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INTRODUCTION |
Interferons
(IFNs)1 are a family of
multifunctional secreted proteins characterized by their abilities to
interfere with virus infection and replication (1, 2). IFNs can
indirectly inhibit viral production by reducing the growth of target
cells and by stimulating their susceptibility to apoptotic processes
(3, 4) or by promoting the recognition and the cytotoxic killing of
infected cells by the immune system (5, 6). IFNs also act directly at
various steps of the viral multiplication cycle through the products of
specific but usually overlapping sets of cellular genes induced in the
target cells and involved in RNA and protein metabolism and signaling
as well (7, 8). Until now, three IFN-regulated pathways have been
considered to be involved in these processes: the double-stranded
RNA-dependent protein kinase R (PKR) (9-11), the
2-5A/RNase L system (12, 13), and the Mx proteins (14-16). PKR is a
serine/threonine kinase that, after binding to dsRNA, phosphorylates
the protein synthesis initiation factor eIF2 and the inhibitor of
nuclear factor
B (I
B), resulting in the inhibition of protein
synthesis and specific transcription regulation (reviewed in Refs.
17-19). RNase L is a dormant cytosolic endoribonuclease that is
activated by short oligoadenylates produced by the 2'-5' oligoadenylate
synthetase after viral infection or IFN exposure (reviewed in Refs. 2 and 13). Degradation of viral RNAs and cleavage of cellular 18 S and 28 S rRNAs by the activated RNase L lead to the inhibition of protein
synthesis, thus preventing viral propagation (2). Mx proteins are
IFN-induced GTPases that interfere with the replication of some
negative-stranded RNA viruses by perturbing the intracellular movement
and functions of viral proteins (reviewed in Refs. 14 and 15). However,
there are now clear evidences for the existence of alternative
antiviral pathways beyond the PKR, 2-5A/RNase L, and Mx systems. These
evidences were obtained by analysis of mice deficient in all three
pathways. Triply deficient mice died 3-4 days earlier than wild type
mice after encephalomyocarditis virus (EMCV) infection. However there
was still an IFN dose-dependent increase in survival time
after viral infection for both wild type and triply deficient mice
(20). Moreover, cultured embryonic fibroblasts lacking RNase L, PKR, or
both proteins still mounted a substantial residual IFN antiviral
response against RNA viruses EMCV or vesicular stomatitis virus (VSV),
suggesting that another presently unknown mechanism contributes to the
IFN-dependent antiviral response (20).
We have isolated a human cDNA encoding an IFN-induced protein,
which we have termed ISG20 for IFN-stimulated gene product of
Mr 20,000 (21, 22). ISG20 is a member of
the 3' to 5' exonuclease superfamily that includes RNases (such as
RNase T and D), the proofreading domains of the polymerase I family of
DNA polymerases, and DNases that exist as independent proteins (23).
Homology within the superfamily is concentrated at three conserved
exonuclease motifs termed ExoI, ExoII, and ExoIII (23). Based on these
observations, we have demonstrated that ISG20 is a 3' to 5' exonuclease
in vitro with specificity for single-stranded RNA and, to a
lesser extent, for DNA, suggesting that ISG20 could be involved in the
antiviral function of IFN against RNA viruses (24). Notably, ISG20 is the second known RNase regulated by IFN, along with RNase L (24).
In this report, we have analyzed the ability of ISG20 to protect cells
against various viral infections. We demonstrate that in the absence of
IFN treatment, ISG20-overexpressing cells showed resistance to
infections by VSV, influenza virus, and EMCV but not to adenovirus. A
single amino acid substitution in the conserved exonuclease motif ExoII
completely abolished both the exonuclease and the antiviral activities
of ISG20, demonstrating that the protective effects were due to ISG20
exonuclease activity. We showed that the antiviral action of IFN
against VSV was reduced in cells expressing the inactive ExoII mutant
of ISG20 protein, suggesting that the antiviral activity of IFN against
VSV is partly mediated by ISG20. Because ISG20 is a new RNase induced
by IFN, these data support the idea that it might represent a novel
antiviral pathway in the mechanism of IFN action.
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EXPERIMENTAL PROCEDURES |
Cell Cultures--
Human HeLa cells and mouse L929 cells were
cultured at 37 °C in Dulbecco's modified Eagle's medium
supplemented with 10% FBS. HeLa cells overexpressing ISG20 (wt-ISG20)
and the inactive mutant of ISG20 (mut-ISG20) were cultured in the same
medium supplemented with 1 mg/ml Geneticin (G418; Invitrogen). Mouse
embryonic fibroblasts (MEFs) triply deficient in PKR, RNase L, and Mx
gene expression were obtained from Dr. R. H. Silverman and
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
FBS. HuIFN-
2a (intron A) was purchased from Schering-Plough. Mouse
type I IFN was obtained from Dr. G. Uze.
Construction of ISG20 Expression Vectors--
The full-length
ISG20 cDNA fragment was inserted into the pCiNeo
expression vector (Promega) to generate the pCiNeo-ISG20 expression
construct. The cDNA encoding the inactive ISG20 mutant protein was
generated by site-specific mutation in the conserved ExoII motif (24)
and subsequently cloned into the pCiNeo vector to generate the
pCiNeo-mut-ISG20 expression vector. For the transient transfection
experiments, the cDNAs coding for wt-ISG20 and mut-ISG20 protein
were subcloned into the pEGFP vector in the same open reading frame as
the GFP to generate the pGFP-wt-ISG20 and pGFP-mut-ISG20 expression
vector, respectively.
ISG20-expressing HeLa Cells--
HeLa cells were transfected by
the calcium phosphate procedure with either the pCiNeo-ISG20 or the
pCiNeo-mut-ISG20 expression vector. Geneticin (G418; 1 mg/ml) was added
to the culture medium 72 h later, and resistant clones were
recovered after an additional 2 weeks of selection. ISG20 and mut-ISG20
protein expressions were tested by Western blot analysis with an
ISG20-specific mouse polyclonal antibody.
Virus Stocks and Virus Yield Assays--
Stocks of VSV (Indiana
strain) and EMCV were prepared from supernatants of virus-infected L929
cells. Influenza virus was obtained from Dr. M. Chelbi-Alix. The
derivative adenovirus strain bearing a
-galactosidase-encoding
reporter gene (
-gal-adenovirus) was obtained from V. Millet.
Typically, 5 × 105 cells were plated on 6-well plates
and infected for 24 h at 37 °C, in Dulbecco's modified
Eagle's medium supplemented with 10% FBS, with VSV, influenza virus,
or EMCV at a multiplicity of infection (MOI) of 1 or 0.1. Cell cultures
were then frozen and thawed three times. The supernatants were serially
diluted, and the virus titers were measured alternatively by a plaque
assay on L929 cells as described previously (25) or by an end point
method (26). Transient transfection experiments were performed in
6-well plates by the LipofectAMINE Plus Reagent method (Invitrogen).
24 h after transfection, cells were washed twice in
phosphate-buffered saline and infected with VSV at an MOI of 0.1 in
Dulbecco's modified Eagle's medium supplemented with 10% FBS.
Infection with adenovirus was performed in 6-well plates at 10 MOI in
RPMI 1640 medium supplemented with 2% FBS. After 2 h, the
cells were washed in phosphate-buffered saline, placed in RPMI 1640 medium supplemented with 10% FBS, and then cultured for 24 h at
37 °C before the
-galactosidase assay using the
-Galactosidase
Enzyme Assay System (Promega).
Antibodies--
Specific ISG20 antibody was developed from the
ISG20-glutathione S-transferase fusion protein produced in
the BL21-DE3 Escherichia coli strain and purified by
affinity chromatography on glutathione-Sepharose. This material was
injected into mice to raise a polyclonal antiserum. The polyclonal
ISG20 antibody was used in Western blot analysis to detect endogenous
ISG20 protein. Rabbit polyclonal anti-VSV antibodies were described
previously (27). Monoclonal antibody against
-tubulin (clone
B-5-1-2) was purchased from Sigma Aldrich.
Immunofluorescence Analysis--
Confocal immunofluorescence was
performed with rabbit polyclonal anti-VSV antibody (27). The cells were
fixed for 5 min in phosphate-buffered saline containing 3.7%
formaldehyde. VSV antigens were detected with a rabbit anti-VSV
antibody (1:500 dilution) and revealed with a fluorescein
isothiocyanate-conjugated secondary antibody (Beckman Coulter,
Marseille, France). Slides were viewed using a Leika microscopic, and
image files were processed with the Adobe Photoshop program.
Western Blotting Analysis--
Cells (1 × 106)
were resuspended in 50 µl of loading buffer (10 mM
Tris-HCl, pH 6.8, 1% SDS, 5 mM EDTA, and 50% glycerol)
and incubated for 5 min at 95 °C. The proteins were fractionated on 10% SDS-PAGE and transferred onto a nitrocellulose membrane. After a
blocking step, the membrane was hybridized with the appropriate antibodies and then revealed by using a chemiluminescent detection system (ECL; Amersham Biosciences).
RNA Preparation and Northern Blot Analysis--
Total RNA was
extracted by lysis in guanidinium isothiocyanate as described
previously (28). RNA aliquots (20 µg) were electrophoresed through
1.5% agarose/10% formaldehyde gel, transferred onto a nylon membrane
(Hybond N+; Amersham Biosciences), and hybridized to
107 cpm/ml 32P-labeled cDNA probe prepared
by random priming (Invitrogen). Membranes were washed to a final
stringency of 0.2× SSC/0.1% SDS (1× SSC = 0.15 M
sodium chloride and 0.015 M sodium citrate, pH 7.0) at
65 °C before autoradiography. The cDNA probes encoding the N,
NS, M, and G protein of VSV were obtained from Dr. D. Blondel (25).
Hybridization to a glyceraldehyde-3-phoshate dehydrogenase probe (28)
was used as an invariant control.
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RESULTS |
ISG20 Confers Resistance to VSV Infection in Transiently
Transfected HeLa Cells--
We have shown previously that ISG20 is a
3' to 5' exonuclease that specifically degrades in vitro
single-stranded RNA substrates (and, to a lesser extent,
single-stranded DNA substrates) (24). Homology within the superfamily
is concentrated at three conserved exonuclease motifs termed ExoI,
ExoII, and ExoIII (23). In accordance with this information, a
site-specific aspartate to glycine residue mutation in the ExoII
conserved motif of ISG20 (mut-ISG20D94G; Fig.
1A) abolished its activity
(24). Because IFN causes a strong induction of ISG20 expression (21,
29), these data suggested that the protein could be an active player in
the antiviral action mediated by IFNs against RNA viruses. The
potential antiviral activity of ISG20 against the VSV, a negative-sense
RNA genomic virus (rhabdovirus), was evaluated by transient
transfection experiments in HeLa cells. The cDNAs coding for both
the wild type ISG20 protein (wt-ISG20) and the inactive ExoII-mutated
ISG20 protein (mut-ISG20) were cloned under the transcriptional
dependence of the cytomegalovirus promoter in the pCiNeo vector. An
empty pCiNeo vector was used as a negative control. The cells were
transfected with 0.5 or 1 µg of each plasmid and then infected
24 h later with VSV at an MOI of 0.1. 24 h later, the
productions of infectious viral particles were determined as described
under "Experimental Procedures." The results of a typical
experiment, presented in Fig. 1B, show that the cells
transfected with the wild type ISG20-expressing construct exhibited a
dose-dependent reduction in virus production (54.2% for
0.5 µg of transfected DNA and 69.4% for 1 µg of transfected DNA),
as compared with cells transfected with the empty pCiNeo vector.
Similar results were obtained in three independent experiments. No
protective effect was observed in HeLa cells transfected with mut-ISG20, confirming the specificity of the ISG20 antiviral activity. The virus yields were significantly higher in these cells as compared with the cells transfected with the empty vector, suggesting that mut-ISG20 protein might exhibit dominant-negative effects in
vivo by inhibiting the basal activity of ISG20. These results also suggest that the antiviral action of ISG20 is mediated by its exonuclease activity.

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Fig. 1.
ISG20 expression confers
resistance to VSV infection in transiently transfected HeLa cells.
A, schematic representation of wt-ISG20 and mut-ISG20
proteins. The three exonuclease motifs are indicated, and
conserved amino acids are underlined. Amino acid
substitution is indicated by an arrow in position 94. B, the cDNAs coding for wt-ISG20 and for the inactive
ExoII-mutated ISG20 protein were cloned under the transcriptional
dependence of the cytomegalovirus promoter in the pCiNeo vector
(pCiNeo-wt-ISG20 and pCiNeo mut-ISG20, respectively). The empty pCiNeo
vector was used as a negative control. The cells were transfected
with 0.5 µg or 1 µg of each plasmid by the LipofectAMINE Plus
Reagent procedure. 24 h after transfections, the cells were
infected with 0.1 MOI of VSV. Viral productions were determined 24 h later as described under "Experimental Procedures." The result of
a typical experiment is presented. For each experiment, the histogram
represents the relative values of viral production calculated by
dividing the number of viral particles obtained after transfections
with the indicated constructions by the number of viral particles
obtained after transfection with the empty pCiNeo vector. For
comparison, the relative virus production obtained after treatment of
parental HeLa cells with 500 units/ml HuIFN- 2a is presented.
C, the cDNAs coding for wt-ISG20 and for the inactive
ExoII-mutated ISG20 protein were cloned in the pEGFP vector to generate
wt-ISG20 (pEGFP-wt-ISG20)- and mut-ISG20
(pEGFP-mut-ISG20)-fused proteins, respectively. Cells
transfected with a vector expressing GFP alone (pEGFP) were
used as control. HeLa cells were transfected with each plasmid and then
infected 24 h later with VSV at 1 MOI. 16 h later,
transfected cells were detected by green fluorescence (GFP),
and VSV antigen expression was monitored by immunofluorescence using a
rabbit polyclonal anti-VSV antibody visualized with Texas red
(VSV). The single confocal image were superimposed
(Merge).
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Because, in transient transfection experiments, only a part of the cell
population is transfected and expresses the transgene, the antiviral
effect observed is underestimated. To circumvent this problem, the
anti-VSV ISG20 activity was determined cell by cell by confocal
immunofluorescence microscopy. To this end, the cDNAs coding for
wt-ISG20 and mut-ISG20 protein were subcloned into the pEGFP vector in
the same open reading frame as the GFP to generate the pGFP-wt-ISG20
and pGFP-mut-ISG20 expression vectors. The empty pEGFP vector
expressing GFP protein alone was used as a negative control. HeLa cells
were transfected with each plasmid and then infected 24 h later
with VSV at 1 MOI. 16 h later, transfected cells were detected by
green fluorescence, and VSV antigen expression was monitored by
immunofluorescence using a rabbit polyclonal anti-VSV antibody
described previously (27). As shown in Fig. 1C, when HeLa
cells were transfected with the pGFP-wt-ISG20 plasmid, the GFP-positive
cells that expressed the GFP-wt-ISG20-fused protein did not express
detectable VSV antigens, whereas GFP-negative cells exhibited a high
level of VSV antigens. At the opposite, all cells expressing either the
GFP alone or the GFP-mut-ISG20-fused protein expressed VSV antigens.
These data clearly demonstrated the VSV antiviral activity of ISG20.
Constitutive Expression of ISG20 Protein Confers Resistance to VSV
Infections--
To further analyze the mechanism of ISG20 antiviral
action, stable HeLa cells constitutively overexpressing the wt-ISG20 or the mut-ISG20 proteins were constructed (see "Experimental
Procedures"). First, mouse specific polyclonal antibodies directed
against recombinant ISG20 protein were developed to characterize cells
overexpressing ISG20 (see "Experimental Procedures"). According to
the modulation of ISG20 mRNA after IFN treatment (21),
the 20-kDa protein detected by the antibodies was induced by IFN in
HeLa cells (Fig. 2A). These
antibodies were used to analyze ISG20 protein expression from stable
clones. After Geneticin selection of HeLa cells transfected with either
wt-ISG20 or mut-ISG20 constructs, clones expressing wild type ISG20
protein (wt-ISG20) or the inactive ISG20-mutated protein (mut-ISG20)
were selected. The data for a representative clone of each population
are presented in Fig. 2A. ISG20 appears to be expressed at a
3.5 times higher level in wt-ISG20 than in HeLa cells
transfected with the empty pCiNeo vector, as determined by densitometry
analysis using National Institutes of Health Image software for signal
quantification. A higher level of expression was obtained with
mut-ISG20 protein, suggesting that the overexpression of wild type
ISG20 protein could be critical for cell survival. Because cell
proliferation could influence viral infection, we wished to exclude the
possibility that variations in virus yields were the result of
differences in the growth rate of selected clones. The growth curves
established for each clone were similar, excluding this possibility
(data not shown).

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Fig. 2.
Constitutive expression of ISG20 protein
confers resistance to VSV infections. A, Western blot
analysis of ISG20 protein expression in constitutively wt-ISG20- and
mut-ISG20- expressing HeLa clones. Total protein extracts from
unstimulated (Ct) or stimulated (IFN) HeLa cells
by 500 units/ml HuIFN- 2a and from constitutively wt-ISG20- or
mut-ISG20-expressing clones were analyzed by immunoblotting with a
specific mouse polyclonal antibody directed against ISG20 recombinant
protein. The sizes of the molecular weight marker (MW) and
ISG20 protein are indicated. Expression of -tubulin was used as an
invariant control. B, the wt-ISG20 and mut-ISG20 clones and
the HeLa cells transfected with the empty pCiNeo vector (HeLa/pCiNeo)
were infected with VSV at 0.1 MOI. 24 h later, the productions of
infectious viral particles were determined as described under
"Experimental Procedures." The histograms represent the log of
virus yield produced by each cell population. The standard deviations
were determined for three independent experiments. C,
HeLa/pCiNeo cells and the wt-ISG20 clones were infected with 0.1 and 1 MOI of VSV, and viral productions were determined as described in
B.
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The selected clones were then infected with 0.1 MOI of VSV. A stable
population of HeLa cells transfected with the empty pCiNeo vector
(HeLa/pCiNeo) was used to determine viral infection efficiencies. 24 h later, the viral productions were determined as described previously. The mean value of three independent experiments presented in Fig. 2B shows a reproducible protective effect, ranging
from a 2- to 3-log reduction of viral production in the wt-ISG20
HeLa cells, as compared with the HeLa/pCiNeo cells. A low ISG20 protein expression was sufficient to promote efficient protection against VSV.
To determine whether the viral resistance observed was dependent on the
amount of viral particles used for infections, an additional experiment
was performed with 1 MOI of VSV. As expected, the amplitude of ISG20
viral protection was decreased when the cells were infected at high MOI
(Fig. 2C). Clearly, these data strengthen our findings in
transient transfection experiments and demonstrate the role of ISG20 in
the antiviral actions of IFNs against VSV. Because VSV particle
production was significantly higher in mut-ISG20 HeLa cells, these data
confirmed the dominant-negative activity of the ExoII inactive mutant
of ISG20 protein.
ISG20 Inhibits VSV RNA and Protein Accumulation--
To determine
whether ISG20 overexpression interfered with viral RNA expression,
empty pCiNeo, wt-ISG20, and mut-ISG20 HeLa cells were infected with VSV
at 1 MOI. 6 h after infection, the cells were collected, and total
RNA was extracted and analyzed by Northern blot for the presence of the
four major VSV mRNAs encoding the viral proteins N, NS, M, and G
(25, 27). As shown in Fig. 3A,
the expression of all the viral mRNAs tested was strongly reduced
in the wt-ISG20 clone, compared with HeLa/pCiNeo cells or with the
mut-ISG20-expressing cells. Hybridization with a
glyceraldehyde-3-phosphate dehydrogenase probe (30) was used as an
invariant control to normalize the Northern blot (Fig. 3A).
The effect of wt-ISG20 was also confirmed by Western blot analysis
using a rabbit polyclonal antibody able to detect the major structural
proteins of VSV, the nucleoproteins N and NS (Mr
40,000), the matrix protein M (Mr 25,000), and
the glycoprotein G (Mr 69,000) (27). In
accordance with the inhibition of VSV mRNA expression, a strong
reduction in VSV antigen expression was observed in the wt-ISG20 clone
as compared with mut-ISG20 and HeLa/pCiNeo cells (Fig. 3B).
The VSV antigens remained undetectable in HeLa-pCiNeo cells treated
with 500 units/ml HuIFN-
2a used as positive control.

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Fig. 3.
Overexpression of ISG20 inhibits viral
mRNA and protein accumulation. A, HeLa/pCiNeo cells
and wt-ISG20- and mut-ISG20-expressing cells were infected with VSV at
1 MOI. 6 h after infection, the cells were collected, and their
total RNA was extracted. RNAs (20 µg/lane) were separated on 1.2%
formaldehyde-agarose gel, transferred onto a nylon membrane, and
hybridized to 32P-labeled cDNA probes corresponding to
the four major VSV mRNAs encoding for the viral proteins N, NS, M,
and G. The same blot was reprobed with a glyceraldehyde-3-phoshate
dehydrogenase (GAPDH) probe to ensure that equal amounts of
RNA were loaded onto each lane. The cDNA probes used are indicated
to the left of the blot. B, HeLa/pCiNeo cells and
wt-ISG20- and mut-ISG20-expressing cells were infected with VSV at 1 MOI. 16 h after infection, the cells were collected, and total
protein extracts were analyzed by immunoblotting with rabbit polyclonal
antibodies directed against the four major structural proteins of VSV,
the nucleoproteins N and NS, the matrix protein M, and the glycoprotein
G. For comparison, the level of viral protein expression in HeLa cells
treated with 500 units/ml HuIFN- 2a is shown. Expression of
-tubulin was used as an invariant control.
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ISG20 Partly Mediates IFN Antiviral Activity against VSV--
We
have previously shown that
90% of ISG20 RNase activity was inhibited
when RNase assays were performed, in vitro, in the presence
of the inactive ExoII-mutated ISG20 protein (24). The fact that the
amounts of VSV produced by transiently or stably transfected HeLa cells
with the mut-ISG20 construct were significantly higher than that
produced by cells transfected with the empty pCiNeo vector strongly
suggests that mut-ISG20 could exhibit dominant-negative effects
in vivo (24). We took advantage of this inhibitory effect to
test the contribution of ISG20 to the IFN-mediated antiviral activity.
To this end, the antiviral action of IFN on mut-ISG20 HeLa cells was
compared with that observed on HeLa/pCiNeo cells. The cells were
treated for 16 h with 500 units/ml HuIFN-
2a and then infected
with 0.1 MOI of VSV. 24 h later, the virus titers were determined
as described under "Experimental Procedures." The results presented
in Fig. 4 show that overexpression of
mut-ISG20 protein reduced the ability of IFN to interfere with VSV
infection. These data confirm that the antiviral activity of IFN
against VSV infection is partly mediated by ISG20.

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Fig. 4.
Overexpression of inactive ISG20 mutated
protein acts as a dominant negative in vivo.
Mut-ISG20 and HeLa/pCiNeo cells were treated with 500 units/ml
HuIFN- 2a for 24 h and then infected with VSV at 0.1 MOI.
24 h later, the productions of infectious viral particles were
determined as described under "Experimental Procedures." The
histograms represent the log of virus yield produced by each cell
line. The standard deviations were determined for three
independent experiments.
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Overexpression of ISG20 Confers Resistance to Influenza Virus and
EMCV Infections but Not to Adenovirus Infection--
Because the
different IFN-induced antiviral pathways described thus far present
some virus specificities, we analyzed the potential ISG20 antiviral
activity against two other RNA genomic viruses, the influenza virus (an
orthomyxovirus) and EMCV (a picornavirus). The wt-ISG20, mut-ISG20, and
HeLa/pCiNeo cells were infected with these viruses at 0.1 MOI. The
viral productions were determined as described for VSV. As shown in
Fig. 5A, a strong reduction in
influenza virus production was observed with the wt-ISG20 clone but not
with mut-ISG20. However, this decrease in viral multiplication was
lower than the one observed with VSV. On the other hand, only a weak
protective effect was observed for EMCV (Fig. 5B). These data show that ISG20 presents antiviral specificities among the viruses
tested and suggest that this protein acts preferentially against VSV in
the IFN-mediated antiviral barrier. Interestingly, the expression of
mut-ISG20 did not affect the action of IFN against influenza virus and
EMCV (data not shown), suggesting that the contribution of ISG20
against these viruses is probably minor in comparison with the other
IFN-induced pathways.

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Fig. 5.
Stable constitutive expression of ISG20
protein confers resistance to influenza virus and EMCV infections but
not to adenovirus infection. HeLa/pCiNeo cells and the wt-ISG20-
and mut-ISG20-expressing cells were infected with influenza virus
(A) or EMCV (B) at 0.1 MOI. 24 h later, the
productions of infectious viral particles were determined as described
under "Experimental Procedures." The histograms represent the log
of virus yield produced by each cell population. The standard
deviations were determined for three independent experiments.
C, HeLa cells and wt-ISG20 and mut-ISG20 clones were
infected for 12 h with 1 MOI of VSV (+VSV), and VSV
antigen expression was monitored by immunofluorescence with rabbit
anti-VSV polyclonal antibodies. The same cells were infected by a
derivative adenovirus strain bearing a -galactosidase encoding
reporter gene (+adenovirus) at 10 MOI. 24 h after
infection, -galactosidase-expressing cells were detected by
histochemical staining and light microscopy analysis. The relative
-galactosidase activities are indicated in milliunits.
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We also addressed the question of the ability of ISG20 to interfere
with the replication of a DNA genomic virus. To this end, the
HeLa/pCiNeo cells and the wt-ISG20 and mut-ISG20 clones were infected
with a derivative adenovirus strain bearing a
-galactosidase reporter gene at an MOI of 10. 24 h after infection,
-galactosidase-expressing cells were detected by histochemical
staining and light microscopy analysis. In addition, cellular extracts
were prepared, and the
-galactosidase activity was determined as
described under "Experimental Procedures." As a control, the cells
were infected for 12 h with 1 MOI of VSV, and VSV antigen
expression was monitored by immunofluorescence. According to our
previous data, a strong inhibition of VSV antigen expression was
observed in the wt-ISG20 clone (Fig. 5C). On the contrary,
no inhibition of adenovirus protein expression was observed in wt-ISG20
HeLa cells, strengthening the idea of virus specificity for ISG20 activity.
Expression of ISG20 in Cells Triply Deficient in PKR, RNase L, and
Mx Proteins Confers Resistance to VSV Infection--
It has been
showed that MEFs triply deficient in PKR, RNase L, and Mx gene
expression retain a substantial residual IFN-mediated antiviral
activity against VSV, suggesting the existence of other alternative
antiviral pathways (20). The ability of ISG20 to interfere with VSV
multiplication prompted us to analyze the regulation of ISG20
expression by IFN and its potential antiviral activity in this cellular
context. To this end, triply deficient MEFs were cultured in the
presence of increasing concentrations of type I IFN and then analyzed
for ISG20 protein expression. Concurrently, the cells were infected for
24 h with 0.1 MOI of VSV, and viral productions were determined as
described previously. As shown in Fig.
6A, mouse ISG20 protein was
detected with the antibody directed against its human homologue (the
mouse protein shares 81% homology with the human protein) and was
inducible by IFN in a dose-dependent manner. The level of
the IFN-mediated antiviral activity (Fig. 6B) correlated
with the induction of ISG20 expression (Fig. 6A). In
addition, MEFs transiently transfected with the wt-ISG20-expressing
vector exhibited a significant reduction in virus production as
compared with cells transfected with the empty pCiNeo vector (Fig.
6C). Taken together, these data demonstrate that the
induction of ISG20 expression by type I IFN does not require functional
PKR, RNase L, or MX gene expressions and suggest that ISG20 might
represent an alternative antiviral pathway against VSV infection.

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Fig. 6.
Expression of ISG20 in cells triply deficient
in PKR, RNase L, and Mx proteins confers resistance to VSV
infection. A, triply deficient MEFs were cultured in
the presence of increasing concentrations of type I IFN. Total protein
extracts were analyzed by immunoblotting with a specific mouse
polyclonal antibody directed against ISG20 recombinant protein. The
size of the molecular weight marker (MW) is indicated.
Expression of -tubulin was used as an invariant control.
B, triply deficient MEFs were cultured in the presence of
increasing concentrations of type I IFN and then infected for 24 h
with 0.1 MOI of VSV. Viral productions were determined as described
previously. C, triply deficient MEFs were transiently
transfected, by the LipofectAMINE Plus Reagent procedure, with 0.5 µg
of wt-ISG20 or the empty pCiNeo vector. 24 h after transfections,
the cells were infected with 0.1 MOI of VSV. Viral productions were
determined 24 h later as described under "Experimental
Procedures." The histogram represents the relative values of viral
production calculated by dividing the number of viral particles
obtained after transfections with the indicated constructions by the
number of viral particles obtained after transfection with the empty
pCiNeo vector. The standard deviations were determined for three
independent experiments.
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DISCUSSION |
The diverse biological activities of IFNs are thought to be
mediated by the induction and activation of specific but usually overlapping sets of proteins (31). In particular, the 2-5A/RNase L
system (reviewed in Refs. 2 and 13), the double-stranded RNA-dependent protein kinase (PKR) (9), and the Mx proteins (14, 15) are the three major described IFN-regulated pathways that are
involved in the cellular protection against viral infections. Attempts
to further resolve the contribution of each of these pathways in the
antiviral activities mediated by IFN have included the establishment of
genetically deficient mice (14, 20, 32-34). Interestingly, fibroblasts
derived from mice triply deficient in PKR, RNase L, and Mx gene
expressions were still protected by IFN against viral infections,
suggesting the existence of additional IFN-induced antiviral pathways
(20). The huge diversity of virus families and the fact that viruses
have developed strategies to circumvent the antiviral activities of IFN
implicate that mammalian cells use various alternative strategies to
interfere with viral multiplication (35). We have recently isolated a
human cDNA encoding a new IFN-induced gene that we have termed
ISG20 (21, 22, 29). We provided biochemical evidence that
ISG20 is a processive 3' to 5' exonuclease specific for single-stranded
RNA (24). In the present report, we analyzed the potential antiviral activity of ISG20.
We demonstrated that stable and constitutive expression of ISG20
confers resistance to VSV, influenza virus, and EMCV infection in HeLa
cells, providing an alternative antiviral pathway against RNA genomic
viruses. The same experiments were performed in transiently transfected
cells, demonstrating that the protective effect was due to ISG20
expression and was not a characteristic of the selected clones.
However, the protective efficiency of ISG20 seems to be variable even
among RNA viruses because protection against influenza virus and EMCV
was less efficient than that against VSV infection. In accordance with
that, an inactive mutant ISG20 protein able to inhibit ISG20
exonuclease activity in vitro significantly reduced the
ability of IFN to block VSV but not EMCV or influenza virus developments. These data strongly suggest that ISG20 partly mediates the IFN antiviral effect against VSV, with a minor contribution against
influenza virus and EMCV infections. ISG20 did not seem to interfere
with the replication of a derivative adenovirus strain bearing a
-galactosidase-encoding reporter gene. This suggests that
single-stranded RNA genomic viruses might be preferential targets for
ISG20. These data are not surprising because the effectiveness with
which the host's antiviral response can clear a virus infection indicates that cooperation between several antiviral pathways is
required. Each of these pathways affects different stages of the viral
life cycle (2, 9, 13, 19, 36). However, the virus specificity of each
of these pathways is not clearly established and seems dependent on the
cell type or the animal model used (32, 37-39). Thus, additional
studies with several other RNA and DNA viruses are needed to precisely
clarify the viral specificities of ISG20.
Our dominant-negative experiments suggest that the antiviral action of
ISG20 is mediated by its exonuclease activity. Throughout its 3' to 5'
exonuclease, it is conceivable that ISG20 might affect viral
development by degrading viral RNA. However, we cannot exclude that
ISG20 acts indirectly on cellular factors required for viral replication or transcription. Like ISG20 and RNase L, some cellular and
extracellular ribonucleases appear to be major contributors to the
protection against various pathogens including viruses and bacteria.
Because dsRNAs are formed in almost all viral infections, they
represent preferential targets for the ribonuclease-mediated antiviral
effect. Indeed, it has been shown that dsRNA duplexes can be
hyper-edited by members of the adenosine deaminase enzyme family,
resulting in up to 50% adenosine to inosine conversion (40).
Hyper-edited dsRNA are specifically cleaved by a cytoplasmic endoribonuclease that requires an RNA structure fitted to hyper-edited RNA (41). The fact that the cytoplasmic isoform of adenosine deaminase
(ADAR1) is inducible by IFN (42) lent weight to the idea that this
process provides an efficient mechanism to remove long, uninterrupted
dsRNAs frequently associated with infection by viruses. In addition,
adenosine to inosine editing dramatically changes the stability of
dsRNA structures, resulting in a stronger vulnerability to attacks by
single-stranded RNAses (43). In particular, inosine-containing
single-stranded RNA and unwinding dsRNA edited by ADAR1 have been
reported to be degraded at a highly accelerated rate by a specific 3'
to 5' exonuclease termed I-RNase (44). The authors speculated that
I-RNase, in concert with ADAR1, might form part of a novel antiviral
defense mechanism that acts to degrade dsRNA. Some extracellular
ribonucleases also display antiviral properties. The eosinophil-derived
neurotoxin protein (EDN or RNase 2) and the eosinophil cationic protein
(ECP or RNase 3), members of the RNase A family found in secretory
granules of human eosinophilic leukocytes, reduce the infectivity of
certain RNA viruses including respiratory syncytial virus (45) and
human immunodeficiency virus (46). These activities are mediated
through an RNase-dependent process. In the same way, human
onconase has been shown to act as a ribonuclease-dependent
antiviral agent (47). Surprisingly, nothing is known about the
contribution of deoxyribonucleases in the control of viral development
in particular against DNA viruses.
More generally, the control of RNA turnover is involved in the
regulation of critical functions such as cell cycling, apoptosis, and stress response. The fact that all these functions appear to be
modulated by IFN and the fact that IFN can regulate the expression of
cellular genes at the RNA level (48, 49) suggest that control of RNA
stability may play a major role in the mechanism of IFN action. Thus,
the identification of cellular targets of ISG20 remains a main
challenge for comprehension of the molecular mechanism of IFN action.
However, how ISG20 can specifically degrade particular viral or
cellular RNAs remains unclear. Indeed, RNases are typically present in
very low amount in cells associated with a specific inhibitor or are
present in an inactive latent form requiring the presence of a specific
activator. This is the case for RNase L, whose activation requires the
presence of oligoadenylates synthesized by 2'-5' oligoadenylate
synthetase in response to replicating dsRNA forms of viruses such as
EMCV (12, 50, 51). It is conceivable that such a mechanism could be
involved in a local activation of ISG20 preventing cell toxicity.
Recently, monitoring of global gene expression of immune cells using
DNA microarrays revealed two clusters of IFN-induced genes that are
coordinately expressed (52). One of them, termed IFN-2, contains genes
that promote resistance to viral infection and reflects a coordinated
effort by cells to escape viral control, including RNase L, PKR, Mx,
and ISG20 genes. Interestingly, we showed that mouse ISG20 protein was
inducible by type I IFN in MEFs triply deficient in RNase L, PKR, and
Mx gene expressions. In addition, these cells transiently transfected
with the wt-ISG20-expressing vector exhibited a protection against VSV
infection. These data greatly strengthened the role of ISG20 as an
alternative antiviral pathway. More recently, ISG20 was shown to be
up-regulated during activation programs induced in human macrophages by
some bacterial strains, providing a more general role of ISG20 against
different kinds of pathogens (53, 54). Additional studies are needed to
precisely determine the biological contribution of ISG20 in these processes.