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during Coxsackievirus Infection
Complementary Roles in Islet Cell Defense
RNA-Dependent Protein Kinase Exert
RNase L and Double-Stranded
Williams, Robert Silverman and Nora Sarvetnick
Stotland, Amy Maday, Devin Tsai, Cody Fine, Bryan
Malin Flodström-Tullberg, Monica Hultcrantz, Alexandr
2005; 174:1171-1177; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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Copyright © 2005 by The American Association of
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The Journal of Immunology
by guest on June 12, 2013
RNase L and Double-Stranded RNA-Dependent Protein Kinase
Exert Complementary Roles in Islet Cell Defense during
Malin Flodstro ¨m-Tullberg,*†Monica Hultcrantz,†Alexandr Stotland,* Amy Maday,*
Devin Tsai,* Cody Fine,* Bryan Williams,‡Robert Silverman,‡and Nora Sarvetnick2*
Coxsackievirus (CV) is an important human pathogen that has been linked to the development of autoimmunity. An intact
pancreatic ? cell IFN response is critical for islet cell survival and protection from type 1 diabetes following CV infection. In this
study, we show that IFNs trigger an antiviral state in ? cells by inducing the expression of proteins involved in intracellular
antiviral defense. Specifically, we demonstrate that 2?,5?-oligoadenylate synthetases (2-5AS), RNase L, and dsRNA-dependent
protein kinase (PKR) are expressed by pancreatic islet cells and that IFNs (IFN-? and IFN-?) increase the expression of 2-5AS
and PKR, but not RNase L. Moreover, our in vitro studies uncovered that these pathways play important roles in providing unique
and complementary antiviral activities that critically regulate the outcome of CV infection. The 2-5AS/RNase L pathway was
critical for IFN-?-mediated islet cell resistance from CV serotype B4 (CVB4) infection and replication, whereas an intact PKR
pathway was required for efficient IFN-?-mediated repression of CVB4 infection and replication. Finally, we show that the
2-5AS/RNase L and the PKR pathways play important roles for host survival during a challenge with CVB4. In conclusion, this
study has dissected the pathways used by distinct antiviral signals and linked their expression to defense against CVB4.
Journal of Immunology, 2005, 174: 1171–1177.
tected in pancreatic tissue, including ? cells, from T1D patients
and individuals that succumbed to a CV infection (3–6). CVs in-
fect ? cells in vitro, often leading to ? cell dysfunction and de-
struction (6–12). Because these viruses have such dramatic effects
on ? cell survival in vitro, it may seem surprising that the majority
of systemic infections pass without the development of T1D (13).
Recent studies have explained this paradox. First, an interesting
study by Chehadeh et al. (14) demonstrated that human pancreatic
? cells survive an in vitro challenge with CV only in the presence
of IFN. Subsequent studies in an animal model showed that an
intact ? cell response to IFNs is indispensable for islet cell survival
in vitro and in vivo and that protection from diabetes following
oxsackievirus (CV)3infections are common in humans
and have been etiologically linked to type 1 diabetes
(T1D) (1, 2). Members of the CV family have been de-
systemic CV infection required an intact islet cell response to IFNs
(11, 15). Taken together, these studies suggest that ? cells survive
a systemic CV infection by responding to IFNs released early dur-
ing the infection. Importantly, the studies also suggest that islet
cell activities directly determine the outcome of an infection with
a diabetogenic virus. Indeed, the efficiency by which ? cells mount
antiviral defense activities may directly regulate an individual’s
risk for developing viral-induced T1D (11, 15). This awareness has
accentuated the need for further studies on ? cell antiviral defense.
IFNs are produced early following a viral encounter, including
infections with picornaviruses (Refs. 16–18, and M. Flodstro ¨m-
Tullberg and N. Sarvetnick, unpublished observation). They acti-
vate the host’s antiviral immune response and, therefore, are often
critical for host survival. Early during viral exposure, cells at the
local site of viral entry rapidly produce and secrete type I IFN
(IFN-?, -?, and -?). The type II family of IFNs, containing a
single member, IFN-?, is elicited at a slightly later stage of infec-
tion. This cytokine is secreted by activated NK cells, CD4?Th
cells, and CD8?CTLs (19–21). Members of both IFN families
contribute to the host’s antiviral defense by up-regulating MHC I
expression, activating NK cells, macrophages, and T cells (19–
21). Besides this, the IFNs act in auto-, para-, and endocrine fash-
ions, triggering the transition of uninfected cells into an antiviral
state and apoptotic cell death in already infected cells (19–23). The
overall goal for these actions is prevention of viral infection, rep-
lication, and dissemination.
The antiviral state aims at lowering a cell’s permissiveness to
infection. This is commonly achieved by the expression of proteins
exhibiting intracellular antiviral activities. For example, RNase L
degrades viral and host RNA. This endonuclease is activated by
2-5A oligoadenylates (2-5A) synthesized by a family of IFN-reg-
ulated enzymes denoted 2-5A synthetases (2-5AS) (24). The
2-5AS enzymes become activated only in the presence of viral
dsRNA intermediates. In addition to 2-5AS, IFNs can induce the
*Department of Immunology, IMM-23, The Scripps Research Institute, La Jolla, CA
92037;†Center for Infectious Medicine, Department of Medicine, Karolinska Insti-
tutet, Huddinge University Hospital, Stockholm, Sweden; and‡Department of Cancer
Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH
Received for publication July 28, 2004. Accepted for publication October 20, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grant R0I: AI42231 (to
N.S.), the Juvenile Diabetes Research Foundation (to M.F.-T.), the Swedish Research
Council (to M.F.-T.), the Swedish Foundation for Strategic Research (to M.F.-T. and
M.S.), Åke Wiberg and Jeansson’s Foundations (to M.F.-T.), and the Swedish Dia-
betes Association Research Foundation (to M.F.-T.). This is manuscript number
16424-IMM from The Scripps Research Institute.
2Address correspondence and reprint requests to Dr. Nora Sarvetnick, Department of
Immunology, IMM-23, The Scripps Research Institute, 10550 North Torrey Pines
Road, La Jolla, CA 92037. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: CV, Coxsackievirus; T1D, type 1 diabetes; PKR,
dsRNA-dependent protein kinase; wt, wild type; p.i., post infection; EM, electron
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
by guest on June 12, 2013
expression of a dsRNA-dependent protein kinase frequently de-
noted PKR (20, 22, 23, 25). In response to viral dsRNA, this
serine/threonine protein kinase phosphorylates and inactivates ri-
bosomal protein eukaryotic initiation factor 2? resulting in a block
in protein translation. The perturbed protein biosynthesis inhibits
viral replication. Other examples of IFN-regulated proteins with
demonstrated antiviral actions are the Mx family of GTPases and
the inducible form of NO synthase (NO synthase 2) (20).
Whether any particular antiviral pathway is favored for IFN-
induced inhibition of viral replication in pancreatic islet cells is
unknown. In the present study, we tested the hypothesis that IFNs
regulate islet cell permissiveness to CV serotype B4 (CVB4) (26),
a serotype associated with the onset of T1D (1, 2), by inducing the
2-5AS/RNase L and PKR pathways. We demonstrate that pancre-
atic islet cells express RNase L, 2-5AS, and PKR and that the
expression of 2-5AS and PKR is increased following exposure to
both IFN-? and -?. We uncovered a critical role for both antiviral
pathways in host survival following CVB4 infection. Interestingly,
our in vitro studies revealed that the 2-5AS/RNase L pathway is
required for IFN-?-mediated islet cell resistance against CVB4
infection. Furthermore, we demonstrate that IFN-?-mediated re-
pression of CVB4 infection of islet cells requires an intact PKR
Materials and Methods
C57BL/6J mice were originally obtained from The Jackson Laboratory or
Taconic Farms. RNase L?/?, PKR?/?, and RNase L?/?? PKR?/?mice
(here denoted DKO mice) were bred and maintained at The Scripps Re-
search Institute or Karolinska Institutet. Because RNase L?/?mice were
originally bred to C57BL/6J mice obtained from The Jackson Laboratory,
C57BL/6J mice from The Jackson Laboratory were used as wild-type (wt)
controls in experiments involving RNase L?/?mice. PKR?/?mice had
originally been bred to C57BL/6J mice from Taconic Farms, and wt control
mice for the experiments involving PKR?/?mice were purchased from
Taconic Farms. RNase L?/?and PKR?/?mice were intercrossed to obtain
RNase L?/?? PKR?/?mice. Because wt C57BL/6J mice obtained from
The Jackson Laboratory demonstrated a slightly higher susceptibility to
CVB4 infections than C57BL/6 mice from Taconic Farms (see Fig. 2),
mice from The Jackson Laboratory were used as wt control mice for ex-
periments involving DKO mice. All mice were kept in a specific pathogen-
free environment. The animal experiments were conducted in accordance
with institutional guidelines for animal care and use.
Pancreatic islet isolation and culture.
Pancreatic islets were isolated from 8- to 12-wk-old C57BL/6J mice (The
Jackson Laboratory) and cultured as previously described (27). The islet
preparations were cultured for at least 6 days before experiments were
performed to remove exocrine tissue and immune cells (11).
Semiquantitative RT-PCR analysis
Pancreatic islets (C57BL/6J mice; The Jackson Laboratory) were exposed
to IFN-? (1000 U/ml, recombinant murine produced in Escherichia coli,
catalog number 407293; Calbiochem), IFN-? (1000 U/ml recombinant mu-
rine, produced by transfected insect cells, catalog number 554587; BD
Biosciences), or vehicle for 6 h. Total RNA was isolated using the RNeasy
kit (Qiagen) according to the manufacturer’s instructions. Using the Su-
perScript first-strand synthesis system for RT-PCR (Invitrogen Life Tech-
nologies), the RNA was converted into cDNA. The cDNA, diluted 1/1, 1/5,
and 1/25, was subjected to PCR analysis using the following primers:
2-5AS, forward 5?-CCCCATCTGCATCAGGAGGTGGAG-3?; reverse
5?-AAGTCATAATACTTTGTCCAGTAG-3? (28); and actin, forward 5?-
GTGGGCCGCCCTAGGCACCA-3?; reverse 5?-CTCTTTGATGTCACG
The 2-5AS primers amplify murine 2-5AS 1A (Oas1a) and 2-5AS 1G
(Oas1g) mRNA. PCRs amplifying 2-5AS and actin were run using one
2-min cycle at 94°C, followed by 33 cycles (94°C/30 s, 58°C/60 s, 72°C/60
s) and 24 cycles (94°C/30 s, 58°C/45 s, 70°C/60 s), for 2-5AS and actin,
respectively. Finally, the samples were incubated at 70°C for 10 min. Pre-
liminary experiments were performed to establish reaction conditions that
allowed reproducible and reliable amplifications. PCR products were run
on 1.5% agarose gels containing 0.5 ?g/ml ethidium bromide. The bands
were visualized by UV light and photographed. The images were saved and
analyzed using NIH Image 1.63. The intensities of the 2-5AS bands were
expressed in arbitrary density units. In all experiments, the density units for
2-5AS were normalized to the actin density units.
Western blot analysis
Protein extracts were isolated from pancreatic islets (C57BL/6J mice; The
Jackson Laboratory) exposed to cytokines (IFN-?, 1000 U/ml or IFN-?,
1000 U/ml; for sources of cytokines, see above) or vehicle for 24 h. Pre-
vious studies had shown that a 24-h exposure of pancreatic islets to IFN-?
or -? was sufficient to obtain a robust protection from CVB4-mediated
destruction (11). Equal amounts of proteins were separated under denatur-
ing and reducing conditions on SDS-PAGE and transferred to nitrocellu-
lose membranes. The membranes were first incubated with a primary Ab
binding RNase L (a rabbit polyclonal Ab generated using the N terminus
of mouse RNase L), and signal detection was accomplished as previously
described (27). The membranes were then stripped for 10 min in strip
buffer (0.5 M acetic acid and 0.5 M NaCl) and incubated with a primary Ab
detecting PKR (Santa Cruz Biotechnology). The signal was detected as
described above. Finally, the membranes were stripped and reblotted with
a mouse mAb to actin to confirm equal protein loading (ICN Biomedicals).
Band intensities were quantified from nonsaturated exposures using the
NIH Image (1.62) program and expressed in arbitrary units of density. In
all experiments, the densities of RNase L and PKR were corrected by
values of actin density.
Virus strain and propagation of viral stocks
CVB4 Edwards strain 2 (E2) (26) was originally obtained from C. Gauntt
(University of Texas, San Antonio, TX). A stock of CVB4 was prepared,
and the titer was determined as previously described (30).
Viral infection in vivo and in vitro
Mice aged 8–10 wk were infected with one i.p. injection of CVB4 (100
PFU diluted in 200 ?l of HBSS) and sacrificed on days 3 or 4 post infection
(p.i.). Alternatively, the mice were monitored for survival for a 24-day
study period. Pancreatic islets were infected as previously described (11).
Briefly, islets (20 islets per condition) were treated with IFN-? (1000
U/ml), IFN-? (1000 U/ml), or vehicle for 24 h. The islets were then washed
once in HBSS and infected with CVB4 in 2 ml of HBSS containing 2 ?
105PFU CVB4/ml (2 ? 104PFU/islet). After 1.5 h of incubation at 37°C,
the islets were washed three times in HBSS and placed in Millicell culture
plate inserts (Millipore) containing fresh medium (1 ml) and fresh IFN. The
plates were incubated at 37°C, and the medium was changed every second
day for up to 6 days p.i. Fresh IFN was added at each medium change.
Previous experiments had demonstrated that addition of fresh IFN to the
cultures every second day is sufficient to provide islets with maximum
protection from CVB4-mediated destruction (Ref. 11, and M. Flodstro ¨m-
Tullberg, unpublished data). Viral titers in culture supernatants were de-
termined by a plaque assay (see below).
Virus recovery from infected pancreatic islets and tissue,
determinations of viral titers
The titers of infectious virus in culture medium from infected pancreatic
islets (retrieved every 48 h p.i.) or pancreata retrieved from infected mice
were quantitated by a standard plaque assay technique in HeLa cells (30).
Viral titers were quantitated as PFU per islet, and results were presented as
log10PFU/islet. Alternatively, viral titers were quantitated as PFU per gram
of wet tissue and presented in the text as log10(PFU per gram of tissue).
The lower detection limit of this assay is 50 PFU/ml islet culture medium
(i.e., 2.5 PFU/islet or 0.4 log10PFU/islet) or 10 PFU/g of tissue.
Ultrastructural analysis of cell death
Infected and control islet cells were subjected to an ultrastructural analysis
by electron microscopy (EM). Islets were fixed in glutaraldehyde (2.5%
glutaraldehyde, 0.1 M sodium cacodylate (pH 7.3), and 1 mM CaCl2) and
processed for Epon/Araldite resin embedding by standard procedures. Ul-
trathin sections were stained with uranyl acetate followed by staining with
Reynold’s lead citrate and examined at the Core Electron Microscope Fa-
cility (The Scripps Research Institute).
Blood glucose determinations
Venous blood glucose concentrations were measured in nonfasting mice
using a Glucometer Elite (Bayer). Animals were considered diabetic if
having a nonfasting blood glucose value ?13.8 mM (250 mg/dl) for at least
two consecutive measurements.
1172PANCREATIC ? CELL ANTIVIRAL DEFENSE
by guest on June 12, 2013
Histology and immunohistochemistry
Paraffin sections of formalin-fixed organs were prepared, cut in 5-?m thick
sections, and stained with H&E or with a primary Ab against insulin,
glucagon (DakoCytomation), or VP-1 (a capsid protein conserved within
the members of the enterovirus family; DakoCytomation) biotinylated in-
house. Bound insulin and glucagon Abs were detected with a biotinylated
secondary Ab (anti-guinea pig IgG or biotinylated anti-rat IgG) in con-
junction with the Vectastatin ABC (peroxidase) kit (Vector Laboratories)
and the chromogen diaminobenzidine (Sigma-Aldrich). Slides were coun-
terstained in Mayer’s hematoxylin.
Results are expressed as means ? SEM. Plaque assay determinations were
performed in duplicate, and the mean of the two values was considered as
one independent observation. The statistical analyses were performed using
Student’s t test (single comparisons), ANOVA (multiple comparisons), or
by Kaplan-Meier life table analysis (survival of infected mice).
IFNs increase the expression of 2-5AS and PKR
We asked whether IFNs promote the antiviral state in pancreatic
islet cells by inducing the expression of key proteins involved in
two intracellular antiviral defense pathways, namely the 2-5AS/
RNase L and PKR pathways; we assessed whether IFNs induced
the expression of RNase L and PKR in pancreatic islets by West-
ern blot analysis. Due to a paucity of commercially available Abs
to 2-5AS, we evaluated the expression of 2-5AS mRNA by RT-
We determined that islet cells expressed a low basal level of
2-5AS mRNA (Fig. 1, A and B). Following exposure to IFN-? or
-?, the 2-5AS mRNA expression level increased 7- and 4.5-fold,
respectively. The cells also expressed RNase L (n ? 4; data not
shown). However, neither IFN-? nor IFN-? altered the expression
level of this protein (C57BL6, n ? 4; data not shown). Finally, we
observed that islet cells expressed PKR, extending previous results
demonstrating PKR mRNA expression by islets (31). Treatment
with IFN-? led to a 2.4-fold increase in PKR expression (Fig. 1, C
and D). A similar exposure to IFN-? resulted in 2.1-fold increase
in PKR expression (Fig. 1, C and D). These observations indicate
that 2-5AS, RNase L, and PKR are expressed at low basal levels
in islet cells. PKR and 2-5AS expression was increased following
IFN stimulation, supporting the hypothesis that IFNs induce islet
cell expression of proteins participating in antiviral defense.
The 2-5AS/RNase L and PKR pathways are important for host
The experiments above demonstrated that IFNs induced 2-5AS
and PKR expression in islet cells. Therefore, we next asked
whether the 2-5AS/RNase L and PKR pathways are important for
survival during infection with CVB4. RNase L is the effector mol-
ecule downstream of 2-5AS, and a deletion of this gene results in
a deficiency in the 2-5AS/RNase L pathway (32). Because the role
for RNase L and PKR in host defense during infection with CVB4
was unexplored, we infected mice lacking these genes and moni-
tored them for survival. We found that RNase L?/?mice showed
enhanced susceptibility to infection compared with wt mice; al-
though 62% of the wt mice survived the infection, only 7% of the
infected RNase L?/?mice survived the 24-day study period (Fig.
2A). Similar to the RNase L?/?mice, animals lacking a functional
PKR gene were less resistant to CVB4 infection than their wt
controls. Only one (1/21) of the PKR?/?mice survived the initial
observation period of 24 days, whereas the majority of the wt mice
cells. A and B, Expression of 2-5AS mRNA in islet cells exposed to ve-
hicle, IFN-?, or IFN-? for 6 h was evaluated by RT-PCR analysis, as
described in Materials and Methods. Each cDNA was diluted 1/1, 1/5, and
1/25 before PCR amplification. The gels that are shown for 2-5AS and
actin mRNA (A) are representative of the four independent experiments
summarized in B. ?, p ? 0.05 and ??, p ? 0.01 vs respective controls,
ANOVA. C and D, Expression of PKR protein in islet cells was evaluated
by Western blot analysis. Cell lysates were prepared from islets exposed to
vehicle, IFN-?, or IFN-? for 24 h. PKR expression was determined using
an anti-PKR Ab. The gel that is shown for PKR and actin is representative
of the four independent experiments summarized in D. ?, p ? 0.05 and
??, p ? 0.01 vs respective controls, ANOVA.
2-5AS, RNase L, and PKR expression by pancreatic islet
roles for host survival during CVB4 infection. Mice were infected with a
single dose of CVB4 (100 PFU). The percentage of surviving RNase L?/?
(A), PKR?/?(B), DKO (C), and their respective wt control mice (see
Materials and Methods) are shown over time. ??, p ? 0.01 vs respective
wt control, Kaplan-Meier life table analysis.
The 2-5AS/RNase L and PKR pathways play important
1173The Journal of Immunology
by guest on June 12, 2013
(17/18) survived the 24 days (Fig. 2B). In separate experiments,
mice lacking both RNase L and PKR were infected with CVB4. Of
the wt mice, 45% survived until 24 days p.i. (Fig. 2C). In contrast,
most of the DKO mice succumbed within 6 days, and none of the
DKO mice survived beyond day 11 p.i. (Fig. 2C). These experi-
ments suggest that the 2-5AS/RNase L and the PKR pathways play
important, yet independent, roles in host defense to CVB4. The
requirement for both pathways in host survival was further dem-
onstrated by the failure of mice deficient in both RNase L and PKR
to survive infection.
The pancreata of RNase L?/?and PKR?/?mice are permissive
to early CVB4 infection
We next asked whether a lack in the 2-5AS/RNase L or PKR
pathways would lead to an early, detectable alteration in pancreatic
islet cell permissiveness to CVB4. In initial experiments, we de-
termined the permissiveness of RNase L?/?and PKR?/?pancre-
ata for CVB4 infection. This was accomplished by measurements
of viral titers in organs harvested from infected mice on day 3 p.i.,
a time point chosen because it coincides with the peak of CVB4
replication in the murine pancreas (33, 34). Moreover, none of the
mice succumbed to the infection before day 3 p.i. The results were
as follows (results presented as log10(PFU per gram tissue);
mean ? SEM): RNase L?/?, 9.9 ? 0.4 (n ? 3 mice); wt control,
9.3 ? 0.2 (n ? 2 mice), p ? 0.41; PKR?/?, 10.5 ? 0.2 (n ? 3
mice); wt control, 10.4 ? 0.1 (n ? 3 mice), p ? 0.81). These
experiments propose that pancreas from RNase L?/?and PKR?/?
are permissive to CVB infection and that there is no difference in
pancreatic viral load between the infected knockout and wt ani-
mals at day 3 p.i.
The pancreatic islets comprise 2–3% of the pancreatic tissue and
may respond distinctly compared with the bulk pancreatic studies
above. We therefore asked whether CVB4 was present in islet cells
of infected mice, because this would suggest a functional role for
the antiviral pathways in regulating permissiveness to early CVB4
infection. CVB4 can be visualized in tissue sections using an Ab
binding a conserved sequence of CV capsid protein VP-1 (11, 14,
15). We harvested pancreata from infected wt control, RNase
L?/?, PKR?/?, and DKO mice on day 3 p.i. The immunohisto-
chemical analysis revealed VP-1-positive cells in exocrine pancre-
atic tissue of all infected mice (RNase L?/?, n ? 3 mice; wt
control, n ? 3 mice; PKR?/?, n ? 3 mice; wt control, n ? 3 mice;
DKO, n ? 3 mice; wt control, n ? 6 mice, data not shown).
However, none of the mice revealed VP-1-positive cells in their
pancreatic islets, which remained intact.
IFN-?-mediated block in CVB4 replication requires an intact
2-5AS/RNase L pathway
To address the role for the 2-5AS/RNase L and PKR pathways in
islet cell defense, we infected IFN-treated pancreatic islets isolated
from RNase L?/?, PKR?/?, and DKO mice with CVB4 and mea-
sured islet viability and virus replication.
CVB4 replicated in untreated pancreatic islets from both wt
(Fig. 3A) and RNase L?/?(Fig. 3B) mice, and the islets gradually
lost their round structure and integrity (as evaluated by light and
EM, data not shown). IFN-? prevented CVB replication in islets
from wt mice (Fig. 3A). Interestingly, a similar protection was
observed in RNase L?/?islets that had been treated with IFN-?
(Fig. 3B), suggesting that IFN-? does not use the RNase L pathway
to protect islets from CVB4 destruction. Indeed, these islets, as
before infection with CVB4. Culture medium was harvested and replaced with fresh medium and IFNs every 48 h for 6 days p.i. Viral titers were measured
in the harvested medium. Islets isolated from wt mice (C57BL6J; The Jackson Laboratory) (n ? 2 mice) (A); RNase L?/?mice (C57BL6J; The Jackson
Laboratory)(n ? 2–3 mice) (B); wt mice (C57BL6J; Taconic Farms) (n ? 3 mice) (C); PKR?/?mice (C57BL6J; Taconic Farms) (n ? 3 mice) (D); wt
mice (C57BL6J; The Jackson Laboratory) (n ? 2 mice) (E); and DKO mice (C57BL6J; The Jackson Laboratory) (n ? 3 mice) (F). ?, p ? 0.05, ??, p ?
0.01, and ???, p ? 0.001 vs respective untreated controls, ANOVA. The lower detection limit of the plaque assay was 0.4 log10PFU/islet. G—L, IFN-?
fails to prevent islet degradation in the absence of PKR. Electron micrographs showing pancreatic islet cells from wt (C57BL6J; The Jackson Laboratory)
(G, I, K) and PKR?/?(C57BL6J; The Jackson Laboratory (H, J, L) mice. The islets were exposed to PBS (I, J) or IFN-? (G, H, K, L) for 24 h and then
mock-infected (G, H), or infected with CVB4 (I–L), washed, and cultured in the absence (I, J) or presence (G, H, K, L) of IFN-? for 4 days with one medium
change on day 2 p.i. On day 4 p.i., the islets were harvested and subjected to EM analysis. Arrows indicate dying islet cells. Images are representative of
two independent experiments. Original magnification, ?3900–5200.
A—F, The role for RNase L and PKR in IFN-induced islet antiviral defense. Pancreatic islets were incubated with IFN-? or -? for 24 h
1174 PANCREATIC ? CELL ANTIVIRAL DEFENSE
by guest on June 12, 2013
well as the IFN-?-treated wt islets, maintained their round struc-
ture for the whole study period demonstrating the protective effect
of IFN-? (data not shown). In contrast, CVB4 replication was not
prevented in IFN-?-treated RNase L?/?islets (Fig. 3B), and these
islets failed to sustain their integrity. Taken together, these results
suggest that IFN-? regulates permissiveness to CVB4 in an RNase
L-independent manner, whereas IFN-? appears to require expres-
sion of the 2-5AS/RNase L pathway to efficiently prevent CVB4
replication and islet destruction.
IFN-? prevents CVB4 replication by a PKR-dependent
We next evaluated whether PKR was important for an intact IFN-
induced defense against CVB4. We found no difference in CVB4
replication between wt islets and islets lacking PKR (Fig. 3, C and
D). As evaluated by EM, CVB4-infected wt and PKR?/?islets
lost their integrity over time, whereas uninfected control and un-
infected IFN-treated islets (PKR?/?and wt) remained intact (Fig.
3, G, H, and K, and data not shown). Furthermore, IFN-? afforded
complete protection from CVB4 replication (Fig. 3D) and islet
destruction (data not shown) in islets from PKR?/?mice. How-
ever, CVB4 replication progressed in an unrestricted manner in
PKR?/?islets treated with IFN-? (Fig. 3D) leading to the degra-
dation of these islets (Fig. 3L). From these observations, we con-
clude that IFN-? uses the PKR pathway to reduce islet cell per-
missiveness to CVB4 infection. Moreover, our results demonstrate
that IFN-? regulates permissiveness to CVB4 in a PKR-indepen-
IFN-induced islet cell antiviral defense is greatly perturbed in
the absence of functional 2-5AS/RNase L and PKR pathways
We isolated and infected islets from DKO mice and their wt con-
trols. In DKO islets (Fig. 3E), the preventative effects of IFN-? or
IFN-? on CVB4 replication were clearly weakened compared with
the effects in wt islets (Fig. 3F). Furthermore, IFNs could not pre-
vent the islets from losing their integrity. As evaluated by light
microscopy, many islets were disintegrating on day 4 p.i., and on
day 6 p.i., most of the islets were completely dispersed into single
cells (data not shown). Uninfected islets (DKO and wt islets, un-
treated or treated with IFNs) maintained their integrity during the
study period. Collectively, our observations propose that the
2-5AS/RNase L and PKR antiviral pathways contribute to IFN-
induced islet cell defense and that islet cell permissiveness to early
CVB4 infection is altered in the absence of both these pathways.
We tested the hypothesis that the 2-5AS/RNase L and PKR path-
ways mediate antiviral activities of IFNs in CVB4-infected pan-
creatic islet cells and made some intriguing observations summa-
rized in Table I. We discovered that RNase L is required for an
intact IFN-?-induced defense against CVB4 in vitro. We also un-
veiled that PKR is indispensable for efficient IFN-?-induced islet
That 2-5AS provide resistance to some picornaviruses was dem-
onstrated in the 1980s (35), but its potential role in regulating
pancreatic islet cell permissiveness to CVB has not been tested
previously. Studies by Bonnevie-Nielsen and colleagues (36, 37)
showed that IFN-? increases 2-5AS activity in cell extracts from
insulin-producing cell lines and rat islet cells. In the present study,
we demonstrate that both IFN-? and -? increase 2-5AS mRNA
expression in islet cells, suggesting that the increased expression
level of 2-5AS can at least, in part, explain the observations made
by Bonnevie-Nielsen et al.
Upon activation by dsRNA, the 2-5AS generate 2-5A. The ac-
cumulation of 2-5A leads to the activation of RNase L, an enzyme
that can regulate viral replication by cleaving viral and cellular
RNA (20, 24, 38). Most tissues studied to date express RNase L,
and increased expression following IFN stimulation has been re-
ported for some cell types (20, 24). However, until now it has not
been known whether islet tissue expresses RNase L and a func-
tional 2-5AS/RNase L pathway. Here we showed that islet cells
express RNase L. We also observed that although IFN-? prevented
CVB4 replication in wt islets, it failed to successfully restrain
CVB4 replication in islets lacking RNase L. Interestingly, the an-
tiviral activity induced by IFN-? correlated with an increased ex-
pression of 2-5AS mRNA but not of RNase L, implying that the
expression level of 2-5AS, rather than RNase L, is a rate-limiting
step for the RNase L-mediated degradation of viral RNA in islet
cells. These data are consistent with other reports demonstrating
enhanced RNase L activity with increased endogenous levels of
2-5AS (35, 39, 40). Considering that IFN-? also increased the
expression of 2-5AS mRNA, it was surprising to find that the
2-5AS/RNase L pathway does not play an important role in IFN-
?-mediated protection from CVB4. The mechanism(s) underlying
this observation remains to be determined.
The present study shows that both IFN-? and -? can augment
PKR protein expression in islet cells. Furthermore, our in vitro
infection studies proposed that PKR is a major effector molecule in
IFN-?-induced islet defense against CVB4 in vitro. Similar obser-
vations have been reported in other cell types (41). Our studies also
indicate that PKR is not used by IFN-? to achieve protection from
CVB4 in vitro. Taken together, our observations suggest that the
2-5AS/RNase L and PKR pathways contribute with exclusive and
complementary anti-CVB4 signals following exposure to IFN-?
and -?, respectively.
Although RNase L plays an important role in IFN-?-mediated
islet cell anti-CVB4 defense in vitro, infections of RNase L?/?
mice did not lead to detectable levels of CVB4 protein in the
pancreatic islet cells. Moreover, our in vitro studies pointed to an
important role for PKR in robust IFN-?-induced protection of islet
cells from CVB4. Still, no CVB4 could be detected in islets from
infected PKR?/?mice. These findings showed that islet cell per-
missiveness to early CVB4 infection in vivo is tightly regulated
even if there is a lack in one of these antiviral pathways. Both type
I and II IFNs are important mediators of the host antiviral defense
and are produced during picornavirus infections (Refs. 18 and 42,
and M. Flodstro ¨m-Tullberg and N. Sarvetnick, unpublished obser-
vation). Hence, it is possible that the presence of both types of
IFNs ensure that islet cells up-regulate efficient antiviral defenses
during CVB4 infection.
Although the anti-CVB4 action of IFN-? and -? was clearly
impaired in islet cells lacking RNase L and PKR, respectively, it
Table I. CVB4 replication in IFN-treated pancreatic islet cells lacking
proteins involved in antiviral defensea
aCompiled from plaque assay data. ?, Efficient anti-CVB4 defense, no difference
in CVB4 replication when compared with wild-type islet cells treated similarly. ?,
Impaired anti-CVB4 defense, high levels of CVB4 replication when compared with
wild-type islet cells treated similarly.
bResidual anti-CVB4 activity found when compared with untreated (i.e. not
treated with IFN-? or IFN-?) islet cells of same genotype.
1175The Journal of Immunology
by guest on June 12, 2013
was not completely lost. Weak residual antiviral activities were
also observed in IFN-treated islets from DKO mice. Collectively,
these observations suggest that other factors besides the 2-5AS/
RNase L and PKR pathways may contribute to the regulation of
islet permissiveness to CVB4. Indeed, these observations highlight
the existence of hitherto undefined IFN-induced anti-CVB4 path-
ways. DKO mice infected with another member of the picorna-
viridae family, encephalomyocarditisvirus, also unveiled antiviral
activities beside the ones mediated by PKR and RNase L (43).
Other IFN-induced proteins suggested to exert antiviral activities
in infected cells are the Mx protein GTPases, inducible NO syn-
thase, the virus stress-inducible protein p56, and the RNA adeno-
sine deaminase 1 deaminase (19–22, 25, 44). We have ruled out
inducible NO synthase as a single determinant for ? cell survival
during CVB4 infection (33). It is also unlikely that Mx protein
activities account for the residual anti-CVB4 activity observed
here, because several mouse strains, including the one used here,
lack a functional Mx gene (45). However, this does not exclude
anti-CVB4 actions by Mx proteins (28) in Mx-positive strains. A
more recently described protein is ISG20 (46), and it remains to be
determined whether this or other antiviral proteins, such as p56 and
RNA adenosine deaminase 1, contribute to the regulation of islet
permissiveness to CVB4. In this context, a potential role for the
IFNs in altering the expression of viral receptors should not be
Aside from playing a role in the antiviral state, PKR, 2-5AS, and
RNase L have been implicated in pathways leading to apoptotic
cell death (22, 23, 25). IFNs can trigger apoptosis in already in-
fected cells and several recent studies have suggested that 2-5AS,
RNase L, and PKR are effector molecules in this death pathway
(22, 23, 25). Interestingly, PKR has been shown to mediate islet
cell apoptosis induced by poly(I:C) (synthetic dsRNA) or
poly(I:C) in combination with IFN-? in vitro (47). Hence, PKR
may be an effector molecule inducing apoptosis under conditions
when already infected islets are exposed to IFNs. That IFNs do not
normally trigger apoptotic cell death in islet cells (47, 48), despite
the constitutive expression of 2-5AS (Refs. 36 and 37, and the
present study), RNase L (present study), and PKR (Ref. 47, and
the present study), is not surprising, because dsRNA is absent in
the uninfected state. In this context, it is noteworthy that our ex-
periments showed that CVB4 replicated equally well in unmanipu-
lated wt islets as in unmanipulated islet from RNase L?/?,
PKR?/?, or DKO mice. These observations suggest that, at basal
expression levels, 2-5AS, RNase L, and PKR do not regulate islet cell
permissiveness to CVB4 replication. Similar observations have been
reported from other experimental systems (e.g., in Ref. 49).
We found that a lack in either antiviral pathway led to dramat-
ically increased host susceptibility to CVB4. We and others (11,
50) have demonstrated the requirement for an intact host response
to IFNs in survival following infection with CV. Therefore, it is
possible that the PKR and 2-5AS/RNase L pathways are important
effector molecules in the early IFN-mediated antiviral defense. In-
terestingly, others have demonstrated that a defective host defense
in mice lacking the PKR and 2-5AS/RNase L pathways can be
attributed both to a defective antiviral defense and to an impaired
induction of apoptosis in infected cells (32, 51–53). Although the
specific mechanism(s) by which RNase L and PKR provides pro-
tection from CVB4-induced death remains to be explored, our ob-
servations suggest that both pathways play independent roles for
host survival and that one cannot fully compensate for the lack of
Over the years, in vitro studies and animal models have revealed
distinct mechanisms to explain how a viral infection can induce
autoimmune disease. Although the majority of these studies fo-
cused on the self-reactive T cell population, recent studies have
suggested that target cell activities may affect the autoimmune pro-
cess (reviewed in Ref. 54). For example, Yasukawa et al. (55)
demonstrated that target cell activities critically contribute to the
prevention of myocarditis during CV infection. Our previous stud-
ies showed that if ? cell antiviral defense fails, then CVB4 will
destroy the ? cells regardless of other antiviral defense mecha-
nisms that may be mobilized by the host to fight the infection (11,
15). The unraveling of mechanism(s) behind islet cell survival dur-
ing systemic viral exposure is a complex task. In the present study,
we identified 2-5AS and PKR as IFN-inducible proteins in pan-
creatic islet cells and linked their induced expression to an en-
hanced defense against CVB4. Interestingly, our studies also sug-
gestedthe existenceof additional
modulating ? cell permissiveness to CVB4 in vivo. It is clear that
further knowledge about what specific antiviral defense mecha-
nism(s) ? cells produce in response to IFNs, as well as on the
specific roles of these defense pathways for islet cell survival dur-
ing CV infection, will lead to possible avenues to modulate the risk
for diabetes development following viral infection.
We thank Dr. Aimin Zhou (Cleveland State University) for the gift of
polyclonal Ab against mouse RNase L, and Dr. W. Leitner and N. Restifo
(National Institutes of Health) for providing the DKO mice from their
colonies. We also thank Dr. M. Wood, The Core Electron Microscope
Facility, The Scripps Research Institute, for assistance with electron and
confocal microscopy, and Drs. A. Kayali, M. Solomon, D. Yadav, and
other members of the Sarvetnick laboratory for discussions and sugges-
tions. L. Tucker and P. Secrest are gratefully acknowledged for excellent
1. Knip, M., and H. K. Akerblom. 1999. Environmental factors in the pathogenesis
of type 1 diabetes mellitus. Exp Clin. Endocrinol. Diabetes. 107:S93.
2. Jaeckel, E., M. Manns, and M. Von Herrath. 2002. Viruses and diabetes. Ann. NY
Acad. Sci. 958:7.
3. Gladisch, R., W. Hofmann, and R. Waldherr. 1976. [Myocarditis and insulitis
following Coxsackie virus infection]. Z. Kardiol. 65:837.
4. Yoon, J. W., M. Austin, T. Onodera, and A. L. Notkins. 1979. Isolation of a virus
from the pancreas of a child with diabetic ketoacidosis. N. Engl. J. Med.
5. Jenson, A. B., H. S. Rosenberg, and A. L. Notkins. 1980. Pancreatic islet-cell
damage in children with fatal viral infections. Lancet 2:354.
6. Ylipaasto, P., K. Klingel, A. M. Lindberg, T. Otonkoski, R. Kandolf, T. Hovi, and
M. Roivainen. 2004. Enterovirus infection in human pancreatic islet cells, islet
tropism in vivo and receptor involvement in cultured islet ? cells. Diabetologia
7. Szopa, T. M., D. M. Dronfield, T. Ward, and K. W. Taylor. 1989. In vivo in-
fection of mice with Coxsackie B4 virus induces long-term functional changes in
pancreatic islets with minimal alteration in blood glucose. Diabetes Med. 6:314.
8. Szopa, T. M., D. R. Gamble, and K. W. Taylor. 1986. Coxsackie B4 virus induces
short-term changes in the metabolic functions of mouse pancreatic islets in vitro.
Cell Biochem. Funct. 4:181.
9. Frisk, G., E. Grapengiesser, and H. Diderholm. 1994. Impaired Ca2?response to
glucose in mouse ?-cells infected with Coxsackie B or Echo virus. Virus Res.
10. Roivainen, M., S. Rasilainen, P. Ylipaasto, R. Nissinen, J. Ustinov, L. Bouwens,
D. L. Eizirik, T. Hovi, and T. Otonkoski. 2000. Mechanisms of Coxsackievirus-
induced damage to human pancreatic ?-cells. J. Clin. Endocrinol. Metab. 85:432.
11. Flodstrom, M., A. Maday, D. Balakrishna, M. M. Cleary, A. Yoshimura, and
N. Sarvetnick. 2002. Target cell defense prevents the development of diabetes
after viral infection. Nat. Immunol. 3:373.
12. Roivainen, M., P. Ylipaasto, C. Savolainen, J. Galama, T. Hovi, and
T. Otonkoski. 2002. Functional impairment and killing of human ? cells by
enteroviruses: the capacity is shared by a wide range of serotypes, but the extent
is a characteristic of individual virus strains. Diabetologia 45:693.
13. Torres, A., J. Garib, and M. L. Recurt. 1984. Coxsackie virus: a review. Bol.
Asoc. Med. P. R. 76:49.
14. Chehadeh, W., J. Kerr-Conte, F. Pattou, G. Alm, J. Lefebvre, P. Wattre, and
D. Hober. 2000. Persistent infection of human pancreatic islets by Coxsackievirus
B is associated with ? interferon synthesis in ? cells. J. Virol. 74:10153.
15. Flodstrom, M., D. Tsai, C. Fine, A. Maday, and N. Sarvetnick. 2003. Diabeto-
genic potential of human pathogens uncovered in experimentally permissive
?-cells. Diabetes 52:2025.
1176 PANCREATIC ? CELL ANTIVIRAL DEFENSE
by guest on June 12, 2013
16. Yoon, J. W., P. R. McClintock, T. Onodera, and A. L. Notkins. 1980. Virus-
induced diabetes mellitus. XVIII. Inhibition by a nondiabetogenic variant of en-
cephalomyocarditis virus. J. Exp. Med. 152:878.
17. Pozzetto, B., and I. Gresser. 1985. Role of sex and early interferon production in
the susceptibility of mice to encephalomyocarditis virus. J. Gen. Virol. 66:701.
18. Nakayama, T., T. Urano, M. Osano, Y. Hayashi, S. Sekine, T. Ando, and
S. Makinom. 1989. Outbreak of herpangina associated with Coxsackievirus B3
infection. Pediatr. Infect. Dis. J. 8:495.
19. Bogdan, C. 2000. The function of type I interferons in antimicrobial immunity.
Curr. Opin. Immunol. 12:419.
20. Samuel, C. E. 2001. Antiviral actions of interferons. Clin. Microbiol. Rev.
21. Chesler, D. A., and C. S. Reiss. 2002. The role of IFN-? in immune responses to
viral infections of the central nervous system. Cytokine Growth Factor Rev.
22. Barber, G. N. 2001. Host defense, viruses and apoptosis. Cell Death Differ.
23. Chawla-Sarkar, M., D. J. Lindner, Y. F. Liu, B. R. Williams, G. C. Sen,
R. H. Silverman, and E. C. Borden. 2003. Apoptosis and interferons: role of
interferon-stimulated genes as mediators of apoptosis. Apoptosis 8:237.
24. Player, M. R., and P. F. Torrence. 1998. The 2-5A system: modulation of viral
and cellular processes through acceleration of RNA degradation. Pharmacol.
25. Saunders, L. R., and G. N. Barber. 2003. The dsRNA binding protein family:
critical roles, diverse cellular functions. FASEB J. 17:961.
26. Hartig, P. C., G. E. Madge, and S. R. Webb. 1983. Diversity within a human
isolate of Coxsackie B4: relationship to viral-induced diabetes. J. Med. Virol.
27. Flodstrom, M., and D. L. Eizirik. 1997. Interferon-?-induced interferon regula-
tory factor-1 (IRF-1) expression in rodent and human islet cells precedes nitric
oxide production. Endocrinology 138:2747.
28. Deonarain, R., A. Alcami, M. Alexiou, M. J. Dallman, D. R. Gewert, and
A. C. Porter. 2000. Impaired antiviral response and ?/? interferon induction in
mice lacking ? interferon. J. Virol. 74:3404.
29. Stephens, L. A., H. E. Thomas, L. Ming, M. Grell, R. Darwiche, L. Volodin, and
T. W. Kay. 1999. Tumor necrosis factor-?-activated cell death pathways in NIT-1
insulinoma cells and primary pancreatic ? cells. Endocrinology 140:3219.
30. Horwitz, M. S., L. M. Bradley, J. Harbertson, T. Krahl, J. Lee, and N. Sarvetnick.
1998. Diabetes induced by Coxsackie virus: initiation by bystander damage and
not molecular mimicry. Nat. Med. 4:781.
31. Blair, L. A., M. R. Heitmeier, A. L. Scarim, L. B. Maggi, Jr., and J. A. Corbett.
2001. Double-stranded RNA-dependent protein kinase is not required for double-
stranded RNA-induced nitric oxide synthase expression or nuclear factor-?B ac-
tivation by islets. Diabetes 50:283.
32. Zhou, A., J. Paranjape, T. L. Brown, H. Nie, S. Naik, B. Dong, A. Chang,
B. Trapp, R. Fairchild, C. Colmenares, and R. H. Silverman. 1997. Interferon
action and apoptosis are defective in mice devoid of 2?,5?-oligoadenylate-depen-
dent RNase L. EMBO (Eur. Mol. Biol. Organ) J. 16:6355.
33. Flodstrom, M., M. S. Horwitz, A. Maday, D. Balakrishna, E. Rodriguez, and
N. Sarvetnick. 2001. A critical role for inducible nitric oxide synthase in host
survival following Coxsackievirus B4 infection. Virology 281:205.
34. Vella, C., and H. Festenstein. 1992. Coxsackievirus B4 infection of the mouse
pancreas: the role of natural killer cells in the control of virus replication and
resistance to infection. J. Gen. Virol. 73:1379.
35. Chebath, J., P. Benech, M. Revel, and M. Vigneron. 1987. Constitutive expres-
sion of (2?,5?)oligo A synthetase confers resistance to picornavirus infection.
36. Bonnevie-Nielsen, V., A. M. Gerdes, J. Fleckner, J. S. Petersen, B. Michelsen,
and T. Dyrberg. 1991. Interferon stimulates the expression of 2?,5?-oligoadeny-
late synthetase and MHC class I antigens in insulin-producing cells. J. Interferon
37. Bonnevie-Nielsen, V., K. Buschard, and T. Dyrberg. 1996. Differential respon-
siveness to interferon-? in ?-cells and non-? cells. Diabetes 45:818.
38. Li, X. L., J. A. Blackford, C. S. Judge, M. Liu, W. Xiao, D. V. Kalvakolanu, and
B. A. Hassel. 2000. RNase-L-dependent destabilization of interferon-induced
mRNAs: a role for the 2-5A system in attenuation of the interferon response.
J. Biol. Chem. 275:8880.
39. Silverman, R. H., J. J. Skehel, T. C. James, D. H. Wreschner, and I. M. Kerr.
1983. rRNA cleavage as an index of ppp(A2?p)nA activity in interferon-treated
encephalomyocarditis virus-infected cells. J. Virol. 46:1051.
40. Behera, A. K., M. Kumar, R. F. Lockey, and S. S. Mohapatra. 2002. 2?,5?-
Oligoadenylate synthetase plays a critical role in interferon-? inhibition of respi-
ratory syncytial virus infection of human epithelial cells. J. Biol. Chem.
41. Yang, Y. L., L. F. Reis, J. Pavlovic, A. Aguzzi, R. Schafer, A. Kumar,
B. R. Williams, M. Aguet, and C. Weissmann. 1995. Deficient signaling in mice
devoid of double-stranded RNA-dependent protein kinase. EMBO (Eur. Mol.
Biol. Organ) J. 14:6095.
42. Horwitz, M. S., A. La Cava, C. Fine, E. Rodriguez, A. Ilic, and N. Sarvetnick.
2000. Pancreatic expression of interferon-? protects mice from lethal Coxsack-
ievirus B3 infection and subsequent myocarditis. Nat. Med. 6:693.
43. Zhou, A., J. M. Paranjape, S. D. Der, B. R. Williams, and R. H. Silverman. 1999.
Interferon action in triply deficient mice reveals the existence of alternative an-
tiviral pathways. Virology 258:435.
44. Hui, D. J., C. R. Bhasker, W. C. Merrick, and G. C. Sen. 2003. Viral stress-
inducible protein p56 inhibits translation by blocking the interaction of eIF3 with
the ternary complex eIF2.GTP.Met-tRNAi. J. Biol. Chem. 278:39477.
45. Haller, O., M. Frese, and G. Kochs. 1998. Mx proteins: mediators of innate
resistance to RNA viruses. Rev. Sci. Tech. 17:220.
46. Espert, L., G. Degols, C. Gongora, D. Blondel, B. R. Williams, R. H. Silverman,
and N. Mechti. 2003. ISG20, a new interferon-induced RNase specific for single-
stranded RNA, defines an alternative antiviral pathway against RNA genomic
viruses. J. Biol. Chem. 278:16151.
47. Scarim, A. L., M. Arnush, L. A. Blair, J. Concepcion, M. R. Heitmeier,
D. Scheuner, R. J. Kaufman, J. Ryerse, R. M. Buller, and J. A. Corbett. 2001.
Mechanisms of ?-cell death in response to double-stranded (ds) RNA and inter-
feron-?: dsRNA-dependent protein kinase apoptosis and nitric oxide-dependent
necrosis. Am. J. Pathol. 159:273.
48. Hostens, K., D. Pavlovic, Y. Zambre, Z. Ling, C. Van Schravendijk, D. L. Eizirik,
and D. G. Pipeleers. 1999. Exposure of human islets to cytokines can result in
disproportionately elevated proinsulin release. J. Clin. Invest. 104:67.
49. Al-khatib, K., B. R. Williams, R. H. Silverman, W. Halford, and D. J. Carr. 2003.
The murine double-stranded RNA-dependent protein kinase PKR and the murine
2?,5?-oligoadenylate synthetase-dependent RNase L are required for IFN-?-me-
diated resistance against herpes simplex virus type 1 in primary trigeminal gan-
glion culture. Virology 313:126.
50. Wessely, R., K. Klingel, K. U. Knowlton, and R. Kandolf. 2001. Cardioselective
infection with Coxsackievirus B3 requires intact type I interferon signaling: im-
plications for mortality and early viral replication. Circulation 103:756.
51. Castelli, J. C., B. A. Hassel, K. A. Wood, X. L. Li, K. Amemiya, M. C. Dalakas,
P. F. Torrence, and R. J. Youle. 1997. A study of the interferon antiviral mech-
anism: apoptosis activation by the 2-5A system. J. Exp. Med. 186:967.
52. Zhou, A., J. M. Paranjape, B. A. Hassel, H. Nie, S. Shah, B. Galinski, and
R. H. Silverman. 1998. Impact of RNase L overexpression on viral and cellular
growth and death. J. Interferon Cytokine Res. 18:953.
53. Zheng, X., R. H. Silverman, A. Zhou, T. Goto, B. S. Kwon, H. E. Kaufman, and
J. M. Hill. 2001. Increased severity of HSV-1 keratitis and mortality in mice
lacking the 2-5A-dependent RNase L gene. Invest. Ophthalmol. Vis. Sci. 42:120.
54. Flodstrom-Tullberg, M. 2003. Viral infections: their elusive role in regulating
susceptibility to autoimmune disease. Microbes Infect. 5:911.
55. Yasukawa, H., T. Yajima, H. Duplain, M. Iwatate, M. Kido, M. Hoshijima,
M. D. Weitzman, T. Nakamura, S. Woodard, D. Xiong, et al. 2003. The sup-
pressor of cytokine signaling-1 (SOCS1) is a novel therapeutic target for entero-
virus-induced cardiac injury. J. Clin. Invest. 111:469.
1177 The Journal of Immunology
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