(Received for publication, April 10, 1995; and in revised form, May 19, 1995)
From the
We previously found that the level of Fas, a cell surface
receptor for an apoptosis signal, increases at the mRNA level in
influenza virus-infected HeLa cells prior to their death by apoptosis.
Here we investigated the mechanism of activation of the Fas-encoding
gene expression upon influenza virus infection. Nucleotide sequences
for the binding of nuclear factor for interleukin-6 expression
(NF-IL6), also known as CCAAT/enhancer-binding protein Viral infection brings about a variety of effects in host cells,
including changes in growth and morphology, transformation, and lytic
death, all of which presumably accompany the altered expression of
cellular genes. Understanding the molecular basis for virus-host
interaction should lead to the development of effective therapeutics
against virus-induced diseases such as cancer and AIDS. Although it has
been accepted for many years that most viruses cause host cell death,
the precise mechanism for this phenomenon remains unknown. Several
reports have shown that host cells infected with bovine herpesvirus (1) , chicken anemia virus(2) , insect
baculovirus(3) , lymphocytic choriomeningitis
virus(4) , Molony murine leukemia virus(5) , human
immunodeficiency virus
(HIV) We found that infection with influenza virus (12, 18) or HIV
Figure 2:
The
nucleotide sequence of the 5`-end region of the human FAS gene. Numbers are nucleotide positions with respect to the
transcription initiation site (shown with an arrow) as +
1. Putative binding sites for NF-IL6, AP-1, and Ets are shaded. The first exon is boxed, and the translated
region is indicated with the corresponding amino acids. The nucleotide
sequence data will appear in the CSDB, DDBJ, EMBL, and NCBI nucleotide
sequence data bases with the accession number
D31968.
Figure 1:
Cloning and assignment of the
transcription initiation site of the human FAS gene. Panel
A, schematic representation of the clone EhFas7. The positions of
restriction enzyme sites are indicated with abbreviated enzyme names: E, EcoRI; H, HindIII; S, SalI; X, XhoI. Shown above the line are the results from Northern hybridization. DNA
fragments of HindIII-HindIII, HindIII-XhoI, and XhoI-HindIII were
used as probes to detect the Fas mRNA in the RNA of KT-3 cells. The
symbols + and - mean that the results were positive and
negative, respectively. The bottomillustration shows
a more precise view of the 5`-end region of the gene. Numbers are
nucleotide positions relative to the transcription initiation site as
+1. The box represents the first exon; open and closed
areas refer to untranslated and translated regions, respectively. The bar and arrow indicate the regions that were used to
prepare probes for Southern hybridization (panelC)
and primer extension (panelB), respectively. Panel B, determination of the transcription initiation site by
primer extension. Experimental procedures were as described under
``Materials and Methods.'' The arrowhead points to
the position of a specific extended product. The thick signals at the bottom of the leftpanel are probably due to
nonspecific extension reactions. The sizes of the markers are indicated
in bases. The nucleotide ladders in the rightpanel were obtained by sequencing a DNA with the same primer. Panel
C, genomic Southern analysis. DNA (10 µg) from human
peripheral blood mononuclear cells was cleaved with HindIII,
separated on an agarose gel, and Southern blotted using the 200-base
pair DNA probe indicated in panelA. EhFas7 DNA was
similarly processed as a positive control. The arrow shows the
position of a specific signal. The positions of the size markers are
shown on the left and are in kilobase
pairs.
To
eliminate the possibility that the EhFas7 clone was derived from a
pseudogene, genomic Southern hybridization was performed using DNA from
human peripheral blood mononuclear cells and a small fragment within
the first exon as a probe (see Fig. 1A). The results in Fig. 1C show a single hybridizing band of the predicted
size. This indicates that EhFas7 is not a pseudogene and that it
contains the 5`-end region of the human FAS gene. The
2.2-kilobase pair HindIII-HindIII DNA fragment was
then sequenced, and we found that this DNA contained the sequence
corresponding to the region between nucleotide positions 1 and 226 of
the FAS cDNA, which was flanked by an additional 132-base pair
transcribed sequence (Fig. 2). The first exon (boxed)
and part of the first intron were included in this clone. The proximal
promoter region, 20-40-base pairs upstream of +1, did not
possess TATA, GC, or CCAAT boxes, but there were sequences for the
binding of AP-1, Ets, and NF-IL6 (also called C/EBP
Figure 3:
Activation of the FAS promoter by
influenza virus infection. Panel A, HeLa cells were
transfected with pFLF1 (2 µg) and then infected with influenza
virus for the indicated periods. The ratio of luciferase activity
between the lysates from virus- and mock-infected cells is shown as
-fold activation. The averages of the results from four independent
experiments are shown with standard deviations. Panel B, HeLa
cells were transfected simultaneously with pFLF1 (2 µg) and
Figure 4:
Stimulation of the FAS promoter
by NF-IL6. Panel A, a gel shift assay of NF-IL6 was conducted
using nuclear extracts from uninfected HeLa cells. Arrows point to the positions of a specific shift-band and a free probe.
Oligo-DNA competitors (25-fold excess over the probe, lanes1-3) and antibodies (1 µg of immunoglobulin G, lanes4-7) were added to the binding reactions
to determine the specificity of the signal: probe, unlabeled
probe DNA; unrelated, oligo-AP4;
Figure 5:
Changes in NF-IL6 activity in influenza
virus-infected HeLa cells. Panel A, nuclear extracts were
prepared from HeLa cells incubated with either influenza virus or
poly(I)
Changes in the
NF-IL6 concentrations in HeLa nuclear extracts caused by either
influenza virus infection or poly(I)
Figure 6:
Changes in the NF-IL6 concentration upon
influenza virus infection. Panel A, nuclear extracts of
uninfected HeLa cells were immunoblotted with an antibody against rat
C/EBP
We previously found that infection with influenza
virus(12, 18) or HIV We found that the DNA-binding activity
of NF-IL6 increases without a change in its molecular number upon
infection with influenza virus or exposure to poly(I) Fas must be bound by a
specific signaling molecule, called Fas-ligand (22, 23) , to transmit an apoptosis signal. Fas-ligand
should thus participate in influenza virus-induced apoptosis. It is
unlikely that HeLa cells constitutively produce Fas-ligand, since
poly(I) The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
D31968[GenBank Link].
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, were
repeated 8 times in the 5`-end region of the human FAS gene,
spanning from -1360 to +320. This region directed the
expression of a downstream marker gene when introduced into HeLa cells
and the activity of the FAS gene promoter was stimulated about
2-fold upon influenza virus infection. Gene expression driven by the FAS promoter was activated when human NF-IL6 was overproduced
in a DNA co-transfection study. Moreover, the DNA-binding activity of
NF-IL6 increased after infection with the virus, whereas the amount of
NF-IL6 seemed unchanged. The results suggest that NF-IL6 is activated
upon influenza virus infection through post-translational modification
and that the modified factor stimulates the transcription of the human FAS gene.
(
)(
)(6, 7, 8, 9, 10, 11) ,
and influenza virus (12, 13) undergo apoptotic death.
The physiological meaning of this virus-induced apoptotic death is not
clearly understood, but the death of virus-infected CD4-positive cells
is implicated in immunodeficiency among HIV-infected
individuals(14, 15, 16, 17) . It is
thus important to study virus-induced apoptosis not only to understand
the mechanism of apoptotic cell death in general but to overcome viral
disease.
augments the production of
the cell surface protein Fas at the mRNA level in host cells prior to
the apoptosis of virus-infected cells. Fas(19, 20) ,
also called APO-1(21) , is a receptor for an
apoptosis-mediating signal molecule termed Fas-ligand (22, 23) . Fas/Fas-ligand is the most characterized
signaling system in eukaryotic apoptosis, and it is believed to play
important roles in a variety of biological events such as the
establishment and function of the immune system(24) . We
reported that human T cell lines become susceptible to the apoptosis
trigger provided by an anti-Fas monoclonal antibody when they are
chronically infected with HIV(25) . Thus, an increase in the
Fas concentration on the surface of virus-infected cells might be a key
cause of the induction of apoptosis. In this study, we investigated the
mechanism of activation of the Fas-encoding gene upon influenza virus
infection and obtained evidence for the involvement of the
transcription factor, nuclear factor for interleukin-6 expression
(NF-IL6).
Cloning the Human FAS Gene
A human FAS cDNA was obtained by reverse transcription-mediated polymerase
chain reaction (26) using RNA from the human T cell lymphoma
cell line KT-3(27) . The nucleotide sequence of the amplified
DNA was identical to that reported previously(20) . The cDNA
was labeled with P by nick translation and used as a probe
to screen a library. A genomic DNA library (28) constructed
with the EMBL3 vector and DNA from human peripheral blood cells was
obtained from the Japanese Cancer Research Resources Bank. About
one-million clones were screened by hybridization with the labeled
probe under standard conditions, and five were selected. These clones
were further hybridized with a DNA fragment corresponding to the region
between nucleotide positions 196 and 346 of FAS cDNA(20) . Three clones gave positive signals and one of
them, named EhFas7, contained the most 5`-end region of FAS cDNA.
Cell Culture
HeLa S3 cells were grown in
Eagle's minimal essential medium (Nissui, Japan) supplemented
with 10% fetal bovine serum (Irvine Scientific). Subconfluent
monolayers were infected with SP626, a wild-type strain of the
influenza A/Udorn/72 (H3N2) virus at a multiplicity of infection of 10
as described previously (29) or exposed to double-stranded
poly(I)poly(C) (Pharmacia Biotech, Japan) at 0.1 mg/ml. The cells
were harvested for further analysis after various periods.
Primer Extension
An oligonucleotide containing the
region between +171 and +187 of the FAS gene (see Fig. 2) was synthesized, labeled with P at the
5`-end, and used as a primer. RNA samples were mixed with the primer,
and the cDNA was synthesized with a reverse transcriptase as described
previously(30) . The cDNA was separated on a 6% polyacrylamide
gel containing 8.3 M urea together with sequence ladders
constructed by sequencing a DNA with the same primer.
DNA Transfection and Luciferase Assay
The DNA
fragment corresponding to the region between -1435 and +236
of the FAS gene was inserted 18-base pairs upstream of the
translation start codon of the firefly luciferase gene in the pGV-B
vector (Toyo Ink, Japan), resulting in pFLF1. Various amounts of pFLF1
were introduced into HeLa cells using DEAE-dextran(31) , and
the cells were cultured for 24 h. The cell lysates were prepared, and
luciferase was assayed using the commercially supplied reagent,
PicaGene (Toyo Ink). Luciferase activity was determined using a
luminometer (Lumat LB9501, Berthold, Germany). HeLa cells were also
transfected with pGV-B, and luciferase activity in the cell lysates was
subtracted as background from that in the lysates of the pFLF1
transfectants. A DNA containing the promoter region of the human
-actin gene fused to the coding sequence of the chloramphenicol
acetyltransferase (CAT) gene (
-actin/CAT) was introduced into HeLa
cells together with pFLF1 as an internal control. The expression of
-actin/CAT was determined by the CAT enzyme assay as described
previously(31) .
Gel Shift Assay
Nuclear
``mini-extracts'' were prepared from HeLa cells (0.5-1
10
)(32) . A double-stranded oligonucleotide
with the sequence of 5`-AGATTGTGCAATCT, which contained the consensus
binding-sequence for NF-IL6, 5`-T(T/G)NNGNAA(T/G)(33) , was
labeled with
P at the 5`-ends and used as a probe. Nuclear
extracts (2 µl) were incubated on ice for 10 min in a reaction
mixture consisting of 12 mM Hepes (pH 7.9), 40 mM KCl, 120 mM NaCl, 0.2 mM EDTA, 0.2 mM
EGTA, 0.4 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, 8% glycerol, and 0.1 mg/ml of
poly[d(I
C)]
poly[d(I
C)] (Sigma).
The probe (0.05 pmol) was then added to the mixture, and the binding
reaction proceeded for 10 min on ice. The mixture was loaded onto a 6%
polyacrylamide gel containing 5% glycerol, and resolved by
electrophoresis at 12.5 V/cm at 4 °C in 25 mM Tris borate
(pH 8.3) containing 0.5 mM EDTA. Oligonucleotide competitors
(25-fold excess over the probe) were incubated with nuclear extracts
before the probe was added. Oligo-AP4, 5`-CCAGCTGTGGAATG, contained the
AP4-binding site of simian virus 40 DNA. Antibodies raised against
synthetic peptides of the rat CCAAT/enhancer-binding protein (C/EBP) (34) (Santa Cruz Biotechnology), which cross-react with the
human homolog, were added after the binding reaction was completed, and
the mixture was further incubated for 1 h on ice. The intensity of the
specific signal was determined using an image analyzer (BA100, Fuji
Photo Film, Japan). A gel shift assay for nuclear factor I was
similarly performed using an oligonucleotide with the sequence
5`-TATACCTTATACTGGACTAGTGCCAATATTAAAATG as a probe as described
previously(35) .
Immunoblots
Nuclear extracts were separated on a
10% polyacrylamide gel containing SDS and electrophoretically
transferred onto a polyvinylidene difluoride membrane (Millipore,
Corp.). The membrane was blocked with 1% bovine serum albumin and
incubated with 0.1 µg/ml of anti-rat C/EBP immunoglobulin G in
a buffer consisting of 10 mM Tris-HCl (pH 8), 0.15 M NaCl, and 0.5% Tween 20. The membrane was washed and reacted with
anti-rabbit immunoglobulin G conjugated with horseradish peroxidase
(Amersham, Corp.). Signals were then visualized using the ECL System
(Amersham, Corp.). To determine the specificity of the reaction, the
first antibody was initially incubated for 30 min with 0.2 µg/ml of
either the peptide used to raise the antibody or that containing the
region between amino acid positions 144 and 157 of human
Fas(20) .
Other Methods
The nucleotide sequence was
determined for both strands by dideoxy chain termination(36) .
Total cellular RNA was extracted according to the method of Chomczynski
and Sacchi (37) and poly(A) RNA was enriched
by means of oligo(dT)-cellulose chromatography. Northern and Southern
blots were performed under standard conditions.
Cloning of the Human FAS Promoter
To obtain a
DNA containing the transcription promoter of the human FAS gene, a genomic DNA library constructed with DNA from human
peripheral blood cells was screened, using the human FAS cDNA
as a probe. Five positive clones were selected from one million
independent clones. One of them, named EhFas7, was further analyzed
since it contained the most 5`-end region of the FAS cDNA. A
restriction map of EhFas7 is shown in Fig. 1A. Various
DNA regions of EhFas7 were used as probes against the RNA from KT-3
cells in a Northern blot. The 0.8-kilobase pair XhoI-HindIII fragment detected Fas mRNA, whereas more
upstream regions failed to do so (data not shown, see Fig. 1A). We then searched for the transcription
initiation site (+1) within this DNA segment. An oligo-DNA primer
was constructed corresponding to a sequence within the region, and
primer extension proceeded using the RNA from the human T cell line
MOLT-4, which contains a relatively large amount of Fas mRNA (data not
shown). A single extended product of about 190 bases was detected with
the RNA that bound oligo(dT)-cellulose (Fig. 1B). The
position of +1 was G, according to the sequence ladders.
) in the more
upstream region. AP-1- and Ets-binding sites were present at around
-490, and there were six binding sequences for NF-IL6 in the
region spanning from -130 to -1360. The first exon
contained two more sequences for NF-IL6 binding.
Stimulation of Fas Promoter Activity by Influenza Virus
Infection
We examined whether the 5`-upstream region of the
cloned FAS gene can direct the transcription of a downstream
sequence. The region between -1435 and +236, which includes
seven out of eight NF-IL6-binding sites, was fused to the coding
sequence of the firefly luciferase gene, and various amounts of the
resulting pFLF1 DNA were introduced into HeLa cells. The lysates from
transfectants contained luciferase activity, and 5 µg of the DNA
gave maximal expression (data not shown). The effect of influenza virus
infection on the activity of the FAS promoter was then
examined. HeLa cells were transfected with pFLF1 and cultured for 24 h.
The cells were then infected with influenza virus, and lysates were
prepared from the infected cells after various periods to determine the
luciferase activity. The expression of the luciferase gene began to
increase soon after infection, reaching a maximal stimulation of about
2-fold at 2 h postinfection, and then it gradually descended to the
control level at 6 h postinfection (Fig. 3A). Transient
stimulation of the FAS promoter coincides with an increase of
the Fas mRNA in influenza virus-infected HeLa cells; the amount of Fas
mRNA increases about 3-fold with a sharp peak at 3 h postinfection (18) . Moreover, the specificity of gene expression in
virus-infected cells (18) was reproduced in a DNA transfection
assay since the human -actin promoter used as an internal negative
control was little affected by influenza virus infection (Fig. 3B).
-actin/CAT (2 µg) DNAs, and then the cells were exposed to
influenza virus for 2 h before harvest. The ratio of marker enzyme
activity between the lysates from mock- and virus-infected cells is
shown as -fold activation. The averages of the results from three
independent experiments are shown with standard
deviations.
Stimulation of the FAS Promoter by NF-IL6
Since
the presence of dispersed multiple binding sites in transcription
regulatory regions is typical of NF-IL6-inducible genes, we considered
that NF-IL6 might be a transcription factor responsible for the
activation of the FAS promoter in cells infected with
influenza virus. We first established a gel shift assay to determine
the DNA-binding activity of NF-IL6 using an oligo-DNA having the
consensus sequence for NF-IL6 binding as a probe. Nuclear extracts of
uninfected HeLa cells showed a shifted band, which disappeared only in
the presence of an excess of the unlabeled probe (Fig. 4A, lanes 1-3). The formation of
this specific complex was almost completely inhibited by an
anti-C/EBP antibody, but it was not affected by an antibody raised
against either C/EBP
or C/EBP
(lanes4-7). These results indicate that the binding
activity in uninfected HeLa cells contains C/EBP
, but not
C/EBP
and C/EBP
, suggesting that it consists of the homodimer
of C/EBP
(NF-IL6). NF-IL6 activity in HeLa cells markedly
increased when HeLa cells were transfected with pEF-NFIL6 that
expresses human NF-IL6(38) , while a control vector showed no
effect (lanes8-13). To assess whether NF-IL6
is involved in the transcription regulation of the human FAS gene, we transfected HeLa cells with pFLF1 and pEF-NFIL6, and
luciferase activity in the lysates from transfectants was determined.
Gene expression driven by the FAS promoter was significantly
augmented in the presence of pEF-NFIL6, whereas the
-actin
promoter, whose DNA was simultaneously introduced into HeLa cells, was
not affected (Fig. 4B). These results indicate that
NF-IL6 is a positive transcription factor for the human FAS gene.
, anti-rat
C/EBP
;
, anti-rat C/EBP
;
,
anti-rat C/EBP
. Nuclear extracts from HeLa cells transfected with
either pEF-NFIL6 (lanes8-10) or
pEF-BOS(58) , a vector with no NF-IL6 sequence (lanes11-13), were also analyzed for NF-IL6 activity. Panel B, HeLa cells were transfected with pFLF1 (1 µg),
-actin/CAT (1 µg), and 1 µg of either pEF-BOS or
pEF-NFIL6. The ratio of marker enzyme activity between the lysates from
cells with pEF-BOS and pEF-NFIL6 is shown as -fold activation. The
averages of the results from three independent experiments are shown
with standard deviations.
Increase of NF-IL6 Activity in Influenza Virus-infected
Cells
We then examined whether NF-IL6 activity changes upon
influenza virus infection using a gel shift assay (Fig. 5, A and B). NF-IL6 activity significantly increased soon
after virus infection, reaching a maximal stimulation of about 3-fold
at 2 h postinfection. It then rapidly descended to below the control
level. This profile coincided with the change in the FAS promoter activity that occurs upon influenza virus infection (see Fig. 3A). On the other hand, poly(I)poly(C),
which also augments the accumulation of the Fas mRNA in HeLa
cells(18) , somewhat differently affected NF-IL6. An increase
of NF-IL6 activity was first detectable at 2 h, and maximal 3-fold
stimulation occurred 6 h after the RNA was added. In contrast to the
experiment with virus infection, NF-IL6 activity did not decrease and
maintained this level even after 10 h. This profile again paralleled
the change in the Fas mRNA in HeLa cells exposed to
poly(I)
poly(C) (18) . NF-IL6 activity did not change when
HeLa cells were simply cultured with no added reagents. The activity of
nuclear factor I was examined in the nuclear extracts prepared from
HeLa cells exposed to poly(I)
poly(C), but no significant change
was found (data not shown). This indicates that poly(I)
poly(C)
does not affect the activity of DNA-binding nuclear factors in general.
The increased binding activity in the extracts of the cells exposed to
either influenza virus or poly(I)
poly(C), was sensitive
specifically to an anti-NF-IL6 antibody (Fig. 5C),
suggesting that only the activity of NF-IL6 increased. These results
indicate that the DNA-binding activity of NF-IL6 is augmented by either
influenza virus infection or by poly(I)
poly(C).
poly(C) for the indicated periods and then examined for
NF-IL6 activity in a gel shift assay. Nuclear extracts from untreated
cells were also examined. Only portions of the gels containing specific
shifted bands are shown. Panel B, the intensity of the signals
in panelA were quantified on an image analyzer and
plotted relative to that at time 0 as 1. Panel C, a gel shift
assay was performed using nuclear extracts from cells at 1 h
postinfection (left) or cells exposed to poly(I)
poly(C)
for 10 h (right), in the presence and absence of anti-C/EBP
antibodies.
poly(C) were analyzed by
immunoblotting. The antibody detected discrete doublet proteins with
apparent molecular masses of 46 and 44 kDa (Fig. 6A),
in line with the data of Nishio et al.(39) . Both
signals significantly decreased when the antibody was first incubated
with an excess of the antigen peptide, whereas a peptide with an
unrelated sequence caused little effect (Fig. 6A). This
indicates that the antibody specifically detected NF-IL6 in HeLa cells.
Three peptides, of which calculated molecular masses are 36, 34, and 16
kDa, are presumably translated from a single mRNA of human NF-IL6 (33) and are thought to be the human homolog of the rat
proteins: liver-enriched activator protein*, liver-enriched activator
protein, and liver-enriched inhibitory protein(40) ,
respectively(41) . Judging from the molecular masses of the two
proteins detected by immunoblotting, they are likely to be the products
from the first two AUG codons(33) . A discrepancy in the
molecular masses of these proteins is probably due to abundant Pro
residues(33) , which could cause improper migration of the
peptides on an SDS gel. The intensity of the two signals did not
significantly change at any time after exposure to either reagent (Fig. 6B). This suggests that the DNA-binding activity
of NF-IL6 is stimulated in those cells by posttranslational
modification with no change in the concentration of the transcription
factor. Such modification seemed to be rapidly reversed in cells
infected with influenza virus but not in those exposed to
poly(I)
poly(C), since NF-IL6 activity in virus-infected cells
sharply peaked at 2 h postinfection (see Fig. 5).
. Indicated at the top of the panel are
peptides used to preadsorb the antibody: C/EBP
, the
peptide used to raise the antibody; unrelated, the peptide
having a 14-amino acid sequence of human Fas. The positions of size
markers are indicated on the left, and the molecular mass is
shown in kDa. Panel B, changes in the amount of NF-IL6 in
nuclear extracts of influenza virus-infected or
poly(I)
poly(C)-treated HeLa cells were determined by
immunoblotting.
augmented the
expression of the Fas-encoding gene in host cells before the onset of
apoptosis. The expression of the FAS gene is also enhanced by
murine leukemia virus in lymphocytes(42) , by ischemia in the
brain(43) , by hypoxia in cultured cardiomyocytes(44) ,
by interferons in various cultured cell
lines(18, 20, 45) , and by activation with
various reagents in human peripheral
lymphocytes(46, 47, 48) . It is thus probable
that apoptotic cell death is regulated, at least in part, through
changes in the amounts of Fas. Structural analysis of the 5`-end region
of the human FAS gene revealed that the gene possesses a
TATA-less promoter and that its 5`-upstream region contains putative
binding sequences for a limited number of transcription factors. In
this study, we focused upon one of these factors, termed NF-IL6, of
which eight recognizable sequences are dispersed in a 1.7-kilobase pair
DNA at the 5`-end region of the FAS gene. We obtained evidence
that NF-IL6 is a positive transcription factor for the human FAS gene. The production of a large amount of Fas mRNA in the cell
line KT-3(20) , which is interleukin-6 dependent(27) ,
can also be attributed to the function of NF-IL6, since NF-IL6
participates in signal transduction in response to
interleukin-6(49) .
poly(C). All
circumstantial evidence supports the notion that the increased activity
of NF-IL6 is responsible for the stimulation of FAS gene
transcription in virus-infected cells. NF-IL6 is considered to be
post-translationally modified upon virus infection. Phosphorylation is
the most likely candidate for such modification, since a protein kinase
inhibitor shuts down an increase of the Fas mRNA in
poly(I)
poly(C)-treated HeLa cells(18) . Moreover, other
investigators have reported that NF-IL6 is activated through
phosphorylation by calcium/calmodulin-dependent protein
kinase(50) , by mitogen-activated kinase(51) , by
protein kinase C(52, 53) , and by protein kinase
A(53) . These enzymes phosphorylate Ser and/or Thr residues at
various positions within NF-IL6. Another protein kinase, termed
double-stranded RNA-activated protein kinase(54) , could be
involved in NF-IL6 phosphorylation upon influenza virus infection
because it is activated in virus-infected cells. This kinase has to be
autophosphorylated to become functional, which only occurs in the
presence of double-stranded RNA(55) . There is no doubt that
double-stranded RNA-activated protein kinase is active in
poly(I)
poly(C)-treated HeLa cells, where the DNA-binding activity
of NF-IL6 increased. Moreover, our preliminary experiments indicated
that an increase of Fas protein upon influenza virus infection was
abolished in the presence of a DNA that expresses a dominant negative
form of double-stranded RNA-activated protein kinase.
(
)We thus suggest that autophosphorylated
double-stranded RNA-activated protein kinase phosphorylates NF-IL6 and
that activated NF-IL6 stimulates FAS gene transcription in
influenza virus-infected or poly(I)
poly(C)-treated cells. The
activation of NF-IL6 occurred only transiently upon virus infection.
This might be due to an inhibitor of double-stranded RNA-activated
protein kinase that is induced in influenza virus-infected
cells(56) . Lee and Esteban (57) have reported that the
ectopic expression of double-stranded RNA-activated protein kinase
using the vaccinia virus vector brought about the apoptosis of HeLa
cells. Although they maintain that HeLa cells undergo apoptotic death
because of an increase in the protein kinase, we speculate that
vaccinia virus is also involved. We showed that poly(I)
poly(C),
which probably leads to the activation of double-stranded RNA-activated
protein kinase, did not cause apoptosis of HeLa cells despite an
increase in the amount of Fas (18) .
poly(C)-treated cells do not undergo apoptosis despite the
presence of a sufficient amount of Fas(18) . We speculate that
the synthesis of Fas-ligand as well as of its receptor, Fas, increases
in virus-infected cells prior to cell death. We are currently assessing
this issue.
We thank Yukito Masamune for discussion and
encouragement throughout the study. We also thank Shizuo Akira for
pEF-NFIL6 and suggestions, Masaaki Tsuda for suggestions, and Shigekazu
Nagata for pEF-BOS.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.