JGV Papers in Press. Published November 4, 2014 as doi:10.1099/vir.0.069914-0
1
JGV paper no. VIR/2014/069914
2
Title: RIG-I from waterfowl and mammals have different abilities to induce antiviral responses
3
against influenza A viruses
4
Running title: Various RIG-Is have different antiviral abilities on IAV
5
Subject Category: Animal Viruses-Negative-strand RNA
6
Word count: 5297
7
Authors: Qiang Shao, Wenpin Xu, Qiang Guo, Li Yan, Lei Rui, Jinhua Liu, Yaofeng
8
Zhao & Zandong Li
9
Affiliation: Correspondence to Zandong Li, PhD, [email protected]
10
Qiang Shao and Wenpin Xu contributed equally to this work.
11
State key Laboratory for Agrobiotechnology, College of Biological Sciences, China
12
Agricultural University, No.2 Yuan Ming Yuan West Road, Beijing, 100193, China
13
Tel.: 86-10-62732144. Fax: 86-10-62895439
14
Abstract
15
Retinoic acid-induced gene I (RIG-I), plays a crucial role in sensing viral RNA and facilitating the
16
production of IFN-β. It varies in length and sequence among different species. The present study
17
assessed the functional differences among RIG-I proteins derived from mammals and birds. The
18
transfection of duck caspase recruitment domains (CARDs) and RIG-I (dCARDs and dRIG-I) and
19
goose CARDs and RIG-I (gCARDs and gRIG-I) into DF-1 cells increased the production of
20
IFN-β mRNA and IFN-stimulated genes and decreased influenza A virus (IAV) replication,
21
whereas, human CARDs and RIG-I (hCARDs and hRIG-I) and mouse CARDs and RIG-I
22
(mCARDs and mRIG-I) had no effect. In 293T and A549 cells, hCARDs had the strongest
23
IFN-inducing activity, followed by mCARDs, dCARDs, and gCARDs. The IFN-inducing activity
24
of hRIG-I was stronger than that of mRIG-I, dRIG-I, and gRIG-I, in that order. The results also
25
showed that although the ability dCARDs to activate IFN was stronger than that of gCARDs in
26
DF-1, 293T, and A549 cells, dRIG-I had a weaker ability to activate IFN than gRIG-I in DF-1
27
with or without IAV infection. Taken together, these data suggest that RIG-I protein has different
28
amino acid sequences and functions among species. This genetic and functional diversity renders
29
it flexible, adaptable, and capable of recognizing many viruses in different species. 1
30
Key words: influenza; H1N1; H9N2; RIG-I; CARDs; IFN;
31 32
Introduction
33
Type-I IFNs and other pro-inflammatory cytokines are produced when host pattern
34
recognition receptors (PRRs), such as Toll-like receptors (TLRs) and retinoic acid-induced gene I
35
(RIG-I)-like receptors (RLRs), recognize pathogen-associated molecular patterns (PAMPs)
36
including nucleic acids or other structural components of microbes (Creagh and O’Neill, 2006;
37
Kawai and Akira, 2009). RIG-I, as a cytoplasmic sensor of viral double strand RNA (dsRNA) and
38
single strand RNA (ssRNA), can be triggered by viral RNA (vRNA). RIG-I is composed of two
39
N-terminal CARDs, a central DExD/H box helicase/ATPase, and a C-terminal regulatory domain
40
(CTD/RD) (Hausmann et al., 2008). Under normal conditions, RIG-I is in an inactive, closed
41
conformation (Takahasi et al., 2008). Viral ssRNA or short dsRNA bearing a 5′-triphosphate can
42
bind to the DExD/H box helicase domain at its carboxy-terminus, which causes RIG-I to undergo
43
a conformational change that exposes the CARDs to the cytoplasm (Saito et al., 2007). The
44
exposure of the N-terminal CARDs of RIG-I leads to the Lys63-linked ubiquitination of Lys172.
45
The ubiquitinated CARDs then allow RIG-I bind to MAVS (also called IPS-1, CARDsif, or VISA)
46
(Kawai et al., 2005; Meylan et al., 2005; Xu et al., 2005). Other components of the pathway are
47
also recruited to this multi-protein signal complex, which activates TBK1 (TANK-binding kinase
48
1) and IKK (IκB kinase) (Meylan et al., 2005; Xu et al., 2005). The activation of these kinases
49
results in the phosphorylation and activation of IRF-3 and IRF-7, which form homodimers and
50
heterodimers, translocate into the nucleus and bind to IFN-stimulated response elements (ISREs),
51
to stimulate the expression of type I IFN genes and a set of IFN-inducible genes (Honda et al.,
52
2005; Nakhaei et al., 2006).
53
RIG-I has been identified in many species, including mammals (humans, mice, pigs, and
54
rabbits), birds (ducks, geese, pigeons, and zebra finches), and fish (trout and carp). The signaling
55
pathways activated by hRIG-I and mRIG-I have been well illustrated previously (Loo and Gale Jr,
56
2011). As natural reservoir of most influenza A strains, waterfowl have a crucial role in the
57
ecology and evolution of IAV (Webster et al., 1992). Similar to hRIG-I and mRIG-I, dRIG-I and
58
gRIG-I also induce type I interferon after influenza infection and poly I:C transfection (Barber et
59
al., 2010; Sun et al., 2013). Although dRIG-I and gRIG-I share similar CARDs and a helicase 2
60
domain with hRIG-I and mRIG-I, S8 and T170, which are critical for phosphorylation, and T55
61
and K172, which are essential for interaction and polyubiqutination by tripartite motif containing
62
25 (TRIM25) in humans, are not conserved in mice, ducks, or geese. Recently, Domingo et al.
63
demonstrated that although TRIM25-mediated ubiquitination occurs at K167 and K193, the
64
activation of dRIG-I is independent of the anchored ubiquitination (Miranzo-Navarro and Magor,
65
2014). These results suggest that the RIG-I signaling pathway functions differently in waterfowl
66
and mammals (Miranzo-Navarro and Magor, 2014).
67
In this study, the ability of the CARDs of RIG-I and full-length RIG-I encoded by humans,
68
mice, ducks, and geese to induce type I IFN in chicken DF-1, human A549, and human 293T cells
69
during IAV infection from mice, swine, and avian species was examined. Results showed that the
70
ability of waterfowl RIG-I to induce the expression of antiviral genes was stronger than
71
mammalian RIG-I in DF-1 cells during IAV infection. However, the ability of mammalian RIG-I
72
to induce the expression of antiviral genes was stronger than that of waterfowl RIG-I in 293T and
73
A549 cells during IAV infection. Waterfowl RIG-I interfered with the replication of IAV more
74
efficiently than mammalian RIG-I in DF-1 cells, whereas, mammalian RIG-I was more efficient
75
than waterfowl RIG-I in A549 and MDCK cells.
76 77
2. Results
78
2.1 Protein expression of RIG-I and CARDs in chicken DF-1 cells and human 293T cells.
79
EGFP-tagged RIG-I and CARDs were generated and transfected into 293T or DF-1 cells.
80
The detection of EGFP-tagged RIG-I and CARDs using fluorescence microscopy indicated that
81
significant levels of EGFP-tagged RIG-I and CARDs accumulated in 293T and DF-1 cells (Fig.
82
1A, B). The fluorescence intensity of waterfowl-derived RIG-I and CARDs was significantly
83
higher than that of mammalian-derived RIG-I and CARDs (Fig. 1A, B), respectively. In addition,
84
the expression of EGFP-tagged CARDs and RIG-I proteins derived from humans, mice, ducks,
85
and geese was detected in DF-1 (Fig. 1C) and 293T cells (Fig. 1D) using western blotting with
86
anti-EGFP antibody. Similar to the fluorescence observation, the expression of hCARDs and
87
mCARDs protein was significantly lower than that of the dCARDs and gCARDs as well as
88
hRIG-I, mRIG-I, dRIG-I, and gRIG-I in DF-1 and 293T cells. These data suggest that
89
waterfowl-derived RIG and CARDs are expressed more efficiently than mammal-derived RIG and 3
90
CARDs in both DF-1 and 293T cells.
91 92
2.2 Waterfowl RIG-Is induce the expression of antiviral genes more strongly than
93
mammalian RIG-I in DF-1 cells during IAV infection
94
To evaluate the differences in IFN-inducing activity between mammal-derived and
95
waterfowl-derived RIG-I and CARDs variants in chicken DF-1 cells, cells were transfected with
96
EGFP-tagged RIG-I and CARDs expression plasmids (Fig. 1A, C), and then infected with
97
influenza viruses or mock-treated. The mRNA expression of IFN-β and the antiviral
98
IFN-stimulated genes Mx1 and PKR were measured using qRT-PCR at 8 h post-infection (p.i.)
99
with A/Swine/Shandong/731/2009 (H1N1) (SW731), A/Chicken/Shandong/ZB/2007 (H9N2)
100
(ZB07), or A/WSN/33 (H1N1) (WSN) viruses. Transfection with dCARDs, gCARDs, dRIG-I,
101
and gRIG-I transfection alone induced significantly more IFN-β, Mx1, and PKR synthesis
102
compared with empty vector transfection with or without IAV infection (Fig. 2A–F). However,
103
hCARDs, mCARDs, hRIG-I, and mRIG-I did not increase the mRNA production of IFN-β, Mx1,
104
or PKR compared with empty vector transfection with or without IAV infection (Fig. 2A–F).
105
Transfection with EGFP-mRIG-I inhibited IFN-β, Mx1, and PKR mRNA synthesis significantly
106
only in WSN infection group (Fig. 2B, D, and F). Although dCARDs induced more IFN-β, Mx1,
107
and PKR mRNA production than did gCARDs, dRIG-I induced significant less IFN-β, Mx1, and
108
PKR mRNA production than did gRIG-I (Fig. 2A–F). ZB07, WSN, and SW731 infection was also
109
blocked the gRIG-I-mediated increase in Mx1 and PKR mRNA compared with the mock-treated
110
group. The inhibitory effects of WSN were the strongest (Fig. 2D, F). These observations suggest
111
that transfected waterfowl RIG-I and CARDs have a stronger ability to induce the expression of
112
antiviral genes than mammalian RIG-I and CARDs in DF-1 cells during IAV infection. Moreover,
113
dCARDs was more efficient in IFN-β mRNA production than gCARDs.
114 115
2.3 Mammalian RIG-Is induce the expression of antiviral genes more strongly than
116
waterfowl RIG-Is in human cells during IAV infection
117
To assess the differences in IFN-inducing activity between mammalian and waterfowl RIG-Is
118
in 293T cells, cells were transfected with EGFP-tagged RIG-I and CARDs expression plasmids
119
(Fig. 1B, D), and then infected with influenza viruses or mock-treated. The mRNA expression of 4
120
IFN-β, Mx, and PKR was detected using qRT-PCR at 8 h p.i. hCARDs and mCARDs induced
121
more IFN-β mRNA production compared with dCARDs and gCARDs in the presence or absence
122
of IAV infection (Fig. 3A). hCARDs had the strongest ability to induce IFN-β, followed by
123
mCARDs, dCARDs, and gCARDs (Fig. 3A). Similarly, hRIG-I had the most pronounced ability
124
to induce IFN-β, followed by mRIG-I, dRIG-I, and gRIG-I (Fig. 3B). Although gRIG-I
125
transfection alone increased IFN-β synthesis (Fig. 3B), it did not further increase the production of
126
IFN-β over empty vector transfection after IAV infection (Fig. 3B). Interestingly, gRIG-I blocked
127
IFN-β mRNA production during WSN or SW731 infection (Fig. 3B).
128
Because of the low expression of Mx and PKR in 293T cells (data not shown), we also
129
performed the transfections and infections in a human lung adenocarcinoma epithelial cell
130
line (A549 cells). Fig.4A and B shows that hRIG-I, mRIG-I, dRIG-I, gRIG-I, hCARDs, mCARDs,
131
dCARDs, and gCARDs had the similar IFN-β-inducing ability in A549 and 293T cells. Fig. 4C
132
and E show that hCARDs induced the highest mRNA levels of Mx and PKR, followed by
133
mCARDs, dCARDs, and gCARDs. Similarly, hRIG-I had the strongest ability to induce IFN-β,
134
followed by mRIG-I, dRIG-I, and gRIG-I (Fig. 4D, F). Although gCARDs facilitated IFN-β, Mx
135
and PKR mRNA production (Fig. 4A, C, and E), gRIG-I inhibited IFN-β, Mx and PKR mRNA
136
synthesis compared with empty vector transfection after IAV infection (Fig. 4B, D, and F).
137
Taken together, these data suggest that the transfected mammalian RIG-Is and CARDs had a
138
stronger ability to induce the expression of antiviral genes in 293T and A549 cells during IAV
139
infection than did waterfowl. In addition, gRIG-I could negatively regulate IFN-β signaling
140
pathway in 293T and A549 cells during IAV infection.
141 142
2.4 Waterfowl RIG-Is reduce the replication of IAV more efficiently than mammalian
143
RIG-Is in DF-1 cells
144
We next evaluated the effects of all the CARDs and RIG-Is on viral replication in DF-1 cells.
145
Cells were transfected with EGFP-tagged RIG-I and CARDs plasmids or empty vector alone, and
146
then were challenged with ZB07, WSN, or SW731 at a multiplicity of infection (MOI) of 0.01
147
(Fig. 5). At 24 and 48 h p.i., the viral titers in the cell cultures were determined as 50% tissue
148
culture infectious doses (TCID50/ml). No differences were observed between the ZB07, WSN, or
149
SW731 viral titers in hCARDs-, mCARDs-, hRIG-I-, mRIG-I-, or empty vector-transfected DF-1 5
150
cells at 24 or 48 h p.i. (Fig. 5A–F). However, significantly decreased ZB07, WSN, and SW731
151
viral titers were observed in dCARDs-, gCARDs-, dRIG-I-, and gRIG-I-transfected DF-1cells
152
compared with empty vector transfected DF-1 cells at both 24 and 48 h p.i. (Fig. 5A–F). Similar to
153
the observations regarding IFN-inducing activity, dCARDs, gCARDs decreased the ZB07, WSN,
154
and SW731 viral titers to a lower level than dRIG-I and gRIG-I in DF-1 cells (Fig. 5A–F).
155
Although dCARDs induced more IFN-β mRNA than did gCARDs did, there were no difference
156
between the ZB07 and WSN viral titers on in dCARDs- and gCARDs- transfected DF-1 cells.
157
dCARDs decreased SW731 titers to a lower level than did gCARDs (Fig. 5A). Taken together,
158
these results suggest that waterfowl RIG-Is reduce IAV replication more efficiently than
159
mammalian RIG-Is in DF-1 cells, and gCARDs have a weaker ability to inhibit replication of IAV
160
than dCARDs.
161 162
2.5 Mammalian RIG-I reduces IAV replication more efficiently than waterfowl RIG-I in
163
A549 cells
164
The effects of mammalian and waterfowl RIG-Is on viral multiplication was evaluated
165
further in A549 cells. Cells were transfected with EGFP-tagged CARDs and RIG-I expression
166
plasmids or empty vector alone, and were then challenged with ZB07, WSN, or SW731 at an MOI
167
of 0.01. The viral titers were determined as TCID50 at 24 and 48 h p.i.. hCARDs and mCARDs
168
exhibited the most significant inhibitory effects on the multiplication of ZB07, WSN, and SW731
169
at both 24 and 48 h (Fig. 6A, C, and E). In contrast, dCARDs only slightly decreased the of WSN
170
and SW731 viral titers at 24 h and ZB07 titers at 24 and 48 h (Fig. 6A, C, and E). No differences
171
were observed between the ZB07, WSN, or SW731 viral titers in A549 cells transfected with
172
gCARDs compared with empty vector at 24 or 48 h (Fig. 6A, C, and E). Consistent with hCARDs
173
and mCARDs, hRIG-I and mRIG-I suppressed the replication of the three influenza viruses at 24
174
and 48 h (Fig. 6B, D, and F). No inhibitory effects on viral titers were observed in dRIG-I- and
175
gRIG-I-transfected cells. Similar results were also observed in transfection and viral multiplication
176
experiments performed in Madin-Darby canine kidney cells (MDCK cells) (Supplementary Fig. 1).
177
These data suggest that mammalian RIG-I and CARDs reduce the replication of IAV in
178
mammalian cells more efficiently than do waterfowl RIG-I and CARDs.
179 6
180
3. Discussion
181
Here we examined the differences in the protein expression of RIG-I and CARDs proteins
182
derived from mammals and birds, as well as their IFN-inducing activity and ability to suppress the
183
replication of influenza viruses in chicken and human cells. QRT-PCR was used to analyze the
184
ability of various RIG-Is and CARDs variants to induce IFN expression in DF-1, 293T, and A549
185
cells. Waterfowl RIG-I and CARDs had a more pronounced ability to induce the expression of
186
antiviral genes than did mammalian in DF-1 cells after IAV infection. Conversely, mammalian
187
RIG-I and CARDs had a more pronounced ability to induce the expression of antiviral genes than
188
did waterfowl RIG-I and CARDs in 293T and A549 cells during IAV infection. Data revealed that
189
waterfowl RIG-I interfered with the replication of IAV more efficiently than did mammalian
190
RIG-I in DF-1 cells, whereas, mammalian RIG-I reduced replication of IAV more efficiently than
191
did waterfowl RIG-I in A549 and MDCK cells. Therefore, RIG-I from waterfowl and mammals
192
had different abilities to induce an antiviral response against IAV.
193
Previous studies revealed that duck and goose CARDs domains activate IFN-β in chicken
194
cells, suggesting that chicken cells contain all the proteins required for the activation and
195
downstream signaling of RIG-I (Miranzo-Navarro and Magor, 2014; Sun et al., 2013). Recent in
196
vitro studies demonstrated that human TRIM25 does not interact with, ubiquitinate, or activate
197
human CARDs domains that were co-transfected into chicken DF-1 cells (Miranzo-Navarro and
198
Magor, 2014). Similarly, dCARDs, gCARDs, dRIG-I, and gRIG-I activated IFN-β production in
199
chicken DF-1 cells, whereas hCARDs, mCARDs, hRIG-I, and mRIG-I did not. In the current
200
study, this suggests that only waterfowl-derived RIG-I are functional in chicken DF-1 cells, and
201
that some factors necessary for mammal-derived RIG-I activation are missing or unrecognizable
202
in chicken cells. For example, RIPLET, which ubiquitinates Lys849 and Lys851 in the CTD of
203
RIG-I (Gao et al., 2009; Oshiumi et al., 2009; Oshiumi et al., 2010), is absent from the chicken
204
and duck genomes (Magor et al., 2013; Rajsbaum et al., 2012).
205
SW731, WSN, and ZB07 have different IFN-inducing activity (Fig.2A and B; Fig. 3A and B).
206
Sequence alignment showed that three viruses have different length of non-structural
207
protein1(NS1) (SW731, 219 amino acid (aa); WSN, 230 aa; ZB07, 217 aa) (data not shown). The
208
most pronounced IFN synthesis of ZB07 infected DF-1 and 293T cells (Fig.2A and B; Fig. 3A and
209
B), may due to the weak suppression of IFN by NS1. Otherwise, mRIG-I, but not mCARD, 7
210
blocked the IFN-β, Mx, and PKR mRNA synthesis only in WSN infected DF-1 cells (Fig.2A–F)..
211
Previously study reported that only avian influenza virus (AIV) derived non-structural protein1
212
(NS1) can bind to chicken TRIM25, thereby suppressing the induction of IFN (Rajsbaum et al.,
213
2012). Mouse-adapted IAV NS1 binds to mouse RIPLET specifically in mouse cells (Rajsbaum et
214
al., 2012). NS1 or other viral protein of WSN might specifically interact with mRIG-I to suppress
215
the induction of IFN in DF-1 cells.
216
Although gRIG-I induced slightly more IFN-β production in DF-1 cells than did dRIG-I with
217
or without IAV infection, the expression of gRIG-I induced significantly more Mx and PKR
218
expression (Fig. 2B, D, and F). This suggests that gRIG-I might induce Mx and PKR in
219
IFN-independent manner. Interestingly, dCARDs share 92.98% amino acid identity with gCARDs;
220
however, dCARDs induced significantly more IFN-β production and IFN-stimulated gene
221
expression than did gCARDs in both DF-1 and 293T cells with or without IAV infection. An
222
alignment of dRIG-I and gRIG-I sequences revealed that the phosphorylation sites for inactivation
223
are not conserved between ducks (S8) and geese (G8). It was reported that the phosphorylation of
224
S8 negatively regulates hRIG-I, possibly by preventing TRIM25 binding (Gack et al., 2010;
225
Nistal-Villán et al., 2010). However, it is unclear whether the S8G mutantion in gRIG-I is
226
responsible for its pronounced IFN-inducing activity.
227
Recent in vitro studies demonstrated that dRIG-I interacts with the unanchored K63-linked
228
polyubiquitin chains generated by hTRIM25 in 293T cells (Miranzo-Navarro and Magor, 2014).
229
Sun et al. demonstrated that NDV infection in gRIG-I-transfected 293T/17 cells resulted in
230
upregulated activity of the IFN-β promoter and IRF-3 and IFIT1 mRNA levels, but decreased viral
231
titers (Sun et al., 2013). Consistent with these reports, dCARDs, gCARDs, dRIG-I, and gRIG-I
232
were promoted IFN expression in mock-treated 293T and A549 cells in the current study.
233
However, the ability of dCARDs and gCARDs to induce IFN production was significantly less
234
pronounced than that of hCARDs and mCARDs. The ability of dRIG-I and gRIG-I to induce IFN
235
production was also reduced significantly compared with hRIG-I and mRIG-I. Surprisingly,
236
gRIG-I blocked IFN-β, Mx1, and PKR mRNA production during influenza infection, which is
237
almost the opposite effect reported for gRIG-I in 293T cells after NDV infection (Sun et al., 2013).
238
One possible cause of this inhibitory effect is that gRIG-I could compete with endogenous hRIG-I
239
to bind influenza vRNA. However, gRIG-I could not activate the IFN pathway as effectively as 8
240
could hRIG-I, which resulted in competitive inhibition.
241
Although mRIG-I protein has 76.57% amino acid identity with hRIG-I, hRIG-I induced
242
significantly more IFN-β production in 293T and A549 cells compared with mRIG-I with or
243
without IAV infection. mCARDs also share 75.09% amino acid identity with hCARDs, but
244
hCARDs induced significantly more IFN production and IFN-stimulated gene expression in 293T
245
and A549 cells than did mCARDs regardless of IAV infection. In the current study, hCARDs and
246
dCARDs have conserved phosphorylation sites (S8 and S168 in duck, S8 and T170 in human), but
247
mCARDs contain N8 and V170. Moreover, T55, which plays a critical role in the interaction, is
248
displaced by serine in mCARDs (Gack et al., 2008). K172, the site that is ubiquitinated by
249
TRIM25 in humans, is not conserved in mRIG-I. This might be responsible for the fact that
250
mCARDs and mRIG-I had reduced ability to induce IFN than did hCARDs and hRIG-I (Jiang et
251
al., 2012; Zeng et al., 2010).
252
In the current study, the effects of various RIG-Is and CARDs variants on virus replication
253
were evaluated in DF-1, A549, and MDCK cells. ZB07, WSN, and SW731 titers in hCARDs-,
254
mCARDs-, hRIG-I-, and mRIG-I-transfected DF-1 cells confirmed the inability of hCARDs,
255
mCARDs, hRIG-I, and mRIG-I to function in chicken DF-1 cells. The inability of dRIG-I and
256
gRIG-I to function in MDCK cells was also demonstrated by the ZB07, WSN, and SW731 titers
257
in dRIG-I- and gRIG-I-transfected A549 and MDCK cells. This suggests that the RIG-I signaling
258
pathway has changed during evolution, possibly because of selection pressure from viruses that
259
disrupted this regulation. Both birds and mammals evolved from reptiles 200 million years ago.
260
RIG-Is derived from humans and mice were not functional in chicken DF-1 cells. Although
261
RIG-Is derived from ducks and geese are functional in human 293T cells, but they do not
262
functions as efficiently as hRIG-I. This might be due to the altered amino acid sequence of RIG-I
263
during evolution and the emergence of novel signal transducers, such as RIPLET. Nevertheless,
264
waterfowl still retain part of the original signal transduction system. Therefore, waterfowl RIG-I
265
are still partially functional in human 293T cells. Taken together, these data suggest that RIG-I can
266
only activate the IFN signaling pathway effectively in its own or very similar cellular systems
267
(dRIG-I and gRIG-I in DF-1, and hRIG-I and mRIG-I in A549, 293T, and MDCK cells). Together,
268
these results suggest that additional attention should be paid to whether transgenes from very
269
evolutionary distant species can function in alternative hosts. 9
270 271
4. Materials and methods
272
4.1 Plasmids.
273
The open reading frame (ORF) of hRIG-I was cloned from cDNA isolated from human A549
274
cells
275
TTTGGACATTTCTGCTGGATCA-3′ based on human RIG-I sequence (GenBank accession no.
276
AF038963). The mRIG-I ORF was cloned from mouse lung cDNA using the primers
277
5′-ATGACCGCGGCGCAGCGGC-3′
278
based on mRIG-I sequence (GenBank accession no. AY553221). The dRIG-I ORF was obtained
279
from
280
5′-ATGACGGCGGACGAGAAGC-3 ′ and 5 ′-CTAAAATGGTGGGTACAAGTTGGAC-3 ′ based
281
on the duck RIG-I sequence (GenBank accession no. EU363349.1). Finally, the gRIG-I ORF was
282
amplified from goose splenic cDNA using the primers 5′-ATGACGGCGGAGGAAAAGC-3′ and
283
5′-CTA AAATGGTGGGTACAAGTTGGAC-3′ based on goose RIG-I sequence (GenBank
284
accession no. JF804977). The homologous arms of pEGFP-N1 were added to the 5′ and 3′ termini
285
of human, mouse, duck, and goose full-length RIG-I and CARDs from human, mouse, duck, and
286
goose RIG-I using PCR with the primers shown in Supplementary Table 1. Human, mouse, duck,
287
and goose full-length RIG-I and CARDs were cloned into mammalian expression vector
288
pEGFP-N1 by homologous recombination in accordance with the manufacturer’s instructions
289
(NovoRec).
290
4.2 Viruses.
using
duck
the
(A.
primers
5 ′-ATGACCACCGAGCAGCGAC-3′
and
5 ′-CTA
platyrhynchos)
and
5′-CTA
TACGGACATTTCTGCAGGATCGA-3 ′
splenic
cDNA
using
the
primers
291
The viruses were amplified in chicken embryos. The titers were determined as TCID50 by
292
monitoring the cytopathic effect (CPE) of endpoint dilutions on MDCK cells in the presence of 1
293
μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Worthington Biochemical,
294
Lakewood, NJ, USA.). All experiments with virus were performed under biosafety level 2 (BSL-2)
295
conditions with investigators wearing appropriate protective equipment and complying with
296
general biosafety standards in the microbiological and biomedical laboratories of Ministry of
297
Health of the People’s Republic of China (WS 233-2002).
298
4.3 Cell culture, transfections, and infections.
299
DF-1, 293T, A549, and MDCK cells were maintained in Dulbecco’s modified Eagle’s 10
300
medium (DMEM), supplemented with 0.6 μg/ml penicillin, 60 μg/ml streptomycin and 10% fetal
301
bovine serum (FBS) (Gibco), in an atmosphere of 5% CO2 at 37°C. For infection of transfected
302
cells, 2.5 × 105 DF-1, 293T, A549, or MDCK cells were seeded into 24-well plates and
303
maintained in DMEM plus 10% FBS overnight. DF-1, 293T, and MDCK cells were transfected
304
with 800 ng of EGFP-tagged plasmids with Lipofectamine 2000 (Invitrogen). A549 cells were
305
transfected with EGFP-tagged plasmids using FuGENE® HD (Promega). Then, 24 h p.i., cells
306
were infected
307
μg/ml)TPCK-treated trypsin was used for infection at in view of the trypsin sensitivity of DF-1
308
and 293T cells, compared with 0.5 μg/ml in A549, and 2 μg/ml in MDCK cells (Lee et al., 2008).
309
4.4 Western blotting.
with SW731, WSN, or ZB07
viruses.
A lower concentration (0.1
310
293T and DF-1 cells were lysed in RIPA buffer containing 20 mM Tris base-HCl (pH 7.5),
311
150 mM NaCl, 1.0 mM EDTA (pH 8.0), 1.0 M megta (pH 8.0), 0.1% SDS, 0.5% DOC, 1% NP-40,
312
protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Roche). Proteins were
313
separated using SDS-PAGE under reducing conditions and analyzed using western blotting with
314
anti-EGFP (Beijing B&M Biotech Co., Ltd, Beijing, China) and anti-GAPDH (Beijing CoWin
315
Biotech, Beijing, China) antibodies followed by HRP-labeled secondary antibodies (BIO-RAD,
316
USA) (Chen et al., 2010).
317
4.5 Quantitative real-time PCR (qRT-PCR)
318
Total RNA was extracted using TRIzol (Invitrogen) and treated with DNase I (Promega) to
319
remove the contaminating DNA. Then, 1 μg of RNA was reverse-transcribed into cDNA using a
320
GoScript reverse transcription system (Promega) in a 20-μl reaction mixture. The cDNA was
321
analyzed using qRT-PCR with SYBR green Master I (Roche). The primers specific for chicken
322
GAPDH, IFN-β, Mx, and PKR have been described previously (Barber et al., 2010). The specific
323
primers for human GAPDH, IFN-β, Mx, and PKR were designed using Primer Express 3.0 and are
324
shown in Supplementary Table 2. qRT-PCR was performed under the following cycling
325
conditions: 95°C for 10 min for activation, followed by 40 cycles of 95°C for 15 sec and 60°C for
326
1 min. This was followed by one cycle of 95°C for 15 sec, 60°C for 15 sec, and 95°C for 15 sec.
327
The final step was to obtain a melting curve for the PCR products to determine the specificity of
328
amplification. All control and infected samples were analyzed in triplicate on the same plate. The
329
relative mRNA abundances were calculated using the 2 -∆∆Ct method with GAPDH as a reference 11
330
and plotted as fold change compared with mock-control samples (Livak and Schmittgen, 2001).
331
4.6 Measurement of virus growth in avian and mammalian cells.
332
DF-1, A549, and MDCK cells transfected with EGFP-tagged plasmids were infected with
333
SW731,WSN, and ZB07 at an MOI of 0.01, and cell cultures were collected at different times (24
334
and 48 h) after infection. The cell culture samples were centrifuged at 5000 × g for 1 min, and the
335
supernatants were stored at −80°C until use. The viral contents in the supernatants were titrated
336
using TCID50 in MDCK cells (Reed and Muench, 1938).
337
4.7 Statistical analysis.
338
All data analyses were performed using SPSS 16.0 software. Independent sample t-tests were
339
used to determine significant differences between different plasmid-transfected groups. P values ≤
340
0.05 were considered significant.
341 342
Acknowledgments
343
This work was supported by the National Basic Research Program (973 Program, number
344
2013CB945000). We thank Dr. Zhiyi Wan for critical reading of the manuscript. We thank LetPub
345
(www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
346 347
References
348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365
Barber, M.R., Aldridge Jr, J.R., Fleming-Canepa, X., Wang, Y.-D., Webster, R.G., Magor, K.E., 2013. Identification of avian RIG-I responsive genes during influenza infection. Molecular immunology 54, 89-97. Barber, M.R., Aldridge, J.R., Webster, R.G., Magor, K.E., 2010. Association of RIG-I with innate immunity of ducks to influenza. Proceedings of the National Academy of Sciences 107, 5913-5918. Chen, C.-J., Chen, G.-W., Wang, C.-H., Huang, C.-H., Wang, Y.-C., Shih, S.-R., 2010. Differential localization and function of PB1-F2 derived from different strains of influenza A virus. Journal of virology 84, 10051-10062. Creagh, E.M., O’Neill, L.A., 2006. TLRs, NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity. Trends in immunology 27, 352-357. Gack, M.U., Kirchhofer, A., Shin, Y.C., Inn, K.-S., Liang, C., Cui, S., Myong, S., Ha, T., Hopfner, K.-P., Jung, J.U., 2008. Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction. Proceedings of the National Academy of Sciences 105, 16743-16748. Gack, M.U., Nistal-Villán, E., Inn, K.-S., García-Sastre, A., Jung, J.U., 2010. Phosphorylation-mediated negative regulation of RIG-I antiviral activity. Journal of virology 84, 3220-3229. Gack, M.U., Shin, Y.C., Joo, C.-H., Urano, T., Liang, C., Sun, L., Takeuchi, O., Akira, S., Chen, Z., Inoue, S., 2007. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446, 916-920. 12
366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409
Gao, D., Yang, Y.-K., Wang, R.-P., Zhou, X., Diao, F.-C., Li, M.-D., Zhai, Z.-H., Jiang, Z.-F., Chen, D.-Y., 2009. REUL is a novel E3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-I. PLoS ONE 4, e5760. Hausmann, S., Marq, J.-B., Tapparel, C., Kolakofsky, D., Garcin, D., 2008. RIG-I and dsRNA-induced IFNβ activation. PLoS ONE 3, e3965. Honda, K., Yanai, H., Negishi, H., Asagiri, M., Sato, M., Mizutani, T., Shimada, N., Ohba, Y., Takaoka, A., Yoshida, N., 2005. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434, 772-777. Jiang, X., Kinch, L.N., Brautigam, C.A., Chen, X., Du, F., Grishin, N.V., Chen, Z.J., 2012. Ubiquitin-induced oligomerization of the RNA sensors RIG-I and MDA5 activates antiviral innate immune response. Immunity 36, 959-973. Kawai, T., Akira, S., 2009. The roles of TLRs, RLRs and NLRs in pathogen recognition ARTICLE. International immunology 21, 317-337. Kawai, T., Takahashi, K., Sato, S., Coban, C., Kumar, H., Kato, H., Ishii, K.J., Takeuchi, O., Akira, S., 2005. IPS-1, an adaptor triggering RIG-I-and Mda5-mediated type I interferon induction. Nature immunology 6, 981-988. Kowalinski, E., Lunardi, T., McCarthy, A.A., Louber, J., Brunel, J., Grigorov, B., Gerlier, D., Cusack, S., 2011. Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA. Cell 147, 423-435. Lee, C.-W., Jung, K., Jadhao, S., Suarez, D., 2008. Evaluation of chicken-origin (DF-1) and quail-origin (QT-6) fibroblast cell lines for replication of avian influenza viruses. Journal of virological methods 153, 22-28. Livak, K.J., Schmittgen, T.D., 2001. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2< sup>− ΔΔCT Method. methods 25, 402-408. Loo, Y.-M., Gale Jr, M., 2011. Immune signaling by RIG-I-like receptors. Immunity 34, 680-692. Magor, K.E., Miranzo Navarro, D., Barber, M.R., Petkau, K., Fleming-Canepa, X., Blyth, G.A., Blaine, A.H., 2013. Defense genes missing from the flight division. Developmental & Comparative Immunology 41, 377-388. Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., Bartenschlager, R., Tschopp, J., 2005. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167-1172. Miranzo-Navarro, D., Magor, K.E., 2014. Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin. PLoS ONE 9, e86968. Nakhaei, P., Paz, S., Hiscott, J., 2006. Through Toll-like Receptor-dependent and-independent Pathways. The Interferons: Characterization and Application. Nistal-Villán, E., Gack, M.U., Martínez-Delgado, G., Maharaj, N.P., Inn, K.-S., Yang, H., Wang, R., Aggarwal, A.K., Jung, J.U., García-Sastre, A., 2010. Negative Role of RIG-I Serine 8 Phosphorylation in the Regulation of Interferon-β Production. Journal of Biological Chemistry 285, 20252-20261. Oshiumi, H., Matsumoto, M., Hatakeyama, S., Seya, T., 2009. Riplet/RNF135, a RING finger protein, ubiquitinates RIG-I to promote interferon-β induction during the early phase of viral infection. Journal of biological chemistry 284, 807-817. Oshiumi, H., Miyashita, M., Inoue, N., Okabe, M., Matsumoto, M., Seya, T., 2010. The ubiquitin ligase Riplet is essential for RIG-I-dependent innate immune responses to RNA virus infection. Cell host & microbe 8, 496-509. 13
410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
Pichlmair, A., Schulz, O., Tan, C.P., Näslund, T.I., Liljeström, P., Weber, F., e Sousa, C.R., 2006. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates. Science 314, 997-1001. Rajsbaum, R., Albrecht, R.A., Wang, M.K., Maharaj, N.P., Versteeg, G.A., Nistal-Villán, E., García-Sastre, A., Gack, M.U., 2012. Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein. PLoS pathogens 8, e1003059. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. American Journal of Epidemiology 27, 493-497. Saito, T., Hirai, R., Loo, Y.-M., Owen, D., Johnson, C.L., Sinha, S.C., Akira, S., Fujita, T., Gale, M., 2007. Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2. Proceedings of the National Academy of Sciences 104, 582-587. Sun, Y., Ding, N., Ding, S.S., Yu, S., Meng, C., Chen, H., Qiu, X., Zhang, S., Yu, Y., Zhan, Y., 2013. Goose RIG-I functions in innate immunity against Newcastle disease virus infections. Molecular immunology 53, 321-327. Takahasi, K., Yoneyama, M., Nishihori, T., Hirai, R., Kumeta, H., Narita, R., Gale Jr, M., Inagaki, F., Fujita, T., 2008. Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses. Molecular cell 29, 428-440. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiological reviews 56, 152-179. Xu, L.-G., Wang, Y.-Y., Han, K.-J., Li, L.-Y., Zhai, Z., Shu, H.-B., 2005. VISA is an adapter protein required for virus-triggered IFN-β signaling. Molecular cell 19, 727-740. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira, K., Akira, S., Fujita, T., 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nature immunology 5, 730-737. Zeng, W., Sun, L., Jiang, X., Chen, X., Hou, F., Adhikari, A., Xu, M., Chen, Z.J., 2010. Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity. Cell 141, 315-330.
438 439
Figure legends
440
Figure 1. Expression levels of RIG-Is and CARDs in DF-1 and 293T cells.
441
(A) DF-1 cells were transfected with EGFP-tagged RIG-I and CARDs expression plasmids or
442
empty vector alone. After 24 h, the expression of all the RIG-Is and CARDs was confirmed using
443
fluorescence microscopy. (a) pEGFP-N1; (b) EGFP-hCARDs; (c) EGFP-mCARDs; (d)
444
EGFP-dCARDs; (e) EGFP-gCARDs; (f) EGFP-hRIG-I; (g) EGFP-mRIG-I; (h) EGFP-dRIG-I,
445
and (i) EGFP-gRIG-I. Scale bars = 100 μm.
446
(B) 293T cells were transfected with EGFP-tagged RIG-I and CARDs expression plasmids or
447
empty vector alone. After 24 h, the expression of RIG-Is and CARDs was confirmed using
448
fluorescence microscopy. (a) pEGFP-N1; (b) EGFP-hCARDs; (c) EGFP-mCARDs; (d) 14
449
EGFP-dCARDs; (e) EGFP-gCARDs; (f) EGFP-hRIG-I; (g) EGFP-mRIG-I; (h) EGFP-dRIG-I,
450
and (i) EGFP-gRIG-I. Scale bars = 100 μm.
451
(C) DF-1 cells were transfected with EGFP-tagged RIG-I and CARD expression plasmids or
452
empty vector alone. After 24 h, cell lysates were separated by SDS-PAGE and probed with
453
anti-EGFP and anti-GAPDH antibodies. Results are representative of two independent
454
experiments.
455
(D) 293T cells were transfected with EGFP-tagged RIG-I and CARD expression plasmids or
456
empty vector alone. After 24 h, cell lysates were separated by SDS-PAGE and probed with
457
anti-EGFP and anti-GAPDH antibodies. Results are representative of two independent
458
experiments.
459
The relative fleorescence intensity was analysed with Image-pro-plus. Data represent mean ± SEM
460
from three wells per group. *P ≤ 0.05 vs. dCARDs, #P ≤ 0.05 vs. dRIG-I, $P ≤ 0.05 vs. gRIG-I.
461
Results are representative of two independent experiments.
462 463
Figure 2. Waterfowl RIG-Is and CARDs induce the innate immune genes more strongly than do
464
mammalian RIG-Is and CARDs in DF-1 cells during IAV infection.
465
(A, B) RIG-I/CARDs-transfected DF-1 cells responded to IAV infection (MOI, 1) by increasing
466
the expression of chicken IFN-β relative to empty vector-transfected and mock treated cells. DF-1
467
cells were transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all
468
plasmid-transfected DF-1 cells were infected with SW731, WSN, or ZB07 viruses, or mock
469
treated. RNA was extracted from cells for qRT-PCR at 8 h p.i..
470
(C, D) RIG-I/CARDs-transfected DF-1 cells respond to IAV infection (MOI, 1) by increasing the
471
expression of chicken Mx relative to empty vector-transfected and mock-treated cells. DF-1 cells
472
were transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all
473
plasmid-transfected DF-1 cells were infected with SW731, WSN, or ZB07 viruses, or mock
474
treated. RNA was extracted from cells for qRT-PCR at 8 h p.i..
475
(E, F) RIG-I/CARDs-transfected DF-1 cells respond to IAV infection (MOI, 1) by increasing the
476
expression of chicken PKR relative to empty vector-transfected and mock-treated cells. DF-1 cells
477
were transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all
478
plasmid-transfected DF-1 cells were infected with SW731, WSN, or ZB07 viruses, or mock 15
479
treated. RNA was extracted from cells for qRT-PCR at 8 h p.i..
480
Data represent mean ± SEM from three wells per group. *P ≤ 0.05 vs. vector; **P ≤ 0.005 vs.
481
vector. Results are representative of two independent experiments.
482 483
Figure 3. Mammalian RIG-Is and CARDs induced type I IFN more strongly than waterfowl in
484
293T cells during IAV infection.
485
(A, B) RIG-I/CARD-transfected 293T cells responded to IAV infection (MOI, 1) with increased
486
expression of IFN-β relative to empty vector-transfected and mock-treated cells. 293T cells were
487
transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all plasmid-transfected
488
293T cells were infected with SW731, WSN, or ZB07 viruses, or mock treated. RNA was
489
extracted from cells for qRT-PCR at 8 h p.i..
490
Data represent mean ± SEM from three wells per group. αP ≤ 0.05 vs. mCARDs, βP ≤ 0.05 vs.
491
dCARDs, γP ≤ 0.05 vs. gCARDs, δP ≤ 0.05 vs. vector, aP ≤ 0.05 vs. mCARDs, bP ≤ 0.05 vs.
492
dCARDs, cP ≤ 0.05 vs. gCARDs, d P ≤ 0.05 vs. vector. Results are representative of two
493
independent experiments.
494
Figure 4. Mammalian RIG-Is and CARDs induced type I IFN more strongly than waterfowl in
495
A549 cells during IAV infection.
496
(A, B) RIG-I/CARDs-transfected A549 cells responded to IAV infection (MOI, 1) with increased
497
expression of IFN-β relative to empty vector-transfected and mock treated cells. A549 cells were
498
transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all plasmid-transfected
499
A549 cells were infected with SW731, WSN, or ZB07 viruses, or mock treated. RNA was
500
extracted from cells for qRT-PCR at 8 h p.i..
501
(C, D) RIG-I/CARDs-transfected A549 cells respond to IAV infection (MOI, 1) with increased
502
expression of Mx relative to empty vector-transfected and mock-treated cells. A549 cells were
503
transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all plasmid-transfected
504
A549 cells were infected with SW731, WSN, or ZB07 viruses, or mock treated. RNA was
505
extracted from cells for qRT-PCR at 8 h p.i..
506
(E, F) RIG-I/CARDs-transfected A549 cells respond to IAV infection (MOI, 1) with increased
507
expression of PKR relative to empty vector-transfected and mock-treated cells. A549 cells were
508
transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all plasmid-transfected 16
509
A549 cells were infected with SW731, WSN, or ZB07 viruses, or mock treated. RNA was
510
extracted from cells for qRT-PCR at 8 h p.i..
511
Data represent mean ± SEM from three wells per group. αP ≤ 0.05 vs. mCARDs, βP ≤ 0.05 vs.
512
dCARDs, γP ≤ 0.05 vs. gCARDs, δP ≤ 0.05 vs. vector, aP ≤ 0.05 vs. mCARDs, bP ≤ 0.05 vs.
513
dCARDs, cP ≤ 0.05 vs. gCARDs, d P ≤ 0.05 vs. vector. Results are representative of two
514
independent experiments.
515 516
Figure 5. Waterfowl RIG-Is and CARDs reduce IAV replication more efficiently than mammalian
517
RIG-Is in DF-1 cells.
518
(A, B) DF-1 cells were transfected with EGFP-tagged plasmids or control vector, and, 24 h later,
519
infected with SW731 at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell cultures
520
were determined as TCID 50.
521
(C, D) DF-1 cells were transfected with EGFP-tagged plasmids or control vector. Then, 24 h later,
522
they were infected with WSN at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell
523
cultures were determined as TCID 50.
524
(E, F) DF-1 cells were transfected with EGFP-tagged plasmids or control vector. Then, 24 h later,
525
they were infected with ZB07 at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell
526
cultures were determined as TCID 50.
527
Data represent mean ± SEM from three wells per group. *P ≤ 0.05 vs. vector, **P ≤ 0.005 vs.
528
vector. Results are representative of two independent experiments.
529 530
Figure 6. Mammalian RIG-I interfere with replication of IAV more efficiently than waterfowl
531
RIG-I in A549 cells.
532
(A, B) A549 cells were transfected with EGFP-tagged plasmids or control vector. Then, 24 h later,
533
they were infected with SW731 at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell
534
cultures were determined as TCID50.
535
(C, D) A549 cells were transfected with EGFP-tagged plasmids or control vector. Then, 24 h later,
536
they were infected with WSN at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell
537
cultures were determined as TCID50.
538
(E, F) A549 cells were transfected with EGFP-tagged plasmids or control vector. Then, 24 h later, 17
539
they were infected with ZB07 at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell
540
cultures were determined as TCID50.
541
Results were analyzed with the independent sample t test (n=3). Data represent mean ± SEM from
542
three wells per group. *P ≤ 0.05 vs. vector, **P ≤ 0.005 vs. vector, #P ≤ 0.05 vs. dCARDs, $P ≤
543
0.05 vs. dRIG-I. Results are representative of two independent experiments.
544
18
1
JGV paper no. VIR/2014/069914
2
Title: RIG-I from waterfowl and mammals have different abilities to induce antiviral responses
3
against influenza A viruses
4
Running title: Various RIG-Is have different antiviral abilities on IAV
5
Subject Category: Animal Viruses-Negative-strand RNA
6
Word count: 5297
7
Authors: Qiang Shao, Wenpin Xu, Qiang Guo, Li Yan, Lei Rui, Jinhua Liu, Yaofeng
8
Zhao & Zandong Li
9
Affiliation: Correspondence to Zandong Li, PhD, [email protected]
10
Qiang Shao and Wenpin Xu contributed equally to this work.
11
State key Laboratory for Agrobiotechnology, College of Biological Sciences, China
12
Agricultural University, No.2 Yuan Ming Yuan West Road, Beijing, 100193, China
13
Tel.: 86-10-62732144. Fax: 86-10-62895439
14
Abstract
15
Retinoic acid-induced gene I (RIG-I), plays a crucial role in sensing viral RNA and facilitating the
16
production of IFN-β. It varies in length and sequence among different species. The present study
17
assessed the functional differences among RIG-I proteins derived from mammals and birds. The
18
transfection of duck caspase recruitment domains (CARDs) and RIG-I (dCARDs and dRIG-I) and
19
goose CARDs and RIG-I (gCARDs and gRIG-I) into DF-1 cells increased the production of
20
IFN-β mRNA and IFN-stimulated genes and decreased influenza A virus (IAV) replication,
21
whereas, human CARDs and RIG-I (hCARDs and hRIG-I) and mouse CARDs and RIG-I
22
(mCARDs and mRIG-I) had no effect. In 293T and A549 cells, hCARDs had the strongest
23
IFN-inducing activity, followed by mCARDs, dCARDs, and gCARDs. The IFN-inducing activity
24
of hRIG-I was stronger than that of mRIG-I, dRIG-I, and gRIG-I, in that order. The results also
25
showed that although the ability dCARDs to activate IFN was stronger than that of gCARDs in
26
DF-1, 293T, and A549 cells, dRIG-I had a weaker ability to activate IFN than gRIG-I in DF-1
27
with or without IAV infection. Taken together, these data suggest that RIG-I protein has different
28
amino acid sequences and functions among species. This genetic and functional diversity renders
29
it flexible, adaptable, and capable of recognizing many viruses in different species. 1
30
Key words: influenza; H1N1; H9N2; RIG-I; CARDs; IFN;
31 32
Introduction
33
Type-I IFNs and other pro-inflammatory cytokines are produced when host pattern
34
recognition receptors (PRRs), such as Toll-like receptors (TLRs) and retinoic acid-induced gene I
35
(RIG-I)-like receptors (RLRs), recognize pathogen-associated molecular patterns (PAMPs)
36
including nucleic acids or other structural components of microbes (Creagh and O’Neill, 2006;
37
Kawai and Akira, 2009). RIG-I, as a cytoplasmic sensor of viral double strand RNA (dsRNA) and
38
single strand RNA (ssRNA), can be triggered by viral RNA (vRNA). RIG-I is composed of two
39
N-terminal CARDs, a central DExD/H box helicase/ATPase, and a C-terminal regulatory domain
40
(CTD/RD) (Hausmann et al., 2008). Under normal conditions, RIG-I is in an inactive, closed
41
conformation (Takahasi et al., 2008). Viral ssRNA or short dsRNA bearing a 5′-triphosphate can
42
bind to the DExD/H box helicase domain at its carboxy-terminus, which causes RIG-I to undergo
43
a conformational change that exposes the CARDs to the cytoplasm (Saito et al., 2007). The
44
exposure of the N-terminal CARDs of RIG-I leads to the Lys63-linked ubiquitination of Lys172.
45
The ubiquitinated CARDs then allow RIG-I bind to MAVS (also called IPS-1, CARDsif, or VISA)
46
(Kawai et al., 2005; Meylan et al., 2005; Xu et al., 2005). Other components of the pathway are
47
also recruited to this multi-protein signal complex, which activates TBK1 (TANK-binding kinase
48
1) and IKK (IκB kinase) (Meylan et al., 2005; Xu et al., 2005). The activation of these kinases
49
results in the phosphorylation and activation of IRF-3 and IRF-7, which form homodimers and
50
heterodimers, translocate into the nucleus and bind to IFN-stimulated response elements (ISREs),
51
to stimulate the expression of type I IFN genes and a set of IFN-inducible genes (Honda et al.,
52
2005; Nakhaei et al., 2006).
53
RIG-I has been identified in many species, including mammals (humans, mice, pigs, and
54
rabbits), birds (ducks, geese, pigeons, and zebra finches), and fish (trout and carp). The signaling
55
pathways activated by hRIG-I and mRIG-I have been well illustrated previously (Loo and Gale Jr,
56
2011). As natural reservoir of most influenza A strains, waterfowl have a crucial role in the
57
ecology and evolution of IAV (Webster et al., 1992). Similar to hRIG-I and mRIG-I, dRIG-I and
58
gRIG-I also induce type I interferon after influenza infection and poly I:C transfection (Barber et
59
al., 2010; Sun et al., 2013). Although dRIG-I and gRIG-I share similar CARDs and a helicase 2
60
domain with hRIG-I and mRIG-I, S8 and T170, which are critical for phosphorylation, and T55
61
and K172, which are essential for interaction and polyubiqutination by tripartite motif containing
62
25 (TRIM25) in humans, are not conserved in mice, ducks, or geese. Recently, Domingo et al.
63
demonstrated that although TRIM25-mediated ubiquitination occurs at K167 and K193, the
64
activation of dRIG-I is independent of the anchored ubiquitination (Miranzo-Navarro and Magor,
65
2014). These results suggest that the RIG-I signaling pathway functions differently in waterfowl
66
and mammals (Miranzo-Navarro and Magor, 2014).
67
In this study, the ability of the CARDs of RIG-I and full-length RIG-I encoded by humans,
68
mice, ducks, and geese to induce type I IFN in chicken DF-1, human A549, and human 293T cells
69
during IAV infection from mice, swine, and avian species was examined. Results showed that the
70
ability of waterfowl RIG-I to induce the expression of antiviral genes was stronger than
71
mammalian RIG-I in DF-1 cells during IAV infection. However, the ability of mammalian RIG-I
72
to induce the expression of antiviral genes was stronger than that of waterfowl RIG-I in 293T and
73
A549 cells during IAV infection. Waterfowl RIG-I interfered with the replication of IAV more
74
efficiently than mammalian RIG-I in DF-1 cells, whereas, mammalian RIG-I was more efficient
75
than waterfowl RIG-I in A549 and MDCK cells.
76 77
2. Results
78
2.1 Protein expression of RIG-I and CARDs in chicken DF-1 cells and human 293T cells.
79
EGFP-tagged RIG-I and CARDs were generated and transfected into 293T or DF-1 cells.
80
The detection of EGFP-tagged RIG-I and CARDs using fluorescence microscopy indicated that
81
significant levels of EGFP-tagged RIG-I and CARDs accumulated in 293T and DF-1 cells (Fig.
82
1A, B). The fluorescence intensity of waterfowl-derived RIG-I and CARDs was significantly
83
higher than that of mammalian-derived RIG-I and CARDs (Fig. 1A, B), respectively. In addition,
84
the expression of EGFP-tagged CARDs and RIG-I proteins derived from humans, mice, ducks,
85
and geese was detected in DF-1 (Fig. 1C) and 293T cells (Fig. 1D) using western blotting with
86
anti-EGFP antibody. Similar to the fluorescence observation, the expression of hCARDs and
87
mCARDs protein was significantly lower than that of the dCARDs and gCARDs as well as
88
hRIG-I, mRIG-I, dRIG-I, and gRIG-I in DF-1 and 293T cells. These data suggest that
89
waterfowl-derived RIG and CARDs are expressed more efficiently than mammal-derived RIG and 3
90
CARDs in both DF-1 and 293T cells.
91 92
2.2 Waterfowl RIG-Is induce the expression of antiviral genes more strongly than
93
mammalian RIG-I in DF-1 cells during IAV infection
94
To evaluate the differences in IFN-inducing activity between mammal-derived and
95
waterfowl-derived RIG-I and CARDs variants in chicken DF-1 cells, cells were transfected with
96
EGFP-tagged RIG-I and CARDs expression plasmids (Fig. 1A, C), and then infected with
97
influenza viruses or mock-treated. The mRNA expression of IFN-β and the antiviral
98
IFN-stimulated genes Mx1 and PKR were measured using qRT-PCR at 8 h post-infection (p.i.)
99
with A/Swine/Shandong/731/2009 (H1N1) (SW731), A/Chicken/Shandong/ZB/2007 (H9N2)
100
(ZB07), or A/WSN/33 (H1N1) (WSN) viruses. Transfection with dCARDs, gCARDs, dRIG-I,
101
and gRIG-I transfection alone induced significantly more IFN-β, Mx1, and PKR synthesis
102
compared with empty vector transfection with or without IAV infection (Fig. 2A–F). However,
103
hCARDs, mCARDs, hRIG-I, and mRIG-I did not increase the mRNA production of IFN-β, Mx1,
104
or PKR compared with empty vector transfection with or without IAV infection (Fig. 2A–F).
105
Transfection with EGFP-mRIG-I inhibited IFN-β, Mx1, and PKR mRNA synthesis significantly
106
only in WSN infection group (Fig. 2B, D, and F). Although dCARDs induced more IFN-β, Mx1,
107
and PKR mRNA production than did gCARDs, dRIG-I induced significant less IFN-β, Mx1, and
108
PKR mRNA production than did gRIG-I (Fig. 2A–F). ZB07, WSN, and SW731 infection was also
109
blocked the gRIG-I-mediated increase in Mx1 and PKR mRNA compared with the mock-treated
110
group. The inhibitory effects of WSN were the strongest (Fig. 2D, F). These observations suggest
111
that transfected waterfowl RIG-I and CARDs have a stronger ability to induce the expression of
112
antiviral genes than mammalian RIG-I and CARDs in DF-1 cells during IAV infection. Moreover,
113
dCARDs was more efficient in IFN-β mRNA production than gCARDs.
114 115
2.3 Mammalian RIG-Is induce the expression of antiviral genes more strongly than
116
waterfowl RIG-Is in human cells during IAV infection
117
To assess the differences in IFN-inducing activity between mammalian and waterfowl RIG-Is
118
in 293T cells, cells were transfected with EGFP-tagged RIG-I and CARDs expression plasmids
119
(Fig. 1B, D), and then infected with influenza viruses or mock-treated. The mRNA expression of 4
120
IFN-β, Mx, and PKR was detected using qRT-PCR at 8 h p.i. hCARDs and mCARDs induced
121
more IFN-β mRNA production compared with dCARDs and gCARDs in the presence or absence
122
of IAV infection (Fig. 3A). hCARDs had the strongest ability to induce IFN-β, followed by
123
mCARDs, dCARDs, and gCARDs (Fig. 3A). Similarly, hRIG-I had the most pronounced ability
124
to induce IFN-β, followed by mRIG-I, dRIG-I, and gRIG-I (Fig. 3B). Although gRIG-I
125
transfection alone increased IFN-β synthesis (Fig. 3B), it did not further increase the production of
126
IFN-β over empty vector transfection after IAV infection (Fig. 3B). Interestingly, gRIG-I blocked
127
IFN-β mRNA production during WSN or SW731 infection (Fig. 3B).
128
Because of the low expression of Mx and PKR in 293T cells (data not shown), we also
129
performed the transfections and infections in a human lung adenocarcinoma epithelial cell
130
line (A549 cells). Fig.4A and B shows that hRIG-I, mRIG-I, dRIG-I, gRIG-I, hCARDs, mCARDs,
131
dCARDs, and gCARDs had the similar IFN-β-inducing ability in A549 and 293T cells. Fig. 4C
132
and E show that hCARDs induced the highest mRNA levels of Mx and PKR, followed by
133
mCARDs, dCARDs, and gCARDs. Similarly, hRIG-I had the strongest ability to induce IFN-β,
134
followed by mRIG-I, dRIG-I, and gRIG-I (Fig. 4D, F). Although gCARDs facilitated IFN-β, Mx
135
and PKR mRNA production (Fig. 4A, C, and E), gRIG-I inhibited IFN-β, Mx and PKR mRNA
136
synthesis compared with empty vector transfection after IAV infection (Fig. 4B, D, and F).
137
Taken together, these data suggest that the transfected mammalian RIG-Is and CARDs had a
138
stronger ability to induce the expression of antiviral genes in 293T and A549 cells during IAV
139
infection than did waterfowl. In addition, gRIG-I could negatively regulate IFN-β signaling
140
pathway in 293T and A549 cells during IAV infection.
141 142
2.4 Waterfowl RIG-Is reduce the replication of IAV more efficiently than mammalian
143
RIG-Is in DF-1 cells
144
We next evaluated the effects of all the CARDs and RIG-Is on viral replication in DF-1 cells.
145
Cells were transfected with EGFP-tagged RIG-I and CARDs plasmids or empty vector alone, and
146
then were challenged with ZB07, WSN, or SW731 at a multiplicity of infection (MOI) of 0.01
147
(Fig. 5). At 24 and 48 h p.i., the viral titers in the cell cultures were determined as 50% tissue
148
culture infectious doses (TCID50/ml). No differences were observed between the ZB07, WSN, or
149
SW731 viral titers in hCARDs-, mCARDs-, hRIG-I-, mRIG-I-, or empty vector-transfected DF-1 5
150
cells at 24 or 48 h p.i. (Fig. 5A–F). However, significantly decreased ZB07, WSN, and SW731
151
viral titers were observed in dCARDs-, gCARDs-, dRIG-I-, and gRIG-I-transfected DF-1cells
152
compared with empty vector transfected DF-1 cells at both 24 and 48 h p.i. (Fig. 5A–F). Similar to
153
the observations regarding IFN-inducing activity, dCARDs, gCARDs decreased the ZB07, WSN,
154
and SW731 viral titers to a lower level than dRIG-I and gRIG-I in DF-1 cells (Fig. 5A–F).
155
Although dCARDs induced more IFN-β mRNA than did gCARDs did, there were no difference
156
between the ZB07 and WSN viral titers on in dCARDs- and gCARDs- transfected DF-1 cells.
157
dCARDs decreased SW731 titers to a lower level than did gCARDs (Fig. 5A). Taken together,
158
these results suggest that waterfowl RIG-Is reduce IAV replication more efficiently than
159
mammalian RIG-Is in DF-1 cells, and gCARDs have a weaker ability to inhibit replication of IAV
160
than dCARDs.
161 162
2.5 Mammalian RIG-I reduces IAV replication more efficiently than waterfowl RIG-I in
163
A549 cells
164
The effects of mammalian and waterfowl RIG-Is on viral multiplication was evaluated
165
further in A549 cells. Cells were transfected with EGFP-tagged CARDs and RIG-I expression
166
plasmids or empty vector alone, and were then challenged with ZB07, WSN, or SW731 at an MOI
167
of 0.01. The viral titers were determined as TCID50 at 24 and 48 h p.i.. hCARDs and mCARDs
168
exhibited the most significant inhibitory effects on the multiplication of ZB07, WSN, and SW731
169
at both 24 and 48 h (Fig. 6A, C, and E). In contrast, dCARDs only slightly decreased the of WSN
170
and SW731 viral titers at 24 h and ZB07 titers at 24 and 48 h (Fig. 6A, C, and E). No differences
171
were observed between the ZB07, WSN, or SW731 viral titers in A549 cells transfected with
172
gCARDs compared with empty vector at 24 or 48 h (Fig. 6A, C, and E). Consistent with hCARDs
173
and mCARDs, hRIG-I and mRIG-I suppressed the replication of the three influenza viruses at 24
174
and 48 h (Fig. 6B, D, and F). No inhibitory effects on viral titers were observed in dRIG-I- and
175
gRIG-I-transfected cells. Similar results were also observed in transfection and viral multiplication
176
experiments performed in Madin-Darby canine kidney cells (MDCK cells) (Supplementary Fig. 1).
177
These data suggest that mammalian RIG-I and CARDs reduce the replication of IAV in
178
mammalian cells more efficiently than do waterfowl RIG-I and CARDs.
179 6
180
3. Discussion
181
Here we examined the differences in the protein expression of RIG-I and CARDs proteins
182
derived from mammals and birds, as well as their IFN-inducing activity and ability to suppress the
183
replication of influenza viruses in chicken and human cells. QRT-PCR was used to analyze the
184
ability of various RIG-Is and CARDs variants to induce IFN expression in DF-1, 293T, and A549
185
cells. Waterfowl RIG-I and CARDs had a more pronounced ability to induce the expression of
186
antiviral genes than did mammalian in DF-1 cells after IAV infection. Conversely, mammalian
187
RIG-I and CARDs had a more pronounced ability to induce the expression of antiviral genes than
188
did waterfowl RIG-I and CARDs in 293T and A549 cells during IAV infection. Data revealed that
189
waterfowl RIG-I interfered with the replication of IAV more efficiently than did mammalian
190
RIG-I in DF-1 cells, whereas, mammalian RIG-I reduced replication of IAV more efficiently than
191
did waterfowl RIG-I in A549 and MDCK cells. Therefore, RIG-I from waterfowl and mammals
192
had different abilities to induce an antiviral response against IAV.
193
Previous studies revealed that duck and goose CARDs domains activate IFN-β in chicken
194
cells, suggesting that chicken cells contain all the proteins required for the activation and
195
downstream signaling of RIG-I (Miranzo-Navarro and Magor, 2014; Sun et al., 2013). Recent in
196
vitro studies demonstrated that human TRIM25 does not interact with, ubiquitinate, or activate
197
human CARDs domains that were co-transfected into chicken DF-1 cells (Miranzo-Navarro and
198
Magor, 2014). Similarly, dCARDs, gCARDs, dRIG-I, and gRIG-I activated IFN-β production in
199
chicken DF-1 cells, whereas hCARDs, mCARDs, hRIG-I, and mRIG-I did not. In the current
200
study, this suggests that only waterfowl-derived RIG-I are functional in chicken DF-1 cells, and
201
that some factors necessary for mammal-derived RIG-I activation are missing or unrecognizable
202
in chicken cells. For example, RIPLET, which ubiquitinates Lys849 and Lys851 in the CTD of
203
RIG-I (Gao et al., 2009; Oshiumi et al., 2009; Oshiumi et al., 2010), is absent from the chicken
204
and duck genomes (Magor et al., 2013; Rajsbaum et al., 2012).
205
SW731, WSN, and ZB07 have different IFN-inducing activity (Fig.2A and B; Fig. 3A and B).
206
Sequence alignment showed that three viruses have different length of non-structural
207
protein1(NS1) (SW731, 219 amino acid (aa); WSN, 230 aa; ZB07, 217 aa) (data not shown). The
208
most pronounced IFN synthesis of ZB07 infected DF-1 and 293T cells (Fig.2A and B; Fig. 3A and
209
B), may due to the weak suppression of IFN by NS1. Otherwise, mRIG-I, but not mCARD, 7
210
blocked the IFN-β, Mx, and PKR mRNA synthesis only in WSN infected DF-1 cells (Fig.2A–F)..
211
Previously study reported that only avian influenza virus (AIV) derived non-structural protein1
212
(NS1) can bind to chicken TRIM25, thereby suppressing the induction of IFN (Rajsbaum et al.,
213
2012). Mouse-adapted IAV NS1 binds to mouse RIPLET specifically in mouse cells (Rajsbaum et
214
al., 2012). NS1 or other viral protein of WSN might specifically interact with mRIG-I to suppress
215
the induction of IFN in DF-1 cells.
216
Although gRIG-I induced slightly more IFN-β production in DF-1 cells than did dRIG-I with
217
or without IAV infection, the expression of gRIG-I induced significantly more Mx and PKR
218
expression (Fig. 2B, D, and F). This suggests that gRIG-I might induce Mx and PKR in
219
IFN-independent manner. Interestingly, dCARDs share 92.98% amino acid identity with gCARDs;
220
however, dCARDs induced significantly more IFN-β production and IFN-stimulated gene
221
expression than did gCARDs in both DF-1 and 293T cells with or without IAV infection. An
222
alignment of dRIG-I and gRIG-I sequences revealed that the phosphorylation sites for inactivation
223
are not conserved between ducks (S8) and geese (G8). It was reported that the phosphorylation of
224
S8 negatively regulates hRIG-I, possibly by preventing TRIM25 binding (Gack et al., 2010;
225
Nistal-Villán et al., 2010). However, it is unclear whether the S8G mutantion in gRIG-I is
226
responsible for its pronounced IFN-inducing activity.
227
Recent in vitro studies demonstrated that dRIG-I interacts with the unanchored K63-linked
228
polyubiquitin chains generated by hTRIM25 in 293T cells (Miranzo-Navarro and Magor, 2014).
229
Sun et al. demonstrated that NDV infection in gRIG-I-transfected 293T/17 cells resulted in
230
upregulated activity of the IFN-β promoter and IRF-3 and IFIT1 mRNA levels, but decreased viral
231
titers (Sun et al., 2013). Consistent with these reports, dCARDs, gCARDs, dRIG-I, and gRIG-I
232
were promoted IFN expression in mock-treated 293T and A549 cells in the current study.
233
However, the ability of dCARDs and gCARDs to induce IFN production was significantly less
234
pronounced than that of hCARDs and mCARDs. The ability of dRIG-I and gRIG-I to induce IFN
235
production was also reduced significantly compared with hRIG-I and mRIG-I. Surprisingly,
236
gRIG-I blocked IFN-β, Mx1, and PKR mRNA production during influenza infection, which is
237
almost the opposite effect reported for gRIG-I in 293T cells after NDV infection (Sun et al., 2013).
238
One possible cause of this inhibitory effect is that gRIG-I could compete with endogenous hRIG-I
239
to bind influenza vRNA. However, gRIG-I could not activate the IFN pathway as effectively as 8
240
could hRIG-I, which resulted in competitive inhibition.
241
Although mRIG-I protein has 76.57% amino acid identity with hRIG-I, hRIG-I induced
242
significantly more IFN-β production in 293T and A549 cells compared with mRIG-I with or
243
without IAV infection. mCARDs also share 75.09% amino acid identity with hCARDs, but
244
hCARDs induced significantly more IFN production and IFN-stimulated gene expression in 293T
245
and A549 cells than did mCARDs regardless of IAV infection. In the current study, hCARDs and
246
dCARDs have conserved phosphorylation sites (S8 and S168 in duck, S8 and T170 in human), but
247
mCARDs contain N8 and V170. Moreover, T55, which plays a critical role in the interaction, is
248
displaced by serine in mCARDs (Gack et al., 2008). K172, the site that is ubiquitinated by
249
TRIM25 in humans, is not conserved in mRIG-I. This might be responsible for the fact that
250
mCARDs and mRIG-I had reduced ability to induce IFN than did hCARDs and hRIG-I (Jiang et
251
al., 2012; Zeng et al., 2010).
252
In the current study, the effects of various RIG-Is and CARDs variants on virus replication
253
were evaluated in DF-1, A549, and MDCK cells. ZB07, WSN, and SW731 titers in hCARDs-,
254
mCARDs-, hRIG-I-, and mRIG-I-transfected DF-1 cells confirmed the inability of hCARDs,
255
mCARDs, hRIG-I, and mRIG-I to function in chicken DF-1 cells. The inability of dRIG-I and
256
gRIG-I to function in MDCK cells was also demonstrated by the ZB07, WSN, and SW731 titers
257
in dRIG-I- and gRIG-I-transfected A549 and MDCK cells. This suggests that the RIG-I signaling
258
pathway has changed during evolution, possibly because of selection pressure from viruses that
259
disrupted this regulation. Both birds and mammals evolved from reptiles 200 million years ago.
260
RIG-Is derived from humans and mice were not functional in chicken DF-1 cells. Although
261
RIG-Is derived from ducks and geese are functional in human 293T cells, but they do not
262
functions as efficiently as hRIG-I. This might be due to the altered amino acid sequence of RIG-I
263
during evolution and the emergence of novel signal transducers, such as RIPLET. Nevertheless,
264
waterfowl still retain part of the original signal transduction system. Therefore, waterfowl RIG-I
265
are still partially functional in human 293T cells. Taken together, these data suggest that RIG-I can
266
only activate the IFN signaling pathway effectively in its own or very similar cellular systems
267
(dRIG-I and gRIG-I in DF-1, and hRIG-I and mRIG-I in A549, 293T, and MDCK cells). Together,
268
these results suggest that additional attention should be paid to whether transgenes from very
269
evolutionary distant species can function in alternative hosts. 9
270 271
4. Materials and methods
272
4.1 Plasmids.
273
The open reading frame (ORF) of hRIG-I was cloned from cDNA isolated from human A549
274
cells
275
TTTGGACATTTCTGCTGGATCA-3′ based on human RIG-I sequence (GenBank accession no.
276
AF038963). The mRIG-I ORF was cloned from mouse lung cDNA using the primers
277
5′-ATGACCGCGGCGCAGCGGC-3′
278
based on mRIG-I sequence (GenBank accession no. AY553221). The dRIG-I ORF was obtained
279
from
280
5′-ATGACGGCGGACGAGAAGC-3 ′ and 5 ′-CTAAAATGGTGGGTACAAGTTGGAC-3 ′ based
281
on the duck RIG-I sequence (GenBank accession no. EU363349.1). Finally, the gRIG-I ORF was
282
amplified from goose splenic cDNA using the primers 5′-ATGACGGCGGAGGAAAAGC-3′ and
283
5′-CTA AAATGGTGGGTACAAGTTGGAC-3′ based on goose RIG-I sequence (GenBank
284
accession no. JF804977). The homologous arms of pEGFP-N1 were added to the 5′ and 3′ termini
285
of human, mouse, duck, and goose full-length RIG-I and CARDs from human, mouse, duck, and
286
goose RIG-I using PCR with the primers shown in Supplementary Table 1. Human, mouse, duck,
287
and goose full-length RIG-I and CARDs were cloned into mammalian expression vector
288
pEGFP-N1 by homologous recombination in accordance with the manufacturer’s instructions
289
(NovoRec).
290
4.2 Viruses.
using
duck
the
(A.
primers
5 ′-ATGACCACCGAGCAGCGAC-3′
and
5 ′-CTA
platyrhynchos)
and
5′-CTA
TACGGACATTTCTGCAGGATCGA-3 ′
splenic
cDNA
using
the
primers
291
The viruses were amplified in chicken embryos. The titers were determined as TCID50 by
292
monitoring the cytopathic effect (CPE) of endpoint dilutions on MDCK cells in the presence of 1
293
μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Worthington Biochemical,
294
Lakewood, NJ, USA.). All experiments with virus were performed under biosafety level 2 (BSL-2)
295
conditions with investigators wearing appropriate protective equipment and complying with
296
general biosafety standards in the microbiological and biomedical laboratories of Ministry of
297
Health of the People’s Republic of China (WS 233-2002).
298
4.3 Cell culture, transfections, and infections.
299
DF-1, 293T, A549, and MDCK cells were maintained in Dulbecco’s modified Eagle’s 10
300
medium (DMEM), supplemented with 0.6 μg/ml penicillin, 60 μg/ml streptomycin and 10% fetal
301
bovine serum (FBS) (Gibco), in an atmosphere of 5% CO2 at 37°C. For infection of transfected
302
cells, 2.5 × 105 DF-1, 293T, A549, or MDCK cells were seeded into 24-well plates and
303
maintained in DMEM plus 10% FBS overnight. DF-1, 293T, and MDCK cells were transfected
304
with 800 ng of EGFP-tagged plasmids with Lipofectamine 2000 (Invitrogen). A549 cells were
305
transfected with EGFP-tagged plasmids using FuGENE® HD (Promega). Then, 24 h p.i., cells
306
were infected
307
μg/ml)TPCK-treated trypsin was used for infection at in view of the trypsin sensitivity of DF-1
308
and 293T cells, compared with 0.5 μg/ml in A549, and 2 μg/ml in MDCK cells (Lee et al., 2008).
309
4.4 Western blotting.
with SW731, WSN, or ZB07
viruses.
A lower concentration (0.1
310
293T and DF-1 cells were lysed in RIPA buffer containing 20 mM Tris base-HCl (pH 7.5),
311
150 mM NaCl, 1.0 mM EDTA (pH 8.0), 1.0 M megta (pH 8.0), 0.1% SDS, 0.5% DOC, 1% NP-40,
312
protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Roche). Proteins were
313
separated using SDS-PAGE under reducing conditions and analyzed using western blotting with
314
anti-EGFP (Beijing B&M Biotech Co., Ltd, Beijing, China) and anti-GAPDH (Beijing CoWin
315
Biotech, Beijing, China) antibodies followed by HRP-labeled secondary antibodies (BIO-RAD,
316
USA) (Chen et al., 2010).
317
4.5 Quantitative real-time PCR (qRT-PCR)
318
Total RNA was extracted using TRIzol (Invitrogen) and treated with DNase I (Promega) to
319
remove the contaminating DNA. Then, 1 μg of RNA was reverse-transcribed into cDNA using a
320
GoScript reverse transcription system (Promega) in a 20-μl reaction mixture. The cDNA was
321
analyzed using qRT-PCR with SYBR green Master I (Roche). The primers specific for chicken
322
GAPDH, IFN-β, Mx, and PKR have been described previously (Barber et al., 2010). The specific
323
primers for human GAPDH, IFN-β, Mx, and PKR were designed using Primer Express 3.0 and are
324
shown in Supplementary Table 2. qRT-PCR was performed under the following cycling
325
conditions: 95°C for 10 min for activation, followed by 40 cycles of 95°C for 15 sec and 60°C for
326
1 min. This was followed by one cycle of 95°C for 15 sec, 60°C for 15 sec, and 95°C for 15 sec.
327
The final step was to obtain a melting curve for the PCR products to determine the specificity of
328
amplification. All control and infected samples were analyzed in triplicate on the same plate. The
329
relative mRNA abundances were calculated using the 2 -∆∆Ct method with GAPDH as a reference 11
330
and plotted as fold change compared with mock-control samples (Livak and Schmittgen, 2001).
331
4.6 Measurement of virus growth in avian and mammalian cells.
332
DF-1, A549, and MDCK cells transfected with EGFP-tagged plasmids were infected with
333
SW731,WSN, and ZB07 at an MOI of 0.01, and cell cultures were collected at different times (24
334
and 48 h) after infection. The cell culture samples were centrifuged at 5000 × g for 1 min, and the
335
supernatants were stored at −80°C until use. The viral contents in the supernatants were titrated
336
using TCID50 in MDCK cells (Reed and Muench, 1938).
337
4.7 Statistical analysis.
338
All data analyses were performed using SPSS 16.0 software. Independent sample t-tests were
339
used to determine significant differences between different plasmid-transfected groups. P values ≤
340
0.05 were considered significant.
341 342
Acknowledgments
343
This work was supported by the National Basic Research Program (973 Program, number
344
2013CB945000). We thank Dr. Zhiyi Wan for critical reading of the manuscript. We thank LetPub
345
(www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
346 347
References
348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365
Barber, M.R., Aldridge Jr, J.R., Fleming-Canepa, X., Wang, Y.-D., Webster, R.G., Magor, K.E., 2013. Identification of avian RIG-I responsive genes during influenza infection. Molecular immunology 54, 89-97. Barber, M.R., Aldridge, J.R., Webster, R.G., Magor, K.E., 2010. Association of RIG-I with innate immunity of ducks to influenza. Proceedings of the National Academy of Sciences 107, 5913-5918. Chen, C.-J., Chen, G.-W., Wang, C.-H., Huang, C.-H., Wang, Y.-C., Shih, S.-R., 2010. Differential localization and function of PB1-F2 derived from different strains of influenza A virus. Journal of virology 84, 10051-10062. Creagh, E.M., O’Neill, L.A., 2006. TLRs, NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity. Trends in immunology 27, 352-357. Gack, M.U., Kirchhofer, A., Shin, Y.C., Inn, K.-S., Liang, C., Cui, S., Myong, S., Ha, T., Hopfner, K.-P., Jung, J.U., 2008. Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction. Proceedings of the National Academy of Sciences 105, 16743-16748. Gack, M.U., Nistal-Villán, E., Inn, K.-S., García-Sastre, A., Jung, J.U., 2010. Phosphorylation-mediated negative regulation of RIG-I antiviral activity. Journal of virology 84, 3220-3229. Gack, M.U., Shin, Y.C., Joo, C.-H., Urano, T., Liang, C., Sun, L., Takeuchi, O., Akira, S., Chen, Z., Inoue, S., 2007. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446, 916-920. 12
366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409
Gao, D., Yang, Y.-K., Wang, R.-P., Zhou, X., Diao, F.-C., Li, M.-D., Zhai, Z.-H., Jiang, Z.-F., Chen, D.-Y., 2009. REUL is a novel E3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-I. PLoS ONE 4, e5760. Hausmann, S., Marq, J.-B., Tapparel, C., Kolakofsky, D., Garcin, D., 2008. RIG-I and dsRNA-induced IFNβ activation. PLoS ONE 3, e3965. Honda, K., Yanai, H., Negishi, H., Asagiri, M., Sato, M., Mizutani, T., Shimada, N., Ohba, Y., Takaoka, A., Yoshida, N., 2005. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434, 772-777. Jiang, X., Kinch, L.N., Brautigam, C.A., Chen, X., Du, F., Grishin, N.V., Chen, Z.J., 2012. Ubiquitin-induced oligomerization of the RNA sensors RIG-I and MDA5 activates antiviral innate immune response. Immunity 36, 959-973. Kawai, T., Akira, S., 2009. The roles of TLRs, RLRs and NLRs in pathogen recognition ARTICLE. International immunology 21, 317-337. Kawai, T., Takahashi, K., Sato, S., Coban, C., Kumar, H., Kato, H., Ishii, K.J., Takeuchi, O., Akira, S., 2005. IPS-1, an adaptor triggering RIG-I-and Mda5-mediated type I interferon induction. Nature immunology 6, 981-988. Kowalinski, E., Lunardi, T., McCarthy, A.A., Louber, J., Brunel, J., Grigorov, B., Gerlier, D., Cusack, S., 2011. Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA. Cell 147, 423-435. Lee, C.-W., Jung, K., Jadhao, S., Suarez, D., 2008. Evaluation of chicken-origin (DF-1) and quail-origin (QT-6) fibroblast cell lines for replication of avian influenza viruses. Journal of virological methods 153, 22-28. Livak, K.J., Schmittgen, T.D., 2001. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2< sup>− ΔΔCT Method. methods 25, 402-408. Loo, Y.-M., Gale Jr, M., 2011. Immune signaling by RIG-I-like receptors. Immunity 34, 680-692. Magor, K.E., Miranzo Navarro, D., Barber, M.R., Petkau, K., Fleming-Canepa, X., Blyth, G.A., Blaine, A.H., 2013. Defense genes missing from the flight division. Developmental & Comparative Immunology 41, 377-388. Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., Bartenschlager, R., Tschopp, J., 2005. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167-1172. Miranzo-Navarro, D., Magor, K.E., 2014. Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin. PLoS ONE 9, e86968. Nakhaei, P., Paz, S., Hiscott, J., 2006. Through Toll-like Receptor-dependent and-independent Pathways. The Interferons: Characterization and Application. Nistal-Villán, E., Gack, M.U., Martínez-Delgado, G., Maharaj, N.P., Inn, K.-S., Yang, H., Wang, R., Aggarwal, A.K., Jung, J.U., García-Sastre, A., 2010. Negative Role of RIG-I Serine 8 Phosphorylation in the Regulation of Interferon-β Production. Journal of Biological Chemistry 285, 20252-20261. Oshiumi, H., Matsumoto, M., Hatakeyama, S., Seya, T., 2009. Riplet/RNF135, a RING finger protein, ubiquitinates RIG-I to promote interferon-β induction during the early phase of viral infection. Journal of biological chemistry 284, 807-817. Oshiumi, H., Miyashita, M., Inoue, N., Okabe, M., Matsumoto, M., Seya, T., 2010. The ubiquitin ligase Riplet is essential for RIG-I-dependent innate immune responses to RNA virus infection. Cell host & microbe 8, 496-509. 13
410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
Pichlmair, A., Schulz, O., Tan, C.P., Näslund, T.I., Liljeström, P., Weber, F., e Sousa, C.R., 2006. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates. Science 314, 997-1001. Rajsbaum, R., Albrecht, R.A., Wang, M.K., Maharaj, N.P., Versteeg, G.A., Nistal-Villán, E., García-Sastre, A., Gack, M.U., 2012. Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein. PLoS pathogens 8, e1003059. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. American Journal of Epidemiology 27, 493-497. Saito, T., Hirai, R., Loo, Y.-M., Owen, D., Johnson, C.L., Sinha, S.C., Akira, S., Fujita, T., Gale, M., 2007. Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2. Proceedings of the National Academy of Sciences 104, 582-587. Sun, Y., Ding, N., Ding, S.S., Yu, S., Meng, C., Chen, H., Qiu, X., Zhang, S., Yu, Y., Zhan, Y., 2013. Goose RIG-I functions in innate immunity against Newcastle disease virus infections. Molecular immunology 53, 321-327. Takahasi, K., Yoneyama, M., Nishihori, T., Hirai, R., Kumeta, H., Narita, R., Gale Jr, M., Inagaki, F., Fujita, T., 2008. Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses. Molecular cell 29, 428-440. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiological reviews 56, 152-179. Xu, L.-G., Wang, Y.-Y., Han, K.-J., Li, L.-Y., Zhai, Z., Shu, H.-B., 2005. VISA is an adapter protein required for virus-triggered IFN-β signaling. Molecular cell 19, 727-740. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira, K., Akira, S., Fujita, T., 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nature immunology 5, 730-737. Zeng, W., Sun, L., Jiang, X., Chen, X., Hou, F., Adhikari, A., Xu, M., Chen, Z.J., 2010. Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity. Cell 141, 315-330.
438 439
Figure legends
440
Figure 1. Expression levels of RIG-Is and CARDs in DF-1 and 293T cells.
441
(A) DF-1 cells were transfected with EGFP-tagged RIG-I and CARDs expression plasmids or
442
empty vector alone. After 24 h, the expression of all the RIG-Is and CARDs was confirmed using
443
fluorescence microscopy. (a) pEGFP-N1; (b) EGFP-hCARDs; (c) EGFP-mCARDs; (d)
444
EGFP-dCARDs; (e) EGFP-gCARDs; (f) EGFP-hRIG-I; (g) EGFP-mRIG-I; (h) EGFP-dRIG-I,
445
and (i) EGFP-gRIG-I. Scale bars = 100 μm.
446
(B) 293T cells were transfected with EGFP-tagged RIG-I and CARDs expression plasmids or
447
empty vector alone. After 24 h, the expression of RIG-Is and CARDs was confirmed using
448
fluorescence microscopy. (a) pEGFP-N1; (b) EGFP-hCARDs; (c) EGFP-mCARDs; (d) 14
449
EGFP-dCARDs; (e) EGFP-gCARDs; (f) EGFP-hRIG-I; (g) EGFP-mRIG-I; (h) EGFP-dRIG-I,
450
and (i) EGFP-gRIG-I. Scale bars = 100 μm.
451
(C) DF-1 cells were transfected with EGFP-tagged RIG-I and CARD expression plasmids or
452
empty vector alone. After 24 h, cell lysates were separated by SDS-PAGE and probed with
453
anti-EGFP and anti-GAPDH antibodies. Results are representative of two independent
454
experiments.
455
(D) 293T cells were transfected with EGFP-tagged RIG-I and CARD expression plasmids or
456
empty vector alone. After 24 h, cell lysates were separated by SDS-PAGE and probed with
457
anti-EGFP and anti-GAPDH antibodies. Results are representative of two independent
458
experiments.
459
The relative fleorescence intensity was analysed with Image-pro-plus. Data represent mean ± SEM
460
from three wells per group. *P ≤ 0.05 vs. dCARDs, #P ≤ 0.05 vs. dRIG-I, $P ≤ 0.05 vs. gRIG-I.
461
Results are representative of two independent experiments.
462 463
Figure 2. Waterfowl RIG-Is and CARDs induce the innate immune genes more strongly than do
464
mammalian RIG-Is and CARDs in DF-1 cells during IAV infection.
465
(A, B) RIG-I/CARDs-transfected DF-1 cells responded to IAV infection (MOI, 1) by increasing
466
the expression of chicken IFN-β relative to empty vector-transfected and mock treated cells. DF-1
467
cells were transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all
468
plasmid-transfected DF-1 cells were infected with SW731, WSN, or ZB07 viruses, or mock
469
treated. RNA was extracted from cells for qRT-PCR at 8 h p.i..
470
(C, D) RIG-I/CARDs-transfected DF-1 cells respond to IAV infection (MOI, 1) by increasing the
471
expression of chicken Mx relative to empty vector-transfected and mock-treated cells. DF-1 cells
472
were transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all
473
plasmid-transfected DF-1 cells were infected with SW731, WSN, or ZB07 viruses, or mock
474
treated. RNA was extracted from cells for qRT-PCR at 8 h p.i..
475
(E, F) RIG-I/CARDs-transfected DF-1 cells respond to IAV infection (MOI, 1) by increasing the
476
expression of chicken PKR relative to empty vector-transfected and mock-treated cells. DF-1 cells
477
were transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all
478
plasmid-transfected DF-1 cells were infected with SW731, WSN, or ZB07 viruses, or mock 15
479
treated. RNA was extracted from cells for qRT-PCR at 8 h p.i..
480
Data represent mean ± SEM from three wells per group. *P ≤ 0.05 vs. vector; **P ≤ 0.005 vs.
481
vector. Results are representative of two independent experiments.
482 483
Figure 3. Mammalian RIG-Is and CARDs induced type I IFN more strongly than waterfowl in
484
293T cells during IAV infection.
485
(A, B) RIG-I/CARD-transfected 293T cells responded to IAV infection (MOI, 1) with increased
486
expression of IFN-β relative to empty vector-transfected and mock-treated cells. 293T cells were
487
transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all plasmid-transfected
488
293T cells were infected with SW731, WSN, or ZB07 viruses, or mock treated. RNA was
489
extracted from cells for qRT-PCR at 8 h p.i..
490
Data represent mean ± SEM from three wells per group. αP ≤ 0.05 vs. mCARDs, βP ≤ 0.05 vs.
491
dCARDs, γP ≤ 0.05 vs. gCARDs, δP ≤ 0.05 vs. vector, aP ≤ 0.05 vs. mCARDs, bP ≤ 0.05 vs.
492
dCARDs, cP ≤ 0.05 vs. gCARDs, d P ≤ 0.05 vs. vector. Results are representative of two
493
independent experiments.
494
Figure 4. Mammalian RIG-Is and CARDs induced type I IFN more strongly than waterfowl in
495
A549 cells during IAV infection.
496
(A, B) RIG-I/CARDs-transfected A549 cells responded to IAV infection (MOI, 1) with increased
497
expression of IFN-β relative to empty vector-transfected and mock treated cells. A549 cells were
498
transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all plasmid-transfected
499
A549 cells were infected with SW731, WSN, or ZB07 viruses, or mock treated. RNA was
500
extracted from cells for qRT-PCR at 8 h p.i..
501
(C, D) RIG-I/CARDs-transfected A549 cells respond to IAV infection (MOI, 1) with increased
502
expression of Mx relative to empty vector-transfected and mock-treated cells. A549 cells were
503
transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all plasmid-transfected
504
A549 cells were infected with SW731, WSN, or ZB07 viruses, or mock treated. RNA was
505
extracted from cells for qRT-PCR at 8 h p.i..
506
(E, F) RIG-I/CARDs-transfected A549 cells respond to IAV infection (MOI, 1) with increased
507
expression of PKR relative to empty vector-transfected and mock-treated cells. A549 cells were
508
transfected with EGFP-tagged plasmids or empty vector. Then, 24 h later, all plasmid-transfected 16
509
A549 cells were infected with SW731, WSN, or ZB07 viruses, or mock treated. RNA was
510
extracted from cells for qRT-PCR at 8 h p.i..
511
Data represent mean ± SEM from three wells per group. αP ≤ 0.05 vs. mCARDs, βP ≤ 0.05 vs.
512
dCARDs, γP ≤ 0.05 vs. gCARDs, δP ≤ 0.05 vs. vector, aP ≤ 0.05 vs. mCARDs, bP ≤ 0.05 vs.
513
dCARDs, cP ≤ 0.05 vs. gCARDs, d P ≤ 0.05 vs. vector. Results are representative of two
514
independent experiments.
515 516
Figure 5. Waterfowl RIG-Is and CARDs reduce IAV replication more efficiently than mammalian
517
RIG-Is in DF-1 cells.
518
(A, B) DF-1 cells were transfected with EGFP-tagged plasmids or control vector, and, 24 h later,
519
infected with SW731 at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell cultures
520
were determined as TCID 50.
521
(C, D) DF-1 cells were transfected with EGFP-tagged plasmids or control vector. Then, 24 h later,
522
they were infected with WSN at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell
523
cultures were determined as TCID 50.
524
(E, F) DF-1 cells were transfected with EGFP-tagged plasmids or control vector. Then, 24 h later,
525
they were infected with ZB07 at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell
526
cultures were determined as TCID 50.
527
Data represent mean ± SEM from three wells per group. *P ≤ 0.05 vs. vector, **P ≤ 0.005 vs.
528
vector. Results are representative of two independent experiments.
529 530
Figure 6. Mammalian RIG-I interfere with replication of IAV more efficiently than waterfowl
531
RIG-I in A549 cells.
532
(A, B) A549 cells were transfected with EGFP-tagged plasmids or control vector. Then, 24 h later,
533
they were infected with SW731 at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell
534
cultures were determined as TCID50.
535
(C, D) A549 cells were transfected with EGFP-tagged plasmids or control vector. Then, 24 h later,
536
they were infected with WSN at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell
537
cultures were determined as TCID50.
538
(E, F) A549 cells were transfected with EGFP-tagged plasmids or control vector. Then, 24 h later, 17
539
they were infected with ZB07 at an MOI of 0.01. At 24 and 48 h p.i., the viral titers in the cell
540
cultures were determined as TCID50.
541
Results were analyzed with the independent sample t test (n=3). Data represent mean ± SEM from
542
three wells per group. *P ≤ 0.05 vs. vector, **P ≤ 0.005 vs. vector, #P ≤ 0.05 vs. dCARDs, $P ≤
543
0.05 vs. dRIG-I. Results are representative of two independent experiments.
544
18
