Molecular Cloning of a New Interferon-induced Factor That Represses Human Immunodeficiency Virus Type 1 Long Terminal Repeat Expression

Transcriptional induction ofgenes is an essential part of the cellular response to interferons. To isolate yet unidentified IFN-regulated genes we have performed a differential screening on a cDNA library prepared from human lymphoblastoid Daudi cells treated for 16 h with human atlp interferon (Hu-atlpIFN). In the course ofthese studies we have isolated a human cDNA which codes for a protein sharing homology with the mouse Rpt-l gene; it will be referred as Staf-50 for Stimulated Trans-Acting Factor of50 kDa. Amino acid sequence analysis revealed that Staf-50 is a member of the Ring finger family and contains all the features of a transcriptional regulator able to initiate a second cascade of gene induction (sec ondary response). Staf·50 is induced by both type I and type II IFN in various cell lines and down-regulates the transcription directed by the long terminal repeat pro moter region of human immunodeficiency virus type 1 in transfected cells. These data are consistent with a role of Staf·50 in the mechanism of transduction of the IFN antiviral action. interferons (lFNs)1 are a family of secreted multifunc tional proteins which a broad spectrum of potent antiviral proper established in


Molecular Cloning of a New Interferon-induced Factor That Represses Human Immunodeficiency Virus Type 1 Long Terminal Repeat Expression*
(Received for publication, December 29, 1994, and in revised form, March 23, 1995) Catherine Tissot and Nadir MechtH Transcriptional induction of genes is an essential part of the cellular response to interferons. To isolate yet unidentified IFN-regulated genes we have performed a differential screening on a cDNA library prepared from human lymphoblastoid Daudi cells treated for 16 h with human atlp interferon (Hu-atlpIFN). In the course of these studies we have isolated a human cDNA which codes for a protein sharing homology with the mouse Rpt-l gene; it will be referred as Staf-50 for Stimulated Trans-Acting Factor of 50 kDa. Amino acid sequence analysis revealed that Staf-50 is a member of the Ring finger family and contains all the features of a transcriptional regulator able to initiate a second cascade of gene induction (secondary response). Staf·50 is induced by both type I and type II IFN in various cell lines and down-regulates the transcription directed by the long terminal repeat promoter region of human immunodeficiency virus type 1 in transfected cells. These data are consistent with a role of Staf·50 in the mechanism of transduction of the IFN antiviral action.
The interferons (lFNs)1 are a family of secreted multifunctional proteins which exert a broad spectrum of biological activities. First characterized for their potent antiviral properties, it has now been established that they are involved in number of regulatory functions such as control of cell proliferation, differentiation, and regulation of the immune system (1). They are subdivided into two types that activate transduction pathways via different cell surface receptors (2,3). Binding of both type I IFN (lFN-a/{:l) and type II IFN (IFN-y) result in the differential activation of latent cytoplasmic transcription factors termed 8tats (for 8ignal Transducer and Activator of Transcription) (4,5) which act at different cis-acting DNA elements. Type I IFN promptly induces the phosphorylation of 8tat-113 (p113 kDa), 8tat-91 (p91 kfra), and 8tat-84 (p84 kDa) (p91 and * This work was supported by grants from the Association pour la Recherche contre Ie Cancer, the Institut National de la Sante et de la Recherche Medicale, the Centre National de la Recherche Scientifique, and the Association Nationale pour la Recherche sur Ie Sida. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequencers) reported in this paper has been submitted to the GenBank™ I EMBL Data Bank with accession numberts) X82200.
Gene induction by type II IFN involves solely the phosphorylation of 8tat-91 by the JAK-2 kinase (a homolog of TYK2). This phosphorylation generates a homodimer of 8tat-91 which is able to bind the IFN-y-activated site (GA8 element) to activate transcription (14)(15)(16).
Of the many IFN activities, the antiviral state has been best characterized at the biochemical level. The IFNs can act directly at various steps of the viral multiplication cycle including cell penetration, transcription, translation, and the assembly of viral particles (17,18). 8everal IFN-induced proteins involved have been described such as the double stranded RNA-dependent p68 (human)/p65 (murine) protein kinase (double stranded-activated protein kinase) (19), the 2-5A synthetases (20)(21)(22), and the product of the Mxl gene (23). In the presence of double stranded RNA, the phosphorylation on a serine residue activates the latent ribosome-associated double stranded-activated protein kinase which then phosphorylates the a-subunit of the eukaryotic initiation factor-2. The phosphorylated form of eukaryotic initiation factor-2a induces an inhibition of protein synthesis giving rise to the establishment of an antiviral state (24). It has been established that the replicating viral RNA of viruses, like encephalomyocarditis virus, is most probably responsible for the activation of double stranded-activated protein kinase during viral infection (22). The second of the two IFN-induced and double stranded RNAactivated enzymes is the 2-5A synthetase which catalyzes the synthesis of adenosine oligomers (2-5A). This 2-5A then activates the RNase L, an endoribonuclease latent in most mammalian cells (17). Various data suggest that the 2-5A synthe-taselRNase L pathway inhibits the replication of picornaviruses such as encephalomyocarditis virus (20,25) and mengovirus (26). The human and mouse Mxl gene have been shown to confer selective innate resistance to influenza virus in cultured cells as well as in mice without affecting the development of many other viruses (18).
IFNs may also act indirectly on viral replication, by favoring the recognition of infected cells by the immune system. For example, IFN-y can control cytomegalovirus (CMV) infection by favoring presentation of viral antigen by the major histocompatibility class I molecules of CMV infected cells to the immune system (27).

Role of Staf50 in the Mechanism of Antiviral Action of IFNs
Since viruses have developed various strategies to circumvent the antiviral activities of IFNs (28), mammalian cells usually make use of several strategies that act in cooperation to interfere with the viral multiplication cycle. The mechanisms of the IFN-induced antiviral state are still far from being understood, and the molecular characterization of the IFNinduced proteins remains a main challenge for the comprehension of the molecular mechanism of IFN action.
We have therefore established a cDNA library from IFNtreated Daudi cells and made use of differential screening to search for yet unidentified IFN-regulated genes. In the course of these studies we have isolated a human cDNA with homologies to the mouse Rpt-l gene (29) which will be referred as Staf-50 for Stimulated Trans-Acting Factor of 50 kDa. We demonstrate that Staf-50 is induced by both type I and type II IFN and that its gene product down-regulates transcription directed by the long terminal repeat (LTR) promoter region of human immunodeficiency virus type 1 (HIV-1). The potential role of Staf-50 in the mechanism of antiviral action of IFNs is discussed.

MATERIALS AND METHODS
Cell Cultures-Human lymphoblastoid Daudi cells were grown in suspension in RPMI 1640 medium, supplemented with 10% (v/v) fetal calf serum. HeLa cells were grown in monolayer cultures in Dulbecco's medium containing 10% (v/v) fetal calf serum. For IFN induction, exponentially growing cells were exposed 16 h to 500 international unitslml of human lymphoblastoid IFN (Hu-IFNalf3 obtained from Hayashibara Biochemical Laboratories Inc.) or 500 units/ml of y-IFN (a gift of Roussel Uclaf, France). SV40-transformed monkey kidney epithelial cells (COS-7 mfi) were grown in monolayer cultures in Dulbecco's medium supplemented with 10% (v/v) fetal calf serum.
RNA Purification and Northern Blot Analysis-For RNA purification the cells were pelleted, washed in phosphate-buffer saline, and total mRNAs were isolated by the guanidine thiocyanate method, as described previously (30). RNAs were fractionated by electrophoresis on a 10% (v/v) formaldehyde-containing 1.2% (w/v) agarose gel and transferred to nylon membranes (Hybond N, Amersham). The multiple tissues Northern blot membrane (Clontech) was a gift of Dr. P. Fort. Prehybridizations were performed at 42°C for 12 h, in a mixture containing 50% (v/v) formamide, 0.75 MNaCl, 50 mM sodium phosphate buffer, pH 7, 1 mM EDTA, 0.2% (w/v) sodium dodecyl sulfate (SDS), 5 x Denhardt's, 10% (w/v) dextran sulfate, and 100 ,..g/ml denatured salmon sperm DNA. An additional 12-h hybridization was performed in the presence of 10 6 cpm/ml of the 32p random primed cDNA probe. Stringent washings were then conducted at 65°C in 0.1 x SSC buffer (0.15 MNaCl, 0.015 M sodium citrate) before autoradiography.
Construction of cDNA Library and Isolation of cDNA Clones-Poly(A +) RNAs were isolated from total mRNAs using the Dynabeads biomagnetic separation system (Biosys S.A) and the cDNA library was constructed in the ]I. ZAP-cDNA synthesis system (Stratagene). The library was plated at low density in order to obtain individual plaques and transferred to nylon membranes (Hybon N, Amersham). A single round screening was performed by successive hybridization of a single filter using 32P-labeled cDNA probes (2 x 10 6 cpm/ml) obtained from poly(A +) RNAs of untreated or IFN-treated Daudi cells. Prehybridization, hybridization, and washing of the filter were performed as described for Northern blot analysis. Clones exhibiting a variation in signal intensity were isolated and the pBluescript phagemid vectors containing inserts were excised using the ExAssit-SORL system (Stratagene), Phagemid DNAs were then extracted and used to probe Northern blot membranes.
Sequence Determination and Characterization of cDNA Clones-Plasmid DNA of individual clones were prepared and their sequences were determined by the Sanger dideoxy sequencing method (T7 sequencing kit from Pharmacia). The complete sequence of Staf-50 cDNA was obtained on both strands by overlap of sequenced fragments of the original clone after subcloning in the pBluescript II KS vector. Search for sequence homologies in the EMBL and GenBank data bases, as well as sequence analyses were performed by using the BISANCE facilities (31).
In Vitro Translation-In vitro transcription-translation of the Staf-50 containing vector was performed in the transcription/translation T-coupled reticulocyte lysate system (Promega) according to the manufacturer's instructions. The [ 35S]methionine-labeled proteins were fractionated by SDS-polyacrylamide gel electrophoresis before autoradiography.
Plasmid Constructions-The pJ-Staf50 and pJ-Staf50as were generated by cloning the XbaI-XbaI fragment, in the sense or the antisense orientation, respectively (see restriction map in Fig. ill), downstream from the CMV promoter in the pJ7D vector (32). The pLTR-luc and the pAcf3-gal vectors were a generous gift of I, Barlat and the pSVf3-gal (33) was a gift of Dr. J. M. Blanchard. The pCMVf3-gal vector expressing the f3-galactosidase gene under the dependence of the CMV promoter was provided by Stratagene.
Transient Transfection Experiments-For transfection experiments, 1-5 x 10 5 exponentially growing COS-7 m6 and HeLa cells were inoculated in 60-mm culture dishes. The following day, the cells were washed twice with phosphate-buffered saline and transfected with 9 ,..g of the appropriate mixture of vectors using the modified bovine serum mammalian transfection kit of Stratagene. The cells were then incubated 48 h at 37°C, washed twice with phosphate-buffered saline, and the luciferase activities were determined using the Luciferase Assay system (Promega) in a Berthold Luminometer counter (Lumat LB 9501). The f3-galactosidase activities were measured as described previously (34).

Construction and Screening of a cDNA Library from Hu-a] {3IFN-treated Daudi Cells-Total
RNAs were extracted from human lymphoblastoid Daudi cells treated for 16 h with 500 international units of Hu-al{3IFN. These conditions were previously described to induce strong antiviral and antiproliferative action in this cell line (35). An oriented cDNA library was constructed using the AZAP-cDNA synthesis kit (Stratagene), 5000 primary recombinant clones were screened successively with single stranded 32P-Iabeled cDNA derived from exponentially growing untreated cells and with cDNA from IFN-treated cells. A single filter was probed sequentially with both cDNA preparations in order to avoid false-positive clones (36). Since a limited number of clones were screened, the comparative analysis of autoradiographic data was performed manually. 105 spots exhibiting a variation in signal intensity were selected and pBluescript phagemid vectors containing inserts were excised using the Stratagen ExAssit-SORL system. DNA were prepared and used to probe a Northern blot containing total RNA extracted from Daudi cells treated for various times with Hu-al{3IFN. Clones exhibiting differential expression upon Northern blotting analysis by comparison with an invariant glyceraldehyde-3-phosphate dehydrogenase (37) probe were selected. Half of the 105 clones were false-positives and partial sequence examination of the others revealed four unknown IFN-regulated genes.
Analysis and Specificity of the Expression of a New IFNinduced RNA-We have focused our interest on a strongly IFN-induced gene which contains a 2.8-kb insert and which will be referred to as Staf-50. The kinetic of expression of the RNA hybridizing to this cDNA probe was analyzed by probing a Northern blot of total RNAs isolated from Daudi cells treated for various times with Hu-al{3IFN. As shown in Fig. lA, the 2.8-kb probe hybridized strongly to a RNA species of the same size which accumulated rapidly after the onset of IFN treatment (2-fold induction after 2 h). A 9-fold increase in its steady state level was reached after 16 h of exposure to IFN. Hybridization to a glyceraldehyde-3-phosphate dehydrogenase probe used as unvariant control confirmed that each lane of the blot contained an equal amount of total RNA (Fig. lA).
The specificity of induction of the 2.8-kb RNA in response to treatment with the various types of IFN was then analyzed. Since Daudi cells failed to respond to Hu--yIFN for the lack of functional receptors, He La cells were treated with 500 units/ml of Hu-al{3IFN or Hu-yIFN and total RNA were extracted and analyzed as described above. The kinetics of induction of Staf-50 mRNA was found to be similar with the two types of IFN (Fig. 1B), although Hu-al{3IFN was revealed to be a    stronger inducer . This induction wa s not dependent on continuou s protein synt hesis since it was unaffected by cycloheximide tre at me nt (Fig. I e ). Th ese data demonstrated that Staf-50 participates in the primary response of IFN action and wa s not th e consequ ence of a second set of gen e induction . Comparison between untreated Daudi a nd HeLa cells showed that a basal level in th e expre ssion of Staf-50 is easily detectable in Daudi cells but not in HeLa cell s. In ord er to determine th e tissue specificity of Staf-50 expre ssion, the 2.8-kb in sert was used to prob e a se t of RNAs isolated from several tis su es (Multiple Tissue Nor th ern from Clontech ). As show n in Fig. 2, Staf-50 is st rongly expressed, in the ab se nce of exogenous IFN treatment, in peripheral blood leukocyt es, in lymphoi d tissu es, such as spleen or thymus, and in ovary . Various basal levels were detected in other tissues. In contrast with the data observed in Da udi a nd HeLa cells, two major RNA species were detected specially in peripheral blood leu kocytes. Th ese results will be discussed later on t he basis of nucleotide seq ue nce a nalysis.
Sequ ence Analysis of Staf-50 eDNA-The complete nucleoti de and amino acid seque nces of t he 2.8-kb insert are pr esente d in Fig . 3. Computer search in the EMBL and GenBank data bases reveal homologies with t he nu cleotidic seq uences of t he mouse Rpt-1 gene (for regulatory protein , T-lym phocyte 1) (29) and the human SS -AIRO autoantige n (38). The Rpt -1 gene   was selectively expressed in quiescent helper/inducer T-cells and was shown to down-regulate gene expression directed by the interleukin 2 receptor-a chain promoter region (CD25) and by the LTR promoter region ofHIV-l. In contrast, the function of the SS-AIRO gene, described in the Sjorgren type A syndrome, remains unknown. The patterns of expression of Staf-50 and Rpt-l are rather similar with a preferential expression in quiescent T-Iymphocytes (data not shown), peripheral blood leukocytes, and in the lymphoid tissues (Fig. 2). However, such correlations do not provide, at the present time, definitive proof that Staf-50 is the human homolog of the Rpt-l gene or a member of a same family of genes, including the human SS-AlRO gene. The full-length cDNA (2811 bp) of Staf-50 contains an open reading frame encoding 442 amino acids (nucleotide 123-1451; Fig. 3), followed by a very long 3' -untranslated region (1360 bp), Analysis ofthe nucleotide sequence of this 1360-bp region revealed the presence of several potential polyadenylation signals (Fig. 3). The presence of additional more distant polyadenylation signals in the 3' part of the gene may explain the occurrence of an additional RNA species, with a longer 3'noncoding region, in peripheral blood leukocytes and in lymphoid tissues (Fig. 2). The predicted molecular mass of the Staf-50 protein (50,123 daltons) was verified by transcription-translation of a pBluescript phagemid containing the 2.8-kDa insert in the transcription/translation coupled reticulocyte lysate system (Promega), Proteins synthesized in the presence of [ 3 5 SJmet hionine were fractionated in a 10% (w/v) SDS-polyacrylamide gel and the labeled proteins were visualized by autoradiography. The Staf-50 clone directs the synthesis of a major polypeptide with an apparent molecular mass of 54,000 daltons (Fig. 4). Differences between the electrophoretic mobilities of proteins and their calculated molecular mass can be attributed either to post-transcriptional modifications of the translated products or to specific amino acid regions (like an arginine-rich polypeptide) which lead to abnormal migration in SDS-polyacrylamide gel (39).
Staf-50 Is a Member of the Ring Finger Family-A search and analysis for amino acid sequence homologies in the Gen-Bank data base revealed that the complete Staf-50 protein shares 44% amino acid homology with the mouse Rpt-l protein and 40.5% with the human SS-AIRO gene product. Some important characteristics of these three proteins can immediately be drawn from the comparison of their amino-terminal sequences whose alignment is presented in Fig. 5. Strong amino acid cluster homology is found in the 130 first amino acids although these proteins exhibit a relatively weak global homology. The strict conservation of motifs between human and mouse proteins is in favor of their role in the biochemical properties of these proteins. A second round of data base analysis using the PROSITE software was then performed in order to identify specific peptide motifs. The analysis revealed the presence of a C3HC4 zinc finger motif (Fig. 5) characteristic of the Ring finger family of proteins, whose functions are known to be mediated through DNA binding (40). Many of them are viral and cellular proteins involved in some aspect of the gene regulation. In particular, the immediate early genes of herpes simplex virus type 1 are implicated in the reactivation oflatent virus in herpes simplex virus type 1 infection (41). Others are involved in activation of DNA recombination and DNA repair Cotrans fection of pLTR-luc with pJ-Staf-50 resu lted in a 60 -90% inhibition of the lucifer ase activity as compared with pJ-Staf-50as and pJ7H (Fig. 6), or wit h pCMV-(3-gal (data not shown). Th e experiment was repeated se vera l times in th e linear range of th e assay a nd with differen t bat ches of DNA. Identi cal data were obta ined wit h both tra nsfection procedu res. In contrast, pJ -Staf-50 had no effect on (3-galactosidase express ion dir ected by t he SV40 promoter (pSV(3-ga D or by the act in promoter (pAc(3gal) (Fig. 6). Recen t find ings report th a t a synthe tic peptide corres ponding to the C3HC4 domain of th e Rin gl gene pr odu ct binds to DNA, in a zinc dependent manner, alt hough weakl y a nd nonspecifically (40). Th ese resul ts strong ly suggest th at ot her peptide moti fs are responsibl e for th e specificity of DNA binding activity. Th e align me nt of th e amino acid seque nces pr esented in Fig . 5 reveal s th e pr esence near the C3HC4 zinc finger motif, of a second pu tative zinc finger structure with a CHC3H2-type signature. Th is motif has already been identified in t hree other memb ers of th e Ring famil y (42), the human a nd mouse Rfp tyro sine kin as e gene prod ucts (43), th e TIS transforming mouse fusion pr otein (44), a nd th e protein encoded by t he human prom yelocytic leukemi a gene (42). For t he latter, the last hi stidine resi du e is not pr esent. Interestingly, in Staf-50, Rpt-l , SSA-IRO, a nd Rfp protein s, t he two zinc fing er st ru ct ure a re se pa ra te d by 40 amino acid residues. In these regions we have identified a conserv ed ba sic motif (t he relative basicity: H +K+R residu esID +E residues = .5) th at we have termed 1M (for in termedi ate moti f) (Fig. 5). Such a basic motifis known to increase t he affinity of DNA-binding protein to th e DNA. Th e pr esenc e of two zinc fingers and t he 1M motif in th e same configu ra tion in th ese four protein s (Fig. 5) suggests that th ey act in synergy to bind DNA targets.
Th e Staf-50 a mino acid seque nce a lso encloses a KRSESWTLKKPKSVSKKLKSV bi-partite motif (see Fig. 3) simila r to th e nu clear location signal pr esent in most nuc lear protein s (45). Thi s observation is consistent with th e proposed DNA binding activity of Staf-50.
Tran s-acting Fun ction of the Star-50 Protein-In order to determine, by ana logy with Rpt-l , th e ability of St af-50 to affect t ra nscri ption dir ected by the LTR prom oter region of HIV-l , cotran sfection experi ments were performed with COS-7 m6 and HeLa cells. To thi s aim, th e IS26-bp XbaI -XbaI fragm ent of th e pBlu escript-Staf-50 cDNA (Fig. ill ) was cloned und er th e transcri ptiona l depend ence of th e CMV promoter in the pJ7f1 vector.
The St a f-50 cDNA was positioned in sense (pJ -Sta f-50) or antisense orie nta tion as a negative control (pJ -Sta f-50as). A luciferase gene und er t he control of the LTR promoter region of HIV-l (pLTR-luc) was used as a reporter gene. Tr an sfection s were perform ed by calcium phosph at e precipitation or by a lipofectamine procedure (Life Technologies, Inc.). Th e cells wer e collected 72 h later, a nd cellul ar extracts were prepa red to det ermine th e luciferase activities as describ ed und er "Materials a nd Methods."

DISCUSSION
Binding ofIFNs to their specific cell surfa ce receptors trigge rs th e rapid nu clea r t ra nslocation of a complex form ed by ass ociation between the various phosphorylated St ats protein s (see Introducti on). Thi s mechani sm is not depend en t on continuous protein synthes is and resu lts in a first set of genes induction or, prim ary IFN response. Some of t hese genes, as 2-5A synthe tases (17), Mxl gene (23), double st ra nded-activated p6S prot ein kinase (19), major histocomp atibility complex class I and class II, or tryptophan yl tRNA synthe tase (46) a re kn own mediators of th e biological functions of IFN s. Oth er genes code for nuclear protein s which share all th e features of tran scription factor s and a re able to initiate a second cascad e of gene induction (secondary respon se) requ iring continued protein synthes is. As an exa mple, th e IRF-l an d IRF-2 genes act, respectively, as tra nscri ptional activato r and repressor of the Hu-(3IFN gene (47). However, t he function of most of IFN -induced genes remain s unknown. In t his report, we describ e th e clonin g and th e partial cha ra cteriza tion of a new IFN-ind uced gene, design at ed as St af-50 and exhibiting prop erties of transcription al regulator.
Th e comparison of t he nucl eotide seque nce of Sta f-50 wit h a ll th e sequ enc es of EMBL a nd GenB ank data ba ses revea led sign ificant homologies betw een Staf-50, mou se Rpt-l, a nd human SS-AIRO autoa ntige n cDNA seque nces . The best score determined wa s bet ween St af-50 and Rpt-l with 64% homo logy in th e coding region . Th e three pr otein s sha re a weak simila r homology at th e a mino a cid level (44% betw een Staf-50 a nd Rpt-l, and 40 .5% betw een Staf-50 a nd SS-AlRO ). In order to estab lish t he family relationship betw een Sta f-50 a nd t he mouse Rpt-l gen e we have compared th eir ti ssu e specificity of mRNA expre ss ion . As described pr eviou sly for Rpt-I (29), Staf-50 mRNA is constitutively expressed in peripheral blood leukocyt es a nd in lymphoid ti ssu es, such as splee n or th ymu s (Fig. 2). Although such data do not pro ve that St af-50 is t he human homo log of Rpt-l or a memb er of a gene family. Staf-50 is also expressed in non-l ymphoid HeLa cells afte r treatm ent wit h typ e I or type II IFN (Fig. 1). Further st udies on th e modulation of the mouse Rpt-l as well as t he human SS-AIRO genes by th e IFNs might be worth performing.
Sequ ence a na lysis indicated th at St af-50 is a new memb er of th e Ring fing er su perfamily of proteins involv ed in gene regulati on , DNA recombi nation, and DNA repair (40). Interestingly, th e alignment of th e a mino acid seque nce ofStaf-50 with severa l members of th e Rin g family, such as th e Rpt-l protein , t he h uma n and mouse Rfp tyro sin e kinases, a nd th e h um an SS-AIRO a utoantigen (Fig. 5) revea led th e pr esence of tw o zinc fing er motifs. Thi s tandem zinc finger domain contain s a C3HC4 conserv ed motif describ ed in t he Rin g finger family (40) and a putative zinc finger motif with a CHC3 H2 type signature. Th e C3H C4 motif is un abl e to confer high specificity a nd affinity for DNA binding (40). It is lik ely th at othe r motifs, like CHC3H2, might be required to gene ra te a high a ffinity complex. However, th e contributi on of th e CHC3 H2 finger to DNA bind ing has not been demonstrated . Th e t wo zinc finger moti fs are join ed by a ba sic domain (36 :t 1 amino acids with a n isoelectric pH = 12) whi ch is also cha racteristic of DNA-binding protein s. We hav e identified , in t his region, a conse rve d FIG. 6. Inhibition of LTR-directed luciferase expression by Staf·50 in transfected COS·7 m6 cells. The relative luciferase activities were calculated by dividing the values measured after cotransfection of LTR-luc reporter and the indicated constructions (pJ-Staf-50as or pJ-StaJ-50) by the values measured after cotransfection of LTR-luc and pJ7D. Values of less than 1 indicate inhibition of LTR-luc and represent the mean and standard deviation for several distinct experiments. pSVJ3-gal and pAcJ3-gal activities were determined the same way.
basic motif that we have termed 1M (for intermediate motif) (Fig. 5). Interestingly, these three motifs are rigorously positioned in the same configuration in Staf-50, Rpt-L, Rfp tyrosine kinase, and SS-AfRO autoantigen. Although it is tempting to speculate that they could act via common mechanisms we have no direct evidence to accredit this hypothesis at the present time.
Knowing the antiviral properties ofIFNs, we have analyzed the capacity of Staf-50 expression to induce an antiviral state in various cell lines. Based on the homology with the mouse Rpt-l gene product we first examined the ability of Staf-50 to down-regulate the transcription directed by the LTR promoter region of HIV-l. Cotransfection experiments performed in different cell lineages showed a significant and reproducible 60-90% inhibition of the luciferase activity used as a reporter gene (Fig. 6). These results strongly suggest that Staf-50 may be involved in the antiviral process of IFNs against retoviral infections. Experiments are underway to determine whether the constitutive Staf-50 expression is able to confer a partial or total protection against HIV-l infection in various cell lines. In a more general way, it will be important to determine whether  (49), three SPI binding sites (49,50), and a tandemly repeated enhancer region recognized by the cellular NF-KB (51) and EBP (52) transcription factors. The transactivation response element responsive for the viral trans-activator protein Tat (53) controls the LTR transcription at the RNA level. The main functional region with the potential to decrease the synthesis of viral RNA is composed by the negative regulatory element (54). This region is recognized by cellular factors including AP-l and NF-AT-I (55).
The two consensus NF-KB binding motifs are very important for the expression of HIV-I at high level in activated CD4 + T lymphocytes (56). The NF-KB-mediated transactivation of the HIV-I LTR promoter is inhibited in IFN-producing cells. This down-regulation is associated with an alteration of the binding pattern ofNF-KB-specific nuclear proteins to the core enhancer element of the HIV-I LTR (57). The induction of HIV-I provirus by herpes simplex virus-l infection involves cooperation between NF-KB and the virus-encoded transactivator ICPO (58). These data suggest that Staf-50 could act as a repressor of the NF-KB activation and interact either directly with NF-KB-binding proteins, thereby altering their affinity for DNA, or indirectly with its DNA target, to modulate HIV-I LTR expression. Staf-50 could also act as an activator of negative regulatory element. However, our results do not provide direct evidence for specific Staf-50-DNA interaction and furthermore, do not exclude the possibility that Staf-50 protein binds to a RNA structure. Sequence homology with the 52-kDa component of the SS-AfRO ribonucleoparticle suggests that Staf-50 may interact with the the transcription response element of LTR promoter to regulate transcription. Experiments are underway to delineate the target of Staf-50 protein. The availability of specific antibodies against this protein is essential to determine its function and their preparation is now in progress.