INTRODUCTION

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The inhibition by alpha interferon (IFN-) and IFN- of viral infection depends upon the full transcriptional activation capacity of Stat1 and Stat2 proteins (9, 37). These transcription factors are latent in the cytoplasm until activated by tyrosine phosphorylation; dimerization, nuclear accumulation, and gene activation follow, with the result that the antiviral state becomes established (9, 37). A great deal has been learned about the functional anatomy of Stats 1 and 2 through mutagenesis of the coding sequences and introduction of mutants into cell lines deficient in one or the other of these proteins. The atomic structures of the core of Stats 1 and 3 and the highly conserved amino terminus have also been described (3, 8), and this information helps to guide such mutagenesis studies. These studies revealed that IFN- treatment results in the activation of both Stat1 and Stat2, which form a heterodimer that interacts with a 48-kDa protein, p48, forming the interferon-stimulated gene factor 3 (ISGF3) DNA-binding complex (18, 31) that activates the IFN- target genes (10, 37). In IFN--stimulated gene expression, the COOH domain of Stat2 is required, while the COOH terminus of Stat1 is not. IFN- treatment results in the activation of only Stat1, which forms a homodimer that activates target genes that contain gamma activation sequences (GAS) in their promoters (6, 10, 36).

The transcriptional activation of ISGF3 depends on the COOH-terminal segment of Stat2 but not that segment of Stat1. IFN--dependent gene activation requires the COOH terminus of Stat1. Thus, at least one transcriptional activation function of the Stats is provided by the COOH terminus (16, 21, 24, 26, 38, 42). The C-terminal transcription activation domain (TAD) is known to interact with CBP-p300 (4, 14, 41) as well as with other proteins which contribute to transcriptional activation (41, 42). We have examined the requirement for and the specificity of the Stat1 COOH terminal domain in transcriptional activation by introducing a variety of recombinant Stat1 constructs transiently or into stable cell lines and correlated results from increasingly more specific assays for transcripitonal activation. The least specific assay was supplementing the yeast Gal4 DNA-binding domain (DBD) (32) with various transactivation domains. Next, the activation of endogenous genes by wild-type Stat1 and various Stat1 chimeric molecules was tested. Finally, induction of the antiviral state, an in vivo response presumably requiring the balanced activation of a set of genes to achieve a physiologic result (1, 27), were tested. We found that considerable variation exists in the ability of the Stat1 constructs with various COOH-terminal activation domains to drive transcription from synthetic promoters, several being more effective than the wild-type Stat1 COOH terminus. However, the Stat1 COOH terminus functions about as well as any other activation domain in stimulating endogenous genes or the antiviral state in response to IFN-. Activation domains other than the native Stat1 COOH domain can, however, support establishment of the antiviral state.

	MATERIALS AND METHODS

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Cell culture and transfection. Human U3A cells (provided by George Stark, Cleveland Clinic Foundation Research Institute, Ohio) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% cosmic calf serum (Hyclone).

Plasmid constructions. Mammalian expression vectors Rc/CMV (Clontech), containing wild-type Stat1 or Stat1(S727A), and the 3×Ly6E-GAS luciferase reporter were described previously (39). Rc/CMV Stat1N-TAD fusions were constructed by replacing the XbaI-ApaI fragment at the COOH end of Stat1 with PCR-amplified TADs of Stats 2, 3, 4, 5a, and 6, VP16, and p53 (12, 20, 29, 43). The same TAD fragments were cloned into pSG424 (32) for generating Gal4 fusion proteins. The PCR regions of all constructs were confirmed by sequencing analysis. The Gal4-Stat1C(711-750) and the Gal4 luciferase reporter (5×Gal4 DB) were provided by J. Zhang (42).

RT-PCR. Reverse transcriptase (RT)-PCR assays were performed on RNAs prepared from stably transfected cell lines with or without IFN- or IFN- treatment as previously described with slight modifications (17). Briefly, total RNAs were isolated using Trizol reagent (Gibco BRL) from subconfluent cells treated with IFN- or IFN- for 4.5 h or left untreated and digested with DNaseI (Promega), followed by reverse transcription with Moloney murine leukemia virus (MMLV) reverse transcriptase (Gibco-BRL) using random primers (Invitrogen). A mock transcription was carried out with no MMLV added (RT). Typically, 5 µg of total RNA was used in each reverse transcription reaction, and 1/20 of the resulting cDNAs was then used as the template for 25 cycles of PCR amplification with radioactive deoxynucleotides using primers specific to the indicated genes. The products were resolved on a 5% polyacrylamide gel and detected by autoradiography. The primers for IRF-1, guanylate-binding proteins (GBP), ISG15, ISG54, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as described (40). The primers for TAP1 were TAP1a (AACGGTTGGCTCCAAGAGC) and TAP1b (CGCACAGGGTTTCCAGAGC).

Antiviral CPE assay. IFN-mediated antiviral response analyzed by a cytopathic effect (CPE) assay was performed as described (15) with modifications. Briefly, cells were plated on 96-well plates 1 day before the assay. Cells were pretreated with 1,000 IU of human IFN- or 25 ng of IFN- per ml for 6 h or left untreated. Encephalomyocarditis virus (EMCV) was diluted in plain DMEM without serum to the desired concentration and added to the cells. After 24 h, the medium was removed, and cells were stained and visualized with 2% methylene blue in 50% ethanol. The absolute absorbance of the methylene blue staining was measured at 630 nm. The killing curve of each test was plotted, and the virus concentration required to kill 50% of the cells was calculated to evaluate the protection efficiency. EMCV was a gift from Robert H. Silverman and was produced and titrated on U3A cells.

	RESULTS

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Activity of the Gal4-TAD fusion proteins. Recombinant DNA constructs encoding the COOH-terminal transactivation domains of Stat 1, 2, 3, 4, 5a, and 6 fused to the Gal4DBD were prepared to assess and compare their transactivation potential in parallel with TADs from two well-studied acidic activators, VP16 and P53 (5). All of these fusion constructs, when transiently transfected into cells, could activate transcription from a Gal4-luciferase reporter construct that has five Gal4 DNA-binding sites, indicating that all of the STAT carboxyl termini have transactivating capacity and thus constitute TADs (Fig. 1).

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Comparison of the transactivation domains of the different STAT COOH termini by fusion to the DNA-binding domain of the yeast transcription factor Gal4. Gal4DBD-TAD fusions which comprise the DNA binding and dimerization domain of Gal4 (residues 1 to 147) and indicated TADs were constructed. These constructs were transiently transfected into U3A cells with a luciferase reporter (5×Gal4DB) with five copies of Gal4 binding sites. Luciferase activities were determined ~40 h after transfection. Representative results of four experiments are shown with the standard error for triplicate samples.

Different transcriptional activity on a reporter with multiple Stat1 binding sites. We next tested the various Stat1 COOH-terminal replacement constructs for their response to IFN- in transient transfections. The human cell line U3A lacks endogenous Stat1 (22, 25) and therefore can be used to assay IFN--induced transcriptional activity after introduction of the various Stat1 constructs along with a cotransfected luciferase reporter with multiple Stat1 binding sites (39) (Fig. 2). The TADs assayed in the previous section were fused with the Stat1 N-terminal domain (residues 1 to 716, referred to as Stat1N hereafter), replacing the wild-type Stat1 COOH terminal TAD. These Stat1N-TAD chimeric proteins showed different activity in mediating IFN- activation of the reporter gene (see below), but all gave transcriptional activation.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Transcription activity of various TADs when fused to Stat1 N-terminal domain. Various TADs as indicated were fused to Stat1N (residues 1 to 716). These constructs were transiently transfected into U3A cells with a luciferase reporter (3×Ly6E-Gas) with three copies of Stat1 binding sites. At 24 h after transfection, cells were treated or left untreated with IFN- for 6 h and harvested for luciferase assays. The experiments were performed five times, each time with triplicate or quadruple samples. A representative experiment is shown, with the mean and standard deviation of triplicate samples. Shown at the bottom is the Western blot analysis with an antibody reactive with Stat1N on equal amounts of cell extracts from the IFN- treated samples. The faint signal in the vector lane at the STAT1 position was due to contamination from the leak of lane 1. *, nonspecific protein band. The untreated samples showed similar protein expression levels (data not shown).

Stat1N-TAD fusion proteins activate endogenous IFN target genes. To further examine the requirement and specificity of the TADs in supporting Stat1 transcriptional activity, activation of IFN--inducible target genes in the chromosome was tested (Fig. 3). Expression vectors encoding the Stat1N-TAD chimeric proteins were permanently transfected into U3A cells. Individual cell lines were selected for expression of the Stat1 COOH-terminal fusion proteins (Fig. 3A). The Stat1N-Stat4C(698-749) cell line was not constructed, since the Stat4 COOH-terminal domain showed very low activity in both of the above assays. To determine how the Stat1 fusions function in mediating IFN- and IFN- activation, a semiquantitative RT-PCR analysis (17) was used to assay activation of several target genes. Representative results are shown in Fig. 3. IRF-1, GBP, and TAP1 (23) can be activated to various degrees by both IFN- and IFN-, whereas ISG15 and ISG54 are activated by IFN- but not IFN- (6).

View larger version (72K): [in this window] [in a new window]   FIG. 3.   Activation of IFN-responsive genes by Stat1N-TAD fusions. U3A stable transfectants expressing wild-type Stat1 or various Stat1N-TAD fusions were selected. (A) Western blot analysis using an antibody against Stat1 N-terminal domain. (B) RT-PCR analysis as detailed in Materials and Methods was performed on the indicated endogenous genes from the Stat1N-TAD stable cell lines treated with IFN- or IFN- or left untreated. In the samples of GAPDH (RT), reverse transcriptase was left out in the reverse transcription.

Stat1N-TAD fusion proteins induce antiviral state. Presumably through activation of a large number of different genes, IFN can induce an antiviral state (37). Using a standard CPE assay of EMCV infection, we next assayed the effectiveness of the various Stat1 chimeras in establishing the IFN-- and IFN--induced antiviral state (33, 44). Antiviral response to both IFN- and IFN- can be reconstituted in U3A cells by permanent transfection of Stat1 (25). Monolayer U3A cells or U3A cells permanently transfected with various Stat1 fusion constructs were treated with a maximally protective dose of either IFN- or IFN-, followed by infection with serially diluted EMCV (Fig. 4A). Protection by IFN- was quantitated by comparing the virus concentrations required to generate 50% of the cell killing as measured by staining of remaining cells (Fig. 4B). All assays were performed on at least two cell lines derived from each construct, and Fig. 4 shows the results from one cell line which were reproduced in several other tests. Without IFN treatment, different cell lines showed slightly different basal levels of susceptibility to EMCV infection, and the maximal protection generated by the IFN- antiviral effect was between ~15- and 50-fold. (It is known that IFN- induces less protection than does IFN- [1], as is evident in Fig. 4A.) The Stat1 fusion proteins protected cells from viral infection in response to IFN- to a similar level as did wild-type Stat1, consistent with the dominant role of Stat2 in mediating the IFN- response (30). Stat1N-Stat3C(709-770) and Stat1N-VP16(413-490) were more effective than or as effective as wild-type Stat1 in mediating the IFN--induced antiviral state (Fig. 4A and B, ~53- and ~17-fold protection, respectively, compared with ~16-fold by Stat1). The Stat1 COOH-terminal mutant Stat1(S727A) was less effective in this assay (Fig. 4A and B, ~3-fold, compared with ~16-fold by Stat1).

View larger version (45K): [in this window] [in a new window]   FIG. 4.   Antiviral responses of U3A cells complemented with various Stat1N-TADs. (A) The indicated cell lines were treated with IFN- or IFN- for 6 h or left untreated. The indicated amount of EMCV was added to the corresponding wells and left on the cells for 24 h. The viable cells left in the wells were visualized by methylene blue staining. Similar results were obtained from several experiments performed in duplicate and cells plated at different densities on at least two cell lines from each construct. (B) The above results were quantitated by measuring the absorbance of methylene blue staining of remaining cells at 630 nm. The IFN- protection efficiency was evaluated by comparing the virus concentrations required to kill 50% of the nontreated cells versus 50% of the IFN--treated cells.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have tested the requirement for and specificity of the Stat1 C-terminal domain in mediating its transcriptional activation function. The first major conclusion from these experiments is that the IFN--mediated protection against virus infection does not specifically require the natural Stat1 COOH-terminal sequences, i.e., other activator sequences can suffice. Similar results have been reported in other systems whereby heterologous activator sequences fused to the DNA-binding domain can mediate in vivo biological responses. For example, VP16 can turn ZEBRA into a more powerful activator in vivo when fused with ZEBRA, although part of the ZEBRA activation domain needs to be present for the fusion protein to work (2). Likewise, the mutant bicoid (Bcd) phenotype (11) could be rescued by injection of Bcd mutant embryos with mRNAs encoding fusion proteins consisting of the DNA-binding domain of Bcd attached to several heterologous acidic activating sequences, including acidic regions derived from yeast GAL4- and Escherichia coli-derived sequences. However, when the Bcd DNA-binding domain was fused to the most potent activator VP16, its mRNA had a deleterious toxic effect even when injected at a low concentration.

Second, it is clear that all of the COOH-terminal domains of the STATs have demonstrable transactivation potential that varies both among the different proteins and according to the assay used in assessing transcription. Perhaps the least specific assay, the ability of Gal4DBD-TADs to activate transcription, is the least specific guide to physiologic function. Significant variation was found when the activities of the TADs as Gal4-DBD fusions and Stat1N-TAD fusions were compared. Stat6C(677-837), either as a Gal4 fusion product or when fused to Stat1N(1-716), seems to be more effective than wild-type Stat1 in activating the reporter constructs in transient-transfection assays, in accord with earlier reports (13, 24). In contrast, the other much stronger TADs determined by Gal4 reporter assay, Stat2C(700-851), VP16(413-490), and P53(2-73), showed activity weaker than wild-type Stat1 when fused with Stat1N instead (Fig. 2). These results suggest caution in use of synthetic promoters to derive physiologic conclusions.

Likewise, differences between the effectiveness of each TAD exist when comparing transient and permanent transfections. Of course, in these cases there is also a difference in the promoters, synthetic promoters being used in the transient transfections and endogenous promoters in the permanent transfections. Perhaps not surprisingly, these results make a case for scoring endogenous gene activation when attempting to determine the possible contribution of a transcription factor to a physiologic decision.

When a more stringent and perhaps physiologically relevant comparison was made, namely, for the capacity to induce the Stat1-mediated antiviral response involving activation of a dozen(s) genes organized in the in vivo chromosomal context (6, 37), again a discrepancy with the transient assay was found. Unrelated TADs from VP16 and P53 could also function in this assay, with VP16-TAD being very effective. There was a reasonable correspondence between induction of several endogenous genes and IFN responsiveness; Stat1 induced both, as did Stat3C(709-770), the closest in sequence to Stat1-TAD. This was the case in spite of their relatively weak activation potential, as assayed by Gal4 fusion. It also appears that different promoters may have different requirements. For example, the TAP1 promoter is less sensitive to the difference in the Stat1N-TAD fusions than IRF1 or GBP [Fig. 4B, compare wild-type Stat1 and Stat1N-Stat2C(700-851)]. Such results may reflect the use of different auxilliary transcriptional activators in the enhanceosomal complexes (7, 28). Nevertheless, no specific Stat1 C-terminal TAD appears to be required to bring about the antiviral state.