JGV Papers in Press. Published April 28, 2015 as doi:10.1099/vir.0.000168
Journal of General Virology Delayed interferon response differentiates replication of West Nile virus and Japanese encephalitis virus in human neuroblastoma and glioblastoma cells --Manuscript Draft-Manuscript Number:
VIR-D-15-00215R1
Full Title:
Delayed interferon response differentiates replication of West Nile virus and Japanese encephalitis virus in human neuroblastoma and glioblastoma cells
Short Title:
Delayed IFN response in WNV- or JEV- infected human neuronal cells
Article Type:
Short Communication
Section/Category:
Animal - Positive-strand RNA Viruses
Corresponding Author:
Yuki Takamatsu, Ph.D., M.D. Institute of Tropical Medicine, Nagasaki University Nagasaki city, Nagasaki JAPAN
First Author:
Yuki Takamatsu, Ph.D., M.D.
Order of Authors:
Yuki Takamatsu, Ph.D., M.D. Leo Uchida, Ph.D., D.V.M. Kouichi Morita, Prof., Ph.D., M.D.
Abstract:
West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important causes of human encephalitis cases that resulted in high-mortality ratio and neurological sequelae after recovery. The mechanism for neuro-pathogenicity in these viruses infection is an important research interest to develop specific anti-viral therapy. Here, we focused on human derived neuronal and glial cells to understand the cellular responses against WNV- and JEV- infection. It was demonstrated that the early IFN-β induction regulates viral replication in glioblastoma T98G cells, whereas delayed IFN-β induction resulted in efficient virus replication in neuroblastoma SK-N-SH cells. Moreover, the concealing of viral dsRNA in the intracellular membrane resulted in the delayed IFN response in SK-N-SH cells. These results which showed different IFN response between human neuronal and glial cells after WNV or JEV infection are expected to contribute in understanding the molecular mechanisms for neuropathology in these viruses infection.
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Delayed interferon response differentiates replication of West Nile
virus
and
Japanese
encephalitis
virus
in
human
neuroblastoma and glioblastoma cells
Yuki Takamatsu1, Leo Uchida1, and Kouichi Morita1*
1) Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
*Corresponding author: Kouichi Morita Address: 1-12-4, Sakamoto, Nagasaki, Nagasaki 852-8524, Japan Phone: +81-95-819-7829, Fax; +81-95-819-7830 E-mail: [email protected]
The word count of abstract: 144 The word count of the main text: 2441
1
1
Abstract
2
West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important
3
causes of human encephalitis cases that resulted in high-mortality ratio and
4
neurological sequelae after recovery. The mechanism for neuro-pathogenicity in
5
these viruses infection is an important research interest to develop specific
6
anti-viral therapy. Here, we focused on human derived neuronal and glial cells to
7
understand the cellular responses against WNV- and JEV- infection. It was
8
demonstrated that the early IFN- induction regulates viral replication in
9
glioblastoma T98G cells, whereas delayed IFN- induction resulted in efficient
10
virus replication in neuroblastoma SK-N-SH cells. Moreover, the concealing of
11
viral dsRNA in the intracellular membrane resulted in the delayed IFN response
12
in SK-N-SH cells. These results which showed different IFN response between
13
human neuronal and glial cells after WNV or JEV infection are expected to
14
contribute in understanding the molecular mechanisms for neuropathology in
15
these viruses infection.
16 17
Short communications
18
West
Nile
virus (WNV) and
Japanese
2
encephalitis virus
(JEV) are
19
mosquito-borne viruses that belong to genus Flavivirus, family Flaviviridae
20
(Gubler et al., 2007; Sips et al., 2012). Most infections due to either viruses are
21
asymptomatic or cause febrile illness in humans, and may lead to encephalitis
22
resulting in high-mortality ratio and neurological sequelae after recovery
23
(Solomon, 2004). The specific anti-viral therapy has not yet been developed
24
(Solomon, 2004). There have been few reports focused on WNV and JEV
25
infection in human cells derived from brain (Kleinschmidt et al., 2007; Kumar et
26
al., 2010), and it has not yet been revealed how these viruses spread in these
27
cells. The mechanism of neuro-pathogenicity in WNV and JEV infection is an
28
important research interest to develop specific anti-viral therapy.
29
WNV and JEV can multiply in several cultured cells which exhibit cytopathic
30
effect (CPE) upon infection (Gubler et al., 2007; Kleinschmidt et al., 2007; Kumar
31
et al., 2010; Parquet et al., 2001; Stim & Henderson, 1969). To understand the
32
cellular responses against WNV- and JEV- infection, we used two kinds of cells
33
which showed different interferon (IFN) response and viral replication after WNV
34
and JEV infection.
35
3
36
The IFN response is the important defense against the early phase of viral
37
infections (Randall & Goodbourn, 2008). The type-I IFN induction is triggered by
38
viral components called pathogen-associated molecular patterns (PAMPs) such
39
as virus derived double-stranded RNA (dsRNA). The endoplasmic reticulum
40
(ER) provides the membrane platform for the formation of the flavivirus
41
replication complex that houses the nonstructural (NS) proteins and the
42
accumulating viral dsRNAs during viral genome synthesis (Gillespie et al., 2010;
43
Westaway et al., 1997). The dsRNA is recognized by pattern recognition
44
receptors (PRR), namely retinoic acid-inducible gene-I (RIG-I) and melanoma
45
differentiation-associated protein 5 (MDA5), and induces the IFN response (Loo
46
& Gale, 2011; Quicke & Suthar, 2013). The induction of IFN- is critical for
47
controlling WNV and JEV replication during the course of infection (Lin et al.,
48
2004; Quicke & Suthar, 2013). It was firstly reported in tick borne encephalitis
49
virus (TBEV) infection that the dsRNA is enclosed in intracellular membrane
50
vesicles, and as a consequence leads to an escape from the host immune
51
system (Overby et al., 2010). It was reported previously in JEV infected porcine
52
cells that viral dsRNA is concealed in intracellular membrane resulting in delayed
53
cytosolic exposure (Espada-Murao & Morita, 2011). The concealing mechanism
4
54
of dsRNA was also reported in Dengue virus (DENV) infected HeLa cells, which
55
resulted in the evasion of IFN response (Uchida et al., 2014). These findings
56
clearly suggest that the concealing of dsRNA plays a crucial role on viral
57
replication by escaping from the host immune system in flaviviruses infection.
58
This study aims to reveal the molecular mechanisms for WNV or JEV
59
dissemination and replication in human neuronal and glial cells.
60 61
Human
62
glioblastoma T98G cells (ATCC; CRL-1690) were used for viral infection. The
63
role of glial cells in WNV and JEV infection has been unrevealed, and this is the
64
first attempt to compare WNV and JEV infection between neuronal cells and glial
65
cells. WNV strains NY99 and Egypt101, JEV strains JaOArS982 and JaNAr0102
66
were used in this study. The viruses were propagated in the African green
67
monkey kidney cells (Vero cells) to generate working stocks. At 72 hour
68
post-inoculation, the culture supernatants were collected and stored in aliquots
69
at -80oC. WNV or JEV infection was performed on a monolayer of cells on
70
24-well plates at a multiplicity of infection (MOI) of 5, and supernatants were
71
harvested at the indicated time points. Virus titers were determined by
neuroblastoma
SK-N-SH
cells
5
(ATCC;
HTB-11),
and
human
72
plaque-forming assays on BHK cells and expressed as PFU /mL (Hayasaka et
73
al., 2009; Takamatsu et al., 2014). The clear CPE (rounding of cells, detachment
74
from the monolayer, and cell shrinkage) was observed in WNV or JEV infected
75
SK-N-SH cells from 3 days post-inoculation. On the other hand, no clear
76
morphology changes were observed in WNV or JEV infected T98G cells up to 5
77
days post-inoculation (data not shown). SK-N-SH cells showed higher virus titers
78
than T98G cells after infection with WNV or JEV. A slower viral growth and a
79
lower peak of viral titer were observed in T98G cells (Fig. 1a). These
80
experiments suggested that SK-N-SH cells are permissive for viral replication,
81
whereas T98G cells have some mechanisms to regulate virus propagation.
82
Next, we focused on cellular innate immune response against viral infection.
83
Real-time quantitative RT-PCR assay for IFN- was performed as described
84
previously (Takamatsu et al., 2014). The primer information was indicated in
85
Table S1. The early IFN- up-regulation was observed both in WNV- and JEV-
86
infected T98G cells from 8 hour post-inoculation (Fig. 1b). The timing of
87
increased viral RNA level and virus production was similar in SK-N-SH cells and
88
T98G cells (Fig. S1). Interestingly, the delayed IFN- induction was shown in
89
SK-N-SH cells, although sufficient viral replication was observed from early time
6
90
of infection. On the other hand, immediate IFN- induction was shown in the
91
absence of sufficient viral replication in T98G cells. There was no significant
92
difference in the basal levels of IFN- (absolute amount) between SK-N-SH and
93
T98G cells prior to infection (data not shown).
94 95
To clarify the role of type-I IFN in virus dissemination and growth, it was blocked
96
using an anti-IFN antibody cocktail. The cells were treated with a combination of
97
an anti-human IFN- (PBL Interferon Source) and an anti-human IFN-R2
98
(PBL Interferon Source) at different concentrations 1 hour before the viral
99
inoculation (Uchida et al., 2014). Focus formation assay was performed on
100
SK-N-SH cells and T98G cells as described previously (Espada-Murao & Morita,
101
2011). Foci of infected cells were larger in WNV- or JEV- infected SK-N-SH and
102
T98G cells treated with anti-IFN cocktail compared to the non-treated group (Fig.
103
2a). These results indicate that IFN response restricts viral replication both in
104
SK-N-SH and T98G cells. To elucidate the role of IFN- on viral replication, an
105
immediate IFN- treatment with 100 or 1000 units/well was performed
106
(Espada-Murao & Morita, 2011). The results showed that an early IFN-
107
treatment significantly reduced viral titers both in SK-N-SH cells and T98G cells
7
108
(Fig. 2b). It is suggested that the delayed IFN- induction impaired IFN response
109
at early time of infection, thereby enhancing WNV and JEV replication in
110
SK-N-SH cells. The cytosolic PRRs are reported to regulate IFNexpression
111
in flavivirus infection (Kato et al., 2006; Loo & Gale, 2011). To compare protein
112
expression in WNV- and JEV- infected cells, cellular extracts were subjected to
113
immunoblotting for RIG-I, MDA5, and for -Actin as an internal control
114
(Takamatsu et al., 2014; Uchida et al., 2014). The RIG-I expression was
115
detectable from 24 hour post-inoculation both in SK-N-SH and T98G cells (Fig.
116
2c), whereas the MDA5 expression was not detectable in the two cells (data not
117
shown). It indicates that the RIG-I is required but not MDA-5 for IFN-induction
118
in JEV infection (Kato et al., 2006), and this is in agreement with the previous
119
report (Uchida et al., 2014).
120
121
The timing of the dsRNA exposure in cytosol has been reported to be important
122
for inducing IFN response in TBEV-, DENV-, and JEV- infected cells
123
(Espada-Murao & Morita, 2011; Overby et al., 2010; Uchida et al., 2014). Two
124
permeabilization methods were applied to differentiate the localization of the
125
dsRNA, either exposed in cytoplasm or concealed in intracellular membrane. 8
126
Nonidet P-40 (NP-40) treatment permeabilizes all cellular membrane structure
127
like the plasma membrane, and ER; digitonin permeabilizes only the plasma
128
membrane (Uchida et al., 2014). WNV or JEV infection was performed on a
129
monolayer of cells on eight-well chamber slides (Nunc) at an MOI of 10. At
130
indicated time points, the cells were subjected to immunostaining as described
131
previously (Espada-Murao & Morita, 2011; Uchida et al., 2014). The mouse
132
IgG2a K1 monoclonal Ab (English & Scientific Consulting Kit) was used to
133
visualize viral dsRNA. The images were captured by LSM 780 confocal laser
134
scanning microscope (Carl Zeiss). The cell number in a field was counted by
135
ImageJ software (Schneider et al., 2012). Interestingly, the dsRNA was
136
predominantly concealed in intra-cellular membrane structures in SK-N-SH cells
137
after WNV infection (Fig. 3a). The ratio of exposed dsRNA in cytoplasm was
138
lower (approximately 10%) in WNV infected SK-N-SH cells, whereas that in
139
T98G cells was higher (approximately 50%) during the course of infection (Fig.
140
3b). A similar finding of concealed dsRNA was noted in JEV- infected SK-N-SH
141
cells, and the ratio of exposed dsRNA was also higher in T98G cells than that in
142
SK-N-SH cells (Fig. 3c). The results suggested that the delayed cytosolic
143
exposure of dsRNA was correlated with the delayed IFN- induction in SK-N-SH
9
144
cells. In our previous report, the dsRNA of DENV was concealed in intracellular
145
membrane but JEV was exposed in the cytoplasm of a human cervical-derived
146
HeLa cells at early infection (Uchida et al., 2014). A similar finding was observed
147
in WNV- infected HeLa cells (data not shown). The specific observation of
148
concealed dsRNA in neuronal cells may contribute to the viral pathogenicity in
149
WNV and JEV infection. It is suggested that the dsRNA leaking from the small
150
pore of replication vesicles at the later phase of infection is recognized by the
151
cytosolic PRRs in TBEV infection (Overby & Weber, 2011). It is possible that the
152
difference in formation of vesicle packets where dsRNA is likely to be concealed
153
(Gillespie et al., 2010) is related to the difference in the levels of dsRNA
154
exposure in cytoplasm between neuronal and glial cells. However, the
155
mechanism of triggering the cytosolic dsRNA exposure has not yet been
156
identified. Therefore, we need further investigations to reveal how WNV and JEV
157
utilize the mechanism for concealing viral dsRNA in association with host cellular
158
factors.
159 160
In conclusion, this is the first report to indicate the concealing mechanisms of
161
dsRNA in WNV- and JEV- infected human neuronal cells. The early IFN-
10
162
induction regulates viral replication in glioblastoma T98G cells, whereas delayed
163
IFN- induction resulted in efficient virus replication in neuroblastoma SK-N-SH
164
cells. These results which showed a difference in the IFN response between
165
human neuronal and glial cells after WNV or JEV infection, are expected to
166
contribute to understanding the molecular mechanisms for neuropathology due
167
to these viruses.
168 169
Acknowledgements
170
We would like to thank Dr. Corazon C. Buerano, and Mr. Gianne Eduard L.
171
Ulanday from the Department of Virology, Institute of Tropical Medicine,
172
Nagasaki University for helping the revision of our manuscript, and all of the
173
members and the recent alumni of the Department of Virology, Institute of
174
Tropical Medicine, Nagasaki University for their support. This study was
175
supported by a Grant-in-Aid for Scientific Research from the Ministry of
176
Education, Culture, Sports, Science and Technology (MEXT), Japan; the
177
Grant-in-Aid for JSPS Fellows (Japan Society for the Promotion of Science),
178
MEXT, Japan; the Global COE program, MEXT, Japan, the Japan Initiative for
179
Global Network on Infectious Diseases (J-GRID), MEXT, Japan; and the
11
180
Grant-in-aid for scientific research from the Ministry of Health, Labour, and
181
Welfare, Japan.
182
12
183
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Lin, R. J., Liao, C. L., Lin, E. & Lin, Y. L. (2004). Blocking of the alpha interferon-induced
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Overby, A. K., Popov, V. L., Niedrig, M. & Weber, F. (2010). Tick-borne encephalitis virus
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delays interferon induction and hides its double-stranded RNA in intracellular
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243 244 245
Figure legend
246
Figure 1 Virus replication and IFN- induction in SK-N-SH cells and T98G
247
cells
248
(a) Each virus (WNV; NY99, Egyot101, JEV; JaOArS982, JaNAr0102) was
14
249
inoculated at MOI of 5 onto SK-N-SH cells and T98G cells. The cell supernatants
250
were harvested at 1, 4, 8, 12, 24, 36, and 48 hour post-inoculation. The results of
251
PFU titration are expressed as the mean of three independent experiments, the
252
error bars indicate standard deviations of the mean. (b) Each virus (WNV; NY99,
253
Egyot101, JEV; JaOArS982, JaNAr0102) was infected on a monolayer of
254
SK-N-SH cells and T98G cells on 24-well plates at an MOI of 5. The number of
255
copies of IFN- mRNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
256
mRNAs was calculated by absolute quantification based on in vitro-transcribed
257
viral RNA, and GAPDH standards. The results for IFN- mRNA were normalized
258
to GAPDH, and expressed as the fold increase over non-infected cells. The
259
results are expressed as the mean of three independent experiments, and the
260
error bars indicate standard deviations of the mean. A group of samples was
261
assessed by Student’s t-tests or Welch tests. A P value of less than 0.05 was
262
considered statistically significant. Double asterisks and a number indicated a P
263
value.
264 265
Figure 2 IFN response and cytosolic PRRs expression
266
(a) WNV (NY99) or JEV (JaOArS982) infection was performed on a monolayer
15
267
of cells on 24-well plates with or without anti-IFN cocktail treatment. The cells
268
were treated by anti-IFN cocktail 1 h before viral inoculation. Culture medium
269
was used for control group of cells. At 5 days post-inoculation, the size of foci
270
was compared by focus forming assay. a; 400 neutralization U/ml of anti-human
271
IFN- and 4 mg/ml of anti-human IFN-R2. b; 2,000 neutralization U/ml of
272
anti-human IFN- and 20 mg/ml of anti-human IFN-R2. (b) WNV (NY99) or
273
JEV (JaOArS982) infection was performed on a monolayer of cells on 24-well
274
plates at an MOI of 5 with or without immediate IFN- treatment (100 or 1000
275
units/well). Culture medium was used for control group of cells. At 24 and 48
276
hour post-inoculation, viral titers were measured by PFU titration. The results are
277
expressed as the mean of three independent experiments, and the error bars
278
indicate standard deviations of the mean. A group of samples was assessed by
279
Student’s t-tests or Welch tests. A P value of less than 0.01 was indicated as
280
asterisks. (c) WNV (NY99) or JEV (JaOArS982) infection was performed on a
281
monolayer of cells on 12-well plates at an MOI of 5. At 24 and 48 hour
282
post-inoculation, the infected cells were harvested and the target proteins were
283
detected by the immunoblotting assay. The -Actin was used as an internal
284
control. All the samples were derived from the same experiment and blotting was
16
285
processed in parallel.
286 287
Figure 3 Delayed cytosolic exposure of dsRNA in SK-N-SH cells
288
WNV (NY99) infection was performed on a monolayer of cells on eight-well
289
chamber slides (Nunc) at an MOI of 10. (a) At the indicated time points, the
290
infected cells were fixed and permeabilized by 1% NP-40 or 0.5mM digitonin.
291
The viral dsRNA was visualized by using the immunofluorescence assay.
292
Nuclear staining was achieved using DAPI. (b, c) The exposed dsRNA ratio in
293
(b) WNV (NY99)- or (c) JEV (JaOArS982)- infected SK-N-SH cells and T98G
294
cells. The ratio expressed in percentage was determined by dividing the number
295
of digitonin-treated cells positive for dsRNA with the number of NP-40 treated
296
cells positive for dsRNA. The images showed comparable numbers of total cells
297
from both NP-40- and digitonin- treated cells. The percentage of cells was
298
calculated in 3 different fields. The error bars indicate standard error of the mean.
299
A group of samples was assessed by Mann Whitney test. A P value of less than
300
0.05 was considered statistically significant, and indicated as asterisks.
17
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Journal of General Virology Delayed interferon response differentiates replication of West Nile virus and Japanese encephalitis virus in human neuroblastoma and glioblastoma cells --Manuscript Draft-Manuscript Number:
VIR-D-15-00215R1
Full Title:
Delayed interferon response differentiates replication of West Nile virus and Japanese encephalitis virus in human neuroblastoma and glioblastoma cells
Short Title:
Delayed IFN response in WNV- or JEV- infected human neuronal cells
Article Type:
Short Communication
Section/Category:
Animal - Positive-strand RNA Viruses
Corresponding Author:
Yuki Takamatsu, Ph.D., M.D. Institute of Tropical Medicine, Nagasaki University Nagasaki city, Nagasaki JAPAN
First Author:
Yuki Takamatsu, Ph.D., M.D.
Order of Authors:
Yuki Takamatsu, Ph.D., M.D. Leo Uchida, Ph.D., D.V.M. Kouichi Morita, Prof., Ph.D., M.D.
Abstract:
West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important causes of human encephalitis cases that resulted in high-mortality ratio and neurological sequelae after recovery. The mechanism for neuro-pathogenicity in these viruses infection is an important research interest to develop specific anti-viral therapy. Here, we focused on human derived neuronal and glial cells to understand the cellular responses against WNV- and JEV- infection. It was demonstrated that the early IFN-β induction regulates viral replication in glioblastoma T98G cells, whereas delayed IFN-β induction resulted in efficient virus replication in neuroblastoma SK-N-SH cells. Moreover, the concealing of viral dsRNA in the intracellular membrane resulted in the delayed IFN response in SK-N-SH cells. These results which showed different IFN response between human neuronal and glial cells after WNV or JEV infection are expected to contribute in understanding the molecular mechanisms for neuropathology in these viruses infection.
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): revised manuscript.docx
Delayed interferon response differentiates replication of West Nile
virus
and
Japanese
encephalitis
virus
in
human
neuroblastoma and glioblastoma cells
Yuki Takamatsu1, Leo Uchida1, and Kouichi Morita1*
1) Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
*Corresponding author: Kouichi Morita Address: 1-12-4, Sakamoto, Nagasaki, Nagasaki 852-8524, Japan Phone: +81-95-819-7829, Fax; +81-95-819-7830 E-mail: [email protected]
The word count of abstract: 144 The word count of the main text: 2441
1
1
Abstract
2
West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important
3
causes of human encephalitis cases that resulted in high-mortality ratio and
4
neurological sequelae after recovery. The mechanism for neuro-pathogenicity in
5
these viruses infection is an important research interest to develop specific
6
anti-viral therapy. Here, we focused on human derived neuronal and glial cells to
7
understand the cellular responses against WNV- and JEV- infection. It was
8
demonstrated that the early IFN- induction regulates viral replication in
9
glioblastoma T98G cells, whereas delayed IFN- induction resulted in efficient
10
virus replication in neuroblastoma SK-N-SH cells. Moreover, the concealing of
11
viral dsRNA in the intracellular membrane resulted in the delayed IFN response
12
in SK-N-SH cells. These results which showed different IFN response between
13
human neuronal and glial cells after WNV or JEV infection are expected to
14
contribute in understanding the molecular mechanisms for neuropathology in
15
these viruses infection.
16 17
Short communications
18
West
Nile
virus (WNV) and
Japanese
2
encephalitis virus
(JEV) are
19
mosquito-borne viruses that belong to genus Flavivirus, family Flaviviridae
20
(Gubler et al., 2007; Sips et al., 2012). Most infections due to either viruses are
21
asymptomatic or cause febrile illness in humans, and may lead to encephalitis
22
resulting in high-mortality ratio and neurological sequelae after recovery
23
(Solomon, 2004). The specific anti-viral therapy has not yet been developed
24
(Solomon, 2004). There have been few reports focused on WNV and JEV
25
infection in human cells derived from brain (Kleinschmidt et al., 2007; Kumar et
26
al., 2010), and it has not yet been revealed how these viruses spread in these
27
cells. The mechanism of neuro-pathogenicity in WNV and JEV infection is an
28
important research interest to develop specific anti-viral therapy.
29
WNV and JEV can multiply in several cultured cells which exhibit cytopathic
30
effect (CPE) upon infection (Gubler et al., 2007; Kleinschmidt et al., 2007; Kumar
31
et al., 2010; Parquet et al., 2001; Stim & Henderson, 1969). To understand the
32
cellular responses against WNV- and JEV- infection, we used two kinds of cells
33
which showed different interferon (IFN) response and viral replication after WNV
34
and JEV infection.
35
3
36
The IFN response is the important defense against the early phase of viral
37
infections (Randall & Goodbourn, 2008). The type-I IFN induction is triggered by
38
viral components called pathogen-associated molecular patterns (PAMPs) such
39
as virus derived double-stranded RNA (dsRNA). The endoplasmic reticulum
40
(ER) provides the membrane platform for the formation of the flavivirus
41
replication complex that houses the nonstructural (NS) proteins and the
42
accumulating viral dsRNAs during viral genome synthesis (Gillespie et al., 2010;
43
Westaway et al., 1997). The dsRNA is recognized by pattern recognition
44
receptors (PRR), namely retinoic acid-inducible gene-I (RIG-I) and melanoma
45
differentiation-associated protein 5 (MDA5), and induces the IFN response (Loo
46
& Gale, 2011; Quicke & Suthar, 2013). The induction of IFN- is critical for
47
controlling WNV and JEV replication during the course of infection (Lin et al.,
48
2004; Quicke & Suthar, 2013). It was firstly reported in tick borne encephalitis
49
virus (TBEV) infection that the dsRNA is enclosed in intracellular membrane
50
vesicles, and as a consequence leads to an escape from the host immune
51
system (Overby et al., 2010). It was reported previously in JEV infected porcine
52
cells that viral dsRNA is concealed in intracellular membrane resulting in delayed
53
cytosolic exposure (Espada-Murao & Morita, 2011). The concealing mechanism
4
54
of dsRNA was also reported in Dengue virus (DENV) infected HeLa cells, which
55
resulted in the evasion of IFN response (Uchida et al., 2014). These findings
56
clearly suggest that the concealing of dsRNA plays a crucial role on viral
57
replication by escaping from the host immune system in flaviviruses infection.
58
This study aims to reveal the molecular mechanisms for WNV or JEV
59
dissemination and replication in human neuronal and glial cells.
60 61
Human
62
glioblastoma T98G cells (ATCC; CRL-1690) were used for viral infection. The
63
role of glial cells in WNV and JEV infection has been unrevealed, and this is the
64
first attempt to compare WNV and JEV infection between neuronal cells and glial
65
cells. WNV strains NY99 and Egypt101, JEV strains JaOArS982 and JaNAr0102
66
were used in this study. The viruses were propagated in the African green
67
monkey kidney cells (Vero cells) to generate working stocks. At 72 hour
68
post-inoculation, the culture supernatants were collected and stored in aliquots
69
at -80oC. WNV or JEV infection was performed on a monolayer of cells on
70
24-well plates at a multiplicity of infection (MOI) of 5, and supernatants were
71
harvested at the indicated time points. Virus titers were determined by
neuroblastoma
SK-N-SH
cells
5
(ATCC;
HTB-11),
and
human
72
plaque-forming assays on BHK cells and expressed as PFU /mL (Hayasaka et
73
al., 2009; Takamatsu et al., 2014). The clear CPE (rounding of cells, detachment
74
from the monolayer, and cell shrinkage) was observed in WNV or JEV infected
75
SK-N-SH cells from 3 days post-inoculation. On the other hand, no clear
76
morphology changes were observed in WNV or JEV infected T98G cells up to 5
77
days post-inoculation (data not shown). SK-N-SH cells showed higher virus titers
78
than T98G cells after infection with WNV or JEV. A slower viral growth and a
79
lower peak of viral titer were observed in T98G cells (Fig. 1a). These
80
experiments suggested that SK-N-SH cells are permissive for viral replication,
81
whereas T98G cells have some mechanisms to regulate virus propagation.
82
Next, we focused on cellular innate immune response against viral infection.
83
Real-time quantitative RT-PCR assay for IFN- was performed as described
84
previously (Takamatsu et al., 2014). The primer information was indicated in
85
Table S1. The early IFN- up-regulation was observed both in WNV- and JEV-
86
infected T98G cells from 8 hour post-inoculation (Fig. 1b). The timing of
87
increased viral RNA level and virus production was similar in SK-N-SH cells and
88
T98G cells (Fig. S1). Interestingly, the delayed IFN- induction was shown in
89
SK-N-SH cells, although sufficient viral replication was observed from early time
6
90
of infection. On the other hand, immediate IFN- induction was shown in the
91
absence of sufficient viral replication in T98G cells. There was no significant
92
difference in the basal levels of IFN- (absolute amount) between SK-N-SH and
93
T98G cells prior to infection (data not shown).
94 95
To clarify the role of type-I IFN in virus dissemination and growth, it was blocked
96
using an anti-IFN antibody cocktail. The cells were treated with a combination of
97
an anti-human IFN- (PBL Interferon Source) and an anti-human IFN-R2
98
(PBL Interferon Source) at different concentrations 1 hour before the viral
99
inoculation (Uchida et al., 2014). Focus formation assay was performed on
100
SK-N-SH cells and T98G cells as described previously (Espada-Murao & Morita,
101
2011). Foci of infected cells were larger in WNV- or JEV- infected SK-N-SH and
102
T98G cells treated with anti-IFN cocktail compared to the non-treated group (Fig.
103
2a). These results indicate that IFN response restricts viral replication both in
104
SK-N-SH and T98G cells. To elucidate the role of IFN- on viral replication, an
105
immediate IFN- treatment with 100 or 1000 units/well was performed
106
(Espada-Murao & Morita, 2011). The results showed that an early IFN-
107
treatment significantly reduced viral titers both in SK-N-SH cells and T98G cells
7
108
(Fig. 2b). It is suggested that the delayed IFN- induction impaired IFN response
109
at early time of infection, thereby enhancing WNV and JEV replication in
110
SK-N-SH cells. The cytosolic PRRs are reported to regulate IFNexpression
111
in flavivirus infection (Kato et al., 2006; Loo & Gale, 2011). To compare protein
112
expression in WNV- and JEV- infected cells, cellular extracts were subjected to
113
immunoblotting for RIG-I, MDA5, and for -Actin as an internal control
114
(Takamatsu et al., 2014; Uchida et al., 2014). The RIG-I expression was
115
detectable from 24 hour post-inoculation both in SK-N-SH and T98G cells (Fig.
116
2c), whereas the MDA5 expression was not detectable in the two cells (data not
117
shown). It indicates that the RIG-I is required but not MDA-5 for IFN-induction
118
in JEV infection (Kato et al., 2006), and this is in agreement with the previous
119
report (Uchida et al., 2014).
120
121
The timing of the dsRNA exposure in cytosol has been reported to be important
122
for inducing IFN response in TBEV-, DENV-, and JEV- infected cells
123
(Espada-Murao & Morita, 2011; Overby et al., 2010; Uchida et al., 2014). Two
124
permeabilization methods were applied to differentiate the localization of the
125
dsRNA, either exposed in cytoplasm or concealed in intracellular membrane. 8
126
Nonidet P-40 (NP-40) treatment permeabilizes all cellular membrane structure
127
like the plasma membrane, and ER; digitonin permeabilizes only the plasma
128
membrane (Uchida et al., 2014). WNV or JEV infection was performed on a
129
monolayer of cells on eight-well chamber slides (Nunc) at an MOI of 10. At
130
indicated time points, the cells were subjected to immunostaining as described
131
previously (Espada-Murao & Morita, 2011; Uchida et al., 2014). The mouse
132
IgG2a K1 monoclonal Ab (English & Scientific Consulting Kit) was used to
133
visualize viral dsRNA. The images were captured by LSM 780 confocal laser
134
scanning microscope (Carl Zeiss). The cell number in a field was counted by
135
ImageJ software (Schneider et al., 2012). Interestingly, the dsRNA was
136
predominantly concealed in intra-cellular membrane structures in SK-N-SH cells
137
after WNV infection (Fig. 3a). The ratio of exposed dsRNA in cytoplasm was
138
lower (approximately 10%) in WNV infected SK-N-SH cells, whereas that in
139
T98G cells was higher (approximately 50%) during the course of infection (Fig.
140
3b). A similar finding of concealed dsRNA was noted in JEV- infected SK-N-SH
141
cells, and the ratio of exposed dsRNA was also higher in T98G cells than that in
142
SK-N-SH cells (Fig. 3c). The results suggested that the delayed cytosolic
143
exposure of dsRNA was correlated with the delayed IFN- induction in SK-N-SH
9
144
cells. In our previous report, the dsRNA of DENV was concealed in intracellular
145
membrane but JEV was exposed in the cytoplasm of a human cervical-derived
146
HeLa cells at early infection (Uchida et al., 2014). A similar finding was observed
147
in WNV- infected HeLa cells (data not shown). The specific observation of
148
concealed dsRNA in neuronal cells may contribute to the viral pathogenicity in
149
WNV and JEV infection. It is suggested that the dsRNA leaking from the small
150
pore of replication vesicles at the later phase of infection is recognized by the
151
cytosolic PRRs in TBEV infection (Overby & Weber, 2011). It is possible that the
152
difference in formation of vesicle packets where dsRNA is likely to be concealed
153
(Gillespie et al., 2010) is related to the difference in the levels of dsRNA
154
exposure in cytoplasm between neuronal and glial cells. However, the
155
mechanism of triggering the cytosolic dsRNA exposure has not yet been
156
identified. Therefore, we need further investigations to reveal how WNV and JEV
157
utilize the mechanism for concealing viral dsRNA in association with host cellular
158
factors.
159 160
In conclusion, this is the first report to indicate the concealing mechanisms of
161
dsRNA in WNV- and JEV- infected human neuronal cells. The early IFN-
10
162
induction regulates viral replication in glioblastoma T98G cells, whereas delayed
163
IFN- induction resulted in efficient virus replication in neuroblastoma SK-N-SH
164
cells. These results which showed a difference in the IFN response between
165
human neuronal and glial cells after WNV or JEV infection, are expected to
166
contribute to understanding the molecular mechanisms for neuropathology due
167
to these viruses.
168 169
Acknowledgements
170
We would like to thank Dr. Corazon C. Buerano, and Mr. Gianne Eduard L.
171
Ulanday from the Department of Virology, Institute of Tropical Medicine,
172
Nagasaki University for helping the revision of our manuscript, and all of the
173
members and the recent alumni of the Department of Virology, Institute of
174
Tropical Medicine, Nagasaki University for their support. This study was
175
supported by a Grant-in-Aid for Scientific Research from the Ministry of
176
Education, Culture, Sports, Science and Technology (MEXT), Japan; the
177
Grant-in-Aid for JSPS Fellows (Japan Society for the Promotion of Science),
178
MEXT, Japan; the Global COE program, MEXT, Japan, the Japan Initiative for
179
Global Network on Infectious Diseases (J-GRID), MEXT, Japan; and the
11
180
Grant-in-aid for scientific research from the Ministry of Health, Labour, and
181
Welfare, Japan.
182
12
183
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Kumar, M., Verma, S. & Nerurkar, V. R. (2010). Pro-inflammatory cytokines derived from
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West Nile virus (WNV)-infected SK-N-SH cells mediate neuroinflammatory markers
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Lin, R. J., Liao, C. L., Lin, E. & Lin, Y. L. (2004). Blocking of the alpha interferon-induced
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243 244 245
Figure legend
246
Figure 1 Virus replication and IFN- induction in SK-N-SH cells and T98G
247
cells
248
(a) Each virus (WNV; NY99, Egyot101, JEV; JaOArS982, JaNAr0102) was
14
249
inoculated at MOI of 5 onto SK-N-SH cells and T98G cells. The cell supernatants
250
were harvested at 1, 4, 8, 12, 24, 36, and 48 hour post-inoculation. The results of
251
PFU titration are expressed as the mean of three independent experiments, the
252
error bars indicate standard deviations of the mean. (b) Each virus (WNV; NY99,
253
Egyot101, JEV; JaOArS982, JaNAr0102) was infected on a monolayer of
254
SK-N-SH cells and T98G cells on 24-well plates at an MOI of 5. The number of
255
copies of IFN- mRNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
256
mRNAs was calculated by absolute quantification based on in vitro-transcribed
257
viral RNA, and GAPDH standards. The results for IFN- mRNA were normalized
258
to GAPDH, and expressed as the fold increase over non-infected cells. The
259
results are expressed as the mean of three independent experiments, and the
260
error bars indicate standard deviations of the mean. A group of samples was
261
assessed by Student’s t-tests or Welch tests. A P value of less than 0.05 was
262
considered statistically significant. Double asterisks and a number indicated a P
263
value.
264 265
Figure 2 IFN response and cytosolic PRRs expression
266
(a) WNV (NY99) or JEV (JaOArS982) infection was performed on a monolayer
15
267
of cells on 24-well plates with or without anti-IFN cocktail treatment. The cells
268
were treated by anti-IFN cocktail 1 h before viral inoculation. Culture medium
269
was used for control group of cells. At 5 days post-inoculation, the size of foci
270
was compared by focus forming assay. a; 400 neutralization U/ml of anti-human
271
IFN- and 4 mg/ml of anti-human IFN-R2. b; 2,000 neutralization U/ml of
272
anti-human IFN- and 20 mg/ml of anti-human IFN-R2. (b) WNV (NY99) or
273
JEV (JaOArS982) infection was performed on a monolayer of cells on 24-well
274
plates at an MOI of 5 with or without immediate IFN- treatment (100 or 1000
275
units/well). Culture medium was used for control group of cells. At 24 and 48
276
hour post-inoculation, viral titers were measured by PFU titration. The results are
277
expressed as the mean of three independent experiments, and the error bars
278
indicate standard deviations of the mean. A group of samples was assessed by
279
Student’s t-tests or Welch tests. A P value of less than 0.01 was indicated as
280
asterisks. (c) WNV (NY99) or JEV (JaOArS982) infection was performed on a
281
monolayer of cells on 12-well plates at an MOI of 5. At 24 and 48 hour
282
post-inoculation, the infected cells were harvested and the target proteins were
283
detected by the immunoblotting assay. The -Actin was used as an internal
284
control. All the samples were derived from the same experiment and blotting was
16
285
processed in parallel.
286 287
Figure 3 Delayed cytosolic exposure of dsRNA in SK-N-SH cells
288
WNV (NY99) infection was performed on a monolayer of cells on eight-well
289
chamber slides (Nunc) at an MOI of 10. (a) At the indicated time points, the
290
infected cells were fixed and permeabilized by 1% NP-40 or 0.5mM digitonin.
291
The viral dsRNA was visualized by using the immunofluorescence assay.
292
Nuclear staining was achieved using DAPI. (b, c) The exposed dsRNA ratio in
293
(b) WNV (NY99)- or (c) JEV (JaOArS982)- infected SK-N-SH cells and T98G
294
cells. The ratio expressed in percentage was determined by dividing the number
295
of digitonin-treated cells positive for dsRNA with the number of NP-40 treated
296
cells positive for dsRNA. The images showed comparable numbers of total cells
297
from both NP-40- and digitonin- treated cells. The percentage of cells was
298
calculated in 3 different fields. The error bars indicate standard error of the mean.
299
A group of samples was assessed by Mann Whitney test. A P value of less than
300
0.05 was considered statistically significant, and indicated as asterisks.
17
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