JGV Papers in Press. Published December 17, 2014 as doi:10.1099/vir.0.000030
Journal of General Virology Influenza A virus Utilizes a Suboptimal Kozak Sequence to Fine-tune Virus Replication and Host Response --Manuscript Draft-Manuscript Number:
VIR-D-14-00300
Full Title:
Influenza A virus Utilizes a Suboptimal Kozak Sequence to Fine-tune Virus Replication and Host Response
Short Title:
A suboptimal Kozak in PB1 regulates virus replication
Article Type:
Standard
Section/Category:
Animal - Negative-strand RNA Viruses
Keywords:
Influenza a virus; segment specific noncoding region; Kozak Sequence; Virus Replication and Host Response
Corresponding Author:
tao deng Institute of Pathogen Biology CHINA
First Author:
Jingfeng Wang
Order of Authors:
Jingfeng Wang Yousong Peng Lili Zhao Mengmeng Cao Tao Hung tao deng
Manuscript Region of Origin:
CHINA
Abstract:
The segment-specific non-coding regions (NCRs) of influenza A virus RNA genome play important roles in controlling viral RNA transcription, replication and genome packaging. In this report, we present, for the first time to our knowledge, a full view of the segment-specific (NCRs) of all influenza A viruses by bioinformatics analysis. Our systematic functional analysis reveal that the eight segment-specific NCRs could differently regulate viral RNA synthesis and protein expression at both transcription and translation levels. Interestingly, a highly conserved suboptimal nucleotide at -3 position of Kozak sequence, that could down-regulate protein expression at translation level, is only present in the segment-specific NCR of PB1. By reverse genetics, we demonstrate that recombinant viruses with an optimized Kozak sequence at -3 position in PB1 results in a significant multiple-cycle replication reduction that is independent of PB1-F2 expression. Our detailed dynamic analysis of the virus infection reveals that, the mutant virus displays slightly altered dynamics from the wild type virus on both viral RNA synthesis and protein production. Furthermore, we demonstrate that the level of PB1 expression is involved in regulating type I interferon production. Together, these data reveal a novel strategy exploited by influenza A virus to fine-tune virus replication dynamics and host anti-viral response through regulating PB1 protein expression.
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1
Influenza A virus Utilizes a Suboptimal Kozak Sequence
2
to Fine-tune Virus Replication and Host Response
3
Jingfeng Wang1, Yousong Peng2, Lili Zhao1, Mengmeng Cao1, Tao Hung1, Tao
4
Deng1*
5
1 MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen
6
Biology, Chinese Academy of Medical Sciences & Peking Union Medical College,
7
Beijing, 100730, P. R. China;
8
2 College of Information Science and Engineering, Hunan University, Changsha,
9
410082, P. R. China
10 11
Running title: A suboptimal Kozak in PB1 regulates virus replication
12
Animal Viruses - Negative-strand RNA
13 14 15
Word Count: 208 (Summary) 7,301 (Text)
16 17 18 19 20 21
*Correspondence
22
[email protected] to:
Tao
Deng,
Tel/Fax:
1
0086-10-67855618;
Email:
23
Summary
24
The segment-specific non-coding regions (NCRs) of influenza A virus RNA genome
25
play important roles in controlling viral RNA transcription, replication and genome
26
packaging. In this report, we present, for the first time to our knowledge, a full view
27
of the segment-specific (NCRs) of all influenza A viruses by bioinformatics analysis.
28
Our systematic functional analysis reveal that the eight segment-specific NCRs could
29
differently regulate viral RNA synthesis and protein expression at both transcription
30
and translation levels. Interestingly, a highly conserved suboptimal nucleotide at -3
31
position of Kozak sequence, that could down-regulate protein expression at
32
translation level, is only present in the segment-specific NCR of PB1. By reverse
33
genetics, we demonstrate that recombinant viruses with an optimized Kozak sequence
34
at -3 position in PB1 results in a significant multiple-cycle replication reduction that is
35
independent of PB1-F2 expression. Our detailed dynamic analysis of the virus
36
infection reveals that, the mutant virus displays slightly altered dynamics from the
37
wild type virus on both viral RNA synthesis and protein production. Furthermore, we
38
demonstrate that the level of PB1 expression is involved in regulating type I
39
interferon production. Together, these data reveal a novel strategy exploited by
40
influenza A virus to fine-tune virus replication dynamics and host anti-viral response
41
through regulating PB1 protein expression.
42 43 44 2
45
Introduction
46
Influenza A virus is a segmented negative-sense RNA virus of Orthomyxoviridae
47
family (Palese & Sham, 2007). The virus genome consists of eight virion RNA
48
(vRNA) segments that encode at least ten proteins. The coding region of each
49
segment is flanked by the segment-specific noncoding region (NCR) and terminal
50
promoter region at both the 3′ and 5′ ends. The highly conserved promoter region is
51
formed by terminal 12 and 13 nucleotides at the 3′ and the 5′ ends that play a critical
52
role in initiating viral RNA transcription and replication (Flick et al., 1996; Fodor et
53
al., 1995; Kim et al., 1997). Adjacent to the promoter region, at both ends, are the
54
segment-specific NCRs that vary in sequence and length among different segments of
55
influenza A virus. It has previously been reported that the segment-specific NCRs of
56
influenza A virus play multiple roles in virus life cycle. They not only act as
57
cis-acting signals to regulate viral transcription, replication and protein expression
58
(Bergmann & Muster, 1996; Zheng et al., 1996; Park & Katze, 1995), but also serve
59
as part of the packaging signals during selective virus genome packaging (Zhao et al.,
60
2014; Hutchinson et al., 2010; Gog et al., 2007).
61 62
The viral ribonucleoproteins (RNPs) of influenza A virus are responsible for
63
synthesizing three viral RNA species (mRNA, cRNA and vRNA) in the nucleus of
64
infected cells. The RdRp is a heterotrimeric complex composed by three subunits
65
(PB1, PB2 and PA) (Palese & Sham, 2007; Resa-Infante et al., 2011). The PB1
66
subunit, encoded by segment 2, is the core of the RdRp (Palese & Sham, 2007). It not 3
67
only acts as the driving force for the assembly of the viral RNA polymerase but also
68
contains active sites for RNA polymerization (Biswas & Nayak, 1994; Braam et al.,
69
1983). In addition, the PB1 is responsible for binding to the viral RNA promoter to
70
initiate viral RNA transcription and replication (Gonzalez & Ortin, 1999a; Gonzalez
71
& Ortin, 1999b).
72 73
Upon influenza A virus infection, the synthesis dynamics of the three RNA species
74
(mRNA, cRNA, vRNA) and all viral proteins are tightly controlled differentially
75
(Park & Katze, 1995; Enami et al., 1985; Varich & Kaverin, 1987; Yamanaka et al.,
76
1985; Yamanaka et al., 1991; Park et al., 1999; Hatada et al., 1989). The significance
77
of such regulation is not only to produce appropriate amount of viral RNAs and
78
proteins for efficient assembly of progeny virions, but also to maintain the fine
79
balance between virus growth rate and host antiviral status (Belicha-Villanueva et al.,
80
2012). Both viral RNAs and viral proteins have been reported to be involved in
81
regulation of the host antiviral status (Rehwinkel et al., 2010; Hale et al., 2008; Talon
82
et al., 2000; Kochs et al., 2007). In addition to the main interferon antiganist NS1, the
83
RdRp was also reported to be involved in host shut-off by targeting host Pol II
84
transcription machinery (Vreede et al., 2010; Graef et al., 2010). Moreover, a short
85
non-structural peptide PB1-F2, expressed from the PB1 segment of influenza A virus,
86
was reported to be involved in modulating host antiviral status (Chen et al., 2001;
87
Wise et al., 2009). Therefore, an understanding of the mechanisms by which influenza
88
virus controls its RNA and protein expression, and the consequences of such 4
89
regulation, is of great significance in order to fully understand influenza A virus
90
replication strategies.
91 92
In the present study, we analyzed the sequences of the segment-specific NCRs of all
93
available influenza A viruses in the NCBI, and investigated the role of these NCRs in
94
differentially regulating viral RNA synthesis and protein expression. Interestingly, we
95
also demonstrated that influenza A virus exceptionally uses a suboptimal Kozak
96
sequence in segment 2 to fine-tune virus infection through modulating PB1 protein
97
expression.
98 99
Results
100
Bioinformatics analysis of the segment-specific non-coding regions of influenza A
101
viruses
102
In order to obtain a full view for segment-specific NCRs of all influenza A viruses,
103
we bioinformatically analyzed all available NCR sequences of all the eight segments
104
from NCBI Influenza Virus Resource database as recently described (Zhao et al.,
105
2014). We used H3 and N2 subtypes for the analsys of HA and NA segments. As
106
shown in Fig. 1a, the lengths and sequences at both 3′ and 5′ ends are
107
segment-specific. It can be seen that, within each segment-specific NCR, most
108
nucleotides are highly conserved, whereas, at certain nucleotide positions, the
109
identities of nucleotides are variable (most of them are transition changes). We further
110
validated these sequence logos by comparing them with the sequence logos derived 5
111
from NCR sequences that were determined by accurate NCR sequencing methods
112
(Wang & Lee, 2009; Wang & Taubenberger, 2013; Wang et al., 2014; Park et al.,
113
2013; Park et al., 2012; Su et al., 2012). We found that majorities of nucleotide
114
conservations/variations at each nucleotide position between them are generally
115
consistent (Fig. 1b), confirming that our bioinformatics analysis of the NCR
116
sequences is accurate and reasonable.
117 118
Segment-specific NCRs mediate the synthesis of viral RNAs and proteins at
119
differential levels.
120
To investigate whether these segment-specific NCRs could act as cis-acting signals to
121
differentially regulate the syntheses of viral RNAs and proteins, we examined their
122
effects in an RNP reconstitution system derived from the PR8 virus. To form an
123
active viral RNPs, the pPolI-HA plasmid was transfected into 293T cells to express
124
the HA vRNA, together with four protein expression plasmids (pcDNA-PB1,
125
pcDNA-PB2, pcDNA-PA and pcDNA-NP). The NCRs of the HA vRNA at both ends
126
in the pPolI-HA plasmid were substituted with the corresponding NCR of the other
127
seven segments, respectively (Fig. 2a). Then the levels of three RNA species were
128
examined by primer extension analysis and the levels of the HA protein expression by
129
Western blots. Fig. 2b and c showed that the different segment-specific NCR
130
substitutions of the HA-NCR led to the synthesis of the three RNA species (mRNA,
131
cRNA and vRNA) at differential levels in comparison with that of the original HA
132
template. In particular, the substitutions with the PB2-NCR led to increased vRNA 6
133
levels; the substitutions with the PB1-NCR led to increased cRNA levels and
134
decreased vRNA levels; the substitutions with the NS-NCR led to increased mRNA
135
levels (Fig. 2b and c). Meanwhile, the levels of the HA protein expression were
136
generally consistent with their corresponding mRNA levels, except for the PB1-NCR
137
substitution (Fig. 2d and e). The mRNA level produced from the PB1-NCR
138
substitution was similar to those produced from other vRNA templates, but the level
139
of protein expressed by the PB1-NCR substitution was much lower than those of the
140
others. We further validated these results using an A/Vietnam/1194/2004 (H5N1
141
subtype) RNP system, (data not shown). This result was also consistent with earlier
142
observations that PB1 NCR mediated low expression level of a reporter gene in RNP
143
reporter systems (Ma et al., 2013; Maeda et al., 2004). Together, these results suggest
144
that the segment-specific NCRs could differentially regulate viral gene expression at
145
transcription level. However, the PB1-NCR, exceptionally, could mediate a
146
significant down-regulation at translation level.
147 148
The unique, suboptimal Kozak sequence in PB1-NCR mediated low expression
149
at translation level
150
It is known that the Kozak sequence has a significant effect on protein expression of
151
eukaryotic cell at translation level (Kozak, 1986). It has been previously reported that
152
the NCR at 3′ end of the PB1 segment of the influenza virus strain A/PR/8/34
153
exceptionally contains an suboptimal “A” at the -3 position (Chen et al., 2001; Kozak,
154
1991). Our bioinformatics analysis confirmed the observation that the PB1 segments 7
155
of all influenza A viruses bear this -3A Kozak sequence in their 3′ NCR (Fig. 3a),
156
suggesting that it might be important for virus replication. In order to confirm that the
157
low expression level of the HA protein was mediated by the suboptimal Kozak
158
sequence of the PB1-NCR in our RNP reconstitution systems, we mutated PB1-NCR
159
in the pPolI-PB1-NCR-HA from suboptimal Kozak into optimal Kozak by mutating
160
the residue from -3U into -3A in the positive sense (Fig. 3b). The effects of the -3A
161
mutation on viral RNA synthesis and protein expression were then examined. The
162
result showed that the HA protein level expressed from PB1(-3A)-NCR substitution
163
was about six times higher than the levels of the protein expressed from
164
PB1(WT)-NCR (Fig. 3c and d). Meanwhile, the levels of mRNAs, vRNA and cRNA
165
synthesized from the PB1(WT)-HA and PB1(-3A)-HA templates were comparable
166
(Fig. 3e and f). These results demonstrated that the nucleotide U at the -3 position of
167
the PB1 mRNA, which weakened the strength of Kozak sequence, mediated low
168
protein expression at the translation level and had a no obvious effect on viral RNA
169
transcription and/or replication.
170 171
Recombinant viruses containing -3 U-A mutation in PB1 were attenuated,
172
independent of the presence or absence of PB1-F2.
173
In order to study the effect of the -3 U-A mutation in the context of virus infection, a
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recombinant influenza A virus WSN-PB1(-3A), bearing a U to A mutation in PB1
175
mRNA at the -3 position, was rescued in an influenza A/WSN/33 background
176
(Hoffmann et al., 2000). The growth properties of recombinant virus WSN-PB1(-3A) 8
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and wild type virus were first assessed in both MDCK cells and A549 cells (MOI,
178
0.001). The results showed that the maximum virus titer of WSN-PB1(-3A) was about
179
1 log lower than WSN-WT in both cell types (Fig. 4a and b). We further confirmed
180
this result in a seasonal influenza A virus A/Hong Kong/1968(H3N2) background, in
181
which the maximum virus titer of HK68-PB1(-3A) was about 0.7 log lower than that
182
of HK68-WT (Fig. 4c). These data indicated that the -3 U-A mutation in PB1 result in
183
virus attenuation.
184 185
Since it has been proposed that the sub-optimal Kozak consensus of PB1, with the
186
unique “U” nucleotide in the –3 position, is involved in regulating the expression of
187
PB1-F2 protein by leaky ribosomal scanning (Chen et al., 2001; Kozak, 1991; Wise et
188
al., 2011), the U to A mutation at the -3 position might be expected to result in
189
defective expression of PB1-F2 protein. In order to examine whether the attenuation
190
of WSN-PB1(-3A) was caused by defective expression of PB1-F2, we made three
191
mutations (T120C, C153G and G291A) according to the literature (Ma et al., 2013;
192
Su et al., 2012) in the coding region of both wild type and the -3 U-A mutant virus to
193
completely eliminate the PB1-F2 expression in both viruses (Fig. 4d) (Le Goffic et al.,
194
2010; Tauber et al., 2011), designated as WSN-WT-del F2 and WSN-PB1(-3A)-del
195
F2 virus. The growth curve of the viruses was then measured with infection at MOI of
196
0.001 in MDCK cells. We found, in consistent with the results that have been
197
previously reported (Wise et al., 2009; Le Goffic et al., 2010), that there were no
198
significant differences in the growth of WSN-WT and WSN-WT-del F2 virus (Fig. 9
199
4e). Similarly, the WSN-PB1(-3A)-del F2 virus showed similar growth properties to
200
WSN-PB1(-3A) virus, indicating that eliminating PB1-F2 expression did not alter
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virus replication of the WSN-PB1(-3A) virus (Fig. 4e). This result suggested that the
202
presence or absence of PB1-F2 was not responsible for attenuation of the
203
WSN-PB1(-3A) virus.
204 205
The WSN-PB1(-3A) virus showed a slightly different protein expression profile
206
from that of the WSN-WT virus
207
In order to investigate the mechanism(s) by which the -3 U-A mutation in the PB1
208
mRNA affected the efficiency of virus replication, we examined the effects of the
209
optimized PB1 Kozak sequence in the course of a single cycle infection. We first
210
examined the viral protein expression profiles in A549 cells infected with
211
WSN-PB1(-3A) and WSN-WT virus at a MOI of 3 (Fig. 5a and b). It can be seen that
212
the level of PB1 in the WSN-PB1(-3A) virus infected cells was significantly higher
213
than that in the wild type (WT) virus infected cells at early times post-infection (4
214
h.p.i.). However, the difference could not be detected at the later time point
215
post-infection (8 and 12 h.p.i.). Meanwhile, we also found that the expression of N40,
216
a shorter form of PB1 protein that could be produced from the PB1 segment (Wise et
217
al., 2009), was decreased in WSN-PB1(-3A) virus infected cells. It has been reported
218
that overexpression of N40 had a detrimental effect on virus replication in cell culture
219
(Tauber et al., 2011). Therefore, the reduced N40 expression was unlikely to
220
contribute the replication reduction observed with the WSN-PB1(-3A) virus. In 10
221
addition, we also detected slightly higher levels of PA and NP in the WSN-PB1(-3A)
222
virus infected cells than those in WT virus infected cells at both time points examined
223
(Fig. 5a), although only the NP was statistically significant (Fig. 5b).
224 225
To further investigate the replication dynamics of the two viruses, we also performed
226
immunofluorescence assay with anti-NP antibody during the course of their single
227
cycle infection (MOI, 2). It showed that the nuclear export of NP occurred earlier in
228
the WSN-PB1(-3A) virus infected cells than that in the WSN-WT virus infected cells
229
(data not shown). These results suggested that the replication cycle of the
230
WSN-PB1(-3A) virus is slightly accelerated in comparison with that of the WSN-WT
231
virus during a single cycle infection.
232 233
Enhanced PB1 expression altered the RNA synthesis dynamics of WSN-PB1(-3A)
234
virus.
235
Since the PB1 is the core subunit of the viral RNA polymerase, the -3 U-A mutation
236
in PB1 mRNA could alter the level of PB1 expression, and subsequently could affect
237
secondary viral RNA syntheses. To further demonstrate the possible mechanism(s)
238
that could cause the attenuation of WSN-PB1(-3A) virus, we performed primer
239
extension analysis to measure the RNA synthesis dynamics of the three viral RNA
240
species of the NA segment in a single cycle infection of the two viruses (Fig. 5c).
241
Marking 6 hours post-infection as a turning point, the rate of synthesis of mRNA,
242
cRNA and vRNA in cells infected with WSN-PB1(-3A) virus was slightly higher than 11
243
that of the wild type before 6 hours post-infection (Fig. 5d). But after that, we found
244
that the levels of mRNA and cRNA fell faster in cells infected with WSN-PB1(-3A)
245
virus than those infected with WSN-WT virus (Fig. 5d). Meanwhile, although the
246
vRNA level in the two virus infected cell continued to increase after 6 hours
247
post-infection, the rate of vRNA synthesis in cells infected with WSN-PB1(-3A) virus
248
also declined faster than that in WSN-WT infected cells (Fig. 5d). Overall, these data
249
demonstrated that enhanced expression of PB1 protein in the early stage of infection
250
resulted in slightly different RNA synthesis dynamics of WSN-PB1(-3A) virus from
251
that of the WSN-WT virus.
252 253
PB1 -3 U-A mutant virus could induce type I interferon production at a higher
254
level than wild type virus.
255
While our research was in progress, Belicha-Villanueva et al. reported that enhanced
256
expression of PB1 increased the number of immunostimulatory RNAs and the
257
production of type I IFN during virus infection (Belicha-Villanueva et al., 2012). So
258
we speculated that PB1 -3 U-A mutant virus might resulted in higher type I IFN
259
production than wild type virus during infection. To answer this question, we first
260
tested the replication properties of WSN-WT/WSN-PB1(-3A) and HK68-WT/
261
HK68-PB1(-3A) viruses in Vero cells, an interferon (IFN) secretion defective cell line.
262
Interestingly, the results showed that virus titers differences between WT and PB1 -3
263
U-A mutant viruses in Vero cells became much smaller than that in MDCK or A549
264
cells (Fig. 6a and 6b). These results suggested that the infection of PB1(-3A) viruses 12
265
may induce more typeⅠIFN than that of WT viruses, which could account for the
266
attenuation of the PB1(-3A) viruses. Meanwhile, due to the fact that replication
267
efficiency of the PB1-3 U-A mutant virus was increased in Vero cells, we could
268
exclude a possibility that the -3 U-A mutation, located in the packaging signal of PB1,
269
might affect the virus replication efficiency in MDCK or A549 cells by affecting PB1
270
segment packaging efficiency.
271 272
To further investigate the properties of the two viruses, 293T cells - transfected with
273
an IFN-β luciferase reporter plasmid, were infected by WSN-WT and WSN-PB1(-3A)
274
virus at MOI of 1, and the luciferase activity of cells were measured 12 hours
275
post-infection. The results showed that infection with WSN-PB1(-3A) virus resulted
276
in an increase of IFN-β promoter activity compared to that with WSN-WT virus
277
infection (Fig. 6c). We also confirmed this result by measuring the endogenous IFN-β
278
mRNA levels in the two virus infected cells respectively by RT-PCR (data not shown).
279
In addition, to elucidate the above result, the IFN-β luciferase promoter assay was
280
performed in 293T cells transfected with the RNP reconstitution system with
281
gradually increasing amount of PB1 expression plasmids. Meanwhile, the levels of
282
three RNA species synthesized by corresponding RNP were also examined by a
283
primer extension assay. It can be seen that the IFN-β promoter was activated to a level
284
in a PB1 dose-dependent manner, whereas no activation of the IFN-β promoter was
285
observed when the PB1 expressing plasmid or HA RNA expression plasmid was
286
omitted (Fig. 6d). Correspondingly, the levels of three RNA species were also 13
287
increased with the increasing amount of the transfected PB1 expressing plasmid (Fig.
288
6e and f), suggesting that viral RNAs acted as the immunostimulatory RNAs to
289
induce type I IFN production in RNP reconstituted cells. Together, these data
290
demonstrated that enhanced PB1 expression in infected cells could induce more
291
IFN-β production.
292 293
Discussion
294
It is known that the segment-specific NCRs of influenza A virus contain critical
295
signals required for transcription, replication and packaging of each viral gene
296
segment. In this report, through our systematic bioinformatics analysis, we present,
297
for the first time to our knowledge, a full view of the segment-specific NCR of all
298
influenza A viruses, which not only fully summarizes the genetic characteristics of the
299
segment-specific
300
conservations/variations at each nucleotide position as a results of natural selections
301
during long-term virus evolution.
NCR,
but
also
directly
shows
the
nucleotide
302 303
We also studied the roles of the segment-specific NCRs in differentially regulating
304
both viral RNA synthesis and protein production in influenza RNP reconstitution
305
systems. At the levels of viral RNA transcription and replication, we observed that the
306
segment-specific NCR of NS segment particularly mediated high level of mRNA
307
production (Fig. 2b and c). This is actually consistent with the fact that high level of
308
NS mRNA is naturally needed because it not only acts as template directly to produce 14
309
the NS1 protein, but also used as pre-mRNA to be spliced into NEP (Nuclear Export
310
Protein) mRNA (Hale et al., 2008). In addition, we found that the NCR of PB2
311
mediated high level of vRNA production, and the NCR of PB1 mediated relative low
312
level of vRNA production but higher levels of cRNA production (Fig. 2b and c). It
313
would be interesting to further elucidate whether these regulations have biological
314
significance during the virus replication.
315 316
Interestingly, at the level of protein translation, we found that a highly conserved
317
nucleotide at -3 position of Kozak sequence in PB1 segment uniquely mediated a
318
significant downregulation of protein expression at translation level (Fig. 3). Although
319
Maeda et al. have previously made an effort to study the biological functions of the
320
-3U of the PB1 vRNA, together with the -3U of the NA vRNA in the context of the
321
WSN virus, they showed negative effects in general (Maeda et al., 2004). However, in
322
the present study, we found that the replication level of the PB1 -3 U-A mutants in
323
both a laboratory adapted WSN virus (WSN-PB1(-3A)) and a seasonal influenza virus
324
(HK68-PB1(-3A) were reduced about 7-10 times compared to wild type viruses in
325
MDCK cells (Fig. 4a, b and c). The reason for the discrepancy is unknown.
326
Nevertheless, Our in-depth studies further revealed that the -3 U-A mutation enhanced
327
the PB1 expression at an early stage of infection and resulted in not only alteration of
328
RNA synthesis dynamics of WSN-PB1(-3A) virus from that of the WSN-WT virus
329
(Fig. 5), but also induction of higher type I IFN level than the WSN-WT during
330
infection (Fig. 6c). 15
331 332
It was reported that single-strand RNA viral genome bearing 5′ triphosphates can be
333
recognized by RIG-I in influenza A virus infected cells, triggering cell-intrinsic innate
334
immune response (Rehwinkel et al., 2010; Davis et al., 2012). Our study showed here
335
that RNPs with increasing amount of PB1 expression, resulted in increased level of
336
three species RNA and increased type I interferon production in a PB1
337
dose-dependent manner (Fig. 6d, e and f). Therefore, the minor changes in the viral
338
RNA synthesis dynamics would correspondingly affect host innate immunity
339
responses in the course of the virus infection. These observations are also consistent
340
with a study of Belicha-Villaueva et al. (Belicha-Villanueva et al., 2012). They
341
showed that enhanced PB1 protein expression, resulting from G3A/C8U mutation in
342
the promoter of PB1 gene, disrupts the stoichiometry of viral RNA synthesis and/or
343
protein expression, and consequently increases the number of immunostimulatory
344
RNAs to induce typeⅠIFN production. Therefore, we propose that the suboptimal
345
Kozak sequence is used by the influenza A virus PB1 gene to not only fine-tune virus
346
replication kinetics, but also to optimize the balance between the virus replication rate
347
and the host antiviral status to make sure the viruses are produced in the most efficient
348
manner.
349 350
In summary, our results revealed that segment-specific NCRs play important roles in
351
differentially regulating viral RNA synthesis and protein expression. Intriguingly, in
352
the course of viral evolution, the PB1 segment of influenza A virus has specifically 16
353
selected a relative weak Kozak sequence in its segment-specific NCR, which mediates
354
a relatively low expression of the PB1 protein. We report here that the suboptimal
355
Kozak sequence is specifically used by the PB1 segment of influenza A virus segment
356
to fine-tune virus replication efficiency and host responses. This finding provided new
357
insights into the regulatory mechanisms of NCR of the influenza A virus genome.
358 359
Methods
360
Cells and antibodies
361
293T, MDCK, A549 and Vero cells were purchased from American type culture
362
collection (ATCC, Manassas, VA) and maintained in Dulbecco’s modified Eagle’s
363
medium
364
penicillin-streptomycin. The Rabbit anti-PA polyclonal antibody was kindly provided
365
by Dr. Ervin Fodor. Goat polyclonal antibody against PB1 was purchased from Santa
366
Cruz Biotechnology; Mouse anti-influenza NP monoclonal antibody (MAB8251)
367
from Millipore (Temecula, CA); Mouse anti-β-Actin monoclonal antibody from Cell
368
Signaling Technology; Rabbit monoclonal anti-influenza virus HA (86001-RM01)
369
from Sino Biological Inc (China).
(DMEM;
Gibco)
with
10%
fetal
bovine
serum
(Gibco)
and
370 371
Plasmids
372
The pcDNA and pPolI-HA plasmids of RNP reconstitution system of influenza
373
A/Puerto Rico/8/34 (PR8) and A/Viet Nam/1194/2004 (H5N1) were povided by Prof.
374
Ervin Fodor (University of Oxford, UK). The series of plasmids pPolI-NCR(1-7)-HA 17
375
(where 1-7 represents PB2, PB1, PA, NP, NP, NA, M, NS), containing a gene cassette
376
for ORF of HA (PR8) flanked by NCR from other segments, were constructed as
377
follows: first, at both ends of HA ORF, NCRs of the other seven segments were
378
extended with the primers containing flu segment-specific NCR sequences and
379
HA-specific sequences by two-step PCRs, the second-step PCR primers contain a Sap
380
I restriction site. The PCR products were then digested with Sap I and cloned into
381
pPolI-Sap I-Rib empty plasmid. pGL3-IFNβ-Luc and pGL3-RLuc plasmids were gifts
382
from Prof. Zhendong Zhao in our institute. The eight pHW plasmids for generation of
383
recombinant A/WSN/33 (H1N1) viruses have been described previously (Hoffmann
384
et al., 2000). The eight pLLB plasmids for generating recombinant A/Hong
385
Kong/1968(H3N2) viruses were kindly provided by Prof. Earl G Brown (University
386
of Ottawa, Canada). The -3A mutation in both pHW-PB1(-3A)-WSN and
387
pLLB-PB1(-3A)-HK68 was generated by site-directed mutagenesis using a
388
QuikChange Site-Directed Mutagenesis kit (Stratagene).
389 390
Bioinformatics analysis of NCRs of influenza A viruses
391
The same bioinformatics analysis was performed as previously described (Zhao et al.,
392
2014). The sequences of all segments (PB2, PB1, PA, H3, NP, N2, M and NS) are
393
obtained
394
(http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) on April 2nd, 2013 (Bao et
395
al., 2004).
from
NCBI
Influenza
396 18
Virus
Resource
database
397
Primer extension and western blot analyses
398
The primer extension analysis was carried out as described previously (Fodor et al.,
399
2002). The primers used for detecting HA(PR8) vRNA and mRNA/cRNA were
400
5′-AGTTCACTGGTGCTTTTGGTC-3′,
401
The primers used for detecting NA(WSN) vRNA and mRNA/cRNA were
402
5′-TCCAGTATGGTTTTGATTTCCG-3′,
403
The primers used for detecting 5S RNA was 5′-TCCCAGGCGGTCTCCCATCC-3′.
404
Western blot was carried out by standard procedures with IRDye Secondary
405
Antibodies (LI-COR Biosciences, Lincoln, NE). Protein expression levels were
406
visualized with an Odyssey Infrared Imaging System (LI-COR Biosciences, USA).
407
The relative protein expression level was analyzed using the integrated software of the
408
Odyssey system.
5′-TGTCACATTCTTCTCGAGCAC-3′,
5′-TGGACTAGTGGGAGCATCAT-3′.
409 410
Virus recue and viral growth kinetics
411
Recombinant viruses (the wild type and mutant viruses) were rescued by the
412
previously described 8-plasmid rescue system (Hoffmann et al., 2000; Liu et al.,
413
2009). The rescued viruses were plaque purified and fully sequenced.
414
growth kinetics of wild type and mutated viruses were performed in various types of
415
cells (A549, MDCK and Vero cells) at an MOI of 0.001 and plaque forming unit
416
(PFU) titers were determined by a standard plaque assay in MDCK cells.
417 418
IFN-β luciferase promoter assay 19
The viral
419
293T cells (2×105) were transfected using Lipofectamine 2000 (Invitrogen) with 200
420
ng pGL3-IFNβ-Luc and 5 ng pGL3-RLuc plasmids. Cells were co-transfected with
421
indicated RNP reconstitution plasmids or infected with WSN-WT or WSN-PB1(-3A)
422
virus respectively for 24 hours. Then, the cells were lysed and luciferase activity was
423
measured by a Dul-Luciferase Reporter Assay System (Promega).
424 425
Acknowledgements
426
We thank Professor George Brownlee (University of Oxford, UK) for critically
427
reading the manuscript. This work is supported by grants from National Natural
428
Science Foundation of China (31070152),Chinese Science and Technology Key
429
Projects (2013ZX10004601, 2013ZX10004611).
430 431
References
432
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575
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576
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577 578
Figure legends
579
Fig. 1. The sequence logos for NCR sequences of each segment of influenza A
580
virus. (a). For segments HA and NA, those of H3 and N2 were used. The conserved
581
promoter region at both the 3' and 5' ends were shaded in grey. The height of the letter
582
in the figure is in proportion to the frequency of the given nucleotide at a given
583
position. The black square refers to the gap in the sequence. The number in the
584
bracket refers to the number of sequences used for generating the NCR logo for this
585
segment. The serial numbers at the top of the logos refer to the positions in the NCR
586
sequence. (b) A comparison between the NCR sequences reported in literatures and
587
those by our bioinformatics analysis. The sequence logo below the line are the NCR
588
sequences reported in literatures, while the sequence logo above the line was derived
589
from (a). “X”s in sequence logo refer to those undetermined or in quasispecies. 27
590 591
Fig. 2. Regulatory roles of NCR in viral RNA and protein synthesis in an RNP
592
reconstitution system. (a) Schematic diagram of chimeric RNAs constructed in
593
pPolI-Sap I-Rib plasmids. The ORF of HA gene is flanked by NCR of other segments
594
of influenza A virus. The black bars represent the 12 and 13 nucleotides of 3′ and 5′
595
NCRs. The segment-specific NCR is shown in dark grey bar. The coding region of
596
HA gene is represented by grey bar. (b) The effect of segment-specific NCR
597
substitutions on viral RNA transcription and replication in the RNP reconstitution
598
system. Every NCR(1-8)-HA RNP was reconstituted in 293T cells and the levels of
599
mRNA, cRNA and vRNA were detected by primer extension analysis at 24h
600
post-transfection. (c) Quantification of the levels of viral RNAs in (b). Results were
601
presented as percentages relative to the viral RNA levels synthesized from the wild
602
type HA RNP system. (d) The effects of segment-specific NCR substitutions on the
603
protein expression. 293T cells were transfected as indicated in (b), cells were
604
harvested at 24h post-transfection, and lysates were analyzed by Western blot with an
605
anti-HA antibody, β-actin was detected as a loading control. (e) Quantification of HA
606
protein level obtained in (d) by densitometric analysis. The values were expressed as
607
percentages relative to the level of the HA protein expressed from the wild type RNP
608
system. Data in (c) and (e) represent the mean ± SD of three independent experiments.
609
Significance was analyzed with two-tailed Student’s t test. *, P < 0.05; **, P < 0.01.
610
As a negative control (NC), the transfection was performed with omitting the PB1
611
expressing plasmid. 28
612 613
Fig. 3. The presence of the “U” residue at the -3 position of the PB1 mRNA leads
614
to low protein expression at translation level. (a) The sequence logos for sequences
615
around the start codon for each segment of influenza A virus, which derived from Fig.
616
1a. The start codon was shaded in grey. The third position upstream of the start codon
617
was highlighted by a red box. (b) Schematic diagram of wild type PB1-HA RNA
618
(upper panel) and mutant PB1(-3A)-HA RNA (lower panel) constructed in pPolI-Sap
619
I-Rib plasmids. The residue at -3 position in the PB1 NCR was shaded in grey. (c)
620
The effect of -3A mutation on protein expression in an RNP reconstitution system.
621
The RNPs of PB1(WT)-HA and PB1(-3A)-HA were reconstituted in 293T cell, the
622
expressed proteins were analyzed by western blotting with indicated antibody. (d)
623
Quantification of HA protein level obtained in (c). The values expressed as
624
percentages relative to the level of the HA protein expressed from the PB1(WT)-HA
625
RNP system. (e) The effect of -3A mutation on viral RNA transcription and
626
replication. PB1(WT)-HA and PB1(-3A)-HA RNPs were reconstituted in 293T cells
627
and synthesized RNAs levels of mRNA, cRNA and vRNA were detected by primer
628
extension assay at 24h post-transfection. Results were representative of three
629
independent experiments. (f) Quantification of mRNA, cRNA and vRNA levels in (e)
630
by phosphorimager analysis. Values were normalized against cellular 5S RNA and
631
expressed as percentages relative to the level of the viral RNA levels from the
632
PB1(WT)-HA RNP system. Data in (d) and (f) were represented as mean ± SD of
29
633
three independent experiments. Significance was analyzed with two-tailed Student’s t
634
test. **, P < 0.01, n. s., not significant.
635 636
Fig. 4. Recombinant viruses containing -3 U-A mutation in PB1 was attenuated
637
which was independent of the presence or absence of PB1-F2. (a and b) Growth
638
curves of WSN-WT and WSN-PB1(-3A) virus in A549 and MDCK cells. (c) Growth
639
curves of HK68-WT and HK68-PB1(-3A) virus in MDCK cells. (d) Schematic
640
diagram of the mutations introduced into PB1 gene to ablate PB1-F2 expression. (e)
641
Growth curves of WSN-WT, WSN-PB1(-3A) and their corresponding PB1-F2
642
deletion mutant virus (WSN-WT-Del F2, WSN-PB1(-3A)-Del F2) in cell culture.
643
Data were represented as mean ± SD of three or two independent experiments with
644
independent virus stocks.
645 646
Fig. 5. The WSN-PB1(-3A) virus showed different protein and RNA synthesis
647
dynamics from that of the WSN-WT virus. (a) Viral proteins synthesized in cells
648
infected with either WSN-WT or WSN-PB1(-3A) virus. A549 cells were mock
649
infected or infected with the two viruses at an MOI of 3. Cell lysates collected at the
650
indicated time points, and subjected to western blotting using indicated antibodies. (b)
651
Quantification of PB1, N40, PA and NP protein level obtained in (a) by densitometry
652
analysis. The results were expressed as relative level to that of the WSN-WT. Data
653
represent the mean ± SD of three independent experiments. Significance was analyzed
654
with two-tailed Student’s t test. *, P < 0.05. (c) RNA synthesis dynamics in the course 30
655
of WSN-WT or WSN-PB1(-3A) virus infection. MDCK cells were infected either
656
with WSN-WT or WSN-PB1(-3A) virus at an MOI of 2, the cells were harvested at
657
indicated time points post-infection, and the levels of vRNA, mRNA and cRNA of the
658
NA segment were measured by primer extension analysis. A representative of three
659
independent experiments with two individually prepared virus stocks is shown. (d)
660
Quantification of mRNA, cRNA and vRNA in (c) by phosphorimager analysis.
661
Values were normalized against cellular 5S RNA.
662 663
Fig. 6. PB1 -3 U-A mutant virus induced more type I interferon than wild type
664
virus. (a) Growth curves of WSN-WT and WSN-PB1(-3A) virus in Vero cells. (b)
665
Growth curves of HK68-WT and HK68-PB1(-3A) virus in Vero cells. (c) The
666
infection of the WSN-PB1(-3A) virus induced more IFN-β than WSN-WT virus. The
667
IFN-β level induced by WSN-WT and WSN-PB1(-3A) infection was measured by
668
IFN-β luciferase promoter assay in 293T cells. Data are represented as means±SD;
669
n=3. Significance was analyzed with two-tailed Student’s t test. *, P < 0.05. (d) The
670
level of PB1 determines the amount of IFN-β production in the RNP reconstitution
671
system. 293T cells were transfected pGL3-IFNβ-Luc and pGL3-RLuc plasmids, along
672
without or with the PR8 RNP reconstituting plasmids with increasing dose of the
673
pcDNA-PB1-PR8 (0.5μg, 1μg, 2μg, 3μg)). Then luciferase activity was measured at
674
24 hours post-transfection. Data represent the mean ± SD of three independent
675
experiments. (e) The increasing amount of PB1 expression led to increasing amount
676
of RNA synthesized in the RNP reconstitution system. RNP was reconstituted in 31
677
293T cells as described in (d), synthesized RNAs levels of mRNA, cRNA and vRNA
678
were detected by primer extension assay after transfection 24 hour. The results was a
679
representative of three independent experiments. (f) Quantitative analysis of viral
680
RNAs in (e). Values of viral RNAs were expressed as fold change relative to the viral
681
RNA levels synthesized from the RNP system with 0.5μg pcDNA-PB1 plasmid. Data
682
represent the mean ± SD of three independent experiments.
32
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