From the
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
The interferons (IFNs)
Gene
induction by type II IFN involves solely the phosphorylation of Stat-91
by the JAK-2 kinase (a homolog of TYK2). This phosphorylation generates
a homodimer of Stat-91 which is able to bind the IFN-
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) . Several 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
Mx1 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
IFNs may also act indirectly on viral
replication, by favoring the recognition of infected cells by the
immune system. For example, IFN-
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 IFN-induced
proteins remains a main challenge for the comprehension of the
molecular mechanism of IFN action.
We have therefore established a
cDNA library from IFN-treated 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-1 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.
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-
The Staf-50 amino acid sequence also encloses a KRSESWTLKKPKSVSKKLKSV bi-partite
motif (see Fig. 3) similar to the nuclear location signal present
in most nuclear proteins(45) . This observation is consistent
with the proposed DNA binding activity of Staf-50.
Binding of IFNs to their specific cell surface receptors
triggers the rapid nuclear translocation of a complex formed by
association between the various phosphorylated Stats proteins (see
Introduction). This mechanism is not dependent on continuous protein
synthesis and results in a first set of genes induction or, primary IFN
response. Some of these genes, as 2-5A synthetases(17) ,
Mx1 gene (23), double stranded-activated p68 protein
kinase(19) , major histocompatibility complex class I and class
II, or tryptophanyl tRNA synthetase (46) are known mediators of
the biological functions of IFNs. Other genes code for nuclear proteins
which share all the features of transcription factors and are able to
initiate a second cascade of gene induction (secondary response)
requiring continued protein synthesis. As an example, the IRF-1 and
IRF-2 genes act, respectively, as transcriptional activator and
repressor of the Hu-
The comparison of the nucleotide sequence of Staf-50 with
all the sequences of EMBL and GenBank data bases revealed significant
homologies between Staf-50, mouse Rpt-1, and human SS-A/RO autoantigen
cDNA sequences. The best score determined was between Staf-50 and Rpt-1
with 64% homology in the coding region. The three proteins share a weak
similar homology at the amino acid level (44% between Staf-50 and
Rpt-1, and 40.5% between Staf-50 and SS-A/RO). In order to establish
the family relationship between Staf-50 and the mouse Rpt-1 gene we
have compared their tissue specificity of mRNA expression. As described
previously for Rpt-1(29) , Staf-50 mRNA is constitutively
expressed in peripheral blood leukocytes and in lymphoid tissues, such
as spleen or thymus (Fig. 2). Although such data do not prove
that Staf-50 is the human homolog of Rpt-1 or a member of a gene
family. Staf-50 is also expressed in non-lymphoid HeLa cells after
treatment with type I or type II IFN (Fig. 1). Further studies on
the modulation of the mouse Rpt-1 as well as the human SS-A/RO genes by
the IFNs might be worth performing.
Sequence analysis indicated that
Staf-50 is a new member of the Ring finger superfamily of proteins
involved in gene regulation, DNA recombination, and DNA
repair(40) . Interestingly, the alignment of the amino acid
sequence of Staf-50 with several members of the Ring family, such as
the Rpt-1 protein, the human and mouse Rfp tyrosine kinases, and the
human SS-A/RO autoantigen (Fig. 5) revealed the presence of two
zinc finger motifs. This tandem zinc finger domain contains a C3HC4
conserved motif described in the Ring finger family (40) and a
putative zinc finger motif with a CHC3H2 type signature. The C3HC4
motif is unable to confer high specificity and affinity for DNA
binding(40) . It is likely that other motifs, like CHC3H2, might
be required to generate a high affinity complex. However, the
contribution of the CHC3H2 finger to DNA binding has not been
demonstrated. The two zinc finger motifs are joined by a basic domain
(36 ± 1 amino acids with an isoelectric pH = 12) which is
also characteristic of DNA-binding proteins. We have identified, in
this region, a conserved basic motif that we have termed IM (for
intermediate motif) (Fig. 5). Interestingly, these three motifs
are rigorously positioned in the same configuration in Staf-50, Rpt-1,
Rfp tyrosine kinase, and SS-A/RO 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 of IFNs, 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-1 gene product we
first examined the ability of Staf-50 to down-regulate the
transcription directed by the LTR promoter region of HIV-1.
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-1 infection in various
cell lines. In a more general way, it will be important to determine
whether the constitutive expression of Staf-50 interferes with the
multiplication cycle of other virus types.
The transcriptional
activity of the LTR-HIV-1 promoter region is controlled by several
regulatory elements acting in either positive or negative fashion (for
review, see Ref. 48). The most influential DNA elements, identified as
positive regulator of basal transcription, include the TATA box element
recognized by TFIID(49) , three SP1 binding
sites(49, 50) , and a tandemly repeated enhancer region
recognized by the cellular NF-
The two consensus NF-
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
This work was preformed in the group of B. Lebleu.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/
interferon (Hu-
/
IFN). In the
course of these studies we have isolated a human cDNA which codes for a
protein sharing homology with the mouse Rpt-1 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.
(
)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 (IFN-
/
) and type II IFN (IFN-
) result in the
differential activation of latent cytoplasmic transcription factors
termed Stats (for Signal Transducer and Activator of Transcription) (4, 5) which act at different cis-acting DNA elements.
Type I IFN promptly induces the phosphorylation of Stat-113 (p113 kDa),
Stat-91 (p91 kDa), and Stat-84 (p84 kDa) (p91 and p84 are generated
from the same gene by alternative splicing) proteins, by tyrosine
phosphorylation involving the
/
IFN receptor-associated
tyrosine kinases TYK2 and JAK1 (6-9). Following phosphorylation,
Stat-113 and Stat-91 or Stat-84 form the transcriptionally active
IFN-stimulated gene factor 3 by association with a 48-kDa subunit which
binds DNA(10, 11) . The specificity of the
transcriptional activation by IFN-stimulated gene factor 3 is mediated
by specific elements termed IFN-stimulatory element located in the
promotor region of IFN-inducible genes(12, 13) .
-activated
site (GAS element) to activate transcription (14-16).
-subunit of the eukaryotic initiation factor-2.
The phosphorylated form of eukaryotic initiation factor-2
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 RNA-activated
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
synthetase/RNase L pathway inhibits the replication of picornaviruses
such as encephalomyocarditis virus (20, 25) and
mengovirus(26) . The human and mouse Mx1 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) .
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) .
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 units/ml of human lymphoblastoid IFN (Hu-IFN/
obtained from Hayashibara Biochemical Laboratories Inc.) or 500
units/ml of
-IFN (a gift of Roussel Uclaf, France).
SV40-transformed monkey kidney epithelial cells (COS-7 m6) 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 M NaCl, 50
mM sodium phosphate buffer, pH 7, 1 mM EDTA, 0.2%
(w/v) sodium dodecyl sulfate (SDS), 5 Denhardt's, 10%
(w/v) dextran sulfate, and 100 µg/ml denatured salmon sperm DNA. An
additional 12-h hybridization was performed in the presence of 10
cpm/ml of the
P random primed cDNA probe. Stringent
washings were then conducted at 65 °C in 0.1
SSC buffer
(0.15 M NaCl, 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
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
P-labeled cDNA
probes (2
10
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 [S]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. 1D), downstream from the CMV
promoter in the pJ7 vector(32) . The pLTR-luc and the
pAc
-gal vectors were a generous gift of I. Barlat and the
pSV
-gal (33) was a gift of Dr. J. M. Blanchard. The
pCMV
-gal vector expressing the
-galactosidase gene under the
dependence of the CMV promoter was provided by Stratagene.
Figure 1:
Northern analysis of IFN-induced
Staf-50 mRNA and restriction map of Staf-50 cDNA. Total RNAs (20
µg/lane) were separated on 1.2% formaldehyde-agarose gel,
transferred to nylon membrane, and hybridized to a P-labeled Staf-50 cDNA probe. The same blots were reprobed
with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe
to ensure that equal amounts of RNA were loaded in each lane. A, time course of Staf-50 mRNA induction with
Hu-
/
IFN. Daudi cells were treated by Hu-
/
IFN (500
units/ml) for the indicated times. The location of the 18 S and 28 S
rRNAs are indicated. B, specificity of Staf-50 mRNA induction
by Hu-
/
IFN and Hu-
IFN. HeLa cells were treated or not by
Hu-
/
IFN (500 units/ml) or Hu-
IFN (500 units/ml) for the
indicated times. C, induction of Staf-50 mRNA during protein
synthesis inhibition. HeLa cells were incubated with cycloheximide (20
µg/ml) for 30 min and then treated (CHX+IFN) or not (CHX) by Hu-
/
IFN (500 units/ml) for 6 h. D,
partial restriction map of Staf-50 cDNA. Restriction enzyme sites EcoRI, XbaI, XhoI, and their nucleotide
location are indicated. The XbaI and XhoI sites at
the extremities are linker-derived sites. The open reading frame is
represented by the solid box (position +123 to
+1448).
Transient Transfection Experiments
For
transfection experiments, 1-5 10
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 µg 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
-galactosidase activities were measured as described
previously(34) .
Construction and Screening of a cDNA Library from
Hu-
Total RNAs were extracted
from human lymphoblastoid Daudi cells treated for 16 h with 500
international units of Hu-/
IFN-treated Daudi Cells
/
IFN. 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
ZAP-cDNA synthesis kit (Stratagene). 5000
primary recombinant clones were screened successively with single
stranded
P-labeled 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-
/
IFN. 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
IFN-induced 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-/
IFN. As shown in Fig. 1A, 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. 1A).
IFN for the
lack of functional receptors, HeLa cells were treated with 500 units/ml
of Hu-
/
IFN or Hu-
IFN 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-
/
IFN was revealed to be
a stronger inducer. This induction was not dependent on continuous
protein synthesis since it was unaffected by cycloheximide treatment (Fig. 1C). These data demonstrated that Staf-50
participates in the primary response of IFN action and was not the
consequence of a second set of gene induction. Comparison between
untreated Daudi and HeLa cells showed that a basal level in the
expression of Staf-50 is easily detectable in Daudi cells but not in
HeLa cells. In order to determine the tissue specificity of Staf-50
expression, the 2.8-kb insert was used to probe a set of RNAs isolated
from several tissues (Multiple Tissue Northern from Clontech). As shown
in Fig. 2, Staf-50 is strongly expressed, in the absence of
exogenous IFN treatment, in peripheral blood leukocytes, in lymphoid
tissues, such as spleen or thymus, and in ovary. Various basal levels
were detected in other tissues. In contrast with the data observed in
Daudi and HeLa cells, two major RNA species were detected specially in
peripheral blood leukocytes. These results will be discussed later on
the basis of nucleotide sequence analysis.
Figure 2:
Tissue specificity of Staf-50 expression.
A multiple tissues Northern blot membrane (Clontech) was hybridized to
the P-labeled Staf-50 cDNA probe. The tissue source of the
mRNA sample in each lane is indicated at the top of the
figure. The position of the molecular weight markers is indicated at
the left.
Sequence Analysis of Staf-50 cDNA
The complete
nucleotide and amino acid sequences of the 2.8-kb insert are presented
in Fig. 3. Computer search in the EMBL and GenBank data bases
reveal homologies with the nucleotidic sequences of the mouse Rpt-1
gene (for regulatory protein, T-lymphocyte 1) (29) and the human
SS-A/RO autoantigen(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- chain promoter region (CD25) and by the LTR promoter
region of HIV-1. In contrast, the function of the SS-A/RO gene,
described in the Sjorgren type A syndrome, remains unknown. The
patterns of expression of Staf-50 and Rpt-1 are rather similar with a
preferential expression in quiescent T-lymphocytes (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-1 gene or a member of a same family of genes, including the human
SS-A/RO gene.
Figure 3:
Nucleotide sequence and predicted amino
acid sequence of Staf-50 cDNA. The complete nucleotide sequence of
Staf-50 cDNA (top line) and the predicted amino acid sequence (bottom line) are shown. The nucleotides are numbered at the right of the sequence. The two zinc finger motif are double underlined. The IM motif is represented in a box. The bi-partite nuclear location signal is underlined and the potential polyadenylation signals founded in the
3`-noncoding region are indicated with dotted lines.
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 of the 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 [S]methionine 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) .
Figure 4:
In vitro expression of the
Staf-50-encoded protein. Transcription-translation of the pBluescript
Staf-50-containing vector was performed in the
transcription/translation-coupled reticulocyte lysate system in the
presence of [S]methionine. The labeled proteins
were fractionated by SDS-polyacrylamide gel electrophoresis before
autoradiography. Size molecular weight marker (M) are
[
C]methylated proteins (Amersham). Staf-50
lane, translational products of Staf-50; and control
lane, translation products obtained with a pBluescript vector
without insert.
Staf-50 Is a Member of the Ring Finger Family
A
search and analysis for amino acid sequence homologies in the GenBank
data base revealed that the complete Staf-50 protein shares 44% amino
acid homology with the mouse Rpt-1 protein and 40.5% with the human
SS-A/RO 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 of latent virus in herpes simplex virus type 1
infection(41) . Others are involved in activation of DNA
recombination and DNA repair (for review, see Ref. 40). These results
would be consistent with a role of Staf-50 in the mechanism of signal
transduction by cytokines like IFN or in gene expression regulation by
IFNs.
Figure 5:
Alignment of the amino-terminal sequences
of Staf-50, Rpt-1, SS-A/RO, and Rfp proteins. The alignment of the
predicted amino-terminal sequence of Staf-50 with several members of
the Ring finger family is shown. Protein names are indicated at the left and the position of amino acids at the right of
the sequences. The two zinc finger motifs and the IM motif are
identified. The position of the conserved amino acids is indicated by
an asterisk (*) and the conserved hydrophobic residues by a
. Cys and His residues in the zinc finger motifs are shown in boldface.
Recent findings report that a synthetic peptide corresponding
to the C3HC4 domain of the Ring1 gene product binds to DNA, in a zinc
dependent manner, although weakly and nonspecifically(40) .
These results strongly suggest that other peptide motifs are
responsible for the specificity of DNA binding activity. The alignment
of the amino acid sequences presented in Fig. 5reveals the
presence near the C3HC4 zinc finger motif, of a second putative zinc
finger structure with a CHC3H2-type signature. This motif has already
been identified in three other members of the Ring family(42) ,
the human and mouse Rfp tyrosine kinase gene products(43) , the
T18 transforming mouse fusion protein (44), and the protein encoded by
the human promyelocytic leukemia gene (42). For the latter, the last
histidine residue is not present. Interestingly, in Staf-50, Rpt-1,
SSA-/RO, and Rfp proteins, the two zinc finger structure are separated
by 40 amino acid residues. In these regions we have identified a
conserved basic motif (the relative basicity: H+K+R
residues/D+E residues = 5) that we have termed IM (for
intermediate motif) (Fig. 5). Such a basic motif is known to
increase the affinity of DNA-binding protein to the DNA. The presence
of two zinc fingers and the IM motif in the same configuration in these
four proteins (Fig. 5) suggests that they act in synergy to bind
DNA targets.
Trans-acting Function of the Staf-50 Protein
In
order to determine, by analogy with Rpt-1, the ability of Staf-50 to
affect transcription directed by the LTR promoter region of HIV-1,
cotransfection experiments were performed with COS-7 m6 and HeLa cells.
To this aim, the 1826-bp XbaI-XbaI fragment of the
pBluescript-Staf-50 cDNA (Fig. 1D) was cloned under the
transcriptional dependence of the CMV promoter in the pJ7 vector.
The Staf-50 cDNA was positioned in sense (pJ-Staf-50) or antisense
orientation as a negative control (pJ-Staf-50as). A luciferase gene
under the control of the LTR promoter region of HIV-1 (pLTR-luc) was
used as a reporter gene. Transfections were performed by calcium
phosphate precipitation or by a lipofectamine procedure (Life
Technologies, Inc.). The cells were collected 72 h later, and cellular
extracts were prepared to determine the luciferase activities as
described under ``Materials and Methods.'' Cotransfection of
pLTR-luc with pJ-Staf-50 resulted in a 60-90% inhibition of the
luciferase activity as compared with pJ-Staf-50as and pJ7
(Fig. 6), or with pCMV-
-gal (data not shown). The experiment
was repeated several times in the linear range of the assay and with
different batches of DNA. Identical data were obtained with both
transfection procedures. In contrast, pJ-Staf-50 had no effect on
-galactosidase expression directed by the SV40 promoter
(pSV
-gal) or by the actin promoter (pAc
-gal) (Fig. 6).
Figure 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-Staf-50) by the values measured after
cotransfection of LTR-luc and pJ7. Values of less than 1 indicate
inhibition of LTR-luc and represent the mean and standard deviation for
several distinct experiments. pSV
-gal and pAc
-gal activities
were determined the same way.
IFN gene(47) . However, the function of
most of IFN-induced genes remains unknown. In this report, we describe
the cloning and the partial characterization of a new IFN-induced gene,
designated as Staf-50 and exhibiting properties of transcriptional
regulator.
B (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-1 and
NF-AT-1(55) .
B binding motifs are
very important for the expression of HIV-1 at high level in activated
CD4
T lymphocytes(56) . The NF-
B-mediated
transactivation of the HIV-1 LTR promoter is inhibited in IFN-producing
cells. This down-regulation is associated with an alteration of the
binding pattern of NF-
B-specific nuclear proteins to the core
enhancer element of the HIV-1 LTR(57) . The induction of HIV-1
provirus by herpes simplex virus-1 infection involves cooperation
between NF-
B and the virus-encoded transactivator
ICP0(58) . These data suggest that Staf-50 could act as a
repressor of the NF-
B activation and interact either directly with
NF-
B-binding proteins, thereby altering their affinity for DNA, or
indirectly with its DNA target, to modulate HIV-1 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-A/RO 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.
/EMBL Data Bank with accession number(s) X82200.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.