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

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al., 1995; Kim et al., 1997). Adjacent to the promoter region, at both ends, are the

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segment-specific NCRs that vary in sequence and length among different segments of

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influenza A virus. It has previously been reported that the segment-specific NCRs of

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influenza A virus play multiple roles in virus life cycle. They not only act as

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cis-acting signals to regulate viral transcription, replication and protein expression

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(Bergmann & Muster, 1996; Zheng et al., 1996; Park & Katze, 1995), but also serve

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as part of the packaging signals during selective virus genome packaging (Zhao et al.,

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2014; Hutchinson et al., 2010; Gog et al., 2007).

61 62

The viral ribonucleoproteins (RNPs) of influenza A virus are responsible for

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synthesizing three viral RNA species (mRNA, cRNA and vRNA) in the nucleus of

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infected cells. The RdRp is a heterotrimeric complex composed by three subunits

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(PB1, PB2 and PA) (Palese & Sham, 2007; Resa-Infante et al., 2011). The PB1

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subunit, encoded by segment 2, is the core of the RdRp (Palese & Sham, 2007). It not 3

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only acts as the driving force for the assembly of the viral RNA polymerase but also

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contains active sites for RNA polymerization (Biswas & Nayak, 1994; Braam et al.,

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1983). In addition, the PB1 is responsible for binding to the viral RNA promoter to

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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

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(mRNA, cRNA, vRNA) and all viral proteins are tightly controlled differentially

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(Park & Katze, 1995; Enami et al., 1985; Varich & Kaverin, 1987; Yamanaka et al.,

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1985; Yamanaka et al., 1991; Park et al., 1999; Hatada et al., 1989). The significance

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of such regulation is not only to produce appropriate amount of viral RNAs and

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proteins for efficient assembly of progeny virions, but also to maintain the fine

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balance between virus growth rate and host antiviral status (Belicha-Villanueva et al.,

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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

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RdRp was also reported to be involved in host shut-off by targeting host Pol II

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transcription machinery (Vreede et al., 2010; Graef et al., 2010). Moreover, a short

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non-structural peptide PB1-F2, expressed from the PB1 segment of influenza A virus,

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was reported to be involved in modulating host antiviral status (Chen et al., 2001;

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Wise et al., 2009). Therefore, an understanding of the mechanisms by which influenza

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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

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replication strategies.

91 92

In the present study, we analyzed the sequences of the segment-specific NCRs of all

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available influenza A viruses in the NCBI, and investigated the role of these NCRs in

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differentially regulating viral RNA synthesis and protein expression. Interestingly, we

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also demonstrated that influenza A virus exceptionally uses a suboptimal Kozak

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sequence in segment 2 to fine-tune virus infection through modulating PB1 protein

97

expression.

98 99

Results

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Bioinformatics analysis of the segment-specific non-coding regions of influenza A

101

viruses

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In order to obtain a full view for segment-specific NCRs of all influenza A viruses,

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we bioinformatically analyzed all available NCR sequences of all the eight segments

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from NCBI Influenza Virus Resource database as recently described (Zhao et al.,

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2014). We used H3 and N2 subtypes for the analsys of HA and NA segments. As

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shown in Fig. 1a, the lengths and sequences at both 3′ and 5′ ends are

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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

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validated these sequence logos by comparing them with the sequence logos derived 5

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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

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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

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substitutions of the HA-NCR led to the synthesis of the three RNA species (mRNA,

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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

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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

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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,

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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

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low expression level of the HA protein was mediated by the suboptimal Kozak

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sequence of the PB1-NCR in our RNP reconstitution systems, we mutated PB1-NCR

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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

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was about six times higher than the levels of the protein expressed from

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PB1(WT)-NCR (Fig. 3c and d). Meanwhile, the levels of mRNAs, vRNA and cRNA

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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

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the PB1 mRNA, which weakened the strength of Kozak sequence, mediated low

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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,

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independent of the presence or absence of PB1-F2.

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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

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mRNA at the -3 position, was rescued in an influenza A/WSN/33 background

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(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

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1 log lower than WSN-WT in both cell types (Fig. 4a and b). We further confirmed

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this result in a seasonal influenza A virus A/Hong Kong/1968(H3N2) background, in

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which the maximum virus titer of HK68-PB1(-3A) was about 0.7 log lower than that

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of HK68-WT (Fig. 4c). These data indicated that the -3 U-A mutation in PB1 result in

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virus attenuation.

184 185

Since it has been proposed that the sub-optimal Kozak consensus of PB1, with the

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unique “U” nucleotide in the –3 position, is involved in regulating the expression of

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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

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defective expression of PB1-F2 protein. In order to examine whether the attenuation

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of WSN-PB1(-3A) was caused by defective expression of PB1-F2, we made three

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mutations (T120C, C153G and G291A) according to the literature (Ma et al., 2013;

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Su et al., 2012) in the coding region of both wild type and the -3 U-A mutant virus to

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completely eliminate the PB1-F2 expression in both viruses (Fig. 4d) (Le Goffic et al.,

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2010; Tauber et al., 2011), designated as WSN-WT-del F2 and WSN-PB1(-3A)-del

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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

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previously reported (Wise et al., 2009; Le Goffic et al., 2010), that there were no

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significant differences in the growth of WSN-WT and WSN-WT-del F2 virus (Fig. 9

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4e). Similarly, the WSN-PB1(-3A)-del F2 virus showed similar growth properties to

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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

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presence or absence of PB1-F2 was not responsible for attenuation of the

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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

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In order to investigate the mechanism(s) by which the -3 U-A mutation in the PB1

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mRNA affected the efficiency of virus replication, we examined the effects of the

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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

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WSN-PB1(-3A) and WSN-WT virus at a MOI of 3 (Fig. 5a and b). It can be seen that

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the level of PB1 in the WSN-PB1(-3A) virus infected cells was significantly higher

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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

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post-infection (8 and 12 h.p.i.). Meanwhile, we also found that the expression of N40,

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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

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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

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addition, we also detected slightly higher levels of PA and NP in the WSN-PB1(-3A)

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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

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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

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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)

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virus.

235

Since the PB1 is the core subunit of the viral RNA polymerase, the -3 U-A mutation

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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

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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,

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cRNA and vRNA in cells infected with WSN-PB1(-3A) virus was slightly higher than 11

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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

Bao, Y., Bolotov, P., Dernovoy, D., Kiryutin, B., Zaslavsky, L., Tatusova, T.,

433

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434

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436

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García-Sastre, A. (2012). Recombinant influenza A viruses with enhanced PB1 and

438

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Bergmann, M. & Muster, T. (1996). Mutations in the nonconserved noncoding

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Regions of Influenza Genomic Sequences Are 5'PPP-Independent Ligands for RIG-I.

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571

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573

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574

Zheng, H., Palese, P., Garcia-Sastre, A. (1996). Nonconserved nucleotides at the 3'

575

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576

replication. Virology 217: 242-251.

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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Influenza A virus utilizes a suboptimal Kozak sequence to fine-tune virus replication and host response.

The segment-specific non-coding regions (NCRs) of influenza A virus RNA genome play important roles in controlling viral RNA transcription, replicatio...
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