Synthesis and analgesic activity evaluation of some agmatine derivatives

Article abstract of DOI:10.1038/sj.cdd.4401833

A series of N,N′-disubstituted-2-nitroethene-1,1-diamine and N,N′-disubstituted-N″-cyanoguanidine derivatives were prepared and evaluated for in vivo analgesic activity. The blood brain barrier (BBB) VolSurf model was used to predict the BBB permeation profiles of our synthesized compounds. Some compounds show both remarkable analgesic activity and good BBB permeation profiles, and these compounds might be developed for treatment of opioid tolerance and dependence.

Full text of DOI:10.1038/sj.cdd.4401833

Cell Death and Differentiation (2006) 13, 393–403
2006 Nature Publishing Group All rights reserved 1350-9047/06
$
30.00
&
www.nature.com/cdd
Review
Viruses, endoplasmic reticulum stress, and interferon
responses
B He*,1
Introduction
1
Virus infection of mammalian cells consists of a series of
Department of Microbiology and Immunology, College of Medicine, The
University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, IL 60612,
USA
events, which involve entry, RNA expression and processing,
polypeptide synthesis and modification, genome replication,
and maturation. Remarkably, as intracellular parasites,
viruses rely on the utilization of cellular machinery and
resource to complete their life cycle. In this complex process,
viruses synthesize double-stranded RNA intermediates and
produce viral proteins within host cells. Consequently, viral
replication elicits cellular responses, such as endoplasmic
reticulum (ER) stress and interferon responses. It is, there-
fore, not surprising that viruses have evolved various
mechanisms to cope with these responses that limit or inhibit
viral replication.
* Corresponding author: B He, Department of Microbiology and Immunology (M/
C 790), College of Medicine, The University of Illinois at Chicago, 835 South
Wolcott Avenue, Chicago, IL 60612, USA. Tel: þ 1 312 996 2391;
Fax: þ 1 312 996 6415; E-mail: tshuo@uic.edu
Received 20.7.05; revised 02.11.05; accepted 04.11.05; published online 06.1.06
Edited by J Yuan
Abstract
Viral infection induces endoplasmic reticulum (ER) stress
and interferon responses. While viral double-stranded RNA
intermediates trigger interferon responses, viral polypeptides
synthesized during infection stimulate ER stress. Among
the interferon-regulated gene products, the double-stranded
RNA-dependent protein kinase (PKR) plays a key role in
limiting viral replication. Thus, to establish productive
infection, viruses have evolved mechanisms to overcome
the deleterious effects of PKR. It has become clear that ER
stress causes translational attenuation and transcriptional
upregulation of genes encoding proteins that facilitate folding
or degradation of proteins. Notably, prolonged ER stress
triggers apoptosis. Therefore, viruses are confronted with the
consequences of ER stress. Emerging evidence suggests
that viruses not only interfere with the interferon system
involving PKR but also manipulate the programs emanating
from the ER in a complex way, which may facilitate viral
replication or pathogenesis. This review highlights recent
progress in these areas.
The role of interferon in antiviral defense has long been
recognized. This family of cytokines is produced in response
to virus infection.1 Once bound to its receptor on the cell
surface, interferon activates the Janus tyrosine kinase/signal
transducer and activator pathway, which induces the
expression of a wide spectrum of cellular genes. Among
these that are extensively characterized is the double-
stranded RNA-dependent protein kinase (PKR), a key
player of antiviral action of interferon.2 In mammalian cells,
the interferon-induced PKR is activated by double-stranded
RNA. When activated, PKR phosphorylates the a subunit
of translation initiation factor eIF-2 (eIF2a). This leads to
the shutoff of protein synthesis and thereby inhibition of
viral replication. Moreover, PKR is involved in cell
growth, differentiation, apoptosis, and possibly ER stress
(Table 1).3–5
Recent evidence has suggested the importance of ER
stress response in virus infection.5–8 As a processing plant for
folding and post-translational modification of proteins, the ER
is an essential organelle for viral replication and maturation.
In the course of productive infection, a large amount of viral
proteins are synthesized in infected cells, where unfolded or
misfolded proteins activate the ER stress response. ER stress
caused by viruses has been observed to modulate various
signaling pathways leading to cell survival or cell death.9–11
Obviously, differential regulation of ER stress dictates the viral
pathogenesis or replication. It has been suggested that in
mammalian cells the ER chaperone immunoglobulin heavy-
chain binding protein (BiP), also known as glucose-regulated
protein 78 (GRP78), works as a master control interacting
with three mediators: PKR-like ER kinase (PERK), activating
transcription factor 6 (ATF6) and the ER transmembrane
protein kinase/endoribonuclease (IRE1).12–14 In response to
ER stress, these components function to reduce the levels of
new proteins translocated into the ER lumen, to enhance the
protein-folding capacity and secretion potential of the ER, and
to facilitate transport and degradation of ER-localized proteins
(Figures 1 and 2).
Cell Death and Differentiation (2006) 13, 393–403.
doi:10.1038/sj.cdd.4401833; published online 6 January 2006
Keywords: virus; endoplasmic reticulum; interferon; unfolded
protein; apoptosis
Abbreviations: ATF4, activating transcription factor 4; ATF6,
activating transcription factor 6; BiP, the ER chaperone immuno-
globulin heavy-chain-binding protein; CHOP, C/EBP homologous
protein; dsDNA, double-stranded DNA; ER, endoplasmic reticu-
lum; GADD34, growth arrest and DNA damage-inducible protein
34; IRE1, ER transmembrane protein kinase/endoribonuclease;
PACT, PKR-activating protein; PERK, PKR-like ER kinase; PKR,
double-stranded RNA-dependent protein kinase; ssRNA, single-
stranded RNA; UPR, the unfolded protein response; XBP1, X-
box-binding protein

Viruses and stress responses
B He
394
Table 1 Viruses, ER stress and PKR-mediated IFN response
Virus
Genome Reported site(s) of interaction
Asfarviridae
African swine fever
virus
dsDNA BiP and PERK
Herpesviridae
Cytomegalovirus
Herpes simples virus 1 dsDNA PERK and PKR
dsDNA XBP-1, ATF4, ATF6, and PKR
Hepadnaviridae
Hepatitis B virus
dsDNA BiP
Papillomaviridae
Papillomavirus
dsDNA GADD34 and PKR
dsDNA PERK and PKR
ssRNA BiP and caspase-12
Poxiviridae
Vaccinia virus
Bunyaviridae
Tula virus
Figure 1 Modulation of UPR by viruses. Upon viral infection, unfolded proteins
bind to the master control protein BiP, which thereby releases ER stress
transducers, including PERK, ATF6, and IRE1. Thus, PERK undergoes
dimerization, autophosphorylation, and subsequent activation. Activated PERK
phosphorylates eIF2a, which results in attenuation of general translation and
induction of GADD34 and CHOP. Release of ATF6 from BiP leads to the
translocation of ATF6 to the Golgi apparatus, where ATF6 is cleaved to yield a
truncated form that is capable of stimulating the expression of chaperone genes
in the nucleus. Release of IRE1 from BiP permits its dimerization and activation.
Activated IRE1 facilitates the splicing of XBP1 mRNA, which encodes a
transcription factor leading to the expression of the UPR target genes. Viruses
encode functions that inhibit one or more steps in these signaling pathways. The
balance between viral stimulation and inhibition determines the pathogenesis or
replication of viral infection. Arrows represent activation of components or
processes in the ER stress pathway upon viral infection
Flaviviridae
Bovine viral diarrhea ssRNA BiP
virus
Hepatitis C virus
ssRNA BiP, PERK, XBP-1, and PKR
Japanese encephalitis ssRNA BiPand CHOP/GADD153
virus
Orthomyxoviridae
Influenza A virus
ssRNA BiP, P58IPK, and PKR
Paramyxoviridae
Respiratory syncytial ssRNA BiP and caspase-12
virus
Simian virus 5
ssRNA BiP and caspase-12
Retroviridae
Mouse retrovirus
ssRNA Bip
Rhabdoviridae
Vesicular stomatitis
virus
ssRNA BiP, PERK and PKR
BiP is a member of heat shock proteins that binds to
properly folded and misfolded proteins.15 In normal cells,
BiP associates with the luminal domains of PERK, ATF6,
and IRE1. Under stress conditions, BiP is sequestered to
misfolded or unfolded proteins in the ER, where PERK, ATF6,
and IRE1 are released. BiP release from PERK or IRE1 leads
to homodimerization of each protein through its luminal
domain, which induces autophosphorylation and subsequent
activation.13,16 Activation of PERK attenuates protein syn-
thesis, whereas activation of IRE1 leads to the transcription
induction of a subset of genes encoding protein degradation
enzymes.17 In parallel, BiP release from ATF6 leads to the
translocation of ATF6 from ER to the Golgi apparatus, where
it is cleaved and activated.14 Activation of ATF6 stimulates
the transcription of genes encoding chaperones that refold
misfolded proteins. However, when cells are unable to recover
from ER stress, apoptosis occurs.
Figure 2 The a subunit of eIF2 connects ER stress and interferon responses.
Viral infection produces signals that activate PKR and PERK pathways,
respectively. Double-stranded RNA produced by virus triggers the production of
interferon, which upregulates PKR expression. Furthermore, double-stranded
RNA binds to and activates PKR, which phosphorylates eIF2a and inhibits
shutoff of protein synthesis. Unfolded protein activates PERK, which also
phosphorylates eIF2a. There are three cellular proteins that regulate PKR and
PERK in response to different signals. PACT, a stress-activated protein, binds to
and activates PKR. P58IKP, an ER stress-inducible protein, is capable of binding
to PKR as well as PERK. Binding of P58IKP to the two kinases suppresses their
activities. GADD34, an ER stress-inducible protein, mediates dephosphorylation
of eIF2a by recruiting protein phosphatase 1. Examples of viruses that inhibit the
interferon response mediated by PKR and ER stress response are listed. Thick
lines denotes inhibition or negative regulation, whereas arrows represent positive
regulation or activation
ER stress and interferon responses reflect different
adaptive cellular processes, which are triggered during virus
infection. Notably, viral double-stranded RNA intermediates
stimulate the interferon response, whereas unfolded or
misfolded proteins impose ER stress. Thus, viruses are faced
Cell Death and Differentiation

Viruses and stress responses
B He
395
with the consequences of these cellular responses. Nume-
rous studies demonstrate that virus infection activates PKR,
which plays a pivotal role in the antiviral action of interferon.2
As expected, PKR becomes a target of many viruses. Some
examples are hepatitis C virus, influenza virus, vaccinia virus,
papillomavirus, herpes simplex virus, and cytomegalovirus.
However, the impact of ER stress on viral infection has only
been recognized recently. Several studies suggested a
connection of the ER stress response with viral replication.
These include members of the flavivirus family, bovine viral
diarrhea virus, Japanese encephalitis virus, and hepatitis C
virus.9–11 Furthermore, other viruses have been shown to
regulate ER stress, such as respiratory syncytial virus, simian
virus 5, Tula virus, African swine fever virus, herpes simplex
virus, and cytomegalovirus.7,8,18–21 This review will summa-
rize recent progress on ER stress relevant to viral replication.
Furthermore, it will highlight the regulation of the interferon
system involving PKR in the context of ER stress upon virus
infection.
this result, mammalian cell line stably expressing the E2
protein has an elevated level of BiP. Hepatitis C virus encodes
a single polypeptide precursor, which is cleaved into the
mature structural (core, E1, and E2) and nonstructural (NS2–
NS5B) proteins. As glycoprotein E2 is an ER resident with its
carboxyl-terminal domains anchored in the ER membrane, it
is postulated that E2 protein activates transcription of BiP
indirectly by influencing an intracellular signaling pathway
rather than acting in the nucleus.27 Upon expression, the E1
and E2 proteins form a heterodimer. In the folding process of
the E1 and E2 proteins, a large portion of these proteins are
trapped in aggregates, which may trigger BiP expression.28
Interestingly, hepatitis C virus replicons expressing only
nonstructural proteins are also capable of stimulating BiP
expression.29 Hence, either the process of viral replication or
nonstructural proteins of hepatitis C virus are capable of
inducing BiP expression.
Early experiments suggested that BiP associates transi-
ently with folding intermediates of viral glycoproteins.25,30,31
By binding to viral proteins, BiP performs at least two distinct
functions in virus-infected cells. It is a chaperone that
facilitates folding or assembly of viral proteins along the
maturation process. Furthermore, it is also a sensor to detect
unfolded or misfolded viral proteins. In simian virus 5-infected
cells, HN specifically associates with BiP during glycoprotein
folding.24,30 Elimination of glycosylation sites in HN renders
the protein incapable of folding into a native conformation.
Immunoprecipitation assays suggest that BiP associates
with WT HN transiently, whereas it becomes more stably
associated with misfolded HN.24 Although the glycosylation
mutant of HN expresses at a lower level, it induces a
comparable level of BiP induction as compared to WT HN. A
similar phenotype is noted with glycoprotein G of vesicular
stomatitis virus, hemagglutinin of influenza virus, and glyco-
protein E2 of hepatitis C virus.25,28,31 Collectively, these
experimental data support a model in which interaction of BiP
with misfolded or unfolded viral proteins triggers the ER stress
response during viral infection. Clearly, additional studies are
required to understand the molecular mechanisms that
underlie these observations.
Interaction with GRP78/BiP: Virus
Triggers
As a resident of ER chaperone, the expression of BiP is
upregulated in response to ER stress in mammalian cells.22
Although the effect of BiP induction on virus replication
is not fully understood, accumulating evidence suggests
that one or more viral proteins trigger BiP expression during
virus infection. This phenotype becomes apparent in cells
infected with paramyxoviruses, such as simian virus 5 and
respiratory syncytial virus.18,23 In addition, infection of cells
with other RNA viruses, for example, flaviviruses and
hantavirus, stimulates BiP expression.10,11,19 Intriguingly,
simian virus 5 induces the synthesis of several cellular
proteins, including a 78-kDa protein, BiP.23 Besides the
hemagglutinin-neuroamindase glycoprotein (HN), simian
virus 5 encodes the fusion glycoprotein, a small nonglycosy-
lated integral membrane protein, the viral membrane protein,
the major nucleocapsid protein, and the nucleocapsid-
associated protein L, P, and V, respectively. However, among
these viral proteins, only synthesis of the HN glycoprotein
stimulates BiP expression.24 In virus-infected cells, the HN
glycoprotein is synthesized on membrane-bound ribosomes,
inserted into the ER, and then transported via the exocytic
pathway to the cell surface. It seems that production of a
specific viral protein in the ER stimulates transcriptional
activation of BiP.
Previous studies with hemagglutinin of influenza virus
revealed that the presence of misfolded viral proteins in the
ER signals the induction of BiP and GRP94.25 Influenza
hemagglutinin is synthesized as a monomer that is translo-
cated across the ER membrane and assembled into a trimer.
During maturation, hemagglutinin mutants which are blocked
of transport from the ER are defective in protein folding. Thus,
unlike wild-type hemagglutinin, the misfolded hemagglutinin
induces the synthesis of Bip and GRP94.26 Recent work by
Liberman et al.27 showed that ecotopic expression of the E2
protein, but not E1, core, and NS3 proteins, from hepatitis C
virus activates the promoter of GRP78/BiP. Consistent with
The PERK Pathway: Virus Modulation
Several lines of evidence have indicated a link of viral
replication to the PERK pathway.5,7,8,10,20,21 In the early
phase of ER stress, accumulation of unfolded or misfoled
protein activates PERK, which then phosphorylates eIF-2a at
serine 51. This leads to inhibition of general protein synthesis
and reduces the protein load in the ER.32 However, eIF-2a
phosphorylation also induces the expression of activating
transcription factor 4 (ATF4), a transcription factor that
stimulates the expression of C/EBP homologous protein
(CHOP), as well as growth arrest and DNA damage-inducible
protein 34 (GADD34).32 CHOP, also known as growth arrest
and DNA damage-inducible protein 153 (GADD153), is a
dominant-negative inhibitor of the CCAAT/enhancer-binding
proteins. When expressed in mammalian cells, CHOP/
GADD153 facilitates apoptosis.33 GADD34 is expressed
under conditions of DNA damage, growth arrest, and
Cell Death and Differentiation

Viruses and stress responses
B He
396
differentiation.34 The biochemical function of GADD34 was
initially identified by genetic analysis.35 When expressed in
the context of herpes simplex virus genome, GADD34
rescued protein synthesis in virus-infected cells. Further clue
as to the cellular target of GADD34 has come from the yeast-
two-hybrid screen, which identified GADD34 as a regulatory
subunit of protein phosphatase 1. Interestingly, the carboxyl-
terminus of GADD34 recruits protein phosphatase 1, forming
phosphorylation later in infection, indicating that PERK is
activated. However, there is only limited extent of
a
phosphorylation of eIF-2a, which coincides with increased
expression of ATF4. Despite phosphorylation of PERK and
eIF-2a, translation is not attenuated by cytomegalovirus
infection. This observation implies that a viral mediated
function may act downstream of eIF-2a phosphorylation.
Currently, it is not clear which gene product(s) is involved in
reducing phosphorylation of eIF-2a during the ER stress.
African swine fever virus is a DNA virus, which uses the ER as
a site for assembly and envelopment. Thus, replication of
African swine fever virus is expected to induce ER stress.
However, in virus-infected cells, African swine fever virus
does not induce PERK activation. Furthermore, African swine
fever virus is capable of blocking the expression of CHOP/
GADD153-mediated dithiothreitol, thapsigargin, and other
agents.8 It will be interesting to identify gene products that
inhibit PERK activation.
In addition, RNA viruses that employ the ER as a site for
viral replication and maturation have been shown to regulate
PERK. For example, a cytopathic strain of bovine viral
diarrhea virus, a member of flaviviruses, activates PERK
and increases eIF-2a phosphorylation.10 During peak times of
virion production, PERK phosphorylation is maximal. This
suggests the level of ER stress signaling increases as viral
gene products accumulate during infection. Accordingly,
infected cells undergo apoptosis with increased expression
of GADD153/CHOP and caspase-12. This phenotype is not
associated with the noncytopathic strain of bovine viral
diarrhea virus, which tends to cause chronic infection. Another
example is the E2 protein encoded by hepatitis C virus.40
When expressed, the E2 protein binds to PERK as a
pseudosubstrate and may sequester it from its normal
substrate eIF2a. Although a direct link between hepatitis C
virus and PERK is not known in infected cells, ecotopic
expression of the E2 protein inhibits PERK phosphorylation
and enhances translation, which is believed to contribute
to persistent hepatitis C virus infection. Additional work is
needed to test this hypothesis. Thus, differential modulation of
the PERK pathway is probably related to the biological
properties of viruses.
a
high-molecular-weight complex that dephosphorylates
eIF-2a.36,37 In uninfected cells, GADD34 is a component of
the PERK pathway that serves to relief translation repression
during ER stress.38 Thus, GADD34 controls ER stress-
induced translation inhibition as well as gene expression
under stress conditions in the ER.
The carboxyl-terminus of GADD34 is highly homologous to
the corresponding region of the g134.5 protein encoded by
herpes simplex viruses. Thus, the domain shared by the two
proteins may perform a common function. An interesting
observation came from the analysis of ER stress in cells
infected with herpes simplex virus 1.20 Herpes simplex virus-1
is a DNA virus whose gene expression is regulated in a
cascade fashion. In cells infected with herpes simplex
virus 1, PERK is activated, as seen by an increase in autophos-
phorylation of PERK over the course of virus infection.
Notably, phosphorylation of PERK is dependent on the
production of viral protein synthesis. As PERK possesses
an ER-luminal regulatory domain and a cytoplasmic kinase
domain, processing or accumulation of viral proteins in the ER
presumably facilitates the oligomerization of PERK. Although
herpes simplex virus 1 infection activates PERK, eIF2a
remains in the unphosphorylated state and viral polypeptide
synthesis is normal in infected cells. This suggests that herpes
simplex virus 1 stimulates and then disarms the activity of
PERK. Indeed, the g134.5 protein, a virulence factor encoded
by herpes simplex viruses, plays a critical role in mediating
eIF2a dephosphorylation.20,36 The expression of the g134.5
protein alleviates the translation arrest in mammalian cells
treated with dithiothreitol and thapsigargin, two compounds
that induce unfolded protein response.20 Like its cellular
homolog GADD34, the carboxyl-terminal domain, the g134.5
protein is required to recruit protein phosphatase 1 to
dephosphorylate eIF-2a and block translation shutoff during
virus infection.36 Thus, the conserved carboxyl-terminal
domain of the g134.5 protein represents a functional module.
A hypothesis derived from these analyses is that, in order to
cope with ER stress, herpes simplex virus acquired the g134.5
protein in order to antagonize the activity of PERK during
its evolution. While this suggests a potential viral mechanism
to modulate ER stress, it remains unknown whether the
g134.5 protein regulates the transcription of host genes
required for ER stress response that may affect viral virulence
in vivo.
Regulation of the ATF6 and IRE1
Pathways by Viruses
Compared to PERK, ATF6 and IRE1 are two components that
function in the late stages of the unfolded protein response
(UPR).41 ATF6 resides in the ER membrane with a cytosolic
amino-terminal domain and an ER luminal carboxyl-terminal
domain. As a result of its activation, the amino-terminal
domain of ATF6 is released by proteolysis. This portion of
ATF6 translocates to the nucleus, where it cooperates with
other proteins to form a complex that induces the expression
of genes coding for chaperones or folding enzymes. ATF6
also upregulates the expression of X-box-binding protein
(XBP1) mRNA, a substrate of IRE1.42 IRE1 is a protein
with an ER luminal amino-terminal domain, a transmembrane
domain, a serine/threonine kinase domain, and carboxyl-
terminal endonuclease domain in the cytoplasm. Under
Recent studies have shown that cytomegalovirus and
African swine fever virus also perturb the PERK path-
way.8,21,39 Cytomegalovirus is a b-herpesvirus, whose gene
expression occurs in an ordered temporal pattern. Compared
to the prototype herpes simplex virus-1, it is a slowly
replicating virus. In cells infected with cytomegalovirus, PERK
is not phosphorylated in the early phase. As viral replication
proceeds, there is an increase in the level of PERK
Cell Death and Differentiation

Viruses and stress responses
B He
397
ER stress, IRE1 oligomerizes, autophosphorylates, and
removes an intron from the XBP1 mRNA, which produces
Viruses and Apoptosis in ER Stress
When unfolded proteins continue to accumulate beyond the
capacity of the ER, apoptosis may occur. Under the ER stress,
CHOP is activated to facilitate cell death. The downstream
targets of CHOP remain unknown, but CHOP-mediated
apoptosis has been coupled to a pathway that suppresses
Bcl-2 expression, depletion of intracellular glutathionine, and
an increase of free radicals.33,45 Another pathway involves the
activity of IRE1, TRAF2, and caspase-12. When activated,
IRE1 recruits TRAF2 and c-Jun N-terminal inhibitory kinase;
thus, IRE1 transmits a signal via apoptosis signaling kinase
c-Jun N-terminal kinase. This cascade triggers caspase-12
activation and subsequent apoptosis.46–48 Several viruses
induce apoptosis mediated by ER stress.10,11,19 Infection of
Japanese encephalitis virus exhibits severe cytopathic effect,
which is associated with apoptosis, as measured by nuclear
condensation and DNA laddering. As Japanese encephalitis
virus infection alters the structure of the ER, it is not surprising
that the expression of CHOP is enhanced. Notably, the level of
CHOP induced seems to correlate with the extent of apoptosis
in infected cells. Overexpression of Bcl-2 reduces the virus
induced cell death. In addition to CHOP, Japanese encepha-
litis virus infection also activates p38 MAPK. Inhibition of
p38 MAPK activity alleviates apoptosis induced by Japanese
encephalitis virus. Similarly, a cytopathic strain of bovine
diarrhea virus induces apoptosis by stimulating CHOP
activation, whereas the virus infection causes phosphoryla-
tion of PERK and eIF-2a. In cells undergoing apoptosis, the
levels of Bcl-2 and glutathione are reduced. Collectively, these
observations suggest that virus infection activates the p38
MAPK, which then acts on CHOP to initiate the death signal in
infected cells.
Recent analysis suggests that Tula virus infection activates
the JNK pathway.19 Notably, Tula virus infection leads to
apparent phosphorylation of JNK1, and JNK2 to a lesser
extent. As viral infection proceeds, both phosphorylated and
nonphosphorylated c-Jun is dramatically increased. Further-
more, addition of JNK inhibitor II reduced the cleavage of
PARP in infected cells. Thus, activation of the JNK pathway
may contribute to the cell death mediated by Tula virus. In
addition, cleavage of BAP31, a proapoptotic protein, corre-
lates well with the activation of caspase-8. Virus infection
also activates capsase-12. This activation is a late event in
response to accumulation of misfolded protein in the ER,
which can be blocked by the inhibitor z-VAD-fmk. Apoptosis
induced by a cytopathic strain of bovine diarrhea virus
coincides with caspase-12 activation. Further, infection with
respiratory syncytial virus also activates caspase-12, and
inhibition of caspase-12 by antisense oligonucleotides mark-
edly suppresses apoptosis induced by respiratory syncytial
virus. In fact, in cells infected with Tula virus, caspase-12,
caspase-8, and caspase-3 are all activated. The kinetics of
caspase-12 activation is earlier than that of caspase-8 and
caspase-3, suggesting that caspase-12 might be an initiator
caspase required for transduction of the death signal from the
ER in infected cells. It is unknown how viruses affect the
upstream modulators leading to caspase-12 activation.
Although virus-mediated apoptosis in ER stress has been
recognized, the biological significance of such a process is not
a
transcription factor that activates target genes, for
example, ER degradation-enhancing a-mannoside-like
protein (EDEM), which facilitates the degradation of misfolded
proteins.
Replication of hepatitis C virus has been shown to stimulate
the ATF6 pathway, but suppress the IRE1–XBP1 path-
way.29,43 In cells containing hepatitis C virus replicons,
subgenomic replication promotes the cleavage of ATF6,
producing a 50-kDa fragment that corresponds to the
amino-terminal domain of ATF6. In correlation, there is an
increased transcriptional level of BiP, an ER luminal chaper-
one. As the ribonucleoprotein complex of hepatitis C virus
is associated with the ER membrane, viral replication may
stimulate expression of BiP/GRP78. As hepatitis C virus
replicons only express the structural proteins, it is not clear
which nonstructural protein is involved in the induction of
ATF6. Recent experiments suggest that subgenomic replica-
tion of hepatitis C virus reduces properly folded major
histocompatibility complex class I, which is attributed to a
decline in protein glycosylation.44 It is possible that unfolded
MHC class I may account for the activation of ATF6. With
respect to regulation of XPB1 by ATF6, it is notable that
hepatitis C virus replicons stimulate accumulation of more
unspliced XBP1 mRNA as compared to cells without hepatitis
C virus replicons. Spliced XBP1 mRNA is also detected
in cells containing hepatitis C virus replicons. Surprisingly,
transactivating activity of XBP1 is inhibited in cells with
hepatitis C virus replicons as measured by reporter assays. In
parallel, ER-associated protein degradation is reduced in cells
carrying hepatitis C virus replicons. A model to reconcile these
findings is that hepatitis C virus encodes a function to block the
effect of IRE1–XBP pathway, which enhances the translation
of viral proteins.
In view of ER stress mediated by ATF6 and IRE1, a different
pattern is observed in cells infected with cytomegalovirus.21
Infection of cytomegalovirus causes a transient increase in
BiP levels at the early phase of viral replication, but BiP
returns to basal levels at the later stage. This coincides
with the appearance of other markers of UPR. In the
early phase of infection, the increased BiP may inhibit the
ER stress response by interacting with PERK, ATF6, and
IRE1. Thus, cytomegalovirus appears to control the level
of BiP that regulates the onset of ER stress. In this regard,
the Us11 gene product physically interacts with BiP.39
The expression of cytomegalovirus Us11 in mammalian
cells is sufficient to trigger UPR, as manifested by up-
regulation of Bip and production of spliced XBP-1 mRNA.
This response is dependent on its interaction with
a
cellular protein Derlin-1 although the underlying mechanism
is not known. Surprisingly, cytomegalovirus infection
does not result in the proteolytic cleavage of ATF6. The
full-length ATF6 is present throughout viral replication.
Nevertheless, there is a limited expression of XBP1 mRNA
in virus-infected cells. It seems that cytomegalovirus
stimulates splicing of XBP1 in the later stages of infection
as measured by RT-PCR analysis. Yet, transcriptional
activation of the XBP1 target gene is inhibited in virus-infected
cells.
Cell Death and Differentiation

Viruses and stress responses
B He
398
well defined. Simian virus 5 infection enhances the expression
of BiP and GRP94. However, this does not trigger apoptosis.7
A mutant simian virus 5 with a truncation of the V protein
induces cell death mediated by ER stress. Accordingly, the
expression of CHOP is drastically increased and caspase-12
is activated. In a mouse model, simian virus 5 replicates
more efficiently as compared to the V deletion mutant. This
experimental model suggests that it is beneficial for simian
virus 5 to prevent host cells from undergoing apoptosis
mediated by ER stress. It should be stressed that the situation
is more complex in other virus systems. For instance, it is
generally less clear why some viruses promote ER-mediated
apoptosis. It has been reported that murine retroviruses cause
a spongiform neurodegenerative disease, which is deter-
mined by properties of viral envelope proteins.6 Infection of
cells with the virulent strain FrCasE stimulates the expression
of BiP and CHOP, whereas infection with an avirulent strain
F43 has no effect. Importantly, the envelope protein from F43
binds to BiP transiently and is processed normally through
the secretory pathway. However, the envelope protein from
FrCasE binds to BiP for a prolonged period and is degraded by
the proteosome. Thus, the virulent strain may cause ER
stress that mediates microglial cell death and consequently
results in neuronal degeneration. These studies suggest that
ER-mediated apoptosis induced by virus may be related to the
pathogenesis of viral infection.
threonine residues. These series of events convert PKR
into an active enzyme, which is capable of catalyzing the
phosphorylation of a number of substrates, including eIF-2a at
serine 51. Phosphorylation of eIF-2a increases its affinity for
guanine nucleotide exchange factor eIF-2B, thus sequester-
ing eIF-2B in an inactive complex with phosphorylated eIF-2
and GDP. Consequently, eIF-2B is not available to catalyze
nucleotide exchange on nonphosphorylated eIF-2, leading to
the shutoff of protein synthesis.
PKR is a multifunctional protein that also regulates
apoptosis, cell proliferation, signal transduction, and differ-
entiation.3 Overexpression of PKR has been suggested to
inhibit cell proliferation in yeast, insect, and mammalian
cells.51–53 Several studies showed that the expression of
PKR mediates apoptosis.54,55 In contrast, the expression of
catalytically inactive mutants of PKR in NIH3T3 cells results
in tumorigenicity in nude mice, which is attributed to a
dominant-negative effect of mutant PKR.53 Mouse embryo
fibroblasts derived from PKRÀ/À mice are resistant to cell
death induced by dsRNA and lipopolysaccharide.56 PKR has
been suggested to be involved in dsRNA signal transduction
pathways leading to NF-kB activation and the p38 mitogen-
acitvated kinase pathway.57,58 In PKRÀ/À, but not wild-type
cells, dsRNA fails to induce NF-kB activation, which corre-
lates with the lack of interferon-b production.
In addition to dsRNA, PKR activity is positively regulated by
a cellular protein called PKR-activating protein (PACT)/
RAX.59,60 This protein was identified as a PKR interacting
protein by the yeast two-hybrid screen. PACT/RAX hetrodi-
merizes with PKR and activates it in the absence of dsRNA
in vitro. PACT/RAX is expressed in most cell types. In
mammalian cells treated with arsenite, thapsigargin, hydro-
gen peroxide, and interleukin-3 deprivation, PACT/RAX is
rapidly phosphorylated and associates with PKR, which is
followed by activation of PKR and eIF-2a phosphorylation. In
this respect, it is notable that PKR is activated in mammalian
cells in response to treatment with tunicamycin or thagasga-
gine, which causes ER stress by inhibiting protein folding.4,5
Therefore, PACT/RAX may be a stress-mediated activator of
PKR.
Studies have pointed to P58IPK as a tetratricopeptide repeat
domain protein that negatively regulates both PKR and
PERK.61–64 P58IPK was originally characterized as an
influenza virus-activated protein that interacts with the kinase
domain of PKR and inhibits its activity. In normal cells, P58IPK
associates with a heat shock protein 40 and forms an
inhibitory complex. Cellular stress or virus infection induces
dissociation of P58IPK from heat shock protein 40. Therefore,
the released P58IPK can bind to PKR and disrupt its activity.
In response to serum starvation or heat shock, P58IPK also
interacts with P52rIPK, a protein homologous to heat shock
protein 90.65 The response of P58IPK activation to both
influenza virus infection and cellular stress suggests that it is a
multifunction protein. Recent work by Katze et al.62 showed
that P58IPK interacts with PERK and inhibits its activity.
Overexpression of P58IPK reduces eIF-2a phosphorylation
mediated by PERK in mouse embryonic stem cells. On the
other hand, deletion of P58IPK increases eIF-2a phosphoryla-
tion and induction of CHOP and BiP. Moreover, P58IPK
expression is induced by tunicamycin as well. This is
ER Stress and Interferon Responses:
The eIF2a Connection
Unfolded proteins stimulate ER stress pathways, whereas
dsRNA produced by viruses triggers the interferon pathway.
These stress-responsive pathways converge at the a subunit
of translation initiation factor 2, which is essential for protein
synthesis. To date, four different eIF2a kinases have been
identified.49,50 These are the heme-regulated inhibitor (HRI),
the homolog of Saccharomyces cerevisae protein kinase
GCN2, PKR, and PERK (also known as pancreatic eIF2a
kinase PEK). These kinases regulate the phosphorylation
state of eIF-2a in response to heme deficiency, amino-acid
starvation, dsRNA, and misfolded protein, respectively.
Among these kinases, PKR as well as PERK are activated
by virus infection. Notably, PKR is a cytosolic as well as
nuclear protein, which acts as an intracellular receptor for
dsRNA produced by viral replication. In contrast, PERK is an
ER-resident membrane protein that transmits ER stress
signal. Thus, PKR and PERK may coordinate to control viral
replication.
PKR is a 68 kDa protein that is subjected to two levels of
regulation.2 First, it is induced by interferon. In normal cells,
PKR is present at a low level and remains inactive. In the
presence of interferon, the expression of PKR is elevated.
Secondly, PKR is activated by dsRNA. Biochemical char-
acterization suggested that PKR contains two copies of
dsRNA-binding domains in the amino-terminus and a serine/
threonine kinase domain in the carboxyl-terminus. PKR binds
dsRNA with high affinity and is activated by low concentra-
tions of dsRNA. Upon binding of dsRNA, PKR forms a
homodimer and autophosphorylates on multiple serine/
Cell Death and Differentiation

Viruses and stress responses
B He
399
consistent with the observation that the P58IPK promoter
bears an element that is commonly found in the promoter
region of other genes induced by UPR. Thus, P58IPK is also a
component of the system that regulates unfolded protein
response.
herpes simplex virus, cytomegalovirus, hepatitis C virus,
vaccinia virus, influenza virus, and perhaps papillommavirus.
Herpes simplex viruses are inherently resistant to interferon
in infected cells. Among herpes simplex virus genes that
interfere with interferon responses, the g134.5 protein is well
characterized. In cell culture, herpes simplex virus infection
leads to activation of PKR, but only infection of the g134.5 null
mutant causes phosphorylation of eIF-2a, and subsequently
attenuation of protein translation.66 A unique feature of herpes
simplex virus is that during virus infection, the g134.5 is
expressed to recruit cellular protein phosphatase 1, forming
As mentioned earlier, GADD34 regulates the phosphoryla-
tion state eIF-2a, which is a physiological substrate for PKR
and PERK. Expression of GADD34 is dependent on phos-
phorylation of eIF-2a, which stimulates translation of tran-
scription factor ATF4.32 When induced, GADD34 recruits
cellular protein phosphatase 1 to mediate eIF-2a phosphory-
lation. Thus, it is a control point of a negative-feedback loop
that terminates the signals from PERK. In general, PKR and
PERK are activated in response to different stimuli. However,
a crosstalk exists between the PKR and PERK pathways.5 In
response to vesicular stomatitis virus infection, phosphoryla-
tion of PKR is diminished in PERKÀ/À mouse fibroblasts
(MEF) as compared to wild-type MEFs. In correlation,
vesicular stomatitis virus replicates very efficiently in
PERKÀ/À MEFs. This result suggests that PERK plays a
role in controlling virus infection. Importantly, tunicamycin
induces phosphorylation of PKR in PERK þ / þ , but less so in
PERKÀ/À MEFs. Therefore, the antiviral action of PERK
is mediated by PKR. The mechanism by which vesicular
stomatitis virus activates PERK has not been elucidated. One
interesting issue is how PERK regulates PKR. It should be
pointed out that PKR may be considered as a member of the
ER stress response system. In support of this notion is the
recent finding that PKR is involved in ER-stress-mediated
apoptosis. In neuroblastoma cells treated with tunicamycin,
levels of phosphorylated PKR is increased in the nucleus.4
Together, these observations imply that coordinated interac-
tion of PERK and PKR plays a critical role in regulating ER
stress or interferon responses during viral replication.
a
high-molecular-weight complex that dephosphorylates
eIF-2a.36,67 Accordingly, the g134.5 protein-mediated eIF-2a
dephosphorylation contributes to viral resistance to the
antiviral effect of interferon-a/b.68 This function maps to the
carboxyl-terminal domain that is homologous to the corre-
sponding domain of GADD34, which functionally substitutes
for the g134.5 protein in the context of herpes simplex virus
genome.35 Whether GADD34 has a role in interferon
response in uninfected cells has not been established.
Nevertheless, herpes simplex virus infection stimulates
transient expression of GADD34. Given the role of GADD34
in ER stress, it is interesting that herpes simplex virus infection
activates PERK and mediates eIF-2a dephosphorylation by
the g134.5 protein.20 These results support the hypothesis
that, during the evolution, the g134.5 protein is adapted from
host cells to cope with ER stress and interferon responses. In
this context, it is noteworthy that, in mammalian cells, the E6
protein of papillomavirus associates with the GADD34–PP1
complex and facilitates eIF-2a dephosphorylation induced by
the activation of PKR.69 Thus, a strategy similar to that used
by herpes simplex virus may be employed by papillomavirus.
Additional studies will be required to determine whether this
mechanism does indeed operate under the conditions of viral
infection.
Herpes simplex virus 1 also encodes the Us11 protein that
binds to and inhibits PKR.70 The carboxyl-terminus of the
Us11 protein contains an RNA-binding motif that prevents
PKR activation. This portion of the Us11 protein also inhibits
the activation of PKR by PACT/RAX in vitro.71 It is not known
whether herpes simplex virus infection regulates PACT/RAX,
which is capable of activating PKR. In virus-infected cells,
Us11 is expressed at the late phase of virus life cycle. When
its expression is shifted from a late to early kinetics, it prevents
the shutoff of protein synthesis in virus-infected cells,
suggesting that herpes simplex virus may have an additional
way to block the antiviral action of PKR.72 It has been
suggested that the Us11 protein functions to inhibit PKR in the
late stage of infection and thereby complements the activity of
the g134.5 protein.70 Further work is required to understand
the precise role of Us11 in herpes simplex virus infection.
Another virus that regulates the activity of PKR and
probably ER stress is vaccinia virus. Vaccinia virus encodes
two gene products, E3L and K3L, both of which confer viral
resistance to interferon.73–75 The E3L protein, synthesized
early during viral infection, contains an amino-terminal Z-
DNA-binding domain and a carboxyl-terminal domain with a
typical dsRNA-binding motif.74 The carboxyl-terminus of E3L
sequesters dsRNA and prevents the activation of PKR and
phosphorylation of eIF-2a. In addition, E3L prevents the
Virus Inhibition of the Interferon
Response Mediated by PKR
It is well established that PKR plays a critical role in the
antiviral action of interferon. As viruses synthesize double-
stranded RNA during their replication, the interferon system
will be activated upon virus infection. This is apparently
detrimental to virus replication in the host cells. To survive,
viruses have evolved a variety of mechanisms to disarm the
interferon system.1,2 This includes inhibition of PKR activa-
tion, the prevention of eIF-2a phosphorylation, or the
degradation of PKR. For example, adenovirus VAI RNA,
vaccinia virus K3L protein, hepatitis C NS5A protein, and
influenza virus-induced p58 protein interact with PKR and
block its activation. The herpes simplex virus g134.5 protein
directs the cellular protein phosphatase 1 to dephosphorylate
eIF-2a, whereas poliovirus employs a cellular proteinase to
degrade PKR. In addition, viruses employ countermeasures
to inhibit interferon production or signal transduction initiated
by interferons.
It is apparent that while suppressing the interferon response
mediated by PKR, viruses also inhibit the ER stress regulated
by PERK. Such viral strategies have been suggested to
operate in some DNA as well as RNA viruses. These include
Cell Death and Differentiation

Viruses and stress responses
B He
400
activation of 20–50 oligoadenylate synthetase. E3L deletion
mutant is highly sensitive to interferon. In cell culture, the
carboxyl-terminus, but not amino-terminus of E3L, is required
for viral infection. However, the full-length of E3L is essential
for viral pathogenesis in mice.76 The K3L protein has
homology to eIF2a and acts as a pseudosubstrate for PKR
in competition with eIF2a, consequently, suppressing phos-
phorylation of eIF2a and the shutoff of protein synthesis.77
Studies suggest that E3L and K3L may determine viral host
range in infected cells.75 The E3L gene is required for viral
replication in HeLa cells, but not in BHK cells. On the other
hand, K3L gene is required for viral replication in BHK cells,
but is dispensable for viral replication in HeLa cells. The
biological basis for this is not fully understood. Interestingly, in
a heterologous system, K3L also binds to PERK and inhibits
its activation. This raises the possibility that K3L interferes
with the ER stress response.78 Consistent with this notion,
vaccinia virus infection does not lead to phosphorylation of
PERK.79 Recent studies with cytomegalovirus revealed that
TRS1 and closely related IRS1 can each rescue a vaccinia
mutant that has deletion of the E3L gene. TRS1 is an RNA-
binding protein that may function like E3L of vaccinia virus.
Thus, cytomegalovirus has two genes that function in blocking
the PKR-mediated antiviral pathway.80
The NS5A protein of hepatitis C virus is a phosphoprotein
that interacts with PKR and inhibits its activation.81 The
involvement of NS5A in hepatitis C virus resistance to
interferon is initially suggested by clinical studies with hepatitis
C virus 1b subtype from a Japanese isolate.82 Notably, a
cluster of amino-acid mutations within a discrete region in
NS5A seems to correlate with increased resistance of HCV-
1b to interferon. This region, termed the interferon-sensitivity
determining region (ISDR), is thought to mediate viral
resistance to interferon therapy.83 In supporting this
observation, NS5A was found to bind to and inactive
PKR in vitro. Furthermore, mutations in the ISDR region
disrupted the interaction between NS5A and PKR.81,84,85
Paradoxically, the involvement of ISDR is less clear based
on clinical studies from Europe and North America.86,87 It is
clear that the clinical response to interferon therapy is a
complex process that likely involves multiple viral as
well as host factors. Nonetheless, expression of NS5A in a
number of cell lines reduces the antiviral effect of interferon on
replication of vesicular stomatitis virus or encephalomyocar-
ditis virus.85 The mechanism by which NS5A works remains
controversial. This may partly result from the fact that NS5A
regulates a number of cellular pathways.88 Recent studies
Finally, it appears that influenza A virus has evolved
mechanisms to cope with PKR and PERK. Influenza A virus
is a negative-stranded RNA virus that possesses a segmen-
ted genome. Of 10 proteins encoded by influenza virus, the
NS1 protein functions as an inhibitor of interferon responses
during cellular infection. The NS1 protein is an RNA-binding
protein, which prevents the activation of PKR by dsRNA.91,92
The ability of dsRNA to stimulate phosphorylation of
PKR is abrogated in the presence of the NS1 protein in vitro
and dimerization of the NS1 protein is essential for its
function. However, it has been reported that the NS1 protein
directly binds to PKR and inhibits its activation.93 Whether
binding of dsRNA is required for NS1 to interact with
PKR is not yet resolved. Genetic analysis suggests that
interaction of NS1 and PKR plays a critical role in replication
of influenza A virus.94 Particularly, a recombinant virus
lacking NS1 exhibits a defective viral growth in interferon-
producing cells, but not in interferon-deficient cells. Thus,
unlike wild-type influenza virus, infection with the NS1 deletion
mutant results in hyperphosphorylation of PKR. Accordingly,
similar to wild-type influenza A virus, the NS1 deletion mutant
replicates efficiently in PKRÀ/À mice. In contrast, the NS1
deletion mutant fails to grow in PKR þ / þ mice.95 Studies also
showed that influenza virus infection leads to the activation
of a cellular protein p58IPK, which binds to and inhibits PKR
activity. These experimental results suggest that influenza A
virus has an additional way of inhibiting PKR activity.63,64 As
p58IPK is induced by ER stress, it is interesting that p58IPK also
interacts with PERK and blocks its activity.62 However, the
possible connection between influenza virus and PERK in
infected cells remains to be established. Available evidence is
consistent with the hypothesis that influenza A virus regulates
both the ER stress and interferon responses during infection.
Such viral strategies would ensure suitable environment for
virus infection.
Conclusions
Virus regulation of cellular responses is a critical step in
determining the consequences of infection. Obviously,
viruses face interferon responses mediated by PKR,
which is subjected to inhibition by many viruses. Moreover,
viruses encounter UPR mediated by one master control
protein BiP and three sensors, PERK, ATF6. and IRE1. These
components respond to viral signals emanating from the
ER during viral infection. Certainly, viruses have different
mechanisms to modulate this response. This complex
regulation by viruses is probably evolved to either optimize
viral replication or regulate the pathological process. Despite
some evidence, it still remains less clear how viruses interact
with each component of UPR over the course of replication.
Available data suggest that PERK plays a role in limiting viral
replication. In this respect, there is a crosstalk between the
PERK pathway and the PKR pathway. Emerging evidence
also suggests that PKR may have a role in ER stress
response. The fact that viruses have developed ways to
subdue both the ER stress and interferon responses suggests
that these cellular pathways are crucial in controlling viral
infection. Further investigation on the molecular interaction
suggest that replication of hepatitis
C virus replicons
corresponds with the ability of NS5A to block the activation
of PKR and interferon regulatory factor 1.89 Mutations in the
PKR-binding domain of NS5A lead to the induction of
regulatory factor 1-dependent antiviral genes and concomi-
tant reduction in efficiency of viral RNA replication, suggesting
that NS5A may contribute to viral persistence. Intriguingly, the
glycoprotein E2 of hepatitis C virus has been reported to
interact with PKR and PERK, inhibiting the activities of the
two kinases.40,90 While the role of the E2 protein in viral
persistence remains to be established, it is possible that
hepatitis C virus encodes multiple functions to regulate the ER
stress and interferon responses.
Cell Death and Differentiation

Viruses and stress responses
B He
401
between viruses, the PKR pathway, and the ER stress may
yield important information.
19. Li XD, Lankinen H, Putkuri N, Vapalahti O and Vaheri A (2005) Tula hantavirus
triggers pro-apoptotic signals of ER stress in Vero E6 cells. Virology 333:
180–189
20. Cheng G, Feng Z and He B (2005) Herpes simplex virus 1 infection activates
the endoplasmic reticulum resident kinase PERK and mediates eIF-2a
dephosphorylation by the g134.5 protein. J. Virol. 79: 1379–1388
21. Isler JA, Skalet AH and Alwine JC (2005) Human cytomegalovirus infection
activates and regulates the unfolded protein response. J. Virol. 79: 6890–6899
22. Morris JA, Dorner AJ, Edwards CA, Hendershot LM and Kaufman RJ (1997)
Immunoglobulin binding protein (BiP) function is required to protect cells from
endoplasmic reticulum stress but is not required for the secretion of selective
proteins. J. Biol. Chem. 272: 4327–4334
Acknowledgements
The work in this laboratory was supported by grant AI 46665 (BH) from
the National Institute of Allergy and Infectious Diseases. I thank Dustin
Verpooten for critical reading of the manuscript.
23. Peluso RW, Lamb RA and Choppin PW (1978) Infection with paramyxoviruses
stimulates synthesis of cellular polypeptides that are also stimulated in cells
transformed by Rous sarcoma virus or deprived of glucose. Proc. Natl. Acad.
Sci. USA 75: 6120–6124
24. Watowich SS, Morimoto RI and Lamb RA (1991) Flux of the paramyxovirus
hemagglutinin-neuraminidase glycoprotein through the endoplasmic reticulum
activates transcription of the GRP78-BiP gene. J. Virol. 65: 3590–3597
25. Hurtley SM, Bole DG, Hoover-Litty H, Helenius A and Copeland CS (1989)
Interactions of misfolded influenza virus hemagglutinin with binding protein
(BiP). J. Cell. Biol. 108: 2117–2126
References
1. Stark GR, Kerr IM, Williams BR, Silverman RH and Schreiber RD (1998) How
cells respond to interferons. Annu. Rev. Biochem. 67: 227–264
2. Gale Jr M and Katze MG (1998) Molecular mechanisms of interferon resistance
mediated by viral-directed inhibition of PKR, the interferon-induced protein
kinase. Pharmacol. Ther. 78: 29–46
3. Proud CG (1995) PKR: a new name and new roles. Trends Biochem. Sci. 20:
241–246
26. Kozutsumi Y, Segal M, Normington K, Gething MJ and Sambrook J (1988) The
presence of malfolded proteins in the endoplasmic reticulum signals the
induction of glucose-regulated proteins. Nature 332: 462–464
27. Liberman E, Fong YL, Selby MJ, Choo QL, Cousens L, Houghton M and Yen
TS (1999) Activation of the grp78 and grp94 promoters by hepatitis C virus E2
envelope protein. J. Virol. 73: 3718–3722
28. Choukhi A, Ung S, Wychowski C and Dubuisson J (1998) Involvement of
endoplasmic reticulum chaperones in the folding of hepatitis C virus
glycoproteins. J. Virol. 72: 3851–3858
29. Tardif KD, Mori K and Siddiqui A (2002) Hepatitis C virus subgenomic replicons
induce endoplasmic reticulum stress activating an intracellular signaling
pathway. J. Virol. 76: 7453–7459
4. Onuki R, Bando Y, Suyama E, Katayama T, Kawasaki T, Baba T, Tohyama M
and Taira K (2004) An RNA-dependent protein kinase is invovled in
tunicamycin-induced apoptosis and Alzheimer’s disease. EMBO J. 25:
959–968
5. Baltzis D, Qu LK, Papadopoulou S, Blais JD, Bell JC, Sonenberg N and
Koromilas AE (2004) Resistance to vesicular stomatitis virus infection requires
a functional cross talk between the eukaryotic translation initiation factor 2alpha
kinases PERK and PKR. J. Virol. 78: 12747–12761
6. Dimcheff DE, Faasse MA, McAtee FJ and Portis JL (2004) Endoplasmic
reticulum (ER) stress induced by a neurovirulent mouse retrovirus is associated
with prolonged BiP binding and retention of a viral protein in the ER. J. Biol.
Chem. 279: 33782–33790
30. Ng DT, Randall RE and Lamb RA (1989) Intracellular maturation and transport
of the SV5 type II glycoprotein hemagglutinin-neuraminidase: specific and
transient association with GRP78-BiP in the endoplasmic reticulum and
extensive internalization from the cell surface. J. Cell. Biol. 109: 3273–3289
31. Machamer CE, Doms RW, Bole DG, Helenius A and Rose JK (1990) Heavy
chain binding protein recognizes incompletely disulfide-bonded forms of
vesicular stomatitis virus G protein. J. Biol. Chem. 265: 6879–6883
32. Novoa I, Zhang Y, Zeng H, Jungreis R, Harding HP and Ron D (2003)
Stress-induced gene expression requires programmed recovery from
translational repression. EMBO J. 22: 1180–1187
7. Sun M, Rothermel TA, Shuman L, Aligo JA, Xu S, Lin Y, Lamb RA and He B
(2004) Conserved cysteine-rich domain of paramyxovirus simian virus 5 V
protein plays an important role in blocking apoptosis. J. Virol. 78: 5068–5078
8. Netherton CL, Parsley JC and Wileman T (2004) African swine fever virus
inhibits induction of the stress-induced proapoptotic transcription factor CHOP/
GADD153. J. Virol. 78: 10825–10828
9. Tardif KD, Waris G and Siddiqui A (2005) Hepatitis C virus, ER stress, and
oxidative stress. Trends Microbiol. 13: 159–163
10. Jordan R, Wang L, Graczyk TM, Block TM and Romano PR (2002) Replication
of a cytopathic strain of bovine viral diarrhea virus activates PERK and induces
endoplasmic reticulum stress-mediated apoptosis of MDBK cells. J. Virol. 76:
9588–9599
11. Su HL, Liao CL and Lin YL (2002) Japanese encephalitis virus infection initiates
endoplasmic reticulum stress and an unfolded protein response. J. Virol. 76:
4162–4171
12. Dorner AJ, Wasley LC and Kaufman RJ (1992) Overexpression of GRP78
mitigates stress induction of glucose regulated proteins and blocks secretion of
selective proteins in Chinese hamster ovary cells. EMBO J. 11: 1563–1571
13. Bertolotti A, Zhang Y, Hendershot LM, Harding HP and Ron D (2000) Dynamic
interaction of BiP and ER stress transducers in the unfolded-protein response.
Nat. Cell Biol. 2: 326–332
14. Shen J, Chen X, Hendershot L and Prywes R (2002) ER stress regulation of
ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of
Golgi localization signals. Dev. Cell 3: 99–111
15. Zhang K and Kaufman RJ (2004) Signaling the unfolded protein response from
the endoplasmic reticulum. J. Biol. Chem. 279: 25935–25938
16. Liu CY, Xu Z and Kaufman RJ (2003) Structure and intermolecular interactions
of the luminal dimerization domain of human IRE1alpha. J. Biol. Chem. 278:
17680–17687
33. Zinszner H, Kuroda M, Wang X, Batchvarova N, Lightfoot RT, Remotti H,
Stevens JL and Ron D (1998) CHOP is implicated in programmed cell death in
response to impaired function of the endoplasmic reticulum. Genes Dev. 12:
982–995
34. Zhan Q, Lord KA, Alamo Jr. I, Hollander MC, Carrier F, Ron D, Kohn KW,
Hoffman B, Liebermann DA and Fornace Jr. AJ (1994) The gadd and MyD
genes define a novel set of mammalian genes encoding acidic proteins that
synergistically suppress cell growth. Mol. Cell. Biol. 14: 2361–2371
35. He B, Chou J, Liebermann DA, Hoffman B and Roizman B (1996) The carboxyl
terminus of the murine MyD116 gene substitutes for the corresponding domain
of the g134.5 gene of herpes simplex virus to preclude the premature shutoff of
total protein synthesis in infected human cells. J. Virol. 70: 84–90
36. He B, Gross M and Roizman B (1997) The g134.5 protein of herpes simplex
virus 1 complexes with protein phosphatase 1a to dephosphorylate the alpha
subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff
of protein synthesis by double-stranded RNA-activated protein kinase. Proc.
Natl. Acad. Sci. USA 94: 843–848
37. Boyce M, Bryant KF, Jousse C, Long K, Harding HP, Scheuner D, Kaufman RJ,
Ma D, Coen DM, Ron D and Yuan J (2005) A selective inhibitor of eIF2alpha
dephosphorylation protects cells from ER stress. Science 307: 935–939
38. Novoa I, Zeng H, Harding HP and Ron D (2001) Feedback inhibition of the
unfolded protein response by GADD34-mediated dephosphorylation of
eIF2alpha. J. Cell. Biol. 153: 1011–1022
17. Harding HP, Calfon M, Urano F, Novoa I and Ron D (2002) Transcriptional and
translational control in the mammalian unfolded protein response. Annu. Rev.
Cell. Dev. Biol. 18: 575–599
18. Bitko V and Barik S (2001) An endoplasmic reticulum-specific stress-activated
caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by
respiratory syncytial virus. J. Cell. Biochem. 80: 441–454
39. Tirosh B, Iwakoshi NN, Lilley BN, Lee AH, Glimcher LH and Ploegh HL (2005)
Human cytomegalovirus protein US11 provokes an unfolded protein response
Cell Death and Differentiation

Viruses and stress responses
B He
402
that may facilitate the degradation of class I major histocompatibility complex
products. J. Virol. 79: 2768–2779
dependent protein kinase, PKR, to regulate its autophosphorylation and
activity. J. Biol. Chem. 271: 1702–1707
40. Pavio N, Romano PR, Graczyk TM, Feinstone SM and Taylor DR (2003)
Protein synthesis and endoplasmic reticulum stress can be modulated by the
hepatitis C virus envelope protein E2 through the eukaryotic initiation factor
2alpha kinase PERK. J. Virol. 77: 3578–3585
62. Yan W, Frank CL, Korth MJ, Sopher BL, Novoa I, Ron D and Katze MG (2002)
Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum
stress-induced molecular chaperone P58IPK. Proc. Natl. Acad. Sci. USA 99:
15920–15925
41. Yoshida H, Matsui T, Hosokawa N, Kaufman RJ, Nagata K and Mori K (2003) A
time-dependent phase shift in the mammalian unfolded protein response. Dev.
Cell. 4: 265–271
42. Yoshida H, Matsui T, Yamamoto A, Okada T and Mori K (2001) XBP1 mRNA is
induced by ATF6 and spliced by IRE1 in response to ER stress to produce a
highly active transcription factor. Cell 107: 881–891
43. Tardif KD, Mori K, Kaufman RJ and Siddiqui A (2004) Hepatitis C virus
suppresses the IRE1–XBP1 pathway of the unfolded protein response. J. Biol.
Chem. 279: 17158–17164
63. Lee TG, Tomita J, Hovanessian AG and Katze MG (1990) Purification and
partial characterization of a cellular inhibitor of the interferon-induced protein
kinase of Mr 68,000 from influenza virus-infected cells. Proc. Natl. Acad. Sci.
USA 87: 6208–6212
64. Lee TG, Tomita J, Hovanessian AG and Katze MG (1992) Characterization and
regulation of the 58 000-dalton cellular inhibitor of the interferon-induced,
dsRNA-activated protein kinase. J. Biol. Chem. 267: 14238–14243
65. Melville MW, Tan SL, Wambach M, Song J, Morimoto RI and Katze MG (1999)
The cellular inhibitor of the PKR protein kinase, P58(IPK), is an influenza virus-
activated co-chaperone that modulates heat shock protein 70 activity. J. Biol.
Chem. 274: 3797–3803
66. Chou J, Chen JJ, Gross M and Roizman B (1995) Association of a M(r) 90 000
phosphoprotein with protein kinase PKR in cells exhibiting enhanced
phosphorylation of translation initiation factor eIF-2a and premature shutoff
of protein synthesis after infection with g134.5- mutants of herpes simplex virus
1. Proc. Natl. Acad. Sci. USA 92: 10516–10520
44. Tardif KD and Siddiqui
A (2003) Cell surface expression of major
histocompatibility complex class I molecules is reduced in hepatitis C virus
subgenomic replicon-expressing cells. J. Virol. 77: 11644–11650
45. McCullough KD, Martindale JL, Klotz LO, Aw TY and Holbrook NJ
(2001) Gadd153 sensitizes cells to endoplasmic reticulum stress by down-
regulating Bcl2 and perturbing the cellular redox state. Mol. Cell. Biol. 21:
1249–1259
46. Yoneda T, Imaizumi K, Oono K, Yui D, Gomi F, Katayama T and Tohyama M
(2001) Activation of caspase-12, an endoplastic reticulum (ER) resident
caspase, through tumor necrosis factor receptor-associated factor 2-dependent
mechanism in response to the ER stress. J. Biol. Chem. 276: 13935–13940
47. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA and Yuan J (2000)
Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and
cytotoxicity by amyloid-beta. Nature 403: 98–103
67. He B, Gross M and Roizman B (1998) The g134.5 protein of herpes simplex
virus 1 has the structural and functional attributes of a protein phosphatase 1
regulatory subunit and is present in a high molecular weight complex with the
enzyme in infected cells. J. Biol. Chem. 273: 20737–20743
68. Cheng G, Brett ME and He B (2001) Val193 and Phe195 of the g134.5 protein of
herpes simplex virus 1 are required for viral resistance to interferona/b. Virology
290: 115–120
48. Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP and Ron D
(2000) Coupling of stress in the ER to activation of JNK protein kinases by
transmembrane protein kinase IRE1. Science 287: 664–666
49. Ron D (2002) Translational control in the endoplasmic reticulum stress
response. J. Clin. Invest. 110: 1383–1388
50. Wek RC (1994) eIF-2 kinases: regulators of general and gene-specific
translation initiation. Trends Biochem. Sci. 19: 491–496
51. Chong KL, Feng L, Schappert K, Meurs E, Donahue TF, Friesen JD,
Hovanessian AG and Williams BR (1992) Human p68 kinase exhibits
growth suppression in yeast and homology to the translational regulator GCN2.
EMBO J. 11: 1553–1562
69. Kazemi S, Papadopoulou S, Li S, Su Q, Wang S, Yoshimura A, Matlashewski
G, Dever TE and Koromilas AE (2004) Control of alpha subunit of eukaryotic
translation initiation factor 2 (eIF2 alpha) phosphorylation by the human
papillomavirus type 18 E6 oncoprotein: implications for eIF2 alpha-dependent
gene expression and cell death. Mol. Cell. Biol. 24: 3415–3429
70. Mulvey M, Camarena V and Mohr I (2004) Full resistance of herpes simplex
virus type 1-infected primary human cells to alpha interferon requires both the
Us11 and g134.5 gene products. J. Virol. 78: 10193–10196
71. Peters GA, Hartmann R, Qin J and Sen GC (2001) Modular structure of
PACT: distinct domains for binding and activating PKR. Mol. Cell. Biol. 21:
1908–1920
52. Barber GN, Tomita J, Garfinkel MS, Meurs E, Hovanessian A and Katze MG
(1992) Detection of protein kinase homologues and viral RNA-binding domains
utilizing polyclonal antiserum prepared against a baculovirus-expressed ds
RNA-activated 68,000-Da protein kinase. Virology 191: 670–679
53. Koromilas AE, Roy S, Barber GN, Katze MG and Sonenberg N (1992)
Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent
protein kinase. Science 257: 1685–1689
54. Lee SB and Esteban M (1994) The interferon-induced double-stranded
RNA-activated protein kinase induces apoptosis. Virology 199: 491–496
55. Srivastava SP, Kumar KU and Kaufman RJ (1998) Phosphorylation of
eukaryotic translation initiation factor 2 mediates apoptosis in response to
activation of the double-stranded RNA-dependent protein kinase. J. Biol.
Chem. 273: 2416–2423
72. Mohr I and Gluzman Y (1996) A herpesvirus genetic element which
affects translation in the absence of the viral GADD34 function. EMBO J. 15:
4759–4766
73. Beattie E, Paoletti E and Tartaglia J (1995) Distinct patterns of IFN sensitivity
observed in cells infected with vaccinia K3L- and E3L- mutant viruses. Virology
210: 254–263
74. Chang HW, Uribe LH and Jacobs BL (1995) Rescue of vaccinia virus lacking
the E3L gene by mutants of E3L. J. Virol. 69: 6605–6608
75. Langland JO and Jacobs BL (2002) The role of the PKR-inhibitory genes, E3L
and K3L, in determining vaccinia virus host range. Virology 299: 133–141
76. Brandt TA and Jacobs BL (2001) Both carboxy- and amino-terminal domains of
the vaccinia virus interferon resistance gene, E3L, are required for
pathogenesis in a mouse model. J. Virol. 75: 850–856
56. Der SD, Yang YL, Weissmann C and Williams BR (1997) A double-stranded
RNA-activated protein kinase-dependent pathway mediating stress-induced
apoptosis. Proc. Natl. Acad. Sci. USA 94: 3279–3283
57. Chu WM, Ostertag D, Li ZW, Chang L, Chen Y, Hu Y, Williams B, Perrault J and
Karin M (1999) JNK2 and IKKbeta are required for activating the innate
response to viral infection. Immunity 11: 721–731
58. Goh KC, deVeer MJ and Williams BR (2000) The protein kinase PKR is
required for p38 MAPK activation and the innate immune response to bacterial
endotoxin. EMBO J. 19: 4292–4297
59. Patel RC and Sen GC (1998) PACT, a protein activator of the interferon-
induced protein kinase, PKR. EMBO J. 17: 4379–4390
60. Ito T, Yang M and May WS (1999) RAX, a cellular activator for double-stranded
RNA-dependent protein kinase during stress signaling. J. Biol. Chem. 274:
15427–15432
61. Polyak SJ, Tang N, Wambach M, Barber GN and Katze MG (1996) The P58
cellular inhibitor complexes with the interferon-induced, double-stranded RNA-
77. Beattie E, Tartaglia J and Paoletti E (1991) Vaccinia virus-encoded eIF-2 alpha
homolog abrogates the antiviral effect of interferon. Virology 183: 419–422
78. Sood R, Porter AC, Ma K, Quilliam LA and Wek RC (2000) Pancreatic
eukaryotic initiation factor-2alpha kinase (PEK) homologues in humans,
Drosophila melanogaster and Caenorhabditis elegans that mediate
translational control in response to endoplasmic reticulum stress. Biochem.
J. 346: 281–293
79. Langland JO and Jacobs BL (2004) Inhibition of PKR by vaccinia virus: role of
the N- and C-terminal domains of E3L. Virology 324: 419–429
80. Child SJ, Hakki M, De Niro KL and Geballe AP (2004) Evasion of cellular
antiviral responses by human cytomegalovirus TRS1 and IRS1. J. Virol. 78:
197–205
81. Gale Jr. MJ, Korth MJ, Tang NM, Tan SL, Hopkins DA, Dever TE, Polyak SJ,
Gretch DR and Katze MG (1997) Evidence that hepatitis C virus resistance to
interferon is mediated through repression of the PKR protein kinase by the
nonstructural 5A protein. Virology 230: 217–227
Cell Death and Differentiation

Viruses and stress responses
B He
403
82. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C,
Izumi N, Marumo F and Sato C (1995) Comparison of full-length sequences of
interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is
conferred by amino acid substitutions in the NS5A region. J. Clin. Invest. 96:
224–230
83. Watanabe H, Enomoto N, Nagayama K, Izumi N, Marumo F, Sato C and
Watanabe M (2001) Number and position of mutations in the interferon (IFN)
sensitivity-determining region of the gene for nonstructural protein 5A correlate
with IFN efficacy in hepatitis C virus genotype 1b infection. J. Infect. Dis. 183:
1195–1203
hepatitis C virus 1b isolates and relation to interferon sensitivity. J. Gen. Virol.
79 (Part 6): 1373–1381
88. Tan SL and Katze MG (2001) How hepatitis C virus counteracts the interferon
response: the jury is still out on NS5A. Virology 284: 1–12
89. Pflugheber J, Fredericksen B, Sumpter Jr. R, Wang C, Ware F, Sodora DL and
Gale Jr. M (2002) Regulation of PKR and IRF-1 during hepatitis C virus RNA
replication. Proc. Natl. Acad. Sci. USA 99: 4650–4655
90. Taylor DR, Shi ST, Romano PR, Barber GN and Lai MM (1999) Inhibition
of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 285:
107–110
84. Gale Jr. M, Blakely CM, Kwieciszewski B, Tan SL, Dossett M, Tang NM,
Korth MJ, Polyak SJ, Gretch DR and Katze MG (1998) Control of
91. Lu Y, Wambach M, Katze MG and Krug RM (1995) Binding of the influenza
virus NS1 protein to double-stranded RNA inhibits the activation of the
protein kinase that phosphorylates the elF-2 translation initiation factor.
Virology 214: 222–228
92. Hatada E and Fukuda R (1992) Binding of influenza A virus NS1 protein to
dsRNA in vitro. J. Gen. Virol. 73: 3325–3329
93. Tan SL and Katze MG (1998) Biochemical and genetic evidence for complex
formation between the influenza A virus NS1 protein and the interferon-induced
PKR protein kinase. J. Interferon Cytokine Res. 18: 757–766
PKR protein kinase by hepatitis
C virus nonstructural 5A protein:
molecular mechanisms of kinase regulation. Mol. Cell. Biol. 18:
5208–5218
85. Gale Jr. M, Kwieciszewski B, Dossett M, Nakao H and Katze MG (1999)
Antiapoptotic and oncogenic potentials of hepatitis C virus are linked to
interferon resistance by viral repression of the PKR protein kinase. J. Virol. 73:
6506–6516
ˆ
86. Nousbaum J, Polyak SJ, Ray SC, Sullivan DG, Larson AM, Carithers Jr. RL and
Gretch DR (2000) Prospective characterization of full-length hepatitis C virus
NS5A quasispecies during induction and combination antiviral therapy. J. Virol.
74: 9028–9038
87. Duverlie G, Khorsi H, Castelain S, Jaillon O, Izopet J, Lunel F, Eb F, Penin F
and Wychowski C (1998) Sequence analysis of the NS5A protein of European
94. Garcaia-Sastre A, Egorov A, Matassov D, Brandt S, Levy DE, Durbin JE,
Palese P and Muster T (1998) Influenza A virus lacking the NS1 gene replicates
in interferon-deficient systems. Virology 252: 324–330
95. Bergmann M, Garcia-Sastre A, Carnero E, Pehamberger H, Wolff K, Palese P
and Muster T (2000) Influenza virus NS1 protein counteracts PKR-mediated
inhibition of replication. J. Virol. 74: 6203–6206
Cell Death and Differentiation