(Received for publication, April 18, 1995; and in revised form, June 21, 1995)
From the
Viral infection results in transcriptional activation of the
cellular interferon /
-stimulated genes (ISGs) independent of
the autocrine action of interferon
/
(IFN-
/
).
Induction of ISG expression by virus appears to be mediated through
production of viral double-stranded RNA (dsRNA). Previously, we
identified two novel dsRNA-activated factors (DRAFs) that bind to the
interferon-stimulated response element (ISRE), the DNA sequence that
mediates transcriptional activation by IFN-
/
. In this report
we define sequences that flank the classical ISRE to be necessary for
DRAF1 binding. More significantly, it is shown that the sequences
required to bind DRAF1 correlate with the ability to mediate ISG
induction by virus. These results strongly suggest that DRAF1 is a
positive regulator of ISG transcription. DRAF1 is shown to bind
selectively to the promoters of those ISGs which are strongly induced
by viral infection, again suggesting the functional significance of
this factor. UV cross-linking experiments indicate that DRAF1 and DRAF2
share a common DNA-binding subunit of approximately 70 kDa which is
referred to as the DRAF binding component (DRAF
).
DRAF
is shown to preexist in the cytoplasm of unstimulated
cells. Consistent with this observation, both DRAF1 and DRAF2 are
activated in the cytoplasm prior to nuclear translocation.
A cell responds to viral infection with the transcriptional
induction of specific genes that can function in the defense against
the invading virus. Expression of the type I interferon genes
(IFNs-/
) (
)during viral infection is thought to be
stimulated by viral double-stranded RNA (dsRNA) generated during the
course of infection (reviewed in (1) ). Once synthesized, the
IFN-
/
cytokines are secreted from the cell, bind to cell
surface receptors, and function in an autocrine or paracrine manner to
confer resistance to viral infection (reviewed in (2) ). The
protective effects of IFNs-
/
are mediated through the
activation of a specific set of genes known as interferon-stimulated
genes (ISGs) whose products function in cellular defense (reviewed in (1, 2, 3, 4) ). It has now become
clear that transcription of many of the ISGs is also directly activated
during viral infection, independent of the synthesis or action of
IFNs(5, 6, 7, 8, 9, 10) .
The direct activation of the ISGs in response to viral infection allows
cells to express the ISG-encoded proteins rapidly, before the
synthesis, secretion, or action of IFNs. Such a response pathway may be
critical to the survival of virally infected cells and an important
component of the immune response against virus. In this study we
examine the basis of direct activation of ISGs by viral infection and
dsRNA, the apparent mediator of induction.
ISGs induced by
IFNs-/
contain a promoter sequence known as the
interferon-stimulated response element, or ISRE (reviewed in Refs. 3
and 4). Following IFN binding to its receptor, a multimeric
transcription factor, the interferon-stimulated gene factor 3 (ISGF3),
is activated by tyrosine phosphorylation in the cytoplasm of the cells
and subsequently translocates to the nucleus to bind to the ISRE.
Despite the potential significance of direct activation of the ISGs by
viral infection, little has been characterized of this pathway at the
molecular level. Preliminary experiments have suggested that a DNA
sequence containing the ISRE can mediate a transcriptional response to
virus(6, 11) . However, ISG induction by viral
infection is independent of ISGF3 activation(6) . Consistent
with these observations, we have identified two novel nuclear factors
rapidly induced by viral infection or dsRNA transfection that recognize
an ISRE-containing DNA sequence (6) . These factors have been
termed dsRNA-activated factor 1 and 2 (DRAF1 and DRAF2).
An
important question concerns the DNA sequence transcriptionally
responsive to viral infection or dsRNA. Experiments suggesting that the
ISRE is sufficient to mediate the transcriptional response to virus
have employed DNA elements that contain sequence flanking the classical
ISRE(6, 11) . In addition, certain ISGs are induced
weakly or undetectably by virus although they are strongly induced by
IFNs-/
(10) . This suggests that the DNA sequence
which determines a strong transcriptional response to virus is
different from the classical ISRE. In this report, we examine the
functional significance of the DRAFs by determining more precisely the
DNA sequence required to mediate viral induction of ISGs and
correlating it with the ability of the induced DRAFs to bind to this
sequence. It is demonstrated that the DNA sequence required to mediate
a response to virus correlates with binding to DRAF1. This is the first
strong evidence that DRAF1 is a positive activator of ISG transcription
during infection. In addition, we provide a characterization of DRAF1
and DRAF2 that indicates that they share a common DNA-binding protein
which preexists in the cytoplasm of untreated cells. DRAF activation is
also demonstrated to initiate in the cytoplasm of the cell. Thus, this
report forms the basis of understanding the nature of a DNA element and
transcription factors which mediate the direct activation of ISGs
during viral infection.
The ISRE-TK and TK-109 plasmids have been described
previously(14) . The ISRE-TK plasmid contains 3 copies of the
ISRE (from -111 to -94 of the human ISG15 promoter) cloned
into the -109 position of the herpes simplex virus thymidine
kinase gene promoter. The dl ISG15-E1B plasmids contain various regions
of the ISG15 promoter (-96, -108, or -115 to
+44) fused to the 3` region of the adenovirus E1B
gene(15) . The SV globin plasmid used to control for
transfection efficiency has been described(16) .
Figure 1:
A, the ISRE is sufficient to
mediate a transcriptional response to virus in a cell line resistant to
IFN-/
. HEC-1B cells were transfected with either the ISRE-TK
plasmid (14) or the TK-109 plasmid (14) along with a
plasmid encoding
-globin as a cotransfection control(16) .
Following transfection, cells were either untreated(-) or
infected with NDV for 12 h (+) (lanes 2-5).
Cytoplasmic RNA was isolated from the cells, and the levels of TK,
endogenous ISG15, globin, and actin mRNA were quantified by T2 RNase
protection assay using radiolabeled antisense RNA probes. Endogenous
ISG15 and actin mRNAs were also assayed in HEC-1B cells treated with
IFN-
(1000 units/ml) (lane 1). A representation of the
ISRE-TK plasmid is depicted at the bottom. B, definition of
the region of the ISG15 promoter required to mediate a transcriptional
response to virus. HEC-1B cells were transfected with either the
-96, -108, or -115 dl ISG15-E1B plasmid (15) along with a plasmid encoding
-globin as a
cotransfection control(16) . Following transfection, cells were
either untreated(-) or infected with NDV for 12 h (+). The
levels of ISG15-E1B, endogenous ISG15, globin, and actin mRNAs were
quantified by T2 RNase protection assay using radiolabeled antisense
RNA probes. A representation of the dl ISG15-E1B constructs is depicted
at the bottom.
The
ISRE-TK plasmid used in Fig. 1A contains an ISRE
oligonucleotide corresponding to positions -111 to -94 of
the ISG15 promoter. Therefore, the sequence from -111 to
-94 is sufficient to support a transcriptional response to virus.
To define more precisely the region of the ISG15 promoter which is
required to mediate a response to virus, three plasmids containing
various regions of the ISG15 promoter were tested. These plasmids,
called dl ISG15-E1B, contain from -96, -108, or -115
to +44 of the ISG15 gene linked to the adenovirus E1B gene as a
reporter. These plasmids have been used previously to define the region
of the ISG15 promoter required to mediate a transcriptional response to
IFN-/
(15, 17) . Each of these plasmids was
transfected into HEC-1B cells along with a plasmid encoding
-globin as a cotransfection control (Fig. 1B).
Following transfection, cells were either left untreated (lanes
1, 3, and 5) or infected with NDV (lanes
2, 4, and 6). Cytoplasmic RNA was isolated from
the cells, and the levels of reporter gene expression were quantified.
As expected, the -96 plasmid, which does not contain the ISRE,
did not mediate any induction of the ISG15-E1B reporter gene (lane
2). The -108 plasmid did respond to virus with a relatively
weak (
2-fold, by densitometry) but reproducible induction of the
reporter gene (lane 4). The -115 plasmid responded
strongly to viral infection, with more than a 5-fold induction of
reporter gene expression (lane 6). It should be noted that the
actual induction is greater than it appears to be, since we have
observed a general destabilization of mRNA following viral infection or
dsRNA treatment (compare actin levels in lanes 2, 4,
and 6 with those in lanes 1, 3, and 5). Thus, if reporter gene mRNA levels are normalized to actin
levels, the viral induction is severalfold greater. The different
responses mediated by the -108 and -115 plasmids is not due
to a difference in the efficiency of viral infection, since the
endogenous ISG15 gene was induced to similar levels in both cases (lanes 4 and 6). These results demonstrate that
several nucleotides upstream of the consensus ISRE (-107 to
-95) are required to mediate a maximal response to virus,
although the ISRE alone (-108 plasmid) is able to mediate modest
transcriptional activation.
Figure 2: A, activation of DRAF1 and DRAF2 by dsRNA transfection. HEC-1B cells were untreated(-) or transfected with dsRNA (dsRNA). Nuclear extracts were prepared and analyzed by gel mobility shift assay with a radiolabeled ISRE probe (-114/-92) derived from the human ISG15 gene promoter(15) . B, definition of the 5` boundary of the DRAF1 and DRAF2 binding sites within the ISG15 promoter. Nuclear extract containing DRAF1 and DRAF2 from dsRNA-treated cells was used in a gel mobility shift assay with a radiolabeled ISRE probe (-114/-92 of ISG15). The DNA binding reactions were performed in the absence (lane 1) or presence (lanes 2-4) of a 100-fold molar excess of unlabeled competitor DNAs. The sequences of the DNAs used as competitors are shown at the bottom. C, definition of the 3` boundary of the DRAF1 and DRAF2 binding sites within the ISG15 promoter. Nuclear extract containing DRAF1 and DRAF2 activity was used in a gel mobility shift assay with a radiolabeled ISRE probe. The DNA binding reactions were performed in the absence (lane 1) or presence (lanes 2-4) of a 100-fold molar excess of unlabeled competitor DNAs. The sequences of the DNAs used as competitors are shown at the bottom.
Since the -115 and -108 plasmids demonstrated a measurable difference in inducibility by viral infection, we determined if efficient binding of DRAF1 and/or DRAF2 to these sequences correlated with gene expression. To define more precisely the 5` boundary of the sequence required to bind DRAF1 and DRAF2, a competitive gel mobility shift assay was performed (Fig. 2B). The DNA binding reactions were performed with a radiolabeled ISG15 ISRE oligonucleotide (-114/-92) in the absence (lane 1) or presence (lanes 2-4) of a 100-fold molar excess of various unlabeled competitor DNAs. The competitor DNAs corresponded to -115/-94, -111/-94, or -108/-94 of the ISG15 promoter. The -115/-94 and -111/-94 DNAs competed efficiently for binding of both DRAF1 and DRAF2 (lanes 2 and 3). However, the -108/-94 DNA only competed efficiently for DRAF2 binding (lane 4). DRAF1 binding therefore requires a nucleotide(s) between -109 and -111, directly adjacent to the ISRE (AAA in the ISG15 promoter). Thus, the stronger induction of reporter gene expression observed for the -115 plasmid compared to that seen with the -108 plasmid (Fig. 1B) appears to be due to the ability of DRAF1 to bind much more efficiently to the -115 plasmid than to the -108 plasmid. This suggests that DRAF1 is the major positive transcriptional regulator, since DRAF1 binding to the ISG15 ISRE is tightly correlated with induced transcription of this gene during viral infection. The DNA binding specificity of DRAF1 is distinct from ISGF3 and suggests that DRAF1 activates a subset of ISGs(14) .
To define the 3` boundary of the DRAF1 and DRAF2 binding sites within the ISG15 promoter, a similar DNA binding competition experiment was performed (Fig. 2C). Each DNA binding reaction contained radiolabeled ISRE probe in the absence (lane 1) or presence (lanes 2-4) of a 100-fold molar excess of unlabeled competitor DNA. The competitor DNAs contained -111/-94 of the ISG15 ISRE or a deletion of nucleotides -98 and -97 (dl 98) or of nucleotides -96 to -94 (dl 96) as shown. As expected, the -111/-94 DNA competed efficiently for binding of DRAF1 and DRAF2 (lane 2). However, neither the dl 98 nor the dl 96 DNAs competed for DRAF1 or DRAF2 binding (lanes 3 and 4). Thus, binding of DRAF1 and DRAF2 requires the AAA nucleotides at -99 to -97 and the CTG sequence between -96 and -94. These residues are highly conserved among the ISREs of various ISGs.
Figure 3: DRAF1 and DRAF2 bind to distinct subsets of ISG promoters. Nuclear extract containing DRAF1 and DRAF2 activity was used in a gel mobility shift assay with a radiolabeled ISRE probe (from the ISG15 promoter). The DNA binding reactions were performed in the absence (lane 1) or presence of a 100-fold (lanes 2, 4, 6, and 8) or 250-fold (lanes 3, 5, 7, and 9) molar excess of unlabeled competitor DNA. Competitor DNAs contained from -111 to -94 of the human ISG15 promoter (ISG15, lanes 2 and 3), -103 to -82 of the human ISG54 promoter (ISG54, lanes 4 and 5), -105 to -85 of the human 2`-5` oligoadenylate synthetase promoter (2`-5` OAS, lanes 6 and 7), or -117 to -96 of the human 6-16 promoter (6-16, lanes 83 and 9). The sequences of the competitor DNAs are shown at the bottom.
Figure 4: DRAF1 and DRAF2 share a common DNA-binding subunit of approximately 70 kDa. Left, a fraction containing partially purified DRAF1, DRAF2, and a faster migrating activity was subjected to gel mobility shift analysis with a radiolabeled ISRE probe which was substituted with 5-bromodeoxyuridine on one strand. The position of the faster migrating activity (see text) is indicated by an arrowhead. Right, the protein-ISRE complexes in the gel were UV-cross-linked for 5 min, and the gel was subjected to autoradiography. Gel slices corresponding to each protein-ISRE complex were excised, and the protein-ISRE complexes were electroeluted directly into an SDS-polyacrylamide gel. The cross-linked product in lane 1 is derived from the faster migrating ISRE binding activity. The DRAF2-ISRE complex was electrophoresed in lane 2, and the DRAF1-ISRE complex was electrophoresed in lane 3. The migration positions of molecular mass marker proteins are indicated to the right of the figure.
To
determine the native molecular mass of the DRAF, a glycerol
gradient sedimentation experiment was performed (Fig. 5A). Affinity-purified DRAF
was
sedimented in a 25-40% glycerol gradient. Fractions were assayed
for DRAF
activity by gel shift with a radiolabeled ISRE
probe. In a parallel gradient, proteins with known sedimentation
coefficients (s) were sedimented, and their positions were
determined by SDS-PAGE analysis of the gradient fractions (data not
shown). As shown in Fig. 5A, DRAF
sedimented very closely to the 150-kDa marker protein. A standard
curve was generated by plotting the s values of the marker
proteins against distance traveled (fraction) (Fig. 5B). This allowed for a determination of the s value for DRAF
, which was used to estimate its
molecular mass as 140 kDa(19) . This suggests that the
DRAF
activity exists in solution as a dimer of a 70-kDa
protein. However, the possibility that DRAF
activity
consists of the 70-kDa cross-linked protein and a distinct protein of
similar size cannot be excluded. Attempts to estimate the native
molecular mass of DRAF1 and DRAF2 by glycerol gradient sedimentation
were unsuccessful, since neither DRAF1 nor DRAF2 activity could be
recovered in the gradients, suggesting dissociation of a multimeric
complex (data not shown).
Figure 5:
A,
glycerol gradient sedimentation analysis of DRAF.
Affinity-purified DRAF
was sedimented in a 25-40%
glycerol gradient. Fractions were collected from the top of the
gradient and analyzed for the presence of DRAF
by gel
mobility shift assay using a radiolabeled ISRE probe. Proteins with
known sedimentation coefficients (s) were sedimented in a
parallel gradient, and their positions were determined by SDS-PAGE
followed by staining with Coomassie Blue. The fractions in which the
marker proteins sedimented are indicated by arrows at the top. Gel shift analysis of the material loaded onto the
gradient is shown at the far left (LOAD). B, standard
curve used to estimate the sedimentation coefficient of
DRAF
. The s values of the marker proteins carbonic
anhydrase (CA), bovine serum albumin (BSA), and
alcohol dehydrogenase (AD) were plotted against their distance
traveled in the gradient (FRACTION).
Figure 6:
A, DRAF exists in the
cytoplasm of unstimulated cells. Nuclear and cytoplasmic extracts were
prepared from untreated HEC-1B and HeLa cells. Gel mobility shift
assays were performed using the same number of cell equivalents of
nuclear (lanes 2 and 3) or cytoplasmic (lanes 4 and 5) protein and a radiolabeled ISRE probe. The DNA
binding reaction for lane 1 was performed with
affinity-purified DRAF
to serve as a marker for migration
distance. B, kinetics of appearance of DRAF1 and DRAF2 in
cytoplasmic and nuclear extracts of dsRNA-treated HEC-1B cells. HEC-1B
cells were either untreated (lanes 1 and 6) or
treated with dsRNA for 10 (lanes 2 and 7), 20 (lanes 3 and 8), 30 (lanes 4 and 9), or 60 (lanes 5 and 10) min. Cytoplasmic
and nuclear extracts were prepared and analyzed for ISRE binding
activity by gel mobility shift assay.
DRAF2 activity appears in the cytoplasm by 10 min after treatment (lane 7) and appears to peak by 20 min (lane 8). DRAF2 is first detectable in nuclear extracts 10 min after treatment (lane 7) and continues to increase through 30 min of treatment (lane 9). Therefore, DRAF2 also appears to be activated in the cytoplasm. Since the gel shifts were performed with 20 µg of cytoplasmic protein and 10 µg of nuclear protein, and not normalized to cell equivalents, the actual percentage of DRAF1 and DRAF2 in the cytoplasmic fraction is greater than shown. This strongly suggests that the presence of DRAF1 and DRAF2 in cytoplasmic fractions is not an artifact of the extract preparation, but that it accurately reflects the cytoplasmic activation of these factors.
The defense response of cells to viral infection or dsRNA
transfection is a rapid activation of latent dsRNA-activated factors
(DRAFs) that recognize a DNA sequence containing the
IFN-/
-stimulated response element (ISRE) (Fig. 2).
Activation of the DRAFs occurs with concomitant induction of the
IFN-
/
-stimulated genes (ISGs)(6) . In this report, we
have addressed several fundamental issues regarding the induction of
ISG transcription by virus. We have defined the DNA sequence which
mediates this induction, and we have shown that this sequence
correlates with the sequence required to bind DRAF1. This result
indicates that DRAF1 functions as a positive regulator of ISG
transcription during viral infection. We also have provided an
explanation for the observation that certain ISGs are transcriptionally
activated to a greater extent than others by demonstrating differential
ISRE binding of DRAF1. In addition, we have provided a preliminary
biochemical characterization of DRAF1 and DRAF2, demonstrating that
they contain a novel 70-kDa ISRE-binding subunit which preexists in the
cytoplasm of unstimulated cells.
It is now apparent that viral
infection can activate expression of ISGs directly through a DNA
sequence containing the ISRE. This conclusion is based on the fact that
NDV (which strongly activates DRAF1) induces expression of a reporter
gene driven by the ISRE (Fig. 1A). Moreover, since this
experiment was performed in the HEC-1B cell line, which does not
respond to IFN-/
, we can definitively rule out the
possibility that the ISRE-dependent transcription observed during viral
infection is due to ISGF3 function.
As further evidence that DRAF1 is the primary mediator of ISG transcriptional activation, we have shown that the region of the ISG15 promoter which is required to mediate a strong transcriptional response to NDV infection correlates with the region of the promoter required for efficient DRAF1 binding (Fig. 1B and Fig. 2B). Previous work by others has shown that viral infection selectively induces the expression of a subset of ISGs. In particular, expression of ISG15 and ISG54 is strongly activated by NDV infection, while expression of 6-16 and 2`-5` oligoadenylate synthetase is induced very weakly(5, 9, 10, 12) . This suggests that the transcription factor(s) which induce ISG expression during viral infection binds selectively to certain ISG promoters. In fact, we have shown that DRAF1 binds efficiently to the promoters of both the ISG15 and ISG54 genes, but it does not bind efficiently to the 6-16 or 2`-5` oligoadenylate synthetase promoters (Fig. 3). Thus, ISG promoters which bind DRAF1 can support a strong transcriptional response to virus, providing additional evidence for the functional significance of this factor. The two ISREs analyzed that bind DRAF1 well (ISG15 and ISG54) are flanked upstream by a stretch of three adenine nucleotides that appear to be critical for DRAF1 binding (Fig. 2B and Fig. 3). Furthermore, the ISRE of another ISG that is strongly activated by virus, ISG56, is also flanked by three adenine nucleotides and therefore would be predicted to bind DRAF1 efficiently(24) .
The fact that DRAF1 does not bind efficiently to all consensus ISREs can account for the observation that only a subset of ISGs are induced strongly by virus. However, the result that DRAF2 binding appears to require just the consensus ISRE suggests that DRAF2 potentially could bind to certain ISG promoters which DRAF1 cannot and activate gene expression (Fig. 2B). At the present time, however, the functional significance of DRAF2 is unclear, and further work is required to elucidate the role of this activity during viral infection. A recent report has shown that dsRNA can induce transcription of the IP-10 gene via an ISRE-containing sequence(11) . However, the authors did not observe binding of DRAF1 to the IP-10 ISRE. This appears to be due to the fact that the IP-10 ISRE does not contain the flanking adenine residues which are important for DRAF1 binding. The factor(s) which is responsible for inducing ISRE-dependent IP-10 transcription remains to be identified and may require new protein synthesis for its appearance.
In an effort to begin to characterize the molecular nature of DRAF1 and DRAF2, the activities were partially purified from dsRNA-treated cells. UV-cross-linking analysis with these preparations indicates that the protein in both DRAF1 and DRAF2 which is in contact with the ISRE is approximately 70 kDa (Fig. 4). The size of this DNA-binding component is distinct from the 48-kDa DNA-binding component of ISGF3, consistent with our previous observation that DRAF1 and DRAF2 do not react with antibodies against the 48-kDa protein(6) . Interestingly, although DRAF1 and DRAF2 appear to contain the same DNA-binding protein, their DNA binding specificities differ (Fig. 2B and Fig. 3). This suggests that additional proteins in the DRAF complexes influence their ISRE binding properties. However, the exact protein composition of DRAF1 and DRAF2 and their relationship to each other remain to be determined.
In the
course of our purification of DRAF1 and DRAF2, we observed a faster
migrating activity which copurified with the DRAFs (Fig. 4).
UV-cross-linking of this activity indicated that it also contained the
same 70-kDa DNA-binding protein as DRAF1 and DRAF2 (Fig. 4). The
native molecular mass of this activity was estimated by glycerol
gradient sedimentation to be approximately 140 kDa (Fig. 5).
This result suggests that this novel activity exists in solution as a
homodimer of the 70-kDa ISRE-binding protein. The activity has thus
been termed DRAF (DRAF DNA binding component).
Interestingly, we were able to detect DRAF
activity in
cytoplasmic extracts from untreated cells (Fig. 6A).
Apparently, DRAF
preexists in the cytoplasm and is capable
of binding to the ISRE without requiring any modification induced by
the presence of dsRNA. Consistent with the cytoplasmic localization of
DRAF
, both DRAF1 and DRAF2 are rapidly activated in the
cytoplasm upon exposure of cells to dsRNA (Fig. 6B).
The molecular details which underlie activation of the DRAFs remain to
be characterized. Our previous studies have indicated that the protein
kinase inhibitor, staurosporine, and the tyrosine kinase inhibitor,
genistein, can block the activation of ISGs by virus, suggesting that
DRAF activation involves a cytoplasmic kinase, possibly a tyrosine
kinase, that is activated by dsRNA(6) . It has not yet been
determined if the 68-kDa dsRNA-dependent kinase (PKR) is involved in
DRAF activation. A PKR inhibitor, 2-aminopurine, blocks ISG induction
by viral infection; however, it also blocks ISG activation by IFNs and
therefore it is not a specific inhibitor of
PKR(12, 25) . In addition, others have shown that
2-aminopurine does not inhibit PKR activity in
vivo(26) .
The fact that both a DNA virus (adenovirus)
and an RNA virus (NDV) can activate DRAF1 suggests that this factor may
be of widespread significance in mediating an important host defense
against viral infection(6) . The mediator of viral activation
of the DRAFs is dsRNA, which can activate these factors within minutes
of treatment. Direct activation of ISG expression in response to virus
presumably allows the infected cell to rapidly establish an antiviral
phenotype via production of ISG-encoded proteins. Viral infection does
elicit the production of IFN-/
, which eventually can activate
ISG expression in the infected cell via an autocrine pathway and in
neighboring cells via a paracrine pathway. However, since the IFNs must
first be synthesized, these pathways induce ISG expression with
significantly slower kinetics in comparison to direct viral
activation(6) . Thus, the direct activation of ISG
transcription via DRAF1 may provide cells with an increased chance of
survival during viral infection and appears to be a primary defense
mechanism of the cell.