Messenger RNA cap methylation influences pathogenesis...

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Messenger RNA cap methylation influences pathogenesis of 2

vesicular stomatitis virus in vivo. 3

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Yuanmei Ma1, Yongwei Wei2, Xiaodong Zhang2, Yu Zhang2, Hui Cai1, Yang Zhu2, Konstantin 5

Shilo3, Michael Oglesbee1, Steven Krakowka1, Sean P. J. Whelan4, Jianrong Li1* 6

7 1Department of Veterinary Biosciences, College of Veterinary Medicine, 2Department of Food 8

Science and Technology, College of Food, Agricultural and Environmental Sciences, 3College of 9

Medicine, The Ohio State University, Columbus, OH, USA; 4Department of Microbiology and 10

Immunobiology, Harvard Medical School, Boston, MA, USA. 11

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13 *Corresponding author 14

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Department of Veterinary Biosciences 16

College of Veterinary Medicine 17

The Ohio State University 18

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1925 Coffey Road 20

Columbus, OH 43210 21

Tel: 614-688-2064 22

Fax: 614-292-6473 23

Email: [email protected] 24

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Running title: VSV mRNA cap methylation and pathogenesis 27

Abstract = 246 words; Text = 8006 words; Total figures = 10; Total Tables = 6 28

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JVI Accepts, published online ahead of print on 26 December 2013J. Virol. doi:10.1128/JVI.03420-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Abstract 33

One role of mRNA cap guanine-N-7 (G-N-7) methylation is to facilitate the efficient 34

translation of mRNA. The role of mRNA cap ribose 2’-O (2’-O) methylation is enigmatic, 35

although recent work has implicated this as a signature to avoid detection of RNA by the innate 36

immune system (Daffis et al, 2010). Working with vesicular stomatitis virus (VSV), we 37

previously showed that a panel of VSVs carrying mutations to a predicted methyltransferase 38

catalytic site (rVSV-K1651A, D1762A, and E1833Q) or S-adenosylmethionine (SAM) binding 39

site (rVSV-G1670A, G1672A, and G4A) were defective in cap methylation and were also 40

attenuated for growth in cell culture. Here, we analyzed the virulence of these recombinants in 41

mice. We found that recombinants rVSV-K1651A, D1762A, and E1833Q, which are defective in 42

both G-N-7 and 2’-O methylation, were highly attenuated in mice. All three viruses elicited a 43

high level of neutralizing antibody, and provided full protection against a challenge with the 44

virulent VSV. In contrast, mice inoculated with recombinants rVSV-G1670A and G1672A that 45

are defective only in G-N-7 methylation, were attenuated in vivo yet retained a low level of 46

virulence. Recombinant rVSV-G4A which is completely defective in both G-N-7 and 2’-O 47

methylation also exhibited low virulence in mice despite the fact that productive viral replication 48

was not detected in lung and brain. Taken together, our results suggest that abrogation of viral 49

mRNA cap methylation can serve as an approach to attenuate VSV, and perhaps other 50

nonsegmented negative-strand RNA viruses for potential application as vaccines and viral 51

vectors. 52

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Significance 56

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Non-segmented negative-sense (NNS) RNA viruses include a wide range of significant 58

human, animal, and plant pathogens. For many of these viruses, there are no vaccines or antiviral 59

drugs available. Messenger RNA cap methylation is essential for mRNA stability and efficient 60

translation. Our current understanding of mRNA modifications of NNS RNA viruses comes 61

largely from studies of vesicular stomatitis virus (VSV). In this study, we showed that 62

recombinant VSVs (rVSV) defective in mRNA cap methylation were attenuated in vitro and in 63

vivo. In addition, these methyltransferase (MTase)-defective rVSVs triggered high levels of 64

antibody responses and provided complete protection against VSV infection. Thus, this study 65

will not only contribute to our understanding of the role of mRNA cap MTase in viral 66

pathogenesis, but also will facilitate the development of new live attenuated vaccines for VSV, 67

and perhaps other NNS RNA viruses, by inhibiting viral mRNA cap methylation. 68

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Introduction 79

Non-segmented negative-sense (NNS) RNA viruses include a wide range of significant 80

human, animal, and plant pathogens. The NNS RNA viruses are classified into four families: the 81

Rhabdoviridae, as exemplified by rabies virus and vesicular stomatitis virus (VSV); the 82

Filoviridae, as exemplified by Ebola and Marburg viruses; the Bornaviridae, as exemplified by 83

Borna disease virus; and the Paramyxoviridae, as exemplified by human respiratory syncytial 84

virus (hRSV), human metapneumovirus (hMPV), human parainfluenza virus type 3 (hPIV3), 85

measles, mumps, Nipah and Hendra viruses. For many of these viruses, there are no vaccines 86

available, and there are no antiviral drugs with proven efficacy against this entire class of 87

viruses. With the exception of the Bornaviridae, the mammalian NNS RNA viruses replicate 88

within the host cell cytoplasm and do not have access to the host mRNA capping machinery. 89

Instead, the viruses have evolved their own mRNA capping machinery. Specifically, the large 90

(L) polymerase protein, is a 220-240 kDa multifunctional protein that possesses nucleotide 91

polymerization (1-3) and mRNA modification activities, including cap addition (4-6), cap 92

methylation (7-11) and polyadenylation(12-15). The enzymatic activities have been mapped 93

within L to motifs that are highly conserved amongst all NNS RNA viruses (16). Specifically, a 94

GDN motif, present within conserved region (CR) III is essential for nucleotide polymerization 95

(1). CR-V contains a GxxT[N]HR motif that is required for mRNA capping catalyzed by a 96

GDP:polyribonucelotidyltransferase (PRNTase) (4-6), and, CR-VI contains a methyltransferase 97

(MTase) motif (KDKE) that catalyzes both guanine-N-7 (G-N-7) and ribose 2’-O (2’-O) 98

methylation (6, 9, 11, 17). The molecular architecture of the VSV L polymerase protein (18) 99

shows that a core ring-like domain contains the RdRP and an appendage of three globular 100

domains contains the cap forming machinery. 101


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Our understanding of mRNA modifications of NNS RNA viruses comes largely from 102

studies of VSV. In response to a specific promoter element provided by the genomic leader 103

region, the polymerase initiates mRNA synthesis at the first gene-start sequence (19-20). The 104

nascent mRNA is capped through an unconventional mechanism in which the GDP:PRNTase of 105

L transfers a monophosphate RNA onto a GDP acceptor through a covalent protein-RNA 106

intermediate (4-6). Following cap formation, VSV mRNAs are sequentially methylated at ribose 107

2’-O position and G-N-7 position (8, 21), which is the opposite order to other known methylation 108

reactions. Unlike traditional cap forming enzymes (22), the VSV capping and methylation 109

machinery require cis-acting signals in the RNA (8, 23-25) and a minimal nascent RNA chain 110

length of 31 nt (26). In response to a gene-end sequence, L polyadenylates and terminates 111

mRNA synthesis by the programmed stuttering of polymerase on a U7 tract (27). 112

Messenger RNA 5′ cap structures are essential for mRNA stability and efficient 113

translation (28). Guanine-N-7 (G-N-7) methylation of the mRNA cap structure is required for 114

recognition of the cap by the rate limiting factor for translation initiation, eIF-4E (28-30) . 115

Although the precise mechanism by which VSV mRNA’s are translated is not fully understood, 116

they utilize a variation of the canonical cap-dependent translational pathway that is 117

hypersensitive to the lack of a ribosomal protein, rpL40 (30-34). In infected cells, host mRNA 118

translation is rapidly inhibited through a suppression of the intracellular pools of eIF-4E by a 119

manipulation of the phosphorylation status of the 4E binding protein (4E-BP1) (35). 120

Nevertheless, in vitro experiments have shown that G-N-7 cap methylation facilitates translation 121

of VSV mRNA (30-31, 33-34). The biological function of mRNA cap 2’-O methylation, 122

however, remains less well understood. Recent work with West Nile virus (WNV), vaccinia 123

virus, and SARS coronavirus showed that 2'-O methylation of viral RNA cap structure functions 124


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to prevent the detection of viral RNA by the innate immune system (36-38). From this 125

perspective, 2'-O-methylation provides a molecular signature for the discrimination of self and 126

non-self mRNA. 127

Previously, we characterized recombinant MTase-defective VSV that contain 128

substitutions to a predicted MTase catalytic motif KDKE or a SAM binding site (9, 17, 21). 129

Those VSVs can be classified into three groups. Viruses completely defective in both G-N-7 and 130

2’-O methylation (rVSV-K1651A, D1762A, K1795A, E1833Q, and G4A), those defective in G-131

N-7, but not 2’-O MTase (rVSV-G1670A and G1672A), and those that require elevated SAM 132

concentrations to permit full methylation (rVSV-G1674A and Y1835A). With the exception of 133

rVSV-G1674A and Y1835A, all MTase-defective VSVs were attenuated in cell culture as 134

judged by diminished viral plaque size, reduced infectious viral progeny release (in single-step 135

growth curves), and decreased levels of viral RNA and protein synthesis (9, 17, 21) . However, 136

whether these MTase-defective VSVs are attenuated in vivo is not known. 137

In this study, we examined the impact of mRNA cap methylation status on VSV 138

pathogenicity in mice. We found that mutations to the predicted MTase catalytic site were highly 139

attenuated, and that substitution to the SAM binding site that led to defects in G-N-7, but not 2’-140

O methylation retained low virulence. Each of those recombinants stimulated high levels of 141

VSV-specific antibody and provided full protection against challenge with wild type virus, 142

demonstrating that such methylation defective viruses might be good vaccine candidates. 143

Remarkably, not all mutations that blocked methylation were highly attenuated, since rVSV-144

G4A which contains 4 amino acid substitutions to the predicted SAM binding site retained low 145

virulence in mice despite the fact that it is completely defective in G-N-7 and 2’-O methylation 146

and lacks evidence of productive viral replication in vivo. Since the transcripts generated by this 147


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virus in vitro lack both G-N-7 and ribose 2’-O methyl groups, we anticipated that this virus 148

would be exquisitely sensitive to control by the innate immune system. The molecular 149

mechanism of the virulence associated with this virus will likely be of interest to determine and 150

may help further our understanding of the innate immune control of virus infection. 151

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Materials and Methods 153

Recombinant VSV. Wild-type recombinant VSV (rVSV) and MTase-defective VSVs (rVSV-154

K1651A, D1762A, E1833Q, G1670A, G1672A, G4A, and G1674A) were recovered from an 155

infectious clone of VSV Indiana strain as described previously (9, 21). All recombinant VSVs 156

were grown on BHK-21 cells in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, 157

Carlsbad, CA) containing 2% fetal bovine serum (FBS, Invitrogen). Virus titers were determined 158

by plaque assay in Vero cells as previously described (9, 21). 159

160

Transcription of viral RNA in vitro and scintillation counting. Viral RNA was synthesized in 161

vitro by using 10 μg of purified virus (rVSV and rVSV-G4A) as described previously (9, 21). 162

Reactions were performed in the presence of 1 mM ATP; 0.5 mM CTP, GTP, and UTP; and 15 163

μCi [3H] S-adenosyl-L-methionine (SAM) (85 Ci/mmol, PerkinElmer, Billerica , MA), and 25% 164

(vol/vol) rabbit reticulocyte lysate. Where indicated, reaction mixtures were supplemented with 1 165

mM S-adenosylhomocysteine (SAH). After incubation at 30 oC for 5 h, RNA was purified by 166

RNeasy Mini Kit (Qiagen, Valencia, CA). Aliquots of purified RNA were mixed with 4 ml of 167

ReadySafe scintillation mixture (Beckman Coulter, Brea, CA), and dpm was measured by 168

scintillation counting using a 1414 series counter (PerkinElmer). 169

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Replication of VSV in different cell lines. Confluent BHK-21, Vero, or HEp-2 cells in 35 mm 171

dishes were infected by rVSV or G4A at an MOI of 0.1. After 1 h of adsorption, the inoculum 172

was removed, the cells were washed three times with DMEM, fresh DMEM (supplemented with 173

2% fetal bovine serum) was added, and infected cells were incubated at 37°C. After 24 h, 174

supernatants were harvested, and virus titers were determined by plaque assay on Vero cells. 175


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Analysis of viral mRNA cap methylation in cell culture. Confluent BHK-21, Vero cells, or 177

HEp-2 in 35 mm dishes were infected by rVSV or G4A at an MOI of 0.1. After 24 h post-178

infection, total RNA was isolated by RNeasy Mini Kit (Qiagen). Subsequently, poly (A)-179

containing RNA were isolated from total RNA using Dynabeads® mRNA isolation Kit 180

(Invitrogen) according to manufacture’s recommendations. To examine G-N-7 methylation, 181

equal amounts of viral RNA were incubated with 10 units of vaccinia virus G-N-7 MTase 182

supplied by m7G Capping System (ScriptCap™) in the presence of 15 μCi [3H]-SAM (85 183

Ci/mmol, PerkinElmer). To examine 2’-O methylation, viral RNA were incubated with 10 units 184

of vaccinia virus G-N-7 MTase in the presence of 100 μM of cold SAM, and RNA were purified 185

and further incubated with vaccinia virus 2’-O MTase (VP39). Finally, RNA was purified by 186

RNeasy Mini Kit (Qiagen), and the methylation of mRNA cap structure was measured by 3H 187

incorporation using a 1414 series scintillation counter (PerkinElmer). 188

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Animal experiments. 190

Animal experiment 1 (pathogenicity): Seventy two four-week-old specific-pathogen-free 191

female BALB/c mice (Charles River laboratories, Wilmington, MA) were randomly divided into 192

nine groups (8 mice per group). These mice were housed within ULAR facilities of The Ohio 193

State University under approved Institutional Laboratory Animal Care and Use Committee 194

(IACUC) guidelines. Each inoculation group was separately housed in rodent cages under BSL-2 195

conditions. Mice in group 1 were inoculated with rVSV and served as a virulent control. Mice in 196

groups 2-8 were inoculated with seven different MTase-defective VSVs (K1651A, D1762A, 197

E1833Q, G1670A, G1672A, G1674A, and G4A). Mice in group 9 were inoculated with DMEM 198


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and served as an uninfected control (the normal control). Each mouse was inoculated intranasally 199

at a dose of 5×106 PFU in a volume of 50µl. After inoculation, the animals were evaluated twice 200

every day for mortality and the presence of any symptoms of VSV infection. The severity of 201

clinical signs associated with VSV infection was scored based on the following criteria: grade 3 202

(severe) was characterized by ruffled fur, hyperexcitability, tremors, circling, and paralysis; 203

grade 2 (moderate) was characterized by ruffled fur with neurological symptoms such as 204

circling; grade 1 (mild) was characterized by ruffled fur, no neurological symptoms; grade 0 205

mice exhibited no symptoms. The body weight for each mouse was monitored on a daily basis. 206

At day 7 post-inoculation, 4 mice from each group were euthanized. At day 14 post-inoculation, 207

the remaining 4 mice from each group were euthanized. The brain, lungs, and spleens from each 208

mouse were collected for both virus isolation and histological analysis as described below. 209

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Animal experiment 2 (pathogenicity): Forty four-week-old specific-pathogen-free female 211

BALB/c mice were randomly divided into 5 groups (8 mice per group). Mice in group 1 were 212

inoculated with DMEM and served as the uninfected control. Mice in group 2 were inoculated 213

with rVSV and served as the virulent control. Mice in groups 3-5 were inoculated with MTase-214

defective rVSV-K1651A, G1670A, and G4A, respectively. All mice were inoculated intranasally 215

at a dose of 5×106 PFU per mouse and were evaluated as described in Animal experiment 1 216

except that euthanasia was performed at days 3 and 5 post-inoculation. 217

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Animal experiment 3 (pathogenicity): Twenty four four-week-old specific-pathogen-free 219

female BALB/c mice were randomly divided into 3 groups (8 mice per group). Mice in groups 1-220

3 were inoculated with DMEM, rVSV, rVSV-G4A, respectively. All mice were inoculated 221


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intranasally at a dose of 5×106 PFU per mouse and were evaluated as described in Animal 222

experiment 1 except that euthanasia was performed at days 1 and 2 post-inoculation. 223

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Animal experiment 4 (LD50): The LD50 of selected recombinant VSVs (rVSV, G1674A, G4A, 225

G1762A, and K1651A) was determined. Briefly four-week-old specific-pathogen-free female 226

BALB/c mice were inoculated with four doses of each recombinant virus (105, 106, 107, and 227

108PFU per mouse, 5 mice per group). Animals were observed every 12 h until 14 days post-228

inoculation. The 50% lethal dose (LD50) for each virus was calculated by the method of Reed 229

and Muench (39). 230

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Animal experiment 5 (immunogenicity): Twenty eight four-week-old specific-pathogen-free 232

female BALB/c mice were randomly divided into seven groups (4 mice per group). Mice in 233

groups 1-5 were immunized with MTase-defective rVSV-K1651A, D1762A, E1833Q, G1670A, 234

and G1672A, respectively. Mice in group 6 were inoculated with DMEM and served as the 235

challenged control. Mice in group 7 were inoculated with DMEM and served as the uninfected 236

control (normal control). All mice were immunized intranasally at a dose of 5×106 PFU per 237

mouse. After immunization, the animals were evaluated daily for body weight, mortality and the 238

presence of any symptoms of VSV infection. Blood samples were collected from each mouse 239

weekly by facial vein bleed, and serum was isolated for antibody detection. At week 4 post-240

immunization, mice in groups 1-6 were challenged with rVSV at a dose of 5×106 PFU per 241

mouse. After challenge, the animals were evaluated twice every day for mortality and the 242

presence of any symptoms of VSV infection. The body weight for each mouse was monitored on 243

a daily basis. At day 7 post-challenge, all mice from each group were euthanized. The brains, 244


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lungs, and spleens from each mouse were collected for virus isolation and histological 245

evaluation. 246

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Animal experiment 6 (immunogenicity): Experimental design was essentially identical to 248

experiment 3 with the exception of performing a booster immunization. Briefly, twenty eight 249

four-week-old SPF female BALB/c mice were randomly divided into seven groups (4 mice per 250

group). Mice in groups 1-6 were immunized intranasally at a dose of 5×106 PFU with rVSV 251

K1651A, E1833Q, D1762A, G1670A, G1672A, and G4A, respectively. At week 2 post-primary 252

immunization, mice were boosted intranasally with 5×106 PFU of the corresponding virus. At 253

week 3 post-boost immunization, mice in groups 1-6 were challenged with rVSV at a dose of 254

5×106 PFU per mouse. At day 7 post-challenge, all mice were euthanized, and brains and lungs 255

were collected for virological and pathological evaluation. 256

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Determination of virus burden in lungs and brains. A brain hemisphere and right lung from 258

each mouse were homogenized in PBS solution, and the presence of infectious virus was 259

determined by plaque assay in Vero cells as described previously (9, 21). The presence of viral 260

RNA in brain and lung tissues was determined by reverse transcription (RT)-PCR. Briefly, total 261

RNA was extracted from lungs and brain using an RNeasy Mini Kit (Qiagen) according to the 262

manufacturer’s instructions. Two primers (5’-ATGTCTGTTACAGTCAAGAG-3’and5’- 263

TCATTTGTCAAATTCTGAC-3’) were designed to target VSV N gene ranging from position 264

64 to position 1332 (numbering refers to the complete VSV Indiana strain genome sequence). 265

RT-PCR was performed using a One Step RT-PCR kit (Qiagen).The amplified products were 266

analyzed on 1% agarose gel. 267


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Quantification of IFN-β mRNA by real-time RT-PCR. Total RNA was extracted from lung or 269

brain tissues using the Qiagen RNeasy kit (Qiagen). The RNA was treated with DNase I 270

(Ambion), and first strand cDNA was synthesized by Superscript III Reverse Transcriptase 271

(Invitrogen) using Oligo dT primers (Promega). Real-time PCR was performed in 20 µl reactions 272

with primers for mouse IFN-β (Forward: 5′-CTC CAG CTC CAA GAA AGG AC-3′; reverse: 5′-273

TGG CAA AGG CAG TGT AAC TC-3′). The mRNA levels of IFN-β were determined using 274

the SYBR Green master mix (Applied Biosystems). The PCR conditions and cycles were as 275

follows: initial DNA denaturation 10 min at 95°C, followed by 45 cycles at 95°C for 10 s, 276

followed by an annealing step at 58°C for 10 s, and then extension at 72°C during 10 s, and then 277

signal detection 81°C for 2 s. A plasmid containing sequence of mouse IFN-β was used as 278

standard. 279

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RT-PCR and sequencing. To determine whether MTase-defective VSVs were genetically 281

stable in mice, viral RNA was isolated from lungs and brain. The entire L gene of each MTase-282

defective VSV was amplified by One Step RT-PCR using four overlapping fragments. In 283

addition, the full-length genome of rVSV-G4A isolated from lung or brain was also amplified. 284

The PCR products were purified and sequenced at The Ohio State University Plant Microbe 285

Genetics Facility to confirm the presence of mutation(s) in MTase region. 286

287

Detection of antibody by ELISA. 96-well plates were first coated with 50 µl of highly purified 288

VSV (30 µg/ml, in 50 mM Na2CO3 buffer, pH 9.6) per well at 4°C overnight. Subsequently, 289

individual serum samples were tested for VSV-specific antibody on antigen-coated plates. 290


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Briefly, serum samples were two-fold serially diluted and added to VSV-coated wells. After 291

incubation at room temperature for 1 h, the plates were washed five times with phosphate 292

buffered saline (PBS)-Tween (0.05%), followed by incubation with 50 µl of goat anti-mouse IgG 293

horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma) at a dilution of 1:80,000 294

for 1 h. Plates were washed and developed with 75 µl of 3,3´,5,5´-tetramethylbenzidine (TMB), 295

and the optical density (OD) at 450 nm was determined using an ELISA plate reader. End point 296

titers were determined as the reciprocal of the highest dilution that had an absorbance value 297

greater than the background level (DMEM control). Antibody titers were calculated by the 298

Geometric Mean Titers (GMT). 299

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Histology: Half of tissues from each experiment were preserved in 4% (v/v) phosphate-buffered 301

paraformaldehyde. After fixation, the brain was removed from the skull. A horizontal brain 302

section containing the olfactory bulb, forebrain, cerebrum, hypocampus and cerebellum was 303

recovered. Fixed tissues were embedded in paraffin, sectioned at 5 microns, and stained with 304

hematoxylin-eosin (HE) for the examination of histological changes by light microscopy. The 305

severity of lung histological change was scored based on the following criteria: grade 3 (severe), 306

prominent lymphoid infiltrates around bronchovascular bundles and venules with extension into 307

interlobular septae and interstitium; grade 2 (moderate), lymphoid infiltrates present around 308

bronchovascular bundles and venules without extension into interlobular septae and interstitium; 309

grade 1 (mild), minimal lymphoplasmacytic infiltrates involving occasional bronchovascular 310

bundles and venules; grade 0, no lesions. The severity of brain histological change was scored 311

according to the following criteria: grade 3 (severe), focally extensive and/or multifocal 312

mononuclear inflammatory cell infiltrates in the meanings, around blood vessels in the 313


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parenchyma (perivascular cuffs) with extension into surrounding neuropil, and evidence of 314

gliosis (i.e., parenchymal reaction); grade 2 (moderate), two to three foci of limited infiltration; 315

grade 1 (mild), one infiltrate or parenchymal reaction; grade 0, no lesions. The histological slides 316

were examined independently by three pathologists. 317

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Quantitative and statistical analyses. Quantitative analysis was performed by either 319

densitometric scanning of autoradiographs or by using a phosphorimager (GE Healthcare, 320

Typhoon) and ImageQuant TL software (GE Healthcare, Piscataway, NJ). Statistical analysis 321

was performed by one-way multiple comparisons using SPSS 8.0 statistical analysis software 322

(SPSS Inc., Chicago, IL). A P value of <0.05 was considered statistically significant. 323

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Results 325

MTase-defective VSVs exhibited a variable degree of attenuation in mice. 326

To determine whether MTase-defective VSVs were attenuated in an in vivo pathogenesis 327

model, we inoculated purified virus into BALB/c mice and monitored body weight changes and 328

scored clinical symptoms. Mice infected with rVSV lost 3-5 g of body weight over a 7-day 329

period and 2/8 mice died (Fig.1). All mice in this group exhibited severe clinical symptoms of 330

VSV infection including ruffled fur, hyperexcitability, tremors, circling, and paralysis. The 331

average score of clinical symptoms in rVSV group was 3.0 (severe) (Table 1). In contrast, mice 332

inoculated with recombinants rVSV-K1651A, D1762A and E1833Q exhibited no clinical signs 333

of VSV infection including weight loss (score 0, no symptoms). The average body weight of 334

mice inoculated with these recombinants was indistinguishable from that of the uninfected 335

control (P>0.05) during the entire experimental period (Fig.1). Mice inoculated with rVSV-G4A, 336


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G1670A, and G1672A showed moderate weight loss (2-3 g during day 3-7 post-inoculation). 337

The average body weights of mice in rVSV-G4A and G1670A groups were indistinguishable 338

(P>0.05) by day 14 but were lower than that of the uninfected control (P<0.05). During days 3-5 339

post-inoculation, mice inoculated with G1672A had significant less weight loss compared to 340

G4A and G1670A groups (p<0.05). In addition, mice in G4A group had a faster weight recovery 341

compared to G1670A and G1672A groups (Fig.1). A few mice inoculated with these 342

recombinants showed mild illnesses such as ruffled coat for 2-3 days but recovered quickly. 343

None of mice in rVSV-G1670A and G1672A had neurological symptoms or died. The average 344

score of clinical symptoms in these groups was 1.0 (mild) (Table 1). However, some mice in 345

rVSV-G4A group exhibited mild neurological symptoms with average score of 1.375, which was 346

not significantly different from mice challenged with rVSV-G1670A and G1672A (P>0.05) 347

(Table 1). Mice inoculated with the rVSV-G1674A group exhibited similar body weight loss to 348

that of rVSV group (P>0.05). In addition, mice in rVSV-G1674A groups showed significant 349

clinical signs of VSV infections including ruffled fur and neurological disorders although the 350

severity was diminished compared to rVSV. Taken together, these data demonstrated that (i) 351

recombinants rVSV-K1651A, D1762A and E1833Q are avirulent in mice; (ii) rVSV-G4A, 352

G1670A and G1672A retain low virulence; and (iii) rVSV- G1674A remains virulent in mice. 353

354

MTase-defective VSV had diminished pathological changes in lung and brain. 355

To examine pathological changes, four out of eight mice from each group were 356

euthanized at day 7 post-inoculation. Lung, spleen, and brain from each mouse were collected 357

for histological examination and antigen detection. At the gross level, lung tissues from rVSV 358

and G1674A-infected mice appeared to be dark-red in color, a signature of inflammation, 359


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whereas lungs from other MTase-defective VSV groups as well as the uninfected control group 360

were pink. The spleen samples from rVSV and G1674A-infected mice were enlarged as 361

compared to those from the uninfected control group. As for brain tissues, no significant gross 362

pathological changes were observed in all infected groups as compared with the uninfected 363

control. Following euthanization of the remaining 4 mice in each group at day 14 post-364

inoculation, no significant gross pathological changes were found in lung, spleen and brain in 365

comparison to the uninfected controls. 366

To evaluate the histopathological changes in the brain and lung tissues, samples were 367

fixed and embedded in paraffin, sectioned, followed by hematoxylin-eosin staining. At day 7 368

post-infection, pulmonary tissues from the rVSV group showed severe histological changes 369

characterized by prominent mononuclear cell infiltrates (lymphocytes and macrophages) around 370

bronchi and bronchiole vascular bundles and venules (Fig.2, and Table 1). This inflammatory 371

response extended into interlobular septae and interstitium resulting in variably severe interstitial 372

pneumonia and mononuclear cell alveolitis (Fig.2). Pulmonary tissue sections from mice of 373

groups rVSV-K1651A, G1670A, G1672A, G4A and G1674A groups had moderate lymphocyte 374

and monocyte cellular infiltrates, while recombinant rVSV-E1833Q only exhibited mild 375

inflammatory cell changes in lung (Fig.2, and Table 1). At day 14 post-infection, the pulmonary 376

infiltrates were diminished and were scored as moderate. In contrast, lung tissue sections from all 377

MTase-defective VSV groups exhibited either no or mild lymphoid infiltrates (Table 1). No 378

inflammation was observed in lung tissues from the uninfected control group. The extent and 379

severity of brain inflammatory cell infiltrates varied by inoculation group. By day 7 post-380

infection, brain sections from the rVSV group showed moderate histological changes 381

characterized by multifocal meningeal mononuclear cell infiltrates with formation of 382


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perivascular cuffs in the parenchyma and evidence of inflammatory cells extending into the 383

neuropil (Fig.3 and Table 1). Brain tissues from the rVSV-G1670A, G1674A and G4A infected 384

mice had mild inflammatory changes, where as those from rVSV-K1651A, E1833Q and 385

G1672A had no apparent histological change (Table 1). By day 14 post-infection, no histological 386

change was found in brain tissues from all groups. Taken together, these data demonstrated that 387

(i) infection with rVSV causes moderate inflammatory changes in both lung and brain tissues; 388

(ii) infection with rVSV-K1651A, E1833Q, and G1672A causes mild histological changes in the 389

lungs but not the brain; and (iii) infection with rVSV-G1670A, G4A and G1674A causes 390

moderate histological changes in the lungs and mild changes in brains. 391

392

Disparate levels of virus burden in the lungs and brains of mice inoculated with MTase-393

defective VSV. 394

Since the above analysis revealed disparate effects in brain and lung pathology following 395

infection of mice with the MTase defective viruses, we examined the amount of virus present in 396

the lung and brains by plaque assay (Table 1). At 7 days post-inoculation, 3 out of 4 mice in the 397

rVSV group had virus in the brain (average titer of 2.6 ×105), whereas no infectious virus 398

particles were detected in all other groups. At 14 days post-inoculation, no virus was detected in 399

any brain or lung tissue. 400

Since we were unable to detect any infectious virus particles at both days 7 and 14 post-401

inoculation in MTase-defective VSV groups, we next tested for viral RNA by RT-PCR. Briefly, 402

after extraction of total RNAs from equal amounts of lung and brain tissue, a 1268 bp fragment 403

spanning the VSV N gene was amplified by RT-PCR and subsequently analyzed by agarose-gel 404

electrophoresis. The presence of the viral N gene was detected in most lung samples from 405


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MTase-defective infected mice at days 7 post-inoculation, however, the amount of the RT-PCR 406

product was lower than that obtained for the rVSV infected mice (Fig.4). Similar observations 407

were obtained with brain samples. While 3 out of 4 brain samples from rVSV-infected mice at 408

day 7 post-inoculation were positive in viral RNA detection, no viral RNA was detected in most 409

of brain samples from mice infected by MTase-defective VSVs (Table 1, Fig.4). At day 7, 2 out 410

of 4 mice in the G4A challenge group had detectable viral RNA in brain whereas no viral RNA 411

was detected in G1670A and G1672A infected animals (Table 1). By day 14, lung samples from 412

the mice infected with MTase-defective VSV were negative for viral RNA, where as those from 413

the rVSV group remained positive. Typically, viral RNA levels in the brain were significantly 414

less than that in lung tissues (Table 1, Fig.4). 415

One possibility underlying our inability to detect infectious virus in most lung and brain 416

samples from mice infected with MTase-defective VSVs is that the virus had already been 417

cleared by day 7 post-inoculation. To determine whether VSV was present in the lung and brain 418

tissues from infected mice at earlier time points, we therefore inoculated mice with rVSV, rVSV-419

K1651A, G1670A and G4A and monitored as above except that the euthanization time points 420

were day 3 and day 5 post-inoculation (Table 2). By day 3 post-inoculation, 6.7 × 105, 1.1×103, 421

3.9×104, and 2.1 × 101 PFU/g of viruses were detected in lung samples from mice infected with 422

rVSV, rVSV-K1651A, G1670A and G4A, respectively. By day 5 post-inoculation, virus titers 423

were diminished to 1.2 ×103 and 6.3 ×101 PFU/g for rVSV and rVSV-G1670A groups 424

respectively, and below the limit of detection for rVSV-K1651A and G4A groups. All brain 425

samples from mice in the rVSV group also had high viral burden at day 3 post-inoculation with 426

an average titer of 8.0 × 105 PFU/g. In comparison, the brains of mice inoculated with rVSV-427

G1670A group contained 1.5×104 PFU/g (P<0.05). By day 5 post-inoculation, the virus load in 428


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brain samples from the rVSV group was reduced to 1.1 ×104 PFU/g, whereas virus was 429

completely cleared in the rVSV-G1670A group. No virus was detected in brain samples from 430

mice infected by rVSV-K1651A and G4A at day 3 or 5 post-inoculation. These data 431

demonstrated that MTase-defective VSVs had much less viral burden in both lungs and brains 432

compared to rVSV. Specifically, viral titer in brain and lung was ranked rVSV > G1670A > 433

K1651A > G4A. These results illustrate a reduced ability of MTase-defective VSVs to replicate 434

and spread in tissues of mice. 435

Recombinant rVSV-G4A induced body weight changes and clinical symptoms that were 436

comparable in severity to rVSV-G1670A and G1672A infected mice, despite the fact that no 437

infectious rVSV-G4A virus was detected in lungs or brains at days 3 and 5 post-inoculation 438

(Table 2). Thus, we performed an additional animal experiment in which mice were euthanized 439

at days 1 and 2 post-inoculation. As shown in Table 3, all of the mice in the rVSV group had 440

105-106 PFU/g of virus in lungs and brain, whereas only 1 of 4 mice in the VSV-G4A group had 441

a low titer at days 1 and 2 post-inoculation. The plaque size of rVSV-G4A isolated from lung or 442

brain (designated rVSV-G4A-M) was indistinguishable from that of the input rVSV-G4A 443

(Fig.5A). To further characterize rVSV-G4A-M, virus-positive brain and lung homogenates were 444

inoculated into BHK-21 cells. The rVSV-G4A-M was purified by sucrose ultracentrifugation as 445

previously (22, 25). To examine the methylation status of rVSV-G4A-M, in vitro transcription 446

reactions were performed in the presence of 20 μM of [3H]S-adenosyl-L-methionine (SAM) as 447

previously (22, 25), and the amount of [3H] SAM incorporated into the mRNA was measured by 448

scintillation counting (Fig.5B). The level of incorporation of [3H]SAM into RNA by rVSV-G4A-449

M was not significantly different from rVSV-G4A and background control (rVSV + SAH) 450


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(P>0.05), suggesting that mRNAs synthesized by rVSV-G4A-M and G4A were completely 451

defective in both G-N-7 and 2’-O methylation. 452

To confirm that MTase-defective VSVs retained the designed mutations after infection, 453

we extracted total RNAs from the lung and brain tissues of each mouse, amplified by RT-PCR 454

and sequenced the L gene. In case of rVSV-G4A-M, the entire genome was sequenced. The 455

results showed that all MTase-defective VSVs recovered from infected mice contained the 456

expected mutations in MTase region, indicating that these viral mutants are genetically stable in 457

mice. No other changes were detected in any of MTase-defective VSVs. 458

One possibility for the low level of G4A replication in vivo is that G4A induces a strong 459

type 1 interferon response. To address this possibility, we determined the level of INF-β mRNA 460

in lungs and brains from mice infected with rVSV, K1651A, G4A, and G1670A at day 3 post-461

infection. As shown in Fig.6, there is no significant difference in INF-β mRNA levels in lung 462

samples among the groups (P>0.05). However, INF-β mRNA levels in brain samples of G4A 463

infected mice were significant lower than the other challenge groups (P<0.05). Therefore, it is 464

unlikely that cytokine responses contribute to the low level of G4A replication in vivo. 465

Next, we determined whether defects in G4A replication is cell type specific. Briefly, 466

Vero, BHK-21, and HEp-2 cells were infected by rVSV and G4A, and virus titers in supernatants 467

were determined by plaque assay. As shown in Fig.7A, G4A replicates much better in Vero and 468

BHK-21 than in HEp-2 cells. The ratio of viral titer between rVSV and G4A in BHK-21, Vero, 469

and HEp-2 cells is 63:1, 126:1, and 3162:1, respectively, suggesting that G4A is a host range 470

mutant. 471

We also determined whether G4A methylation defects are cell type specific. Briefly, 472

Vero, BHK-21, and HEp-2 cells were infected by rVSV or G4A, and viral mRNAs for each 473


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recombinant were harvested. Equal amounts of viral mRNA from each VSV mutant was trans-474

methylated by vaccinia virus mRNA cap G-N-7 MTase in the presence of [3H]-SAM, and the 475

extent of methylation was determined by scintillation counting. As shown in Fig.7B, rVSV 476

mRNAs isolated in Vero, BHK-21 and HEp-2 cells were not methylated by vaccinia virus G-N-7 477

MTase, which is consistent with the fact that rVSV produces fully methylated mRNA. In 478

contrast, G4A mRNAs were efficiently methylated by vaccinia virus G-N-7 MTase. There was 479

no significant difference in the level of G-N-7 methylation of viral mRNA isolated from Vero, 480

BHK-21, and HEp-2 cells (P>0.05). Similarly, mRNAs of G4A but not rVSV were efficiently 481

methylated by vaccinia virus 2’-O MTase, VP39 (Fig.7C), suggesting that G4A was equally 482

defective in 2’-O methylation in all three cell lines (P>0.05). Together, these results suggest that 483

G4A produced unmethylated in each cell line tested and that differences in methylation do not 484

account for the different levels of G4A replication. 485

The above results showed that MTase-defective VSVs can be classified into four 486

pathotypes in mice. (i) MTase-defective VSVs carrying mutations in the catalytic site, i.e. rVSV-487

K1651A, D1762A and E1833Q, were highly attenuated in mice. These recombinant viruses did 488

not cause any detectable weight loss nor any clinical sign as well as any pathological change in 489

lung and brain. In addition, no infectious virus particles were detected in lung and brain tissues 490

after 3 days post-infection. (ii) MTase-defective VSVs carrying mutations in the SAM binding 491

site, rVSV-G1670A and G1672A, were moderately attenuated in mice. These two recombinant 492

viruses only caused mild clinical signs and moderate histopathological changes in lung, and 493

viruses were detected at early time points (up to 5 days) after inoculation, but were cleared 494

quickly. (iii) Recombinant G4A virus retains low virulence, although no infectious virus was 495

detected in lung and brain tissues except day 1 post-inoculation. (iv) Recombinant G1674A was 496


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still pathogenic to mice but appeared to be less virulent than rVSV. This recombinant virus led to 497

significant weight loss, moderate clinical signs and histopathological changes in lung and brain, 498

and replicated to high titer in lung and brain tissues. 499

500

LD50 of selected MTase-defective VSVs. To further verify the differences in virulence between 501

the MTase defective viruses, the LD50 for selected mutants (rVSV, G1674A, G4A, G1670A, and 502

K1651A) was determined (Table 4). Briefly, mice were inoculated with four doses of each 503

recombinant VSV (105, 106, 107, and 108PFU per mouse, 5 mice per group). Under our 504

experimental conditions, the LD50 for rVSV and G1674A was 2.37×106 and 2.37×107, 505

respectively. However, no mortality was observed in G4A, G1670A, and K1651A groups at the 506

dose of 108PFU. Therefore, the LD50 for these mutants are above 108 PFU. However, it should be 507

emphasized that mice inoculated with G4A and G1670A showed mild symptoms (such as ruffled 508

fur) of VSV infection but recovered by day 14 post-infection. Mice inoculated with rVSV-509

K1651A did not show any symptoms at a dose of 108 PFU. Infection at higher doses was not 510

feasible due to limitations in stock viral titers. These data further support the conclusion that 511

rVSV-G1674A is virulent, G4A and G1670A retain low virulence, and K1651A is highly 512

attenuated. 513

514

Immunogenicity of MTase-defective VSVs in mice. 515

We next examined whether the limited replication of the MTase defective viruses in mice 516

was sufficient to induce a protective immune response. To do this, 4 mice were immunized with 517

5×106 PFU of each MTase-defective VSV, and challenged 4 weeks later with 5×106 PFU of 518

rVSV. Protection was evaluated based on changes in the body weight, clinical symptoms, and 519


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viral burden in lung and brain, and pathological changes in lung and brain. Prior to challenge, 520

mice vaccinated with rVSV-K1651A, D1762A, and E1833Q did not lose body weight or show 521

clinical signs of infection (Fig.8, Table 5). Mice vaccinated with rVSV-G1670A and G1672A 522

only had mild clinical signs (such as ruffled fur) at days 3 post-vaccination but recovered at day 523

5. Mice vaccinated with these two mutant viruses lost 2-3 g of body weight at days 3-7 post-524

vaccination, indicating that these two MTase-defective VSVs retained low virulence to mice 525

(Fig.8). 526

To evaluate whether MTase-defective VSVs can induce humoral immunity, serum 527

samples were isolated from each mouse and serum IgG antibody response was determined by 528

ELISA, and Geometric Mean Titers (GMT) of VSV antibodies were calculated for each group of 529

mice. Prior to inoculation, all mice were negative (GMT <10) for VSV antibody (data not 530

shown). Mice inoculated with MTase-defective VSVs triggered strong antibody responses (Fig 531

10A, Table 5). At one week post-vaccination, all mice inoculated with MTase-defective viruses 532

had a high level of serum IgG with an average GMT ranging from 1023 to 25704. The antibody 533

level gradually increased during week 2-3 post-inoculation. By 3 weeks post-vaccination, the 534

antibody titer in mice infected with MTase-defective virus reached an average GMT of 15,241-535

215,278. Recombinant rVSV-G1670A triggered the highest antibody titer whereas rVSV-536

D1762A had the lowest antibody titer. There was no significant difference in the antibody titer 537

among mice infected with rVSV-G1670A, G1672A, K1651A, and E1833Q. Control mice 538

inoculated with DMEM did not have any detectable anti-VSV antibody response during the 539

experiment period. 540

Following challenge with virulent VSV, mice in the unimmunized group exhibited 541

typical symptoms of infection, and their body weight dropped rapidly (Table 5). In contrast, mice 542


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immunized with MTase-defective VSVs did not exhibit any clinical sign or weight loss (Table 5, 543

Fig.8). After euthanization at day 7 post-challenge, lungs from unimmunized but challenged 544

group were dark-red in color, whereas lungs from mice in MTase-defective VSVs-vaccinated 545

groups were a normal pink color. Viral RNA load in lung and brain samples was determined by 546

RT-PCR which revealed a high level of viral RNA in the challenge control samples (data not 547

shown). In contrast, levels of viral RNA were at the limit of detection in samples from mice 548

immunized with MTase-defective viruses (Table 5). Histologic analyses showed that lung and 549

brain tissues from mice vaccinated with MTase-defective VSVs only had mild histopathological 550

changes following rVSV challenge, compared to moderate changes in unimmunized rVSV 551

challenged mice (Table 5). Taken together, these data demonstrated that a single dose 552

immunization of MTase-defective VSVs elicited a strong VSV-specific immunity and 553

completely protected mice from virulent challenge. 554

We also evaluated the safety and immunogenicity following a booster immunization for 555

each MTase-defective VSV. Briefly, 4-week-old mice were first immunized with 5×106 PFU of 556

each MTase-defective VSV, and then boosted with the same MTase-defective VSV at the same 557

dose at week 4 post-primary immunization. After 3 weeks post-booster immunization, mice were 558

challenged with 5×106 PFU of rVSV. As show in Fig.9, booster immunization did not cause any 559

weight loss or VSV infection symptom, suggesting that these MTase-defective VSVs are safe to 560

mice. In addition, the level of VSV-specific antibody further increased upon the booster 561

immunization (Fig.10B). The antibody titer in rVSV-K1651A group was significantly higher 562

than those of other MTase-defective VSV groups (P<0.05). After virulent challenge, mice in the 563

challenge control group had severe weight loss and exhibited typical VSV symptoms (Fig.9, 564

Table 6). In contrast, none of mice in the vaccination groups had weight loss or any clinical signs 565


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of VSV infection, indicating that mice were protected from virulent challenge. Lastly, MTase-566

defective VSV-vaccinated mice did not have any gross pathological changes in lung at day 7 567

post-challenge whereas those from the challenge control group were dark-red in color, indicative 568

of inflammation. Moreover, neither infectious virus particle nor viral RNA was detected in lung 569

and brain tissues from MTase-defective VSV-immunized mice (Table 6). However, high viral 570

RNA load in lung and brain was detected in mice from the challenge control group. Furthermore, 571

histological examination found that lung and brain tissues from the challenge control were 572

moderately inflamed whereas those from MTase-defective VSV-vaccinated groups only had 573

mild inflammatory change (Table 6). Taken together, these data demonstrated that booster 574

immunization of MTase-defective were not only safe to mice, but also elicited strong VSV-575

specific immunity, and completely protected mice from virulent challenge. 576

577

Discussion 578

In this study, we evaluated the impact of mRNA cap methylation in VSV pathogenesis in 579

vivo. We found that VSV mutants, rVSV-K1651A, D1762A, and E1833Q, which have 580

mutations in the MTase catalytic site and are defective in both G-N-7 and 2’O methylation, were 581

highly attenuated in mice. Recombinant rVSV-G1670A and G1672A, which have mutations in 582

the SAM binding site and are defective in G-N-7 but not 2’-O methylation, retained low 583

virulence in mice. In contrast, rVSV-G4A, a mutant producing unmethylated mRNA cap 584

structures in vitro, induced weight loss and clinical signs that were indistinguishable from rVSV-585

G1670A and G1672A infections despite the extremely low level of rVSV-G4A replication in 586

vivo. Three major conclusions are apparent from our data: (i) inactivation of the predicted 587

catalytic residues attenuates the virus to a greater extent in vivo, than does inactivation of the 588


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predicted SAM binding site; (ii) the relationship between mRNA cap methylation and viral 589

pathogenesis is not clear cut in VSV infections; and (iii) some MTase-defective VSV may serve 590

as useful vaccine candidates. 591

592

The impact of mRNA cap methylation status on VSV pathogenesis. 593

VSV infects a wide range of wild and domestic animals such as cattle, horses, deer and 594

pigs, characterized by vesicular lesions in the mouth, tongue, lips, gums, teats, and feet. 595

Although VSV does not cause vesicular diseases in mice, the mouse represents an excellent 596

small animal model to understand pathogenesis because VSV causes systemic infection and fatal 597

encephalitis (40-43). After intranasal inoculation, VSV infects olfactory neurons in the nasal 598

mucosa and subsequently enters the central nervous system (CNS) through the olfactory nerves 599

(44-45). The virus is then disseminated to other areas in the brain through retrograde and 600

possibly anterograde trans-neuronal transport, ultimately causing an acute brain infection. The 601

mortality, pathology, and viral burden in lung and brain tissues are dependent on the age and 602

species of mice (40-42, 46). 603

Based on the pathogenesis in mice, MTase-defective VSVs can be classified into three 604

pathotypes: highly attenuated, lowly virulent, and virulent. With the exception of rVSV-G4A, the 605

pathotype of MTase-defective VSVs in mice is essentially consistent with the phenotypes 606

observed in cell cultures. VSV mutants rVSV-K1651A, D1762A, and E1833Q, which carry 607

point mutations in the MTase catalytic site and are defective in both G-N-7 and 2’-O 608

methylation, were highly attenuated in mice. This is consistent with our previous finding that 609

these recombinants were highly attenuated in cell culture (9). Specifically, these mutant viruses 610

had decreased virus titers (2-3 logs) and reduced levels of protein and RNA synthesis (by 30-611


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50%) as compared to rVSV. In contrast, mutations in the SAM binding site impact viral 612

pathogenesis differentially. VSV mutants specifically defective in G-N-7 but not 2’-O MTase, 613

including rVSV-G1670A and G1672A, retained low virulence. These findings suggest that VSV 614

mutants with defect only in G-N-7 methylation are insufficiently attenuated. In cell culture, these 615

two mutants only led to decreases of 1-2 log in virus titer while the levels of viral protein and 616

RNA synthesis were comparable to those of rVSV (21). Recombinant West Nile virus (WNV-617

K61A and K182A) defective in 2′-O but not the G-N-7 MTase activity is also attenuated in vitro as 618

well as in vivo (47-49). In this case, inhibition of 2’-O methylation is sufficient to attenuate 619

WNV. As predicted, rVSV-G1674A remains virulent to mice although the virulence is reduced 620

compared to rVSV. This mutant was fully methylated in cap structure only at high SAM 621

concentrations (200 μM) whereas rVSV only requires 20 μM of SAM for full methylation (21). 622

It replicated as efficiently as rVSV and had no defect in RNA and protein synthesis in cell 623

culture. 624

Most striking in our study was the phenotype associated with infections by recombinant 625

rVSV-G4A, a virus which is completely defective in both G-N-7 and 2’-O methylation. 626

Recombinant rVSV-G4A induced body weigh losses and clinical signs that were comparable to 627

those associated with G1670A and G1672A infections. However, unlike G1670A and G1672A, 628

rVSV-G4A was highly attenuated for replication and/or spread in vivo. Reduction in brain and 629

lung viral titers was not attributed to enhanced production of antiviral cytokines, such as IFN-β 630

(Fig.6). The phenotype (small plaque and no cap methylation) of rVSV-G4A-M isolated from 631

mice was identical to that of rVSV-G4A original stock (Fig.5). Also, no additional mutations 632

were identified in rVSV-G4A-M. In fact, rVSV-G4A is the most attenuated mutant in cell 633

culture among MTase-defective VSVs (21). We also showed that G4A is a host range mutant 634


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which had a much poorer replication in HEp-2 cells compared to BHK-21 and Vero cells. G4A 635

produces unmethylated mRNA in all three cell lines, suggesting that the differences in 636

replication amongst cell lines do not reflect a compensatory cellular methylase modifying the 637

viral mRNA. Also, it is unlikely that cytokine responses contribute to the low level replication of 638

G4A in vivo (Fig.6). The molecular mechanisms underlying the “apparent” discrepancy between 639

replication in cell culture and in vivo pathogenesis remain to be elucidated. One feature of the 640

gene expression strategy of NNS RNA viruses is that the polymerase complex, controls two 641

distinct RNA synthetic events: genomic RNA replication and mRNA transcription (19, 50-51). 642

Previously, we showed that regulation of those activities is perturbed for rVSV-G4A such that 643

genomic replication is enhanced 4-fold (21). Perhaps, the defects in mRNA cap methylation and 644

the alternation of the polymerase function collectively contribute to the unique phenotype of 645

G4A. 646

647

MTase-defective viruses as potential live vaccine candidates 648

In this study, we demonstrated that ablating viral mRNA cap methylation is a means to 649

attenuate VSV, and by extrapolation perhaps other NNS RNA viruses, for development of live 650

attenuated vaccines. Our results showed that MTase-defective VSVs were able to induce high 651

levels of VSV-specific antibody in mice and provided full protection against a virulent challenge 652

with the VSV Indiana serotype. Recombinants rVSV-K1651A, D1762A, and E1833Q are 653

attractive vaccine candidates since they are not only highly attenuated but also retain high 654

immunogenicity. Although recombinants rVSV-G1670A and G1672A retained low virulence to 655

mice, their pathogenicity was significantly reduced compared to rVSV. The safety of using these 656

two viruses as live vaccine candidates remains to be further investigated. 657


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This study extends the approaches that have been employed to attenuate VSV, including 658

gene rearrangement, M gene mutation, G protein cytoplasmic tail truncations, G protein 659

deletions, and combinations thereof (52-55). We now add to this list, mutations in the MTase 660

domain of L, which we show can result in attenuated and immunogenic viruses. Since our 661

sequence data indicates that the viruses isolated from lung and brain tissues retained the original 662

mutation(s), reversion may be less of a problem than anticipated. Moreover, by combining 663

mutations in this region of L, it may be possible to diminish further the chance of selecting 664

virulent revertants. We do not understand the basis for the virulence of G4A, which lacks the 665

ability to methylate mRNA in vitro. Consequently, we cannot yet conclude that elimination of 666

the MTase activities of L is an effective vaccine strategy. Resolving why some MTase-defective 667

VSVs are highly attenuated whereas others are not, may facilitate a strategy to develop viral 668

vectors for vaccines, oncolytic therapy, and gene delivery. 669

Our studies on MTase-defective VSVs also shed light on developing live vaccine 670

candidates for other NNS RNA viruses, particularly paramyxoviruses. Live attenuated vaccines 671

are the most promising vaccine candidates for paramyxoviruses such as hMPV, RSV, and PIV3 672

(54, 56), which account for the majority of respiratory diseases infants, children, and the elderly. 673

However, it has been technically challenging to isolate viruses with low virulence but retaining 674

high immunogenicity. Introducing mutations in the MTase may provide a novel approach to 675

generate live attenuated viruses for these viruses. Recently, we generated a panel of MTase-676

defective hMPVs which showed only 0.5-1 log reductions in growth relative to the parent virus 677

(Zhang and Li, unpublished data). These recombinants were highly attenuated and immunogenic 678

in cotton rats. Thus, it is feasible to utilize MTase-defective viruses as vaccine candidates for 679

paramyxoviruses. 680


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Recently, the concept of using MTase-defective viruses as live vaccine candidates has 681

also been demonstrated in flaviviruses such as Japanese encephalitis virus (JEV) and dengue 682

virus (57-58). In both cases, 2′-O methylation-defective flaviviruses were shown to be promising 683

vaccine candidates. For example, 2′-O methylation-defective JEV was attenuated in mice, 684

elicited robust humoral and cellular immune responses, and was genetically stable in vivo (57). 685

A single immunization induced full protection against lethal challenge with JEV strains in mice. 686

Similarly, dengue virus mutants lacking 2’-O MTase activity are attenuated in mice and rhesus 687

monkeys and elicit a strong adaptive immune response (58). 688

689

Biological role of 2’-O methylation: discrimination of self and non-self mRNA? 690

While it is firmly established that G-N-7 methylation is essential for mRNA stability as 691

well as efficient translation, the role(s) of ribose 2’-O methylation have proved more elusive. 692

Recent studies on West Nile virus (WNV) suggest that the 2′-O methylation of the 5′ cap of viral 693

RNA functions to evade innate host antiviral responses through escape of the suppression of 694

interferon-stimulated genes, tetratricopeptide repeats (IFIT) (38). A WNV MTase mutant 695

(E218A), which is defective in 2′-O MTase activity, was attenuated in wild-type C57BL/6 mice 696

but remained pathogenic in mice that lack a type I interferon (IFN) signaling pathway. A 697

vaccinia virus mutant (J3-K175R) and mouse hepatitis virus mutant MHV-D130A, both of 698

which lack 2′-O MTase activity, also exhibited enhanced sensitivities to the antiviral actions of 699

IFN mediated by IFIT proteins (38). Similarly, 2’-O methylation of mouse and human 700

coronavirus RNA seems to facilitate evasion from detection by the cytoplasmic RNA sensor 701

Mda5 (36). Those studies suggest that 2’-O methylation of viral RNA provides a molecular 702

signature for the discrimination of self and non-self mRNA. The finding here that VSV-G4A 703


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produces unmethylated mRNA yet remains as virulent as G1670A and G1672A underscores that 704

the recognition of non-2’-O methylated mRNA is only one aspect of the immune response to 705

virus infection. 706

In summary, the present study highlights a major gap in our understanding of the impact 707

of mRNA cap methylation in VSV pathogenesis. Our work suggests that abrogation of viral 708

mRNA cap methylation can serve as a novel approach to attenuate VSV, and perhaps other NNS 709

RNA viruses, for use as vaccines and viral vectors. It will be of interest to determine the role of 710

mRNA cap methylation in innate immunity of NNS RNA viruses. 711

712

Acknowledgments 713

This study was supported in part by grants from NIH (AI090060) and USDA Agriculture 714

and Food Research Initiative (2010-65119-20602) and a pilot grant from OSU Center for 715

Clinical and Translational Science (CCTS) to J.L., and by a grant from NIH (AI059371) to 716

S.P.J.W. Yu Zhang is a fellow of Center for RNA Biology at OSU. We thank members of Li’s 717

laboratory for critical review of this manuscript. 718

719


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References 720 721

722

1. Sleat DE, Banerjee AK. 1993. Transcriptional Activity and Mutational Analysis of 723 Recombinant Vesicular Stomatitis Virus RNA Polymerase. J Virol 67:1334-1339. 724

2. Emerson SU, Yu YH. 1975. Both NS and L Proteins Are Required for In vitro RNA 725 Synthesis by Vesicular Stomatitis Virus. J Virol 15:1348-1356. 726

3. Emerson SU, Wagner RR. 1973. L Protein Requirement for InVitro RNA Synthesis by 727 Vesicular Stomatitis Virus. J Virol 12:1325-1335. 728

4. Ogino T, Yadav SP, Banerjee AK. 2010. Histidine-mediated RNA transfer to GDP for 729 unique mRNA capping by vesicular stomatitis virus RNA polymerase. Proc Natl Acad 730 Sci USA 107:3463-3468. 731

5. Ogino T, Banerjee AK. 2007. Unconventional mechanism of mRNA capping by the 732 RNA-dependent RNA polymerase of vesicular stomatitis virus. Mol Cell 25:85-97. 733

6. Li J, Rahmeh A, Morelli M, Whelan SPJ. 2008. A conserved motif in region V of the 734 large polymerase proteins of nonsegmented negative-sense RNA viruses that is essential 735 for mRNA capping. J Virol 82:775-784. 736

7. Testa D, Banerjee AK. 1977. Two methyltransferase activities in the purified virions of 737 vesicular stomatitis virus. J Virol 24:786-793. 738

8. Rahmeh AA, Li JR, Kranzusch PJ, Whelan SPJ. 2009. Ribose 2 '-O Methylation of 739 the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-740 N-7 Methylation by the Large Polymerase Protein. J Virol 83:11043-11050. 741

9. Li JR, Fontaine-Rodriguez EC, Whelan SPJ. 2005. Amino acid residues within 742 conserved domain VI of the vesicular stomatitis virus large polymerase protein essential 743 for mRNA cap methyltransferase activity. J Virol 79:13373-13384. 744

10. Hercyk N, Horikami SM, Moyer SA. 1988. The vesicular stomatitis virus L protein 745 possesses the mRNA methyltransferase activities. Virology 163:222-225. 746

11. Grdzelishvili VZ, Smallwood S, Tower D, Hall RL, Hunt DM, Moyer SA. 2005. A 747 single amino acid change in the L-polymerase protein of vesicular stomatitis virus 748 completely abolishes viral mRNA cap methylation. J Virol 79:7327-7337. 749

12. Li JR, Rahmeh A, Brusic V, Whelan SPJ. 2009. Opposing Effects of Inhibiting Cap 750 Addition and Cap Methylation on Polyadenylation during Vesicular Stomatitis Virus 751 mRNA Synthesis. J Virol 83:1930-1940. 752

13. Hunt DM, Mehta R, Hutchinson KL. 1988. The L Protein of Vesicular Stomatitis 753 Virus Modulates the Response of the Polyadenylic Acid Polymerase to S-754 Adenosylhomocysteine. J Gen Virol 69:2555-2561. 755

14. Galloway SE, Wertz GW. 2008. S-Adenosyl Homocysteine-Induced 756 Hyperpolyadenylation of Vesicular Stomatitis Virus mRNA Requires the 757 Methyltransferase Activity of L Protein. J Virol 82:12280-12290. 758

15. Banerjee AK, Rhodes DP. 1973. In Vitro Synthesis of RNA That Contains 759 Polyadenylate by Virion Associated RNA Polymerase of Vesicular Stomatitis Virus. Proc 760 Natl Acad Sci USA 70:3566-3570. 761

16. Poch O, Blumberg BM, Bougueleret L, Tordo N. 1990. Sequence comparison of five 762 polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical 763 assignment of functional domains. J Gen Virol 71 ( Pt 5):1153-1162. 764


.org/D

ownloaded from

http://jvi.asm.org/

34

17. Zhang XD, Wei YW, Ma YM, Hu SH, Li JR. 2010. Identification of aromatic amino 765 acid residues in conserved region VI of the large polymerase of vesicular stomatitis virus 766 is essential for both guanine-N-7 and ribose 2 '-O methyltransferases. Virology 408:241-767 252. 768

18. Rahmeh AA, Schenk AD, Danek EI, Kranzusch PJ, Liang B, Walz T, Whelan SPJ. 769 2010. Molecular architecture of the vesicular stomatitis virus RNA polymerase. Proc Natl 770 Acad Sci USA 107:20075-20080. 771

19. Whelan SPJ, Wertz GW. 2002. Transcription and replication initiate at separate sites on 772 the vesicular stomatitis virus genome. Proc Natl Acad Sci USA 99:9178-9183. 773

20. Whelan SP, Wertz GW. 1999. Regulation of RNA synthesis by the genomic termini of 774 vesicular stomatitis virus: identification of distinct sequences essential for transcription 775 but not replication. J Virol 73:297-306. 776

21. Li JR, Wang JT, Whelan SPJ. 2006. A unique strategy for mRNA cap methylation 777 used by vesicular stomatitis virus. Proc Natl Acad Sci USA 103:8493-8498. 778

22. Hagler J, Shuman S. 1992. A freeze-frame view of eukaryotic transcription during 779 elongation and capping of nascent mRNA. Science 255:983-986. 780

23. Wang JT, McElvain LE, Whelan SP. 2007. Vesicular stomatitis virus mRNA capping 781 machinery requires specific cis-acting signals in the RNA. J Virol 81:11499-11506. 782

24. Stillman EA, Whitt MA. 1999. Transcript initiation and 5'-end modifications are 783 separable events during vesicular stomatitis virus transcription. J Virol 73:7199-7209. 784

25. Stillman EA, Whitt MA. 1997. Mutational analyses of the intergenic dinucleotide and 785 the transcriptional start sequence of vesicular stomatitis virus (VSV) define sequences 786 required for efficient termination and initiation of VSV transcripts. J Virol 71:2127-2137. 787

26. Tekes G, Rahmeh AA, Whelan SP. 2011. A freeze frame view of vesicular stomatitis 788 virus transcription defines a minimal length of RNA for 5' processing. PLoS Pathog 789 7:e1002073. 790

27. Barr JN, Whelan SPJ, Wertz GW. 2002. Transcriptional control of the RNA-791 dependent RNA polymerase of vesicular stomatitis virus. BBA-Gene Struct Expr 792 1577:337-353. 793

28. Furuichi Y, Shatkin AJ. 2000. Viral and cellular mRNA capping: past and prospects. 794 Adv Virus Res 55:135-184. 795

29. Muthukrishnan S, Both GW, Furuichi Y, Shatkin AJ. 1975. 5'-Terminal 7-796 methylguanosine in eukaryotic mRNA is required for translation. Nature 255:33-37. 797

30. Both GW, Banerjee AK, Shatkin AJ. 1975. Methylation-dependent translation of viral 798 messenger RNAs in vitro. Proc Natl Acad Sci U S A 72:1189-1193. 799

31. Mudd JA, Summers DF. 1970. Protein Synthesis in Vesicular Stomatitis Virus-Infected 800 Hela Cells. Virology 42:328-&. 801

32. Makarow M, Nevalainen LT, Kaariainen L. 1986. Expression of the RNA Genome of 802 an Animal Virus in Saccharomyces Cerevisiae. Proc Natl Acad Sci USA 83:8117-8121. 803

33. Lodish HF, Porter M. 1980. Translational Control of Protein Synthesis after Infection 804 by Vesicular Stomatitis Virus. J Virol 36:719-733. 805

34. Dratewka-Kos E, Kiss I, Lucas-Lenard J, Mehta HB, Woodley CL, Wahba AJ. 806 1984. Catalytic utilization of eIF-2 and mRNA binding proteins are limiting in lysates 807 from vesicular stomatitis virus infected L cells. Biochemistry 23:6184-6190. 808


.org/D

ownloaded from

http://jvi.asm.org/

35

35. Connor JH, Lyles DS. 2002. Vesicular stomatitis virus infection alters the eIF4F 809 translation initiation complex and causes dephosphorylation of the eIF4E binding protein 810 4E-BP1. J Virol 76:10177-10187. 811

36. Zust R, Dong H, Li XF, Chang DC, Zhang B, Balakrishnan T, Toh YX, Jiang T, Li 812 SH, Deng YQ, Ellis BR, Ellis EM, Poidinger M, Zolezzi F, Qin CF, Shi PY, Fink K. 813 2013. Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants 814 are highly attenuated and immunogenic in mice and macaques. PLoS Pathog 9:e1003521. 815

37. Garcia-Sastre A. 2011. 2 methylate or not 2 methylate: viral evasion of the type I 816 interferon response. Nat Immunol 12:114-115. 817

38. Daffis S, Szretter KJ, Schriewer J, Li JQ, Youn S, Errett J, Lin TY, Schneller S, 818 Zust R, Dong HP, Thiel V, Sen GC, Fensterl V, Klimstra WB, Pierson TC, Buller 819 RM, Gale M, Shi PY, Diamond MS. 2010. 2 '-O methylation of the viral mRNA cap 820 evades host restriction by IFIT family members. Nature 468:452-456. 821

39. Reed LJ, Muench H. 1938. A simple method of estimating fifty per cent endpoints. AM 822 J Epidemiol 27:493-497. 823

40. Sabin AB, Olitsky PK. 1938. Influence of Host Factors on Neuroinvasiveness of 824 Vesicular Stomatitis Virus : Iii. Effect of Age and Pathway of Infection on the Character 825 and Localization of Lesions in the Central Nervous System. J Exp Med 67:201-228. 826

41. Sabin AB, Olitsky PK. 1938. Influence of Host Factors on Neuroinvasiveness of 827 Vesicular Stomatitis Virus : Iv. Variations in Neuroinvasiveness in Difierent Species. J 828 Exp Med 67:229-249. 829

42. Sabin AB, Olitsky PK. 1937. Influence of Host Factors on Neuroinvasiveness of 830 Vesicular Stomatitis Virus : I. Effect of Age on the Invasion of the Brain by Virus 831 Instilled in the Nose. J Exp Med 66:15-34. 832

43. Slavin HB, Hale HW, Berry GP. 1946. Passive Protection of the Central Nervous 833 System of Mice against Viruses That Pursue the Pathway of the Olfactory Nerves after 834 Intranasal Instillation - Vesicular Stomatitis and St. Louis Encephalitis. J Immunol 835 54:179-188. 836

44. Plakhov IV, Arlund EE, Aoki C, Reiss CS. 1995. The Earliest Events in Vesicular 837 Stomatitis Virus Infection of the Murine Olfactory Neuroepithelium and Entry of the 838 Central Nervous System. Virology 209:257-262. 839

45. Huneycutt BS, Plakhov IV, Shusterman Z, Bartido SM, Huang A, Reiss CS, Aoki C. 840 1994. Distribution of vesicular stomatitis virus proteins in the brains of BALB/c mice 841 following intranasal inoculation: an immunohistochemical analysis. Brain Res 635:81-95. 842

46. Cornish TE, Stallknecht DE, Brown CC, Seal BS, Howerth EW. 2001. Pathogenesis 843 of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer 844 mouse (Peromyscus maniculatus). Vet Pathol 38:396-406. 845

47. Zhou YS, Ray D, Zhao YW, Dong HP, Ren SP, Li Z, Guo Y, Bernard KA, Shi PY, 846 Li HM. 2007. Structure and function of flavivirus NS5 methyltransferase. J Virol 847 81:3891-3903. 848

48. Ray D, Shah A, Tilgner M, Guo Y, Zhao YW, Dong HP, Deas TS, Zhou YS, Li HM, 849 Shi PY. 2006. West nile virus 5 '-cap structure is formed by sequential guanine N-7 and 850 ribose 2 '-O methylations by nonstructural protein 5. J Virol 80:8362-8370. 851

49. Dong HP, Ren SP, Zhang B, Zhou YS, Puig-Basagoiti F, Li HM, Shi PY. 2008. West 852 nile virus methyltransferase catalyzes two methylations of the viral RNA cap through a 853 substrate-repositioning mechanism. J Virol 82:4295-4307. 854


.org/D

ownloaded from

http://jvi.asm.org/

36

50. Rose JK, Whitt MA. 2001. Rhabdoviridae: the viruses and their replication, Fields 855 virology, 4 ed, vol. 1. Lippincott Williams & Wilkins Co., New York, N.Y. 856

51. Qanungo KR, Shaji D, Mathur M, Banerjee AK. 2004. Two RNA polymerase 857 complexes from vesicular stomatitis virus-infected cells that carry out transcription and 858 replication of genome RNA. Proc Natl Acad Sci USA 101:5952-5957. 859

52. Publicover J, Ramsburg E, Rose JK. 2004. Characterization of nonpathogenic, live, 860 viral vaccine vectors inducing potent cellular immune responses. J Virol 78:9317-9324. 861

53. Flanagan EB, Schoeb TR, Wertz GW. 2003. Vesicular stomatitis viruses with 862 rearranged genomes have altered invasiveness and neuropathogenesis in mice. J Virol 863 77:5740-5748. 864

54. Bukreyev A, Skiadopoulos MH, Murphy BR, Collins PL. 2006. Nonsegmented 865 negative-strand viruses as vaccine vectors. J Virol 80:10293-10306. 866

55. Ahmed M, McKenzie MO, Puckett S, Hojnacki M, Poliquin L, Lyles DS. 2003. 867 Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene 868 expression is genetically correlated with the inhibition of host RNA and protein 869 synthesis. J Virol 77:4646-4657. 870

56. van Bleek GM, Osterhaus AD, de Swart RL. 2011. RSV 2010: Recent advances in 871 research on respiratory syncytial virus and other pneumoviruses. Vaccine 29:7285-7291. 872

57. Zust R, Cervantes-Barragan L, Habjan M, Maier R, Neuman BW, Ziebuhr J, 873 Szretter KJ, Baker SC, Barchet W, Diamond MS, Siddell SG, Ludewig B, Thiel V. 874 2011. Ribose 2 '-O-methylation provides a molecular signature for the distinction of self 875 and non-self mRNA dependent on the RNA sensor Mda5. Nat Immunol 12:137-U146. 876

58. Li SH, Dong H, Li XF, Xie X, Zhao H, Deng YQ, Wang XY, Ye Q, Zhu SY, Wang 877 HJ, Zhang B, Leng QB, Zuest R, Qin ED, Qin CF, Shi PY. 2013. Rational design of a 878 flavivirus vaccine by abolishing viral RNA 2'-O methylation. J Virol 87:5812-5819. 879

880 881

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Figure legend 883

Figure 1. Dynamics of mouse body weight change after VSV infection. Four-week-old SPF 884

female BALB/c mice (8 mice per group) were inoculated with each MTase-defective VSVs 885

intranasally at a dose of 5×106 PFU. At day 7 post-inoculation, 4 mice from each group were 886

euthanized. The body weight of the remaining 4 mice in each group was monitored until day 14 887

post-inoculation. The average body weight for each group was shown. 888

889

Figure 2. Histopathological changes in lungs after VSV infection. Four-week-old SPF female 890

BALB/c mice were inoculated with each MTase-defective VSVs intranasally at a dose of 5×106 891

PFU. Mice from each group were euthanized at day 7 post-inoculation. Right lung from each 892

mouse was fixed in 4% (v/v) phosphate-buffered paraformaldehyde, embedded in paraffin, 893

sectioned at 5 microns, and stained with hematoxylin-eosin (HE). Detailed criteria for evaluation 894

of lung histological change was described in Materials and Methods. Representative lung 895

histology from each group was shown. DMEM control: grade 0, no lesion; rVSV: grade 3, 896

severe; E1833Q: grade 1, mild; G1670A: grade 2, moderate; and G4A: grade 3, severe. 897

898

Figure 3. Histopathological changes in brain after VSV infection. Mice were euthanized at 899

day 7 post-inoculation. A horizontal brain section containing the olfactory bulb, forebrain, 900

cerebrum, hypocampus and cerebellum was fixed in 4% (v/v) phosphate-buffered 901

paraformaldehyde, followed by HE straining. Detailed criteria for evaluation of brain 902


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histological change was described in Materials and Methods. DMEM control: grade 0, no lesion; 903

K1651A: grade 1, mild; G4A: grade 1, mild; and rVSV: grade 3, severe. 904

905

Figure 4. Detection of viral RNA in lung and brain by RT-PCR. Equal amount (0.5g) of lung 906

and brain tissues was homogenized in 0.5 ml of DMEM. Total RNA was extracted from each 907

sample, followed by RT-PCR to amplify VSV N gene. The amplified products were analyzed on 908

1% agarose gel. 909

910

Figure 5. Phenotype of rVSV-G4A isolated from mice. (A) Plaque morphology of rVSV-G4A 911

on Vero cells. rVSV-G4A-M: virus isolated from lungs of rVSV-G4A infected mice. rVSV-M: 912

virus isolated from lungs of rVSV infected mice. Plaques of rVSV and rVSV-M were developed 913

after 24 h; those of rVSV-G4A, and rVSV-G4A-M were developed after 96 h. (B) [3H]SAM 914

incorporation monitored by scintillation counting. Viral RNA was synthesized in vitro by using 915

10 μg of purified virus. The amount of [3H]SAM incorporated into the RNA was determined by 916

scintillation counting and the dpm normalized to the amount of RNA synthesized. Three 917

independent experiments were used to generate the graph shown. 918

919

920

Figure 6. Quantification of IFN-β mRNA by real-time RT-PCR. Mice were infected with 921

rVSV, G4A, K1651A, and G1670A at dose of 5×106 PFU/mouse. At days 3 post-infection, 922


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lung and brain tissues were collected. Total RNA was extracted from lung or brain tissues and 923

was treated with DNase I (Ambion). The IFN-β mRNA in each tissue was quantified by real-924

time RT-PCR. The average IFN-β mRNA of 5 mice for each group was shown. 925

926

Figure 7. Replication and methylation of VSV in different cell lines. (A) Replication rVSV-927

G4A is cell type dependent. Confluent BHK-21, Vero, or HEp-2 cells were infected by rVSV 928

or G4A at an MOI of 0.1. After 24 h, supernatants were harvested, and virus titers were 929

determined by plaque assay. (B) rVSV-G4A is defective in G-N-7 methylation in virus-930

infected cells. Confluent BHK-21, Vero cells, or HEp-2 in 35 mm dishes were infected by rVSV 931

or G4A at an MOI of 0.1. After 24 h post-infection, poly (A)-containing viral mRNA were 932

isolated. Equal amounts of viral RNA were incubated with 10 units of vaccinia virus G-N-7 933

MTase supplied by m7G Capping System (ScriptCap™) in the presence of 15 μCi [3H]-SAM 934

(85 Ci/mmol, PerkinElmer). The G-N-7 methylation was measured by 3H incorporation using a 935

1414 series scintillation counter (PerkinElmer). (C) rVSV-G4A is defective in 2’-O 936

methylation in virus-infected cells. Poly (A)-containing viral mRNA were incubated with 10 937

units of vaccinia virus G-N-7 MTase in the presence of 100 μM of cold SAM. RNA were 938

purified and incubated with vaccinia virus 2’-O MTase (VP39) in the presence of 15 μCi [3H]-939

SAM. The 2’-O methylation was measured by 3H incorporation using a 1414 series scintillation 940

counter. 941

942

Figure 8. Dynamics of mouse body weight change after primary immunization followed by 943

virulent challenge. Four-week-old SPF female BALB/c mice were immunized with MTase-944

defective VSVs intranasally at a dose of 5×106 PFU per mouse. At week 4 post-immunization, 945


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mice were challenged with rVSV at a dose of 5×106 PFU per mouse. The body weight for each 946

mouse was monitored every 3 days. The average body weight of four mice was shown. 947

948

Figure 9. Dynamics of mouse body weight change after booster immunization followed by 949

virulent challenge. Four-week-old SPF female BALB/c mice were immunized with MTase-950

defective VSVs intranasally at a dose of 5×106 PFU per mouse. At week 2 post-primary 951

immunization, mice were boosted intranasally with 5×106 PFU of the corresponding virus. At 952

week 3 post-boost immunization, mice were challenged with rVSV at a dose of 5×106 PFU per 953

mouse. The body weight for each mouse was monitored every 3 days. The average body weight 954

of four mice was shown. 955

956

Figure 10. MTase-defective VSVs triggered high level of antibody responses in mice. Blood 957

samples were collected from each mouse weekly, and serum was isolated for antibody detection 958

by ELISA. Antibody titers were calculated by the Geometric Mean Titers (GMT). (A) Antibody 959

titer after primary immunization. (B) Antibody titer after booster immunization. 960

961

962


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Table 1. Pathogenicity of MTase-defective VSV in mice

Group mice Number of sick mice

Lung histologyB Brain histologyC Viral RNA in lung

Viral RNA in brain

Viral titer at day 7 (PFU/g)D

Day 7 A Day 14 Day 7 Day 14 Day 7 Day 14 Day 7 Day 14 Day 7 Day 14 Lung Brain

DMEM 8 0/8 (0) 0/4 0 0 0 0 0/4 0/4 0/4 0/4 0 0

rVSV 8 8/8 (3.0a) 1/4 3.0a 2.5a 2.5a 0 4/4 4/4 3/4 2/4 0 2.6 ×105

K1651A 8 0/8 (0) 0/4 2.0b 1.5b 0 0 3/4 0/4 0/4 1/4 0 0

E1833Q 8 0/8 (0) 0/4 1.0c 0.5c 0 0 4/4 0/4 0/4 0/4 0 0

G1670A 8 6/8 (1.0c) 0/4 2.0b 1.5b 0.5b 0 3/4 0/4 0/4 0/4 0 0

G1672A 8 5/8 (1.0c) 0/4 2.0b 1.0b 0 0 4/4 0/4 0/4 0/4 0 0

G1674A 8 8/8 (2.25b) 0/4 3.0a 2.0a 0.5b 0 4/4 0/4 1/4 1/4 0 0

G4A 8 8/8 (1.375c) 0/4 3.0a 2.0a 0.5b 0 4/4 0/4 2/4 1/4 0 0

A: The severity of VSV symptoms at day 7 was scored as following: grade 3 (severe); grade 2

(moderate), grade 1 (mild), and grade 0 (no symptoms). Average score for each group was

shown. Value within a column followed by the different lowercase letters (a, b, and c) are

significantly different (P<0.05).

B: The severity of lung histological change was scored based on the extent of lymphoid

infiltrates as described in Materials and Methods: grade 3 (severe), grade 2 (moderate), grade 1

(mild), and grade 0 (no lesion). The average score of four mice was shown. Value within a

column followed by the different lowercase letters (a, b, and c) are significantly different

(P<0.05).

C: The severity of brain histological change was scored based on the extent of inflammatory cell

infiltrates and gliosis as described in Materials and Methods: grade 3 (severe), grade 2

(moderate), grade 1 (mild), and grade 0 (no lesion). The average score of four mice was shown.

Value within a column followed by the different lowercase letters (a, b, and c) are significantly

different (P<0.05).

C: Viral titer was determined by plaque assay. 3 out of 4 mice in rVSV group had average titer of

2.6 ×105 in brains at day 5 post-infection. All other groups did not have detectable infectious

VSV. “0” indicates viral titer is undetectable by plaque assay.


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Table 2. Viral titer in lungs and brain at days 3 and 5 post-infection

Group

mice

Number of sick miceA Viral titer in lung (PFU/g)B

Viral titer in brain (PFU/g)

Day 3 Day 5 Day 3 Day 5 Day 3 Day 5

DMEM 8 0/8 (0) 0/4 (0) 0 (0/4) 0 (0/4) 0 (0/4) 0(0/4)

rVSV 8 8/8 (3.0a) 4/4 (3.0a) 6.7 × 105(4/4) 1.2 ×103(4/4) 8.0 × 105(4/4) 1.1 ×104(4/4)

K1651A 8 0/8 (0) 0/4 (0) 1.1×103(3/4) 0 (0/4) 0 (0/4) 0 (0/4)

G1670A 8 8/8 (1.0c) 2/4 (1.0b) 3.9×104(4/4) 6.3×101(1/4) 1.5 × 104 (4/4) 0 (0/4)

G4A 8 8/8(1.5b) 4/4 (1.25b) 2.1 × 101(1/4) 0 (0/4) 0 (0/4) 0 (0/4)

A: The severity of VSV symptoms at days 3 and 5 was scored as following: grade 3 (severe);

grade 2 (moderate), grade 1 (mild), and grade 0 (no symptoms). Average score for each group

was shown. Value within a column followed by the different lowercase letters (a, b, and c) are


B: Viral titer was determined by plaque assay. The average titer of four mice was shown. “0”

indicates viral titer is undetectable by plaque assay.


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Table 3. Viral titer in lungs and brain at days 1 and 2 post-infection

Group

mice

Number of sick miceA

Viral titer in lung (PFU/g)B

Viral titer in brain (PFU/g)

Day 1 Day 2 Day 1 Day 2 Day 1 Day 2

DMEM 8 0/8 (0) 0/4 (0) 0 (0/4) 0 (0/4) 0 (0/4) 0 (0/4)

rVSV 8 8/8 (2.0a) 4/4 (3.0a) 1.2 × 106(4/4) 5.5 ×105(4/4) 9.6 × 105(4/4) 6.6 ×105(4/4)

G4A 8 8/8 (0.5b) 4/4 (1.0b) 2.7 × 102 (1/4) 5 (1/4) 2.3 × 101(1/4) 0 (0/4)

A: The severity of VSV symptoms at days 1 and 2 was scored as following: grade 3 (severe);

grade 2 (moderate), grade 1 (mild), and grade 0 (no symptoms). Average score for each group

was shown. Value within a column followed by the different lowercase letters (a and b) are


B: Viral titer was determined by plaque assay. The average titer of four mice was shown. “0”

indicates viral titer is undetectable by plaque assay.


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Table 4. LD50 of MTase-defective VSVs

Group LD50A Phenotype B

rVSV 2.37×106 Highly virulent

G1674A 2.37×107 Virulent

G4A >108 Attenuated , low virulence

G1670A >108 Attenuated , low virulence

K1651A >108 Highly attenuated, avirulent

A: No mortality is observed in G4A, G1670A, and K1651A groups at the highest dose (108PFU per mouse).

B: The phenotype is based on the Animal Experiments 1-4.


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Table 5. Immunogenicity of MTase-defective VSV in mice (primary immunization)

Group mice Antibody at week 3 (log10 GMT)A

Number of sick mice

Lung histology B

Brain histology

Viral RNA in lung

Viral RNA in brain

Protection rateC

DMEM 4 0 0/4 0 0 0/4 0/4 NA

DMEMC 4 0 4/4 3.0a 3.0 4/4 4/4 0/4

K1651A 4 5.31 0/4 1.0b 0 0/4 0/4 4/4

E1833Q 4 5.01 0/4 1.0b 0 0/4 0/4 4/4

D1762A 4 4.48 0/4 1.0b 0 0/4 1/4 4/4

G1670A 4 5.04 0/4 1.0b 0 0/4 0/4 4/4

G1672A 4 4.88 0/4 1.0b 0 0/4 0/4 4/4

A: Antibody titer was determined by ELISA. The Geometric Mean Titers (GMT) of four mice

was shown.

B: Value within a column followed by the different lowercase letters (a, b) are significantly

different (P<0.05).

C: Protection is based on the presence of VSV symptoms, mouse body weight change, lung, and

brain histology after virulent challenge. “NA” indicates not applicable.


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Table 6. Immunogenicity of MTase-defective VSV in mice (booster immunization)

Group mice Antibody at week 5 (log10 GMT)

Number of sick mice

Lung histology

Brain histology

Viral RNA in lung

Viral RNA in brain

Protection rate

DMEM 4 0 0/4 0 0 0/4 0/4 NA

DMEMC 4 0 4/4 3.0a 3.0 4/4 4/4 0/4

K1651A 4 6.03 0/4 0.5b 0 0/4 0/4 4/4

E1833Q 4 5.28 0/4 1.0b 0 0/4 0/4 4/4

D1762A 4 5.35 0/4 1.0b 0 0/4 0/4 4/4

G1670A 4 5.21 0/4 1.0b 0 0/4 0/4 4/4

G1672A 4 5.43 0/4 1.0b 0 0/4 0/4 4/4


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