1 5' triphosphate-sirna: potent inhibition of influenza a virus

1

5’ triphosphate-siRNA: potent inhibition of influenza A virus infection by gene silencing 1

and RIG-I activation 2

Li Lin1, Qiang Liu1, Nathalie Berube1, Susan Detmer2, Yan Zhou1* 3

1Vaccine and Infectious Disease Organization 4

2Veterinary Pathology, Western College of Veterinary Medicine 5

University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 5E3 6

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Running title: 3p-siRNA inhibits influenza A virus infection 8

Abstract word count: 212 9

Text word count: 5119 10

*Corresponding author: Yan Zhou Ph.D. 11

Vaccine and Infectious Disease Organization 12

University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK, Canada, S7N 5E3 13

Phone: 306-966-7716 14

Fax: 306-966-7478 15

Email: [email protected] 16

17

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00665-12 JVI Accepts, published online ahead of print on 11 July 2012

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Abstract 18

Limited protection of current vaccines and antiviral drugs against influenza A virus 19

infection underscores the urgent need for development of novel anti-influenza virus interventions. 20

While short interfering RNA (siRNA) has been shown to be able to inhibit influenza virus 21

infection in a gene specific manner, activation of retinoic acid-inducible gene I protein (RIG-I) 22

pathway has an anti-viral effect in a gene non-specific mode. In this study, we designed and 23

tested the anti-influenza virus effect of a short double stranded RNA, designated 3p-mNP1496-24

siRNA that possesses dual functions: a siRNA targeting influenza NP gene and an agonist for 25

RIG-I activation. This double stranded siRNA possesses a triphosphate group at the 5’ end of 26

the sense strand and is blunt ended. Our study showed that 3p-mNP1496-siRNA could potently 27

inhibit influenza A virus infection both in cell culture and in mice. The strong inhibition effect 28

was attributed to its siRNA function as well as its ability to activate RIG-I pathway. To the best 29

of our knowledge, this is the first report that the combination of siRNA together with RIG-I 30

pathway activation can synergistically inhibit influenza A virus infection. The development of 31

such dual functional RNA molecules will greatly contribute to the arsenal of tools to combat not 32

only influenza viruses but also other important viral pathogens. 33

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Introduction 35

Influenza viruses cause annual epidemics and occasional pandemics that have severe 36

consequences for human health and the globe economy. An average of 200,000 hospitalizations 37

occur each year in the United States due to respiratory and heart conditions illness associated 38

with influenza virus infections (28). Most human influenza infections are caused by influenza A 39

viruses (IAV) of the orthomyxovirus family, with a single-stranded, negative-sense, segmented 40

RNA genome (18). In order to evade the immune response and antiviral interventions, these 41

viruses continue to evolve through genetic mutations caused by the error prone RNA dependent 42

RNA polymerase and reassortment of gene segments between viruses. Vaccination and antivirals 43

are the major interventions for prophylaxis and treatment of influenza. However, there are 44

limitations on both measures. Annual vaccine programs can provide protection to most members 45

of the population, but they are less effective for vulnerable groups such as very young, elderly 46

and immunocompromised individuals. From the therapeutic perspective, antivirals are available 47

to treat influenza infection based on M2 or NA inhibition. Unfortunately, the emergence of 48

antiviral resistant influenza strains continues to be on the rise, limiting their efficacy in the long 49

term (10). The rapid global spread of the 2009 pandemic H1N1 virus and the continued threat of 50

avian influenza virus to humans underscore the urgent need to develop novel therapeutic 51

strategies to treat influenza. 52

Short interfering RNAs (siRNAs) are found in many eukaryotes. They are short double-53

stranded RNAs of usually 21- and 22- nucleotides (nt) long with a 2-nt overhang at the 3' end (4). 54

Within cells, each siRNA unwinds into two single-stranded (ss) RNAs: the sense strand and the 55

guide strand (antisense strand). The guide strand is then incorporated into the RNA-induced 56

silencing complex (RISC) which degrades the target mRNA, and the sense strand will be 57


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degraded (13,19).Transfection of synthetic 21-nt siRNAs into mammalian cells can activate the 58

siRNA process and degrade targeting mRNA. Several studies have shown that siRNAs hold a 59

great potential as medical applications against the important human viral pathogens, such as 60

influenza virus (5,6,29), human immunodeficiency virus (2,11,17), hepatitis B virus (7), hepatitis 61

C virus (20) and dengue virus (1). 62

Within the host, the innate immune system is an important defense against viral 63

infections. One of the major mechanisms of innate immune responses is to activate intracellular 64

retinoic acid-inducible gene I protein (RIG-I) and its downstream pathways. This leads to type I 65

interferon production and activation of host antiviral activity. As a member of the DExD/H 66

helicase protein group, RIG-I contains a helicase domain at its C-terminus and two tandem 67

caspase recruitment domains (CARDs) at the N-terminus. Binding of dsRNA to the C terminal 68

RNA helicase domain of RIG-I induces a conformational change that exposes the N-terminal 69

CARD domains to recruit mitochondrial antiviral signaling protein (MAVS), resulting in the 70

activation of host innate immune responses (3,27). 71

The exact structures of RNA agonist for RIG-I activation have been controversial (14). 72

Recently, using fully chemical synthetic 5′-triphosphate RNAs, two groups independently 73

identified the exact molecular features of RNA that are required for RIG-I recognition (22,23). 74

These results demonstrated that for RNA to act as an agonist the following three structures must 75

be in place: i) a triphosphate group (3p-) at the 5′ end of the sense strand of the dsRNA; ii) the 76

length of the dsRNA is more than 22 nucleotides; and iii) the 5’ triphosphate end of the dsRNA 77

is blunt (22,23). Based on these findings, we rationalized that a combination of these two 78

antiviral approaches, namely suppression of influenza virus replication by siRNA targeting a 79

viral gene and triggering the host innate immune response by RIG-I activation, should lead to a 80


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more effective inhibition of influenza virus infection. In this study, we designed and generated a 81

3p-siRNA that simultaneously silences influenza NP gene and activates RIG-I mediated 82

interferon pathway. We report its potent inhibition effect on IAV infection. 83

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Materials and Methods 86

Cells and viruses. A549 and 293T cells were maintained in Dulbecco’s modified Eagle’s 87

medium (DMEM; Thermo scientific, Rockford, IL) containing 10% fetal bovine serum (FBS; 88

Gibco, Carlbad, CA). Madin-Darby canine kidney (MDCK) cells were cultivated in minimal 89

essential medium (MEM; Sigma-Aldrich, St-Louis, MO) supplemented with 10% FBS. 90

Influenza A/Puerto Rico/8/34 H1N1 (PR8), A/Texas/36/91H1N1 (Tx91), and A/ 91

Halifax/210/2009 H1N1 (Halifax210) were propagated in 11-day-old embryonated chicken eggs 92

as previously described (24). 93

Antibodies. Rabbit polyclonal NS1 and NP antibodies were generated in our laboratory 94

as previously described (25,26). The other antibodies were purchased from different companies 95

indicated in parenthesis as follows: rabbit polyclonal anti-human RIG-I antibody (Enzo life 96

sciences, Farmingdale, NY), monoclonal anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, 97

CA), IRDye 800-conjugated donkey polyclonal anti-mouse IgG and IRDye 680-conjugated goat 98

anti-rabbit polyclonal IgG (LI-COR Biosciences, Lincoln, NE). 99

RNA synthesis. Based on the previous reports (5,6,22,23), the following three single 100

stranded RNA oligonucleotides were chemically synthesized by Eurogentec (Liege, Belgium): 101

mNP1496-AS-RNA (5’-GUCUCCGAAGAAAUAAGAUCCUU-3’), mNP1496-S-RNA (5’- 102

AAGGAUCUUAUUUCUUCGGAGACUU-3’), and 3p-mNP1496-S-RNA (5’-ppp-103

AAGGAUCUUAUUUCUUCGGAGACUU-3’). The above mNP1496-AS-RNA represents 104

antisense RNA strand (AS-RNA) or guide RNA strand, mNP1496-S-RNA and 3p-mNP1496-S-105

RNA are sense RNA strand (S-RNA). Especially, 3p-mNP1496-S-RNA containing triphosphate 106

at 5’ end was synthesized from mNP1496-S-RNA using standard phosphoramidite solid phase 107

synthesis as described previously (23). NP1496-siRNA (sense strand: 5’-108


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GGAUCUUAUUUCUUCGGAGdTdT-3’; guide strand: 5’-109

CUCCGAAGAAAUAAGAUCCdTdT-3’) was purchased from Qiagen (Valencia, CA), polyI:C 110

was obtained from Sigma-Aldrich (St-Louis, MO). 111

Double stranded siRNA (ds-siRNA) annealing. Ds-siRNAs were annealed as 112

previously described (6) with minor modifications. Briefly, all chemically synthesized ss-RNAs 113

were dissolved in RNase free water to make a final solution of 10 µg/µl. The mNP1496-S-RNA 114

or 3p-mNP1496-S-RNA was mixed with the same amount of mNP1496-AS-RNA. The mixture 115

was incubated in a beaker containing one liter of 95°C water and was allowed to cool down 116

gradually to 25 °C followed by a further incubate at 25 °C for 3 hours. The annealed dsRNAs 117

were subjected to 16% TBE-acrylamide gel electrophoresis at 100 volts in 0.5 × TBE buffer 118

(44.5mM Tris, 44.5mM Boric acid, 1 mM EDTA, pH8.0) at 4°C. RNA bands were visualized by 119

staining with ethidium bromide. The annealed siRNAs were aliquoted and stored at -80°C until 120

use. 121

SiRNA transfection and infection. A549 cells or Vero cells (7×104/well of 24 well plate) 122

were transfected with siRNAs using X-tremeGene siRNA transfection reagent (Roche, Basel, 123

Switzerland) as described previously (12) . Briefly, various amounts of siRNAs and X-124

tremeGene siRNA transfection reagents were diluted in Opti-MEM (Gibco, Carlbad, CA) in two 125

separate vials. The diluents were mixed immediately and the mixture was further incubated at 126

room temperature for 20 minutes before addition to cells at 30-40% confluence. Medium was 127

changed to Opti-MEM 6 hours post transfection, and 24 hours post transfection, cells were 128

infected with IAV at MOI of 1. Then, 24 hours post infection, supernatants and cell lysates were 129

harvested and subjected to various assays. 130


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SiRNA transfection and RIG-I expression. A549 cells at 30-40% confluence in 24-well 131

plate were transfected with 10 pmol of each siRNA using X-tremeGene siRNA transfection 132

reagent as described above. Total cell lysates were harvested at the indicated time points and 133

RIG-I protein expression was detected with rabbit anti-human RIG-I specific antibody. 134

IFN-β Luciferase assay. To examine whether 3p-mNP1496-siRNA would trigger 135

interferon-β activity, 293T cells (5×105) were transfected with 0.5 µg of pLuc125 plasmid which 136

encodes luciferase gene under the control of IFN-β promoter, 0.1 μg of pTK-rLuc which encodes 137

renilla lucifease, together with 10 pmol of siRNAs or 500 ng of polyI:C using Lipofectamine 138

2000 Reagent (Invitrogen, Grand Island, NY) as per manufacturer’s instruction. Twenty-four 139

hours post transfection, luciferase activity was measured using Dual-Luciferase Reporter Assay 140

System (Promega, Madison, WI). Relative luciferase activities were calculated as the ratio of 141

firefly to renilla luciferase light unit. Each luciferase activity value is the average of three 142

independent experiments. 143

In vitro RIG-I RNA binding and ATPase activity assay. RIG-I ATPase activity assay 144

was performed as described previously (31) with minor modifications. Briefly, Flag-tagged 145

human RIG-I protein was purified from 293T cells transfected with pCMV2-3×Flag-RIG-I 146

plasmid encoding Flag tagged human RIG-I protein gene. Various amount of dsRNAs were 147

incubated with 1 μg of the Flag-tagged RIG-I protein in a 50 μl RNA binding buffer (20 mM 148

Tris-HCl, pH 8.0, 1.5 mM MgCl2, 5% glycerol (v/v) and 1.5 mM DTT) in 96 well plate at 37 °C 149

for 1 hour. Thereafter, fresh ATP was added to each well at 1 mM final concentration, and 150

further incubated at 37 °C for 15 min. Subsequently, 100 μl of BIOMOL Green reagent (Enzo 151

life science, Farmingdale, NY) was added to each well and incubated at room temperature for 152

another 30 minutes to allow fully development of green color. Meanwhile, serial dilutions of 153


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phosphate standard in the binding buffer were also included. Signals were measured at OD 655 154

nm using micro-plate reader (Bio-Rad, Hercules, CA). 155

In vivo study. To assess the inhibitory effect of 3p-mNP1496-siRNA, six- to seven- 156

week old female Balb/c mice (Charles River Laboratories, Wilmington, MA) were divided into 157

five groups (Table 1). Mice in each group (n=8) were intravenously injected with 100 μg of 3p-158

mNP1496-siRNA, mNP1496-siRNA, off-target siRNA (Invitrogen, Grand Island, NY) or PBS 159

mixed with in vivo DNA or RNA delivery reagent in vivo-jetPEI (Polyplus-transfection Inc., 160

New York, NY) in a volume of 100 μl according to the manufacturer's protocol. On day 1 post 161

siRNAs treatment, mice in group 1-4 were infected with 5000 PFU PR8 intranasally. Mice in 162

group 5 that received off target siRNA mixed with in vivo-jetPEI were mock infected with PBS. 163

On day 3 post infection, mice were humanely euthanized and lungs of mice were collected for 164

further analysis. 165

Histopathology, immunohistochemistry (IHC) and virus isolation from mice lung. 166

Tissue samples of left lung lobes were collected from all mice on day 3 post virus infection. 167

These were fixed in 10% neutral phosphate buffered formalin, routinely processed and stained 168

with hematoxylin and eosin (H&E) for histopathologic examination. The immunohistochemical 169

staining was conducted at Prairie Diagnostic Services, Saskatoon, SK using a technique adapted 170

for an automated slide stainer as previously described (8). For these tissues, protease XIV (Sigma 171

Chemical Co., St. Louis, MO) digestion was used for epitope retrieval and the primary antibody 172

(Goat anti RNP Type A Influenza, V304-501-157; National Institute of Allergy and Infectious 173

Diseases; Bethesda, MD) was used at a dilution of 1:5,000 and 1:10,000. Binding of the primary 174

antibody was detected using biotinylated rabbit anti-goat immunoglobulins and an avidin-biotin 175


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immunoperoxidase complex reagent with 3,3’-diaminobenzidine tetrahydrochloride (DAB) 176

(Electron Microscopy Science, Ft. Washington, PA) for the chromogen. 177

For virus isolation, lung tissue was weighed and homogenized in MEM supplemented 178

with antibiotic/antimycotic solution as described previously (15). Virus titer was determined by 179

plaque assay of the supernatant of the homogenized tissue on MDCK cells. 180

RNA isolation from mice lung and RT-qPCR. Total RNA from mouse lung tissues was 181

isolated using Trizol (Invitrogen, Grand Island, NY) per manufacturer’s instructions. The 182

isolated RNA was further treated with RNase free DNase I to remove trace of genomic DNA. 183

Total RNA were further purified with RNeasy kit (Qiagen, Valencia, CA). The mRNA level of 184

RIG-I, IFN-β and GAPDH was measured by RT-qPCR conducted in an iCycler IQTM5 185

Multicolor real-time PCR detection system (Bio-Rad, Hercules, CA). Briefly, 2.5 μg of each 186

RNA was reverse transcribed using Oligo (dT) followed by PCR using specific primers: Mouse 187

RIG-I-forward: 5’-CCACCTACATCCTCAGCTACATGA-3’; Mouse RIG-I-reverse: 5’-188

TGGGCCCTTGTTGTTCTTCT-3’; Mouse IFN-β-forward: 5’-189

GGAGATGACGGAGAAGATGC-3’; Mouse IFN-β-reverse: 5’-190

CCCAGTGCTGGAGAAATTGT-3’; Mouse GAPDH-forward: 5’-191

AACTTTGGCATTGTGGAAGG-3’; Mouse GAPDH-reverse: 5’-192

ACACATTGGGGGTAGGAACA-3’. RIG-I and IFN-β expression was normalized to GAPDH 193

expression in the same sample. Data are presented as relative gene expression to that of untreated 194

cells using the formula 2(ΔCt of gene- ΔCt of GAPDH) (9). All RNA determinations have been assayed in 195

triplicate and repeated three times. 196

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Results 198

Design of dual functional siRNAs. It has been reported that chemically synthesized 199

siRNAs specific for conserved regions of viral genome can potently inhibit IAV infection in both 200

cell lines and mice (5,6,29). Among these siRNAs, NP1496-siRNA that targets NP gene showed 201

the most profound effect on inhibition of virus replication (6). Our design of dual functional 202

siRNA was therefore based on NP1496-siRNA sequence. Although NP1496-siRNA is an 203

optimized siRNA, it does not have any features to serve as an RIG-I agonist (Fig. 1A). Indeed, 204

Ge et al. have demonstrated that the inhibition of viral RNA accumulation by siRNA is not 205

because of a cellular interferon response (6). To generate a dual functional siRNA, we 206

chemically synthesized three ssRNAs. mNP1496-S-RNA is based on the NP1496-siRNA sense 207

strand with two nt (AA) added to the 5’ end and two nt (AC) extension at the 3’end prior to the 208

UU sequence of NP1496-siRNA sense strand (Fig. 1B). mNP1496-AS-RNA is modified from 209

NP1496-siRNA guide strand with two nt (GU) added to the 5’ end of NP1496-siRNA guide 210

strand (Fig. 1B). 3p-mNP1496-S-RNA has the same nt sequence to mNP1496-S-RNA, except it 211

contains triphosphate at the 5’ end (Fig. 1C). Annealing of mNP1496-S-RNA and mNP1496-AS-212

RNA will generate a 23 base paired ds-siRNA (Fig. 1B). Annealing of 3p-mNP1496-S-RNA and 213

mNP-1496-AS-RNA will generate a 23 base paired ds-siRNA with triphosphate and blunt end at 214

its 5’ end, in regards to RNA sense orientation (Fig. 1C), this molecule designated 3p-mNP1496-215

siRNA should fulfill the dual function of siRNA and RIG-I agonist. 216

All products were purified by HPLC and verified by MALDI-TOF MS (Fig. 1 D, E and 217

F). It is worthy to note that the yield efficiency of 3p-mNP1496-S-RNA was about 30% of the 218

total input of mNP1496-S-RNA. The synthesized ssRNA and annealed dsRNA were subjected to 219

16% TBE-acrylamide gel electrophoresis and were visualized by ethidium bromide staining. Fig. 220


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1G showed that the annealed ds-siRNA migrated at different position than ssRNA. Since 221

ethidium bromide binding ability to dsRNA is much stronger than that to ssRNA, the dsRNA 222

bands are much brighter than the ssRNA bands. It is also clear that almost all ssRNAs have been 223

converted to dsRNAs (Fig. 1G). 224

Modified mNP1496-siRNA executed inhibition on influenza A virus infection. To 225

make mNP1496-siRNA, we have modified NP1496-siRNA as follows: basing on the conserved 226

region of NP RNA segment, we extended the length of dsRNA from 19 nt to 23 nt and generated 227

a blunt end at the 5’end of sense strand (Fig.1 A and B). We then asked whether the modified 228

siRNA has any inhibition effects on IAV infection. A549 cells were transfected with 0, 5, 10, 15, 229

20 pmol of each siRNA, 24 hours post transfection, the treated cells were infected with PR8 at 230

MOI of 1. Twenty four hours post infection, cell lysates were prepared and subjected to the 231

Western blotting with antibodies specific for NP and NS1 proteins. Levels of β-actin were 232

monitored as loading control. As shown in Fig. 2, significant amount of NP and NS1 proteins 233

were detected in the siRNA untreated and virus infected cells (lane 1), these levels were 234

normalized to that of the β-actin in the same sample and were set as references (100%). 235

Transfection of NP1496-siRNA and mNP1496-siRNA led to a dose dependent inhibition of viral 236

protein synthesis, where at 20 pmol, the inhibition effect is the most profound (lanes 5 and 9). 237

The modified mNP1496-siRNA showed similar inhibition capability to that of optimized 238

NP1496-siRNA. Virus titers in the supernatant of 5 and 10 pmol treated samples were evaluated 239

by plaque assay. As shown in Fig. 3B, the virus titer in NP1496-siRNA and mNP1496-siRNA 240

treated cells were similar (5.3×106 versus 4.6×106 PFU/ml at 5 pmol, and 8.0×105 versus 7.0×105 241

PFU/ml at 10 pmol). These titers are significantly lower than that in untreated samples (1.1×107 242


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PFU/ml). These results demonstrated that the extension of dsRNA and bunt end modifications 243

did not change the inhibition efficiency of the mNP1496-siRNAs. 244

3p-mNP1496-siRNA showed stronger inhibition effect on IAV infection than that of 245

mNP1496-siRNA in A549 cells. We have shown that mNP1496-siRNA had similar inhibition 246

efficiency as that of optimized NP1496-siRNA. Motivated by these results, we synthesized 3p-247

mNP1496-siRNA which was further modified from mNP1496-siRNA by addition of 248

triphosphates at the 5’end of the sense strand. Since the 3p-mNP1496-siRNA meets all the basic 249

criteria for fully RIG-I binding and activation, we speculated that 3p-mNP1496-siRNA should 250

exert dual functions of siRNA as well as RIG-I agonist and thus would inhibit IAV infection 251

more potently. We therefore tested the efficiency of 3p-mNP1496-siRNA in inhibiting IAV 252

infection. 253

A549 cells were transfected with various amounts of 3p-mNP1496-siRNA or mNP1496-254

siRNA. Twenty-four hours post transfection, cells were infected with PR8 at MOI of 1. Twenty 255

four hours post infection, cell lysates were subjected to the Western blotting with polyclonal 256

antibodies specific for NP or NS1. Virus titers in the supernatant were determined by plaque 257

assay. As seen in Fig. 3A, treatment of cells with 0.6 and 1.2 pmol of mNP1496-siRNA did not 258

have significant inhibition on viral protein synthesis (lanes 2 and 3). With 5 and 10 pmol of 259

mNP1496-siRNA treatment, viral protein synthesis was reduced to about 55% and 20% (lanes 4 260

and 5) compared to untreated cells (lane 1), respectively. In contrast, while treatment of cells 261

with 0.6 pmol of 3p-mNP1496-siRNA led to a 40% reduction of viral protein synthesis, 1.2 pmol 262

of 3p-mNP1496-siRNA could significantly inhibit viral protein synthesis to about 20% (lanes 7). 263

Furthermore, viral proteins were barely detectable in the samples that were treated with 5 or 10 264

pmol of 3p-mNP1496-siRNA (lanes 8 and 9). Consistent with these results, Fig. 3B showed that 265


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virus titer in 3p-mNP1496-siRNA treated samples are dramatically decreased than that in 266

mNP1496-siRNA treated samples (7.3×105 versus 9.4×106 PFU/ml at 1.2 pmol; 1.9×105 versus 267

4.6×106 PFU/ml at 5 pmol, and 1.1×105 versus 7.0×105 PFU/ml at 10 pmol). 268

In order to test whether 3p-mNP1496-siRNA would inhibit other strains of IAV infection, 269

A549 cells were treated with 5 pmol of 3p-mNP1496-siRNA and were infected with Halifax 210 270

and Tx91 at an MOI of 1, viral protein synthesis was determined by Western blot analysis. As 271

seen in Fig. 3C, in agreement with the results obtained by using PR8 virus. Transfection of 3p-272

mNP1496-siRNA resulted in a significant reduction in viral NP protein and NS1 protein 273

synthesis. Cellular β-actin levels were not altered by siRNA transfection and virus infection. 274

3p-mNP1496-siRNA induces RIG-I activation and IFN-β transcription. Since 3p-275

mNP1496-siRNA and mNP1496-siRNA contain the same guide strand (Fig.1B and C) and this is 276

incorporated into RISC to degrade target mRNA, they should have the same siRNA effects. 277

However, we have shown that the inhibition activity of 3p-mNP1496-siRNA was much stronger 278

than that of mNP1496-siRNA. In order to investigate if this effect on inhibition was due to the 279

activation of RIG-I and IFN-β, two experiments were performed to assess RIG-I activation. First, 280

A549 cells were transfected with 5 pmol of 3p-mNP1496-siRNAs and harvested at the indicated 281

times. Total cell lysates were subject to western blotting with antibodies specific for RIG-I and 282

β-actin. As seen in Fig. 4A, RIG-I protein was detectable at 4 hours post transfection, and the 283

elevated level of RIG-I was sustained until 60 hours post transfection. By contrast, both off-284

target siRNA and mNP1496-siRNA were not able to induce RIG-I expression (Fig. 4B). In the 285

second experiment, we evaluated RIG-I ATPase activity, which is critical for antiviral responses 286

(16). Binding of dsRNA to RIG-I activates its helicase ATPase, which will convert ATP to ADP. 287

The free phosphates released from the ATPase hydrolysis was measured with the BIOMOL 288


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Green Reagent. As seen in Fig. 4C, incubation of 3p-mNP1496-siRNA with RIG-I led to an 289

increased activity of ATPase in a dose dependent manner. In contrast, binding of mNP1496-290

siRNA did not induce any RIG-I ATPase activity. 291

We further investigated whether the interferon-β pathway was activated. To this end, 292

293T cells were transfected with pLuc125 plasmid and pTK-rLuc, together with different 293

siRNAs. polyI:C is known to be an inducer of IFN-β (31), thus is included as positive control. 294

Luciferase activity was determined at 24 hours post transfection. Fold induction of the promoter 295

activity was obtained by normalizing to that in the mock siRNA treated cells. As shown in Fig. 296

4D, polyI:C and 3p-mNP1496-siRNA transfection led to a significant induction of IFN-β 297

promoter activity. Specifically, 7.3- and 13.3- fold of induction were obtained in polyI:C and 3p-298

mNP1496siRNA transfected cells, respectively. In contrast, transfection of off target siRNA and 299

NP1496-siRNA did not induce IFN-β promoter activity. These data suggest that the stronger 300

inhibition effect of 3p-mNP1496-siRNA is attributed to RIG-I and IFN-β activation. 301

3p-mNP1496-siRNA showed similar inhibition effect on IAV replication as that of 302

mNP-1496-siRNA in Vero cells. We have shown that 3p-mNP1496-siRNA had stronger 303

inhibition of IAV infection than that of mNP1496-siRNA on A549 cells, and it is very likely 304

correlated with RIG-I mediated IFN-β secretion pathway. To further support this finding, Vero 305

cells which are defective in IFN-β expression were used. These Vero cells were transfected 306

either with different amount of mNP1496-siRNA or 3p-mNP1496-siRNA, 24 hours later, cells 307

were infected with PR8 virus at MOI of 1. Twenty four hours post infection, supernatant was 308

harvested for virus titration and cell lysates were subjected to Western blotting using antibodies 309

specific for NP or NS1 protein. As seen in Fig. 5A, at the concentration of 10 pmol, both 3p-310

mNP1496-siRNA and mNP1496-siRNA reduced viral protein synthesis to about 40% (lanes 3 vs 311


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6); and at 20 pmol, both siRNAs could completely inhibit viral protein synthesis (lanes 4 vs 7). 312

Fig. 5B showed that virus titers are similar in both mNP1496-siRNA and 3p-mNP1496-siRNA 313

treated Vero cells (5.13×105 versus 5.17×105 PFU/ml at 5 pmol, and 2.47×105 versus 2.53×105 314

PFU/ml at 10 pmol). 315

3p-mNP1496-siRNA treatment also showed potent inhibition of IAV replication in 316

mice. In order to test whether 3p-mNP1496-siRNA had strong inhibition of IAV infection in 317

vivo, mice were intravenously injected with 100 μg of 3p-mNP1496-siRNA, mNP1496-siRNA, 318

off-target siRNA or PBS mixed with in vivo-jetPEI. Twenty-four hours post siRNA treatment, 319

mice in group 1-4 were intranasally infected with PR8 (Table 1). Three days post virus infection, 320

mice lung were collected and subjected to the assays to detect virus titer, viral protein expression, 321

RNA extraction and pathology. During the course of the experiment, mice in group 5 that only 322

received treatment of off target siRNA did not show any side effect in terms of the daily activity 323

and weight loss (data not shown). Mice that received mNP1496-siRNA had a lung virus titer 324

that is about 4-fold lower than those in PBS treated and off target siRNA treated groups 325

(1.45×108 PFU/ml/g vs. 6.33 ×108PFU/ml/g and 6.21×108 PFU/ml/g; Fig. 6A). Mice that 326

received 3p-mNP1496-siRNA treatment had a 10-fold decrease in lung titer compared to those in 327

the PBS and off target treatment groups (5.13×107 PFU/ml/g vs. 6.33 ×108PFU/ml/g and 328

6.21×108 PFU/ml/g; Fig 6A). In agreement with these results, Western blotting with lung 329

homogenates showed that NP and NS1 protein levels were reduced in both mNP1496-siRNA 330

and 3p-mNP1496-siRNA treated groups. However, 3p-mNP1496-siRNA has more profound 331

inhibition effect (Fig. 6B). Body weight of mice after virus infection was monitored. Fig. 6C 332

showed that PR8 virus caused rapid weight loss in mice that received pretreatment of PBS or off 333

target siRNA. In contrast, pretreatment of mice with mNP1496-siRNA and 3p-mNP1496-siRNA 334


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protected mice from dramatic weight loss and 3p-mNP1496-siRNA had more profound 335

protection. 336

Inhibition of IAV infection by 3p-mNP1496-siRNA was also evaluated by 337

histopathology. Representative results are shown in Fig. 7. Mice in the PBS and off-target 338

siRNA treated and virus infected groups developed characteristic influenza lesions including 339

severe necrotizing bronchiolitis and interstitial pneumonia (Fig.7A, PBS treated shown). 340

Comparatively, mice in the mNP1496-siRNA treated group developed moderate necrotizing 341

bronchiolitis and interstitial pneumonia (Fig.7B). Mice from the 3p-NP1496-siRNA treated 342

group and the negative control group (off target siRNA treated and non-infected group) had no 343

inflammation and minimal changes to the bronchiolar epithelial cells, including apoptosis and 344

sloughing (Fig.7C, 3p-NP1496-siRNA treated group shown). The lung sections were also 345

evaluated for the presence of IAV specific antigen using IHC staining. There was strong 346

bronchiolar epithelial and interstitial immunoreactivity in lung for the untreated and off-target 347

siRNA treated mice (Fig.7D), and moderate bronchiolar epithelial and interstitial 348

immunoreactivity in lung from mNP1496-siRNA treated mice (Fig. 7E). There was rare 349

bronchiolar epithelial immunoreactivity in lung of 3p-NP1496-siRNA treated mice (Fig.7F) and 350

no immunoreactivity in the lung of the negative control (group 5, data not shown). 351

3p-mNP1496-siRNA treatment also induced RIG-I and IFN-β expression in mice. 352

Total RNA was isolated from mouse lung. Induction of RIG-I and IFN-β mRNA was measured 353

by real-time PCR. As seen in Fig. 8, injection of 3p-mNP1496-siRNA into mice induced 23.8-354

fold increase of RIG-I mRNA (Fig. 8A) and 54.2-fold of IFN-β mRNA expression (Fig. 8B). 355

Injection of off target siRNA as well as mNP1496-siRNA did not induce any RIG-I and IFN-β 356


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mRNA expression. Thus, in vivo experiments demonstrated that 3p-mNP1496-siRNA induced 357

RIG-I and IFN-β expression and has more potent inhibition effect on IAV replication. 358

Therapeutic effect of 3p-mNP1496-siRNA on ongoing influenza virus infection. To 359

investigate the therapeutic effect of 3p-mNP1496-siRNA on virus infection, A549 cells were 360

first infected by PR8 at MOI of 1. At different hours post infection, cells were transfected with 5 361

pmol of 3p-mNP1496-siRNA. At 24 hours post infection, supernatant was harvested for virus 362

titration and cells were lysed for Western blotting. As seen in Fig. 9, when 3p-NP1496-siRNA 363

was administered at 2 hours post infection, viral protein synthesis (Fig. 9A, lane 2) as well as 364

virus yield (Fig. 9B) were significantly inhibited compared to those in non-treated, infected cells. 365

When 3p-mNP1496-siRNA was applied at 4 and 6 hours post infection, the inhibition effects 366

were reduced in a time dependent manner. No inhibitory effect was observed at 8 hour post 367

infection. Fig. 9A also showed that compared to the RIG-I expression level in 2 hours post 368

infection sample, it decreased to 71% and 21% in samples of 6 and 8 hours post infection, 369

respectively. 370

371

Discussion 372

Human influenza infections result in an estimated 3-5 million cases of severe illness and 373

between 250,000 and 500,000 deaths every year around the world. Although current vaccination 374

programs and antiviral drugs could provide some protection against influenza virus infection, 375

development of new prophylactic and therapeutic tools is still needed. SiRNA is a powerful tool 376

that can specifically inhibit gene expression through degradation of target mRNA. Ge et al. 377

designed and tested a total of 20 siRNAs targeting different influenza genes and found that those 378

that target NP are especially effective (6). Further in vivo studies showed that siRNA targeting 379


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NP nt 1496-1514 (NP1496-siRNA) could inhibit various strains of influenza virus infection in 380

mice (5,29). The inhibition was sequences and virus specific, indicating that the antiviral effects 381

of the siRNA are not mediated by IFN-β. Recently, using T7 RNA polymerase synthesized 382

partial double strand 5'ppp-RNA, Ranjan et al. has shown that the 5'ppp-RNA can inhibit drug-383

resistant avian H5N1, 1918 and 2009 pandemic influenza viruses in a RIG-I and type I IFN 384

dependant manner in cells and in mice (21). However, the inhibition effect was not in a siRNA 385

dependent manner, since the sequences of the 5’ppp-RNA are not complementary to any 386

influenza virus mRNAs. 387

Here, we are interested in developing a special siRNA which can play dual antiviral roles: 388

viral gene specific silencing and gene non-specific RIG-I activation. To achieve this goal, we 389

chemically synthesized three short RNA molecules, after annealing they formed two dsRNA 390

molecules, namely mNP1496-siRNA and 3p-mNP1496-siRNA. The only difference between 391

these two dsRNA molecules is that mNP1496-siRNA bears a hydroxyl group at the 5’ end of 392

sense strand, whereas 3p-mNP1496-siRNA possesses a triphosphate at the 5’ end of the sense 393

strand. 394

We first tested whether the modified mNP1496-siRNA still retained the typical siRNA 395

function. By using NP1496-siRNA (29) as a positive control, dose titration experiments showed 396

that while mNP1496-siRNA had similar inhibition effect as that of NP1496-siRNA (Fig. 2), 3p-397

mNP1496-siRNA exerted the most profound inhibition activity (Fig. 3). These results suggested 398

that the aforementioned modifications of mNP1496-siRNA did not change its siRNA inhibition 399

efficiency and 3p-mNP1496-siRNA might exert dual antiviral functions. To further demonstrate 400

that 3p-mNP1496-siRNA could activate RIG-I mediated IFN-β pathway, we assessed RIG-I 401

protein levels after transfection of siRNAs; RIG-I ATPase activity after binding to siRNA, and 402


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IFN-β promoter activity after stimulating with siRNA. Our results demonstrated that 3p-403

mNP1496-siRNA, but not mNP1496-siRNA, could induce RIG-I and IFN-β activities, which 404

contribute to the efficient inhibition of influenza virus infection (Fig. 4). The results that both 405

mNP1496-siRNA and 3p-mNP1496-siRNA exerted similar inhibition effect of IAV on Vero 406

cells (Fig. 5) further confirmed this notion. 407

In animal study, we have demonstrated the pre-treatment of mice with dual functional 3p-408

mNP1496NP-siRNA could significantly reduce virus load and virus induced pathogenesis (Fig. 6 409

and 7). Again, the in vivo experiment was in line with the in vitro results, that dual functional 410

siRNA exerted more profound effect than the monofunctional siRNA. It is noticeable that when 411

chemically adding triphosphate group into the 5’ end of an RNA molecule, the efficiency is only 412

about 30%, i.e. the yield of 3p-mNP1496-S-RNA was only about 30% of the total input of 413

mNP1496-S-RNA. This is mainly due to inherent low efficiency of the chemical modification. 414

Taking this into consideration, it is conceivable that a more pure formulation of 3p-siRNA would 415

have more potent antiviral effect. 416

We also investigated the therapeutic effect of 3p-mNP1496-siRNA on inhibition of 417

influenza virus infection. The inhibitory effect was achieved when 3p-mNP1496-siRNA was 418

given at 2, 4 and 6 hours post virus infection. When 3p-mNP1496-siRNA was given at 8 hours 419

post virus infection, no viral inhibition was achieved. Concomitantly, RIG-I expression was 420

reduced in this sample, this might contribute to the no inhibition effect. In addition, at 8 hours 421

post infection virus has completed one life cycle, the increased amount of mRNA made siRNA 422

function of 3p-mNP1496-siRNA less efficient. 423

One challenge in the use of the siRNA for the treatment of IAV infection is how to 424

deliver the siRNA to the target tissues efficiently. Administration of 3p-siRNA would result in 425


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an induction of innate immune responses of the host, therefore even if less 3p-siRNA is delivered 426

to the virus replication site, it will still exert antiviral activity through the RIG-I activation. Of 427

course, improvement of RNA delivery will greatly enhance the antiviral effect of dual functional 428

siRNA. Transfection reagents and specific vector are two major media for delivering siRNA into 429

animals. The relatively higher price limited the use of cationic polymers based transfection 430

reagents in animals. It has been reported that lentivirus vector based shRNAs showed good 431

delivery of NP1496-siRNA into mice (5,29), therefore how to establish a vector system in which 432

3p-mNP1496-siRNA can be highly expressed and delivered need to be investigated in the future. 433

The other challenge in the use of 3p-siRNA is the cost of chemical synthesis of 3p-RNA. 434

Although T7 polymerase could synthesize RNA bearing triphosphate at 5’ end, the self-coded 3’-435

extension run-off transcription of T7 RNA polymerase (30) makes it difficult to control the 3’ 436

end and get homogenous structure for our studies. Currently, chemical synthesis and 437

modification is still the best way to obtain homogenous 3p-mNP1496-siRNA. 438

It is worthy to note that even though we developed 3p-mNP1496-siRNA which is 439

specific for anti IAV infection, the strategy can be implemented to other important viral 440

pathogens. 441

442

Figure Legend 443

Fig. 1. Schematic representations and characterization of siRNAs. (A) Sequence and 444

structure of NP1496-siRNA. Both strands are 21-nt in length forming a 19-bp double strand with 445

two dTdT overhangs at both 3’ ends. (B) Sequence and structure of mNP1496-siRNA. Sense 446

strand and guide strand is 25-nt and 23-nt in length respectively, forming a 23-bp dsRNA with a 447

UU overhang at the 3’ end of sense strand. The ds-siRNA is blunt ended at the 5’ end of the 448


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sense strand . (C) Sequence and structure of 3p-mNP1496-siRNA. The sequence and structure is 449

quite similar to that of mNP1496-siRNA except that the sense strand contains a triphosphate at 450

5’ end. (D) MALDI-TOF analysis of mNP1496-AS-RNA. The major peak at 7321.8 represents 451

mNP1496-AS-RNA (calculated molecular weight is 7316). (E) MALDI-TOF analysis of 452

mNP1496-S-RNA, the single peak at 7922.8 represents mNP1496-S-RNA (the expected 453

molecular weight is 7922). (F) MALDI-TOF analysis of 3p-mNP1496-S-RNA, the expected 454

molecular weight is 8162, the peak at 8157.1kd represents 3p-mNP1496-S-RNA. There are 455

several other peaks representing mNP1496-S-RNA without or with mono- or bi- phosphate at its 456

5’ end. The tripohsphate mNP1496-S-RNA constitutes about 30% of the final product. (G) 457

Ethidium bromide staining of annealed dsRNAs, the positions of dsRNA and ssRNA are 458

indicated. 459

Fig. 2. Inhibition of IAV infection by mNP1496-siRNA and 3p-mNP1496-siRNA. 460

A549 cells were transfected with mNP1496-siRNA and NP1496-siRNA at the concentrations of 461

0, 5, 10, 15 or 20 pmol in a 24-well plate. Twenty four hours later, cells were infected with PR8 462

virus at MOI of 1. Twenty four hours post infection, cells were harvested and the expression of 463

viral NP and NS1 proteins was assayed by Western blotting. Meanwhile, the levels of β-actin 464

were assessed as internal loading control. NP and NS1 protein levels were first normalized to the 465

level of β-actin in each sample and then compared to that in the sample without any siRNA 466

treatment and expressed as percentage of changes. 467

Fig. 3. Inhibition of IAV infection by 3p-mNP1496-siRNA is more profound. (A) 468

A549 cells were transfected with 3p-mNP1496-siRNA or mNP1496-siRNAs at the 469

concentrations of 0, 0.6, 1.2, 5, or 10 pmol in a 24-well plate. Twenty four hours later, cells 470

were infected with PR8 virus at MOI of 1. Twenty four hours post infection, cells were harvested 471


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and the levels of NP, NS1 and β-actin were evaluated by Western blotting. The percentage of 472

protein level changes was calculated as described in legend to Fig. 2. (B) Virus titres in the 473

supernatant harvested from above experiments were determined by plaque assay. Error bars 474

represent the standard deviation of three independent experiments. (C) A549 cells were 475

transfected with 5 pmol off-target siRNA or 3p- mNP1496 siRNA in a 24-well plate. Twenty-476

four hours post transfection, cells were infected with TX91 or Hallifax at MOI of 1. Twenty-four 477

hours post infection, NP, NS1 and β-actin levels were evaluated by Western blotting using 478

specific antibodies. 479

Fig. 4. Activation of RIG-I and IFN-β pathway by 3p-mNP1496-siRNA. A549 cells 480

were transfected with 5 pmol of 3p-mNP1496-siRNA (A) or mNP1496-siRNA (B) and harvested 481

at the indicated times. Total cell lysates were subject to western blotting with antibodies specific 482

for RIG-I and β-actin. (C) RIG-I ATPase activities after incubating with 3p-mNP1496-siRNAs 483

or mNP1496-siRNA were measured by BIOMOL Green reagent. Three independent 484

experiments were performed and the mean values are plotted with the error bars representing 485

standard deviation. (D) 293T cells were transfected with plasmids pLuc125 and pTK-rLuc 486

together with 10 pmol of siRNAs or 500 ng of polyI:C. Twenty four hours later, luciferase 487

activity was measured using Dual-Luciferase Reporter Assay System. Relative luciferase 488

activities were calculated as the ratio of firefly to renilla luciferase light unit. Fold induction was 489

calculated as the ratio of relative luciferase activity in each sample to that in siRNA 490

untransfected cells. 491

Fig. 5. Viral inhibition effect of 3p-mNP1496-siRNA in Vero cells. Vero cells were 492

transfected with different amount of mNP1496-siRNA or 3p-mNP1496-siRNA for 24 hours. 493

Cells were then infected by PR8 virus for another 24 hours. (A) NP, NS1 and β-actin levels in 494


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the cells were determined by Western blotting. NP and NS1 protein levels were measured as 495

described in the legend to Fig. 2. (B) Virus titres in the supernatant were determined by plaque 496

assay. Error bars represent the standard deviation of three independent experiments. 497

Fig. 6. Inhibition of influenza A virus infection by siRNAs in mice. Mice were 498

intravenously administered respective siRNAs and then intranasally infected by PR8 virus. On 499

day 3 post infection, lung tissue was collected and homogenized. (A) Virus titers were 500

determined on MDCK cells by plaque assay. Mean titer from each group (n=8) were plotted with 501

the error bars representing standard deviation. Comparison of mouse lung virus titer between 502

control group and testing groups was performed by one way ANOVA in Prism. ***, p<0.001. (B) 503

NP, NS1 and β-actin levels were determined by Western blotting. (C) Changes of body weight 504

after virus infection. Error bars represent the standard deviation of 8 mice in one group. 505

Fig. 7. Microscopic lung lesions at 3 days post infection. (A) A lung section from a 506

mouse in PBS treated and virus infected group, with severe necrotizing bronchiolitis and 507

interstitial pneumonia. (B) A lung section from a mouse in mNP1496-siRNA treated and virus 508

infected group, with moderate necrotizing bronchiolitis and interstitial pneumonia. (C) A lung 509

section from a mouse in 3p-NP1496-siRNA treated and virus infected group, no severe 510

pathological changes were observed. (D) Serial section of PBS treated mouse with strong 511

bronchiolar epithelial and interstitial immunoreactivity in lung. (E) Serial section of a mNP1496-512

siRNA treated mouse with moderate bronchiolar epithelial and interstitial immunoreactivity in 513

lung. (F) Serial section of a 3p-mNP1496-siRNA treated mouse with rare bronchiolar 514

immunoreactivity (arrowhead). In panels A, B and C, micrographs are of H&E stained sections, 515

whereas, in panels D, E and F are IHC using anti-influenza antibodies. Scale bars, 200μm. 516


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Fig. 8. Expression of RIG-I and IFN-β mRNAs in the lung. Total RNA was extracted 517

from mice lung. RIG-I (A) and IFN-β mRNA (B) levels were measured by quantitative real-time 518

RT-PCR and were normalized to GAPDH expression in the same sample. Fold induction was 519

achieved by setting the PBS treated and virus infected group as reference. 520

Fig. 9. Therapeutic effect of 3p-mNP1496-siRNA. A549 cells were infected with PR8 521

at MOI of 1. At indicated times post infection cells were transfected with 5 pmol of 3p-522

mNP1496-siRNA. At 24 hours post infection, cells were lysed and subjected to Western blotting 523

using antibodies specific for NP, NS1, RIG-I and β-actin (A). Virus titers in the supernatant were 524

determined on MDCK cells by plaque assay. Error bars represent the standard deviation of three 525

independent experiments (B). 526

527

Acknowledgement 528

We are grateful to the animal care staff at the Vaccine and Infectious Disease Organization for 529

assistance with mice experiments. This work was supported by a grant from CIHR to Y.Z. 530

531


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Table 1. Assignment of mice in each group 532

Group

N=8

Treatment

(IV)

Infection

(IN)

1 3p-mNP1496-siRNA PR8 (5000PFU)

2 mNP1496-siRNA PR8 (5000PFU)

3 off-target siRNA PR8 (5000PFU)

4 PBS PR8 (5000PFU)

5 off-target siRNA PBS

533

IV: intravenous 534

IN: intranasal 535

536

537


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Fig1.

A

B

C

D E

FG

NP-1496 sense strand, 21nt

NP-1496 guide strand, 21nt

G G A U C U U A U U U C U U C G G A G dTdT

dTdT C C U A G A A U A A A G A A G C C U C

NP1496-siRNA

mNP1496-siRNA

G G A U C U U A U U U C U U C G G A G A CUU

U UC C U A G A A U A A A G A A G C C U C U G

A A mNP1496-S-RNA, 25nt

mNP1496-AS-RNA, 23nt

G G A U C U U A U U U C U U C G G A G A CUU

U UC C U A G A A U A A A G A A G C C U C U G

ppp- A A 3p-mNP1496-S-RNA, 25nt

mNP-1496-AS-RNA, 23nt

3p-mNP1496-siRNA

mNP1496-A

S-RNA

mNP1496-S

-RNA

3p-mNP1496-S

-RNA

mNP1496-s

iRNA

3p-mNP1496-s

iRNA

1 2 3 4 5

dsRNA

ssRNA


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Fig. 2

WB: NP

WB: NS1

WB: β-actin

0 5 10 15 20 5 10 15 20 pmol

NP1496-siRNA mNP1496-siRNA

NP changes (%) 100 61 27 12 - 46 25 6 -

NS1 changes (%) 100 73 55 21 0.2 71 43 20 0.3

1 2 3 4 5 6 7 8 9


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Fig. 3

C

WB: NP

WB: NS1

WB: β-actin

0 0.6 1.2 5 10 0.6 1.2 5 10 pmol

3p-siRNAmNP1496-siRNAA

NP changes (%) 100 96 97 55 19 58 18 - -

NS1 changes (%) 100 98 97 58 21 61 20 - -

1 2 3 4 5 6 7 8 9

B

Vir

us t

iter

(Lo

g1

0 P

FU

/ml)

1.0E7

1.0E6

1.0E5

0 1.2 5 10

WB: NP

WB: NS1

WB: β-actin

TX

91

Ha

lifa

x

Ha

lifa

x

TX

91

Off target 3p-siRNA

siRNA (pmol)

No treatment

NP-siRNA

mNP-siRNA

3p-mNP-siRNA

1.0E8


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Fig. 4

WB: RIG-I

WB: β-actinWB: RIG-I

WB: β-actin

0 4 6 8 12 24 48 60 hr

3p-mNP1496-siRNA

SiO

T

3p

-siR

NA

m-s

iRN

A

24hr

A

B

C

D

siRNA binding and RIG-I ATPase activity

0 100 200 300 400 500 600 7000

200

400

600

800mNP

3p-mNP

siRNA concentration (nM)

OD

65

5nm

untrea

ted

Off

targ

et

mNP

3p-m

NP

polyI:C

0

5

10

15untreated

Off target

mNP

3p-mNP

polyI:C

Treatment

Fo

ld i

nd

ucti

on


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WB: NP

WB: NS1

WB: β-actin

3p-siRNAmNP-siRNA

0 5 10 20 5 10 20 pmol

Fig. 5

NP changes (%) 100 71 37 7 77 41 8

NS1 changes (%) 100 79 38 4 88 42 9

1 2 3 4 5 6 7

A

B

Vir

us t

iter

(Lo

g1

0 P

FU

/ml)

0 5 10

siRNA (pmol)

no treatment

mNP-siRNA

3pmNP-siRNA

1.0E7

1.0E6

1.0E5

1.0E4


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Fig. 6

WB: NP

WB: NS1

WB: β-actin

PB

S

mN

P-s

iRN

A

Off ta

rge

t

3p

-siR

NA

A

B

mouse lung virus titer

mNP

PBS

Off target

3p-mNP

Treatment

Vir

us

tit

er

(Lo

g1

0 P

FU

/ml/g

)

C

0 1 2 3

90

95

100

105

PBS/PR8

Off/PR8

NP/PR8

3p-NP/PR8

Off/PBS

Days post infection

Bo

dy w

eig

ht

(%)

***

***

3pNP/P

R8

PBS/P

R8

off/PR

8

NP/P

R8

1.0E7

1.0E9

1.0E8


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Fig. 7


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Fig. 8

RIG-I mRNA

PBS

Off

targ

et

mNP

3p-m

NP

0

10

20

30PBS

Off target

mNP

3p-mNP

Treatment

Fo

ld in

du

cti

on

IFN-β mRNA

PBS

Off

targ

et

mNP

3p-m

NP

0

20

40

60

Off target

PBS

mNP

3p-mNP

Treatment

Fo

ld in

du

cti

on

A

B


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Fig. 9

A

WB: NP

WB: NS1

WB: β-actin

NP changes (%) 10 19 49 95 100

PR8

NS1 changes (%) 11 21 52 96 100

WB: RIG-I

- + + + + +

3p-siRNA - 2 4 6 8 -

B

no trea

tmen

t

SiRNA treatment (hrs post infection)

2 4 6 8

1 2 3 4 5 6

h.p.i.

RIG-I changes (%) 100 99 71 21

1.0E5

1.0E6

1.0E7

1.0E8

Vir

us

tit

er

(Lo

g1

0 P

FU

/ml/g

)


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1 5' triphosphate-sirna: potent inhibition of influenza a virus

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