Signal Transduction

in SARS-CoV-Infected Cells
T. MIZUTANI
Department of Virology 1, National Institute of Infectious Diseases,
Musashimurayama, Tokyo, Japan

ABSTRACT: Severe acute respiratory syndrome (SARS) is a newly found

infectious disease that is caused by a novel human coronavirus, SARS
coronavirus (SARS-CoV). Because the mortality rate of SARS patients
is very high, understanding the pathological mechanisms of SARS not
only in vivo but in vitro is important for the prevention of SARS. Acti-
vation of signaling pathways caused by SARS-CoV infection leads to the
phosphorylation and activation of downstream molecules. Two conflict-
ing cellular programs, apoptosis to eliminate virus-infected cells and sur-
vival to delay apoptosis by producing antiviral cytokines, occur in SARS
patients. Recent studies regarding SARS and SARS-CoV have clarified
that activation of mitogen-activated protein kinases (MAPKs) plays im-
portant roles in upregulation of cytokine expression and apoptosis both
in vitro and in vivo. Both Akt and p38 MAPK are keys for determina-
tion of cell survival or death in SARS-CoV-infected cells in vitro. Agents
being developed to target these signaling cascades may be important
for the design of anti-SARS-CoV drugs. This review highlights recent
progress regarding SARS-CoV biology, especially signal transduction in
SARS-CoV-infected cells.

KEYWORDS: SARS; apoptosis; MAPK

INTRODUCTION

Severe acute respiratory syndrome (SARS) is a newly found infectious

disease that is caused by a novel coronavirus, SARS coronavirus (SARS-
CoV).26,34 In late 2002, SARS spread from China to more than 30 countries,
causing severe outbreaks of atypical pneumonia. Although the mechanism of
SARS pathogenesis in vivo may involves both the effects of viral replication
in the target cells and immune responses to viral antigens, there is a lack of
molecular pathological data, including data regarding the signaling pathways
of SARS-CoV infection. As viral virulence and the mortality rate of SARS
Address for correspondence: T. Mizutani, Department of Virology 1, National Institute of Infectious
Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011, Japan. Voice: +81-42-561-0771; fax:
+81-42-564-4881.
tmizutan@nih.go.jp

Ann. N.Y. Acad. Sci. 1102: 86–95 (2007). 

C 2007 New York Academy of Sciences.

doi: 10.1196/annals.1408.006
86
MIZUTANI 87

patients are very high, understanding the pathological mechanisms of SARS

is important for the prevention of SARS.
Generally, both proapoptotic and prosurvival signaling pathways are acti-
vated during viral replication. Many pro- and antiapoptotic proteins are in-
volved in these pathways in cells. Several reports indicated that activation
of physiological intracellular signaling cascades caused by SARS-CoV infec-
tion leads to the phosphorylation and activation of downstream molecules.
For example, mitogen-activated protein kinases (MAPKs) are well-known sig-
nal transducers that respond to extracellular stimulation by cytokines, growth
factors, viral infection, and stress, and in turn regulate cell differentiation, pro-
liferation, survival, and apoptosis.15,23,40 Recent studies regarding SARS and
SARS-CoV have clarified that activation of MAPKs plays important roles in
upregulation of cytokine expression and apoptosis both in vitro and in vivo.
This review highlights progress regarding SARS-CoV biology, especially sig-
nal transduction in SARS-CoV-infected cells (FIG. 1).

p38 MAPK

Apoptosis, which is fundamentally different from necrosis, is an active and

physiological type of cell death, which can be induced by viral infection, vi-
ral replication, and production of viral proteins. The monkey kidney cell line,
Vero E6, is widely used in SARS-CoV research due to its high sensitivity
to infection with the virus. Several studies have shown that SARS-CoV in-
fection of Vero E6 cells induces apoptosis, detected by DNA fragmentation
and caspase activation.28,42 p38 MAPK is strongly activated by stress and
inflammatory cytokines. Mouse hepatitis virus (MHV), a prototype coron-
avirus, was able to induce activation of p38 MAPK into virus-infected cells.1
p38 MAPK activation in CD14 monocytes was also observed in SARS pa-
tients.24 Phosphorylation of p38 MAPK was significantly upregulated at 18 h
postinfection (h.p.i.) in SARS-CoV-infected Vero E6 cells.28 Cytopathic effects
of SARS-CoV-infection were partially prevented by adding the p38 MAPK-
specific inhibitor, SB203580, to the cells. Although p38 MAPK can promote
both cell death and survival,10 the results of inhibitor studies indicated that
the p38 MAPK signaling pathway may be involved mainly in cell death in
SARS-CoV-infected Vero E6 cells. Several downstream targets of p38 MAPK
were phosphorylated in virus-infected cells, and SB203580 effectively inhib-
ited phosphorylation of these proteins in SARS-CoV-infected cells. MAPK-
activated protein kinase (MAPKAPK)-2, which is activated in response to
stress and growth factors,12,13 was phosphorylated in virus-infected cells. Hsp-
27, which is a substrate of MAPKAPK-2 and is known to show antiapoptotic
activity by inhibiting apoptosome formation,14 survival factors,20 cAMP re-
sponse element-binding protein (CREB), and activation of transcription fac-
tor (ATF)-1, was also phosphorylated in virus-infected cells. Phosphorylation
of Hsp-27, CREB, and ATF-1 may induce an antiapoptotic environment in
88 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

FIGURE 1. Schematic representation of signal transduction on SARS-CoV infection.

SARS-CoV-infected cells. Nucleocapsid (N) protein was able to induce phos-

phorylation of Hsp-27 and CREB in transfected cells as described below.36 The
translation initiation factor, eukaryotic initiation factor 4E (eIF4E), is known
to enhance translation rates of cap-containing mRNAs.16 The phosphoryla-
tion of eIF4E was utilized to promote virus-specific protein synthesis in the
case of MHV.1 Although the levels of phosphorylated eIF4E were increased
by SARS-CoV infection, the activated eIF4E was not advantageous for viral
protein synthesis as demonstrated by the similar kinetics of viral protein accu-
mulation in infected Vero E6 cells in the presence and absence of SB 203580.
There may be other substrates of p38 MAPK that are inducible on apopto-
sis of Vero E6 cells caused by SARS-CoV infection. As each downstream
target molecule of p38 MAPK has a role in the induction of cell death or
survival in response to SARS-CoV infection, determination of the apoptosis-
inducible target molecules of p38 MAPK is important for the development of
anti-SARS-CoV drugs.

ERK1/2

Extracellular signal-regulated kinase (ERK) 1/2 was phosphorylated in

SARS-CoV-infected Vero E6 cells,27 whereas ERK1/2 was downregulated in
N protein-expressing COS-1 cells as described below.36
MIZUTANI 89

JNK

c-Jun N-terminal kinase (JNK) was phosphorylated in SARS-CoV-infected

Vero E6 cells.27 A recent study indicated that persistent infection was es-
tablished after most of the SARS-CoV-infected Vero E6 cells had died by
apoptosis.30 SP600125, an inhibitor of JNK, and LY294002, an inhibitor of
phosphatidylinositol 3-kinase (PI3K), inhibited the establishment of persis-
tence, whereas PD98059, an inhibitor of MEK1/2, and SB203580, an inhibitor
of p38 MAPK, did not. Thus, two signaling pathways of JNK and PI3K are
important for the establishment of persistence in Vero E6 cells.

STAT3

In Vero E6 cells, signal transducer and activator of transcription (STAT)

3 is constitutively phosphorylated at tyrosine (Tyr)-705 and is slightly phos-
phorylated at serine (Ser)-727.27 However, SARS-CoV replication induced de-
phosphorylation of STAT3 at Tyr-705. As STAT3 is a major transcription factor
activated in response to cytokines, such as interleukin-6 (IL-6) and IL-10, inhi-
bition of STAT3 signaling by dominant negative and antisense oligonucleotides
against STAT3 resulted in decreases in cell viability and apoptosis.17,31 Thus,
STAT3 is thought to act as an antiapoptotic transcription factor. Although
inhibitors of MEK and JNK had no effect on the phosphorylation status of
STAT3 in virus-infected cells, two inhibitors of p38 MAPK (SB203580 and
SB202190) partially inhibited dephosphorylation of STAT3 at Tyr-705, sug-
gesting that the p38 MAPK signaling pathway is upstream of Tyr-705 dephos-
phorylation of STAT3 in Vero E6 cells. The level of STAT3 phosphorylation
at Ser-727 was increased in virus-infected cells. Although the effect of Ser-
727 phosphorylation on the function of STAT3 remains unresolved, it was
reported that phosphorylation of Ser-727 of STAT3 negatively modulated its
Tyr phosphorylation.6 Interestingly, Tyr-705 dephosphorylation and Ser-727
phosphorylation showed almost the same timing in SARS-CoV-infected cells.
STAT3 phosphorylated at Tyr-705 was localized mainly in the nucleus in mock-
infected cells, whereas STAT3 disappeared from the nucleus in virus-infected
cells. As STAT3 acts as an activator of transcription in the nucleus, STAT3
may lack its transcriptional activity in SARS-CoV-infected Vero E6 cells, and
thus result in a decrease in antiapoptotic activity in the cells.
As other STAT signal transduction pathways are involved in SARS-CoV
infection, there have been many reports that treatment with interferons (IFNs)
can inhibit viral replication in vivo and in vitro. However, there have been no
detailed studies of IFN signal transduction in SARS-CoV-infected cells. IFN-
alpha receptor recognizes STAT1 and STAT2 via Jak1 and Tyk2, whereas the
IFN-gamma receptor recognizes STAT1 via Jak1 and Jak2. Signal-transducing
adaptor molecule 1 (STAM1) was upregulated in SARS-CoV-infected Vero E6
90 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

cells.25 As STAM1 is known to associate with Jak2 and 3 via the immunore-
ceptor Tyr-based activation motif, it may play an important role in signal trans-
duction of cytokine receptors by SARS-CoV infection.

AKT

As mentioned above, the PI3K signaling pathway (including Akt) plays im-
portant roles in establishing persistent infection by SARS-CoV. Akt is phospho-
rylated at both Ser-473 and threonine (Thr)-308 residues via a PI3K-dependent
mechanism on stimulation by growth factors, insulin, and hormones.39 One
of the most important functions of activated Akt in cells is the prevention of
apoptosis. Akt has many downstream targets and it induces cell survival via
phosphorylation of the forkhead transcription factor (FKHR) family, glycogen
synthase kinase 3ß (GSK-3ß), caspase-9, and Bad.2,8,9 In SARS-CoV-infected
confluent Vero E6 cells, Ser-473 of Akt was phosphorylated at 8 h.p.i. and
maximal phosphorylation was observed at 18 h.p.i., whereas phosphorylation
of Thr-308 was not observed. As Akt is thought to show its full activity when
phosphorylated at both Ser-473 and Thr-308, the total activity of Akt may be
low in Vero E6 cells. Therefore, the phosphorylated Akt in SARS-CoV-infected
cells cannot prevent apoptosis.
Interestingly, tissue inhibitor of metalloproteinase 2 (TIMP2), which acti-
vates Ras and leads to Ras/PI3K complex formation, was shown to be downreg-
ulated in virus-infected Vero E6 cells at 12 h.p.i. by microarray analysis.25 The
acute Ser dephosphorylation of Akt at 24 h.p.i. after tentative phosphorylation
in virus-infected cells may be due to downregulation of TIMP2.

PKC

Protein kinase C (PKC) is one of the major cellular mediators of biological

functions. The PKC superfamily is classified as subsuperfamilies based on
activation profiles: conventional PKC (cPKC , I, II,  novel PKC (nPKC
, , , ), atypical PKC (aPKC ,
/) PKC PKD and PKC . PKC , which
¨ 32
was discovered as a unique PKC isotype, is thought to be one of the most
important PKC, because PI3K-dependent activation of PKC mediates B-
cell survival by nerve growth factor.22 Akt has been reported to interact with
PKC .21 PKC was phosphorylated in SARS-CoV-infected Vero E6 cells,27
suggesting that this molecule is activated as an antiapoptotic response to SARS-
CoV infection.

TRANSCRIPTION FACTORS

Human intestinal epithelial Caco-2 cells, which are highly permissive

to SARS-CoV, have been used for analysis of cellular gene expression
MIZUTANI 91

by microarray analysis. The proinflammatory chemokines, interleukin 8

(CXCL8), and interferon- -inducible protein 10 (CXCL10), were upregulated
in SARS-CoV-infected Caco-2 cells.7 These two chemokines are regulated
by AP-1 and NF-B, which were also activated by viral infection. CXCL10
levels were significantly elevated in the blood of SARS patients38,41 and in
macrophages in vitro.5 As described above and below, CREB was phosphory-
lated in virus-infected Vero E6 cells and N protein-expressing COS-1 cells.

APOPTOSIS-INDUCIBLE VIRAL GENES

The genome of SARS-CoV is approximately 30 kb in length and contains 14

potential open-reading frames (ORFs), including nine viral proteins with no
homologues in other coronaviruses. The N protein, which is a 423 amino acid
predicted phospho-protein of 46 kDa with a short Ser-rich stretch and a putative
bipartite nuclear localization signal, is known to show little homology with
molecules in other coronaviruses. The N protein has been shown to undergo
self-association through the C-terminal 209 amino acid region35 and was able to
induce apoptosis of COS-1 cells in the absence of growth factors.36 Both JNK
and p38 MAPK were upregulated in the transfected cells, whereas ERK and
Akt were downregulated. Activated p38 MAPK by N protein induced actin
reorganization into cells in the absence of growth factors. The downstream
targets of p38 MAPK, MAPKAPK-2, and Hsp-27, were phosphorylated in the
cells as well as in the virus-infected Vero E6 cells. Downregulation of focal
adhesion kinase (FAK) activity and fibronectin expression was observed due
to N protein expression, supporting the hypothesis that apoptosis induced by
N protein is caused by interference with the integrin signaling pathway, and
that serum factors are able to inhibit apoptosis by maintaining the activity of
FAK through alternative pathways. Caspase-3 and -7 were activated only in
the absence of growth factors in N-expressing cells. However, SB203580, an
inhibitor of p38 MAPK, failed to inhibit caspase-3 activation. Thus, there are
two independent functional properties of N protein, p38 MAPK-dependent
actin reorganization, and caspase activation. Another group showed that the
levels of transcription factors, c-Fos, ATF2, CREB, and FosB, are increased by
expression of N in Vero and Huh-7 cells.18 Spike (S) protein of SARS-CoV
also induced activation of AP-1 and IL-8 promoter, possibly via activation of
MAPKs, in A549 and HFL-1 cells.4 As high serum levels of IL-8 were observed
in patients in the acute stage of SARS,19 activation of the IL-8 promoter via
AP-1 induced by S protein may explain these clinical observations. The binding
of S protein to a viral receptor, angiotensin-converting enzyme (ACE)-2, may
be a trigger for activation of these MAPKs.
Two unique proteins of SARS-CoV, 3a (U274, X1) and 7a (U122, X4), have
been studied. The 3a protein is located mainly in the Golgi apparatus and
contains three transmembrane regions,43 whereas 7a encodes a protein of 122
92 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

amino acids containing a probable cleaved signal sequence and a C-terminal

transmembrane helix. As the C-terminal tail also contains a typical endoplas-
mic reticulum (ER) retrieval motif, 7a is localized to the perinuclear region in
both SARS-CoV-infected and -transfected cells.11 The overexpression of 7a,
but not of 3a, induced apoptosis via a caspase-dependent pathway.37

CONCLUSIONS

In SARS patients, two conflicting cellular programs occur: “apoptosis” to

eliminate virus-infected cells and “survival” to delay apoptosis by producing
antiviral cytokines. The control mechanisms balancing cell survival against
cell death in vitro are important for understanding the pathology in vivo. As
mentioned above, activation of p38 MAPK is involved in the regulation of IL-8
in vivo. Both Akt and p38 MAPK are keys for determination of cell survival
or death in SARS-CoV-infected cells in vitro. Activation of the p38 MAPK
signaling pathway and dephosphorylation of STAT3 via p38 MAPK induced
by SARS-CoV infection have partially proapoptotic roles in Vero E6 cells. On
the other hand, the levels of Akt, which inactivates proapoptotic pathways, are
too low to block apoptosis signaling pathways. As Akt activation is necessary
to establish persistently infected cells that escape from apoptotic cell death on
viral infection, Akt plays an important role in delaying apoptosis in SARS-CoV-
infected cells. Agents being developed to target p38 MAPK and its downstream
molecules (for downregulation) and Akt (for upregulation) may be important
for the design of anti-SARS-CoV drugs.

ADDENDUM

In recent papers, Surjit et al. reported that phosphorylated N protein shut-

tled from nucleus to cytoplasm by binding to 14-3-3 (J. Virol. 2005. 79:
11476–11486) and N protein blocked S phase progression (J. Biol. Chem.
2006. 281: 10669–10681). Yuan et al. reported that 7a protein blocked cell
cycle progression at G0/G1 phase (Virology 2006. 346. 74–85). Inhibition
of cell proliferation was caused by dephosphorylation of Akt (by us; FEMS
Immunol. Med. Microbiol. 2006. 46: 236–243). Okabayashi et al. reported
that production and response of IFN were not suppressed by SARS-CoV-
infection (J. Med. Virol. 2006. 78: 417–424). We reported regulation 90 kDa
ribosomal S6 kinase phosphorylation (FEBS Lett. 2006. 580: 1417–1424),
importance of Bcl-2 and Bcl-xL for the establishment of persistent infection
(Biochem.Biophys.Res.Commun. 2006. 347: 261–265), and enhancement of
cytotoxity against Vero E6 cells persistently infected with SARS-CoV by My-
coplasma fermentans (Arch. Virol. In press).
MIZUTANI 93

ACKNOWLEDGMENTS

I thank Drs. S. Fukushi, M. Saijo, M. Ogata, K. Sakai, I. Kurane, and

S. Morikawa (National Institute of Infectious Diseases, Japan) for useful sug-
gestions. We acknowledge funding from a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports and Culture of Japan, a grant-
in-aid from the Ministry of Health, Labor, and Welfare of Japan, and the Japan,
Health Science Foundation, Tokyo, Japan.

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