2 is that the interaction, in yeast cells, of SFV-1 Tas with a single copy of its minimal internal promoter binding site resulted in ~12 times more activation of the linked lacZ indicator gene than did the interaction of Bel-1 with a comparable single-copy sequence from the HFV internal promoter. One possible explanation for this phenomenon is that the HFV internal promoter binding site used in these constructs is incomplete. However, the observation that an extended internal promoter Bel-1 binding site is only a slightly more effective target for Bel-1 in vivo (Table 1) makes this explanation unlikely. An alternative possibility is that the transcription activation domain of SFV-1 Tas is significantly more active than the equivalent domain in HFV Bel-1. To examine this possibility, we linked the previously mapped (3, 12, 16, 27, 32) transcription activation domains of Bel-1 and of Tas to the DNA binding domain of GAL4 to determine whether expression of these fusion proteins in yeast cells would induce different levels of activation of a integrated yeast indicator gene bearing GAL4 DNA binding sites. Plasmids expressing fusion proteins consisting of the GAL4 DNA binding domain (aa 1 to 117) linked to various activation domains were introduced into the yeast strain Y190. After 3 days of selection, -Gal activity was determined as previously described (3). The GAL4 DNA binding domain alone induced a -Gal activity of 1 mOD/ml, while the GAL4-VP16 fusion protein induced had a -Gal activity of 5,645 mOD/ml. As previously reported (3), the Bel-1 activation domain, while clearly functional, is nevertheless ~90-fold less active (-Gal activity of 61 mOD/ml) than the potent activation domain present in the VP16 transcription factor when tested either in yeast cells, as in this case, or in mammalian cells. In contrast, the Tas activation domain was found to be ~14-fold more active than the Bel-1 activation domain when tested in this yeast assay system (-Gal activity of 850 milli-optical density units [mOD]/ml). Therefore, it appears that the ~12-fold difference in the activities of Bel-1 and Tas noted in Table 2 is likely primarily caused by a comparable difference in activation domain function. However, this finding does not exclude the possibility that Tas may also bind its internal promoter target site with a somewhat higher affinity than does Bel-1.

	DISCUSSION

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The expression of genes contained by the primate foamy viruses is regulated by the interplay of two viral promoter elements, located in the LTR and at the 3' end of the env gene, with the viral Bel-1/Tas transcriptional transactivator (29). Early after infection, the internal promoter is activated, resulting in the synthesis of mRNAs encoding the foamy virus accessory proteins, including Bel-1/Tas (21, 25). Subsequently, the LTR promoter is activated, presumably as a result of Bel-1/Tas expression, and mRNAs encoding the Gag, Pol, and Env structural proteins begin to accumulate. Therefore, although foamy viruses are not known to encode posttranscriptional regulatory proteins equivalent to the Rev and Rex proteins found in other primate complex retroviruses (7, 19), they nevertheless appear to be similar in that they display a temporal regulation of viral gene expression. However, the basis for this temporal order has been unclear, given that the internal promoter does not appear to be significantly more active than the LTR promoter in either the presence or the absence of the relevant Bel-1/Tas protein (5, 22, 25).

In this article, we describe a series of experiments designed to shed further light on the interaction of HFV Bel-1 with both the LTR and internal promoter elements. An interesting result to emerge from this analysis is that the Bel-1 DNA binding site present in the HFV internal promoter element is a significantly higher-affinity binding site for Bel-1 both in vitro and in vivo than is the cap-proximal Bel-1 binding site located in the HFV LTR promoter element. In particular, when analyzed by EMSA under identical assay conditions, Bel-1 was found to shift a higher percentage of an internal promoter probe than of a similar LTR promoter probe (Fig. 1). Similarly, internal promoter sequences also proved able to compete more effectively for Bel-1 DNA binding than did LTR promoter sequences when analyzed in a quantitative EMSA (Fig. 3). Finally, when assayed for in vivo DNA binding in yeast cells by a previously described assay, Bel-1 was able to activate a linked lacZ indicator gene ~200-fold more effectively when targeted to the internal promoter Bel-1 DNA binding site than when targeted to an equivalent LTR-derived Bel-1 binding site (Table 1). Taken together, these data suggest that activation of the foamy virus internal promoter may precede activation of the LTR promoter during the foamy virus life cycle because the internal promoter acts as a more effective Bel-1 protein binding site when Bel-1 levels are limiting, as they are predicted to be early in infection. We caution, however, that this analysis only examined binding to the cap-proximal BRE present in the HFV LTR and did not address the affinity of Bel-1 for other proposed LTR BRE elements (8, 20, 33). Although the cap-proximal BRE is fully sufficient to direct essentially wild-type levels of LTR-driven transcription in the presence of the Bel-1 protein in mammalian cells (18), these more 5' putative BREs could potentially play an important role in modulating the level of response of the LTR promoter to Bel-1, particularly given the observation (Table 2) that Bel-1 binding sites can display functional synergy.

Using modification interference and EMSA, we were able to identify several purine residues within both the LTR and internal promoters that are critical for Bel-1 binding in vitro (Fig. 4). We were then able to use this information to align these two sites (Fig. 5) and to propose a potential minimal, 25-bp DNA binding site for Bel-1. This minimal site was then shown to in fact function as an effective Bel-1 DNA binding site both in vitro (Fig. 6) and in vivo (Tables 1 and 2). The identification of this minimal Bel-1 DNA binding site will permit the future mutational definition of individual bases that form a functional binding site and should also allow the identification of residues that attenuate Bel-1 binding to the LTR promoter compared to the internal promoter. It will clearly be of interest to test whether an enhancement in the affinity of the HFV LTR binding site for Bel-1 results in a disruption of the normal temporal order of HFV gene expression.

As part of this analysis, we also tested whether a 25-bp sequence derived from the SFV-1 internal promoter, first identified as important for Tas binding by DNase I footprinting (34), was sufficient to function as an effective Tas binding site in vivo. As shown in Table 2, this short sequence indeed proved fully sufficient to bind Tas efficiently. This observation allowed us to determine whether the inability of Tas to function effectively via HFV promoter elements in mammalian cells was due to an inability of Tas to bind these HFV DNA sequences or, instead, reflected the absence of a critical Tas cofactor binding site. As shown in Table 2, Tas was able to interact only poorly with the minimal internal promoter Bel-1 target site. It is therefore apparent that Tas and Bel-1 have evolved distinct DNA sequence specificities over time. The future definition of these differences will clearly be of interest.

A final interesting result is that the activation domain present in SFV-1 Tas was found to be significantly more active than the one present in Bel-1. This finding explains the earlier observation (3) that expression of SFV-1 Tas from a multicopy plasmid in yeast cells is significantly more deleterious for yeast growth than is expression of HFV Bel-1, in that it has previously been shown that the level of toxicity observed in yeast upon overexpression of an acidic transcription activation domain is a function of the activity of the tested domain (2). This difference also appears to explain the finding that the level of indicator gene expression induced by the binding of Tas to its minimal binding site in yeast cells is significantly higher than the level observed when using Bel-1 and its minimal DNA binding site (Table 2). The question of whether this difference in activation domain function observed in yeast cells is relevant to the effectiveness of transcriptional activation by Tas and Bel-1 in mammalian cells remains to be explored.