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Rice Stripe Tenuivirus NSvc2 Glycoproteins Targeted to Golgi 1

Body by N-Terminal Transmembrane Domain and Adjacent 2

Cytosolic 24 Amino-Acids via COP I- and COP II-Dependent 3

Secretion Pathway 4

Min Yao 1 †, Xiaofan Liu 1 †, Shuo Li 2 †, Yi Xu 3 †, Yijun Zhou 2 *, Xueping Zhou 3,4 * and 5

Xiaorong Tao 1 * 6

1 Key Laboratory for the Integrated Management of Crop Diseases and Pests, Ministry of 8

Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P. R. 9

China; 10

2 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P. R. 11

China; 12

3 State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, 13

Hangzhou 310029, P. R. China; 14

4 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, 15

Chinese Academy of Agricultural Sciences, Beijing, P. R. China. 16

*Corresponding authors: Xiaorong Tao (taoxiaorong@njau.edu.cn); Xueping Zhou 18

(zzhou@zju.edu.cn) and Yijun Zhou (yjzhou@jass.ac.cn). 19

† These authors contributed equally to this study. 21

Running title: Requirements for Golgi targeting of RSV glycoproteins 23

Word count: Abstract, 203 25

Main body of the text, 4988 26

JVI Accepts, published online ahead of print on 3 January 2014

J. Virol. doi:10.1128/JVI.03006-13

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Abstract 28

The NSvc2 glycoproteins encoded by Rice stripe tenuivirus (RSV) share many 29

characteristics common to the glycoproteins found among Bunyaviridae. Within this 30

viral family, glycoproteins targeting to the Golgi apparatus play a pivotal role in the 31

maturation of the enveloped spherical particles. RSV particles, however, adopt a long 32

filamentous morphology. Recently, RSV NSvc2 glycoproteins were shown to localize 33

exclusively to the ER in Sf9 insect cells. Here, we demonstrate that the 34

amino-terminal NSvc2 (NSvc2-N) targets to the Golgi apparatus in Nicotiana 35

benthamiana cells, whereas the carboxyl-terminal NSvc2 (NSvc2-C) accumulates in 36

the ER. Upon co-expression, NSvc2-N redirects NSvc2-C from the ER to the Golgi. 37

The NSvc2 glycoproteins move together with the Golgi stacks along the ER/actin 38

network. The targeting of the NSvc2 glycoproteins to the Golgi was strictly dependent 39

on functional anterograde traffic out of the ER to the Golgi or on a retrograde 40

transport route from the Golgi apparatus. The analysis of truncated and chimeric 41

NSvc2 proteins demonstrates that the Golgi targeting signal comprises amino acids 42

269-315 of NSvc2-N, encompassing the transmembrane domain and 24 adjacent 43

amino acids in the cytosolic tail. Our findings demonstrate for the first time that the 44

glycoproteins from an unenveloped Tenuivirus could target into Golgi bodies in plant 45

cells. 46

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Importance 51

NSvc2 glycoprotein encoded by unenveloped Rice stripe tenuivirus (RSV) share 52

many characteristics in common with glycoprotein found among Bunyaviridae in 53

which all members have membrane-enveloped sphere particle. Recently, RSV NSvc2 54

glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. In this 55

study, we demonstrated that the RSV glycoproteins could target into Golgi in plant 56

cells. The targeting of NSvc2 glycoproteins to the Golgi was dependent on active 57

COP II or COP I. The Golgi targeting signal was mapped to the 23-amino-acids 58

transmembrane domain and the adjacent 24-amino-acids of the cytosolic tail of the 59

NSvc2-N. In light of the evidence from viruses in Bunyavidae that targeting into 60

Golgi is important for the viral particle assembly and vector transmission, we propose 61

that targeting of RSV glycoproteins into Golgi in plant cells represents a 62

physiologically relevant mechanism in the maturation of RSV particle complex for 63

insect vector transmission. 64

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INTRODUCTION 73

Rice stripe virus (RSV) is the type member of the genus Tenuivirus (1). RSV has 74

caused severe damage to rice crops in China and is known to be transmitted by 75

Laodelphax striatellus in a persistent, circulative-propagative manner (2). The RSV 76

genome consists of four negative-sense single-stranded RNA segments, designated 77

RNA1, 2, 3 and 4, which encode seven ORFs using a negative or ambisense coding 78

strategy (3). RNA1 is negative sense and encodes an RNA-dependent RNA 79

polymerase (RdRp) (4). The other three segments adopt an ambisense coding strategy. 80

RNA2 encodes a 22.8 kDa protein (NSs2) from the viral RNA (vRNA) and a 94 kDa 81

protein (NSvc2) from the viral complementary RNA (vcRNA) (5). RNA3 encodes a 82

viral suppressor (NSs3, 23.9 kDa) from the vRNA (6) and a nucleocapsid protein 83

(NSvc3, 35 kDa) from the vcRNA (7, 8). RNA4 encodes a 20.5 kDa protein (NSs4) 84

from the vRNA and a movement protein (NSvc4, 32 kDa) from the vcRNA (9). 85

Based on phylogenetic relationship and their genome organization and gene 87

expression strategies, tenuiviruses are more closely related to the animal-infecting 88

viruses in the genus Phlebovirus of the family Bunyaviridae than they are to plant 89

tospoviruses (10). The NSvc2 protein encoded by RSV (hereinafter the NSvc2 90

glycoprotein) shares many characteristics in common with the glycoproteins found in 91

the Bunyaviridae family of viruses in which all members adopt an enveloped 92

spherical virion form (10). The glycoprotein encoded by the Bunyaviridae viruses is 93

processed into two proteins, Gn (the amino-terminal glycoprotein) and Gc (the 94

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carboxyl-terminal glycoprotein), which together form the surface spikes of the mature 95

enveloped virion (11-14). The Gn protein of several viruses, including Uukuniemi 96

virus (UUKV) (15), the Punta toroviruses (16), and Rift valley fever virus (RVFV) 97

(17) in the genus Phlebovirus, as well as Tomato spotted wilt tospovirus (TSWV) (18), 98

has been shown to accumulate in the Golgi apparatus, while the Gc protein localizes 99

to the endoplasmic reticulum (ER). Upon co-expression, both glycoproteins localize 100

to the Golgi apparatus (16-19), suggesting that Gn can re-target Gc from the ER to the 101

Golgi. The targeting of the viral glycoproteins to the Golgi apparatus plays a pivotal 102

role in the maturation of the viral particles. The NSvc2 glycoprotein encoded by RSV 103

was predicted to be functionally similar to the glycoproteins found on other 104

Bunyaviridae viruses. RSV particles, however, adopt a long filamentous morphology 105

unenveloped (19, 20). The enveloped nature of Bunyaviridae versus the unenveloped 106

nature of Tenuivirus raises the question of what common or unique strategies have 107

evolved for them to form different morphology of viral particle. Zhao et al. (2012) 108

recently reported that the NSvc2 protein, or its two processing products, the 109

amino-terminus of NSvc2 (NSvc2-N) and the carboxyl-terminus of NSvc2 (NSvc2-C), 110

exclusively localized to the ER membrane in Spodoptera frugiperda (Sf9) insect cells 111

(21). It remains poorly understood whether the ER localization (the inability to target 112

to the Golgi apparatus) of the NSvc2 glycoproteins is the key step determining the 113

adoption of a long filamentous particle in RSV. It is also unknown why does a 114

nonenveloped teniuvirus encode glycoproteins. 115

116

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RSV systemically infects Nicotiana benthamiana by mechanical inoculation (9, 22). 117

In this study, the subcellular targeting of the NSvc2 glycoproteins and the 118

requirements for their targeting were extensively characterized in N. benthamiana. 119

We demonstrated that the NSvc2-N glycoprotein alone is able to target to the Golgi 120

apparatus in N. benthamiana, whereas NSvc2-C localizes to the ER membrane in the 121

absence of NSvc2-N. Upon co-expression, NSvc2-N redirects NSvc2-C to the Golgi 122

apparatus. The NSvc2 glycoproteins were found to move together with the Golgi 123

stacks along the ER/actin network in N. benthamiana epidermal cells. Using 124

dominant-negative mutants, we demonstrated that the targeting of the NSvc2 proteins 125

from the ER to the Golgi was strictly dependent on COP I and COP II early secretion 126

pathways. The analysis of truncated and chimeric NSvc2 proteins demonstrated that 127

the Golgi targeting signal localized to amino acids 269-315, encompassing the 128

23-amino acid transmembrane domain and the 24 adjacent amino acids of the 129

cytosolic tail. Our findings provide novel insights into the cellular properties of RSV 130

glycoproteins in plant cells. 131

132

MATERIALS AND METHODS 133

Plasmid constructs and organelle markers 134

p1300S-NSvc2-N-YFP and p1300S-NSvc2-C-YFP. NSvc2-N and NSvc2-C were 135

amplified from total RNA isolated from rice infected by RSV using RT-PCR and the 136

primers XT746/XT747 and XT800/XT388 (Supplemental Table S1). The NSvc2-N 137

and NSvc2-C PCR fragments were digested with Kpn I and BamH I and inserted into 138

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p1300S-YFP using the same restriction sites to obtain p1300S-NSvc2-N-YFP and 139

p1300S-NSvc2-C-YFP, respectively. 140

141

p1300S-NSvc2-Intron-YFP. A potato ST-LS1 intron (23) was inserted into the 142

AG/GT site at nucleotide (nt) position 1182 of NSvc2. The ST-LS1 intron, N-terminal 143

fragment (1182 nt) and C-terminal fragment (1423 nt) of NSvc2 were amplified using 144

the primers XT957/XT958, XT746/XT959 and XT960/XT388, respectively. The 145

three PCR fragments were mixed and amplified using XT746/XT388 to obtain 146

NSvc2-Intron, which was then digested with Kpn I and BamH I and inserted into 147

p1300S-YFP using the same restriction sites. 148

149

p1300S-NSvc2-N-46del-YFP and p1300S-NSvc2-N-63del-YFP. NSvc2-N 150

containing either a 46 or 63 amino acid deletion at the C-terminus was amplified 151

using the primer pairs XT746/XT807 or XT746/XT835, and the PCR products were 152

inserted into the Kpn I and BamH I sites of p1300S-YFP, respectively. 153

154

p1300S-SSNTMDNCTN-YFP, p1300S-SSNTMDNCTNdel46-YFP and 155

p1300S-SSNTMDNCTNdel63-YFP. The signal peptide (SSN), transmembrane domain 156

(TMDN) containing the full-length cytosolic domain (CTN), TMDN containing the 157

CTN with a 46 amino acid deletion and the TMDN with the CTN containing a 63 158

amino acid deletion at the C-terminus of NSvc2-N were amplified using the 159

corresponding primer pairs (XT746/XT837, XT836/XT747, XT836/XT807 and 160

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XT836/XT835). The SSNTMDNCTN, SSNTMDNCTNdel46, and SSNTMDNCTNdel63 161

fragments were fused using overlap PCR and the primers XT746/XT747, 162

XT746/XT807 and XT746/XT835, and were inserted into the Kpn I and BamH I sites 163

of p1300S-YFP, respectively. 164

165

p1300S-NSvc-C(TMDNCTN)-YFP and p1300S-NSvc-C(TMD-CT-del46)-YFP. A 166

fragment of NSvc2-C lacking the TMDC and the CTC was amplified using the primers 167

XT800 and XT869. The TMDN fragment with the full-length CTN and the TMDN 168

fragment with the CTN containing a 46 amino acid deletion at the C-terminus of 169

NSvc2-N were amplified with the primer pairs XT747/XT868 and XT807/XT868. 170

They were then fused using overlap PCR and the primers XT800/XT747 and 171

XT800/XT807, respectively. The products of overlap PCR were digested with Kpn I 172

and BamH I and cloned into p1300S-YFP. 173

174

p1300S-CFP-Sec24 and p1300S-Arf1-CFP. The full-length Sec24 (AT3G07100) 175

and Arf1 genes were amplified using RT-PCR and the total RNA extracted from the 176

Col ecotype of Arabidopsis thaliana using the primers XT743/XT754 and 177

XT784/XT785, respectively. The Sec24 PCR fragments were digested with BamH I 178

and cloned into the Bgl II site of p1300S-CFP, while Arf1 was digested with BamH I 179

and cloned into the BamH I site of p1300S-CFP. 180

181

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p1300S-Arf1 (T31N). To construct p1300S-ArfI (T31N), site-directed mutagenesis 182

was used to introduce the mutation into Arf1 using the primers XT784/XT795 and 183

XT794/XT785 and overlap PCR. The PCR product was digested with BamH I and 184

cloned into p1300S. 185

186

The ER marker mCherry-HDEL (24) and the Golgi marker Man49-mCherry (24) 187

were obtained from the Arabidopsis Biological Resource Center (ABRC). The Sar1 188

dominant-negative mutant construct Sar1 (H74L) was kindly provided by Professor 189

Taiyun Wei (25). 190

191

Plant material, transient expression and treatment 192

RSV (Jiangsu isolate) was collected from infected rice in a field in Nanjing and frozen 193

at -80°C until use. All transient expression experiments were performed using six- to 194

eight-week old N. benthamiana plants. Agrobacterium tumefaciens cells (C58C1 195

containing various RSV constructs and organelle markers) were grown using 196

kanamycin selection. The Agrobacterium cells were treated with infiltration buffer (10 197

mM MgCl2, 10 mM MES, pH 5.9, and 150 μM acetosyringone) for 3 hr at room 198

temperature before being infiltrated (OD600 = 0.5) into the abaxial surface of N. 199

benthamiana leaves. All agroinfiltrated plants were grown in growth chambers 200

(Model GXZ500D, Jiangnan Motor Factory, Ningbo, P. R. China) under a 16 h light/8 201

h dark cycle and a constant temperature of 25°C. The agroinfiltrated leaves were 202

examined for fluorescence expression between 24-72 hpi. When applicable, LatB 203

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(Sigma) was infiltrated at a final concentration of 10 ȝM into N. benthamiana leaves 204

before fluorescence observation. 205

206

Confocal laser scanning microscopy 207

Leaf discs were dissected from the agroinfiltrated leaf area of N. benthamiana leaves 208

and mounted in water between two cover slips. Images and movies were captured 209

using a Carl Zeiss LSM 710 confocal laser scanning microscope and 20, 63 oil or 210

63 water immersion objective lenses. CFP fluorescence was excited at 405 nm and 211

emission captured at 440-470 nm, YFP were excited at 488 nm and emission captured 212

at 497-520 nm, and mCherry was excited at 561 nm and emission captured at 585-615 213

nm. Images were processed using the Zeiss 710 CLSM and Adobe Photoshop 214

programs (San Jose, CA, USA). Movies were edited using the Corel Video Studio Pro 215

X4 software (Ottawa, Ontario, Canada). 216

217

Western blot analysis 218

Plant leaves from N. benthamiana agroinfiltrated with NSvc2-N-YFP, NSvc2-C-YFP 219

and NSvc2-YFP constructs were ground in a 1:3 (w/v; 0.1 g/300 μL) ratio of 220

extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% 221

glycerol, 0.1% Triton X-100 and 1plant protease inhibitor). After centrifugation for 222

10 min at 3,000 × g, the supernatant of the total protein preparation was separated by 223

SDS-polyacrylamide gel electrophoresis for immunoblot analysis. The blots were 224

probed with anti-YFP (Polyclonal antibody, 1:1,000 dilution; Biyuntian, Shanghai, 225

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China) and visualized with AP conjugated Goat anti-rabbit secondary antibodies 226

(1:1,000 dilution; Biyuntian, Shanghai, China) followed by nitro-blue tetrazolium 227

(NBT) and 5-bromo-4-chloro-3'-indolyphosphate (BCIP) staining (ready-made 228

solutions; Shenggong, Shanghai, China). 229

230

For subcellular fractionations, the soluble and microsomal fractions were isolated 231

from N. benthamiana leaves agroinfiltrated with NSvc2-N-YFP, NSvc2-C-YFP and 232

NSvc2-YFP constructs as described by Peremyslov et al. (2004) (26). The antigens on 233

the membranes were blotted with anti-YFP (rabbit). It was detected by DyLight 234

680-coupled goat anti-rabbit antibodies (1:10,000 dilution; Pierce, IL USA) and then 235

visualized by Licor Odyssey scanner. 236

237

RESULTS 238

The NSvc2-N protein is targeted to the Golgi apparatus 239

N. benthamiana is an ideal plant species in which to assess the subcellular 240

localization of viral proteins. To characterize the subcellular target of the NSvc2 241

glycoproteins in plant cells, we first fused the yellow fluorescent protein (YFP) to the 242

C-terminus of NSvc2-N (Fig. 1) and then agroinfiltrated the construct into N. 243

benthamiana epidermal cells. Western blot analysis showed that NSvc2-N-YFP fusion 244

protein was expressed as a size of 68kDa protein (Fig. 2A), indicating a proper 245

expression of the NSvc2-N-YFP construct. To investigate the intracellular localization 246

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of the NSvc2-N-YFP protein, we isolated soluble (S30) and microsomal (P30) protein 247

fractions from N. benthamiana leaves agroinfiltrated with NSvc2-N-YFP. We found 248

that NSvc2-N-YFP was localized exclusively in microsomal fractions that are known 249

to contain ER membrane structures and Golgi bodies (Fig. 2B). 250

251

To further characterize the subcellular localization of NSvc2-N-YFP, the infiltrated 252

leaves were examined using Zeiss 710 confocal laser scanning microscopy. At 36 253

hours post-infiltration (hpi), NSvc2-N-YFP was observed as numerous small bodies 254

in the cortical cytoplasm of the cells (Fig. 2C). To determine whether NSvc2-N 255

accumulated in the ER membrane, we co-expressed the NSvc2-N-YFP protein with 256

the HDEL signal fused to the N-terminus of mCherry (mCherry-HDEL) in N. 257

benthamiana (24). The merge of NSvc2-N-YFP with mCherry-HDEL images 258

revealed that the NSvc2-N-YFP signal did not colocalize with the ER marker, while 259

those NSvc2-N-YFP punctate bodies were still associated with the ER membrane (Fig. 260

2C-E). 261

262

To determine whether the NSvc2-N-YFP bodies co-localized with the Golgi stacks, 263

we co-infiltrated the Golgi marker construct Man49-mCherry (24) with 264

NSvc2-N-YFP in N. benthamiana epidermal cells. At 36 hpi, we found that the 265

NSvc2-N-YFP bodies co-localized with the Golgi stacks (Fig. 2F-H), suggesting that 266

the NSvc2-N-YFP protein targets to the Golgi apparatus. We then examined the 267

NSvc2-N-YFP protein signal at three time points, 24, 48 and 72 hpi, and found that 268

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NSvc2-N-YFP was targeted to the Golgi body as early as 24 hpi. 269

270

The NSvc2-C protein accumulates in the ER membrane 271

We also fused NSvc2-C protein with YFP at its C-terminus (Fig. 1) and infiltrated the 272

construct into N. benthamiana epidermal cells. Immunoblot analysis showed that 273

NSvc2-C-YFP protein expressed as 78kDa protein which is same as the predicted size 274

of NSvc2-C-YFP fusion protein (Fig. 3A). Fractionation analysis revealed that 275

NSvc2-C-YFP protein was localized only in the microsomal membrane fractions (Fig. 276

3B). To precisely define the intracellular distribution of NSvc2-C, the infiltrated 277

leaves were characterized using confocal laser scanning microscopy. The green 278

fluorescent signal of the NSvc2-C-YFP fusion protein appeared to be very weak, but 279

was still detectable in an ER-like network structure observed at 36 hpi (Fig. 3C). To 280

determine whether these fluorescent signals co-localized with the ER structure, the 281

cortical ER marker mCherry-HDEL was co-infiltrated with NSvc2-C-YFP. As shown 282

in Fig. 3C-E, the NSvc2-C-YFP protein co-localized with the ER membrane network. 283

284

To examine whether NSvc2-C-YFP accumulated in the Golgi stacks, we co-infiltrated 285

N. benthamiana cells with NSvc2-C-YFP and the Golgi marker Man49-mCherry. As 286

shown in Fig. 3F-H, no fluorescent signal associated with NSvc2-C-YFP was found to 287

accumulate in the Golgi apparatus. To confirm whether NSvc2-C-YFP exhibits any 288

accumulation in the Golgi stacks, we checked the fluorescent signal of NSvc2-C-YFP 289

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at 24, 48 and 72 hpi. The NSvc2-C-YFP protein did not form any small bodies that 290

could target to the Golgi body at the three time points examined. These results suggest 291

that NSvc2-C-YFP was arrested in the ER in N. benthamiana. 292

293

The NSvc2-N protein recruits NSvc2-C from the ER to the Golgi apparatus 294

To determine the localization and trafficking of the NSvc2 glycoproteins when 295

expressed from their precursor, we fused YFP to the C-terminus of the NSvc2 296

precursor protein. However, the construct containing the full-length NSvc2 gene 297

cannot grow in E. coli cells, suggesting that the full-length NSvc2 gene is toxic to E. 298

coli. We therefore inserted a potato ST-LS1 intron (23) into the AG/GT site at 299

nucleotide (nt) position 1182 of NSvc2. The intron-containing construct, 300

NSvc2-Intron-YFP (Fig. 1), can successfully generate a green fluorescence signal in N. 301

benthamiana epidermal cells after agroinfiltration. Total RNA was then isolated from 302

infiltrated leaves and the NSvc2-Intron-YFP RT-PCR products were sequenced to 303

confirm that the intron had been precisely processed from the inserted site of NSvc2 304

(NSvc2-Intron-YFP is hereinafter referred to as NSvc2-YFP). Immunoblot analysis 305

showed that NSvc2-C-YFP has been efficiently processed from precursor protein 306

NSvc2-YFP and expressed as 78 kDa protein (Fig. 4A). The processed protein was 307

distributed exclusively in the microsomal fractions which are known to contain ER 308

membranes and Golgi bodies (Fig. 4B). 309

310

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We then co-expressed NSvc2-YFP with the ER marker mCherry-HDEL in N. 311

benthamiana and the infiltrated leaves were examined using Zeiss confocal laser 312

scanning microscopy. Monitoring of NSvc2-C-YFP (NSvc2-C-YFP processed from 313

the NSvc2 precursor) showed that the fluorescent signal highlighted by NSvc2-C-YFP 314

co-localized in the ER network at 24-48 hpi. At 48-72 hpi, NSvc2-C-YFP began to 315

induce punctate structures along the ER membrane in the presence of NSvc2-N (Fig. 316

4C-E). To identify whether the newly formed bodies targeted to the Golgi apparatus, 317

we co-infiltrated N. benthamiana with NSvc2-YFP and the Golgi marker 318

Man49-mCherry. As shown in Fig. 4F-H, NSvc2-C-YFP bodies were indeed found to 319

be targeted to the Golgi apparatus. These results strongly suggest that NSvc2-N is 320

able to recruit NSvc2-C from the ER to the Golgi apparatus. 321

322

Targeted NSvc2 glycoproteins move together with the Golgi stacks in N. 323

benthamiana 324

In tobacco leaf cells, Golgi bodies traffic on an underlying ER track in an 325

actin-dependent manner (27, 28). To examine whether the targeted RSV NSvc2 326

glycoproteins move with the Golgi bodies, we utilized time-lapse confocal 327

microscopy to monitor the movement of NSvc2-N-YFP or NSvc2-N/NSvc2-C-YFP 328

(processed from the NSvc2-YFP precursor) in the presence of the Golgi marker. Fig. 329

5A-C and D-F show examples of the movement of the NSvc2-N-YFP and 330

NSvc2-N/NSvc2-C-YFP bodies with the Golgi stacks, and the arrows mark the 331

progressive movement of these bodies in each sequence. We found that both 332

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NSvc2-N-YFP and NSvc2-N/NSvc2-C-YFP moved together with the Golgi bodies 333

(Fig. 5A-C and D-F; Supplemental Video S1 and S2). 334

335

To determine whether the movement of bodies labeled with NSvc2-N-YFP or 336

NSvc2-N/NSvc2-C-YFP is dependent on similar forces driving the movement of the 337

Golgi bodies, we treated agroinfiltrated leaves at 48 hpi with 10 μM latrunculin B, an 338

actin depolymerizing agent (29). After 3 h of chemical treatment, we found that 339

movement of the NSvc2-N-YFP or NSvc2-N/NSvc2-C-YFP as well as Golgi bodies 340

was completely inhibited. However, NSvc2-N-YFP, NSvc2-N/NSvc2-C-YFP and the 341

Golgi bodies remained co-localized (Supplemental Video S3 and S4). These data 342

suggest that the NSvc2-N-YFP or NSvc2-N/NSvc2-C-YFP bodies move together with 343

the Golgi stacks along the ER/actin network. 344

345

ER-to-Golgi targeting of NSvc2 glycoproteins is dependent on a functional COP 346

II complex 347

Given that the RSV NSvc2-N-YFP and NSvc2-YFP fusion proteins targeted to the 348

Golgi, we ask whether the Golgi targeting of viral glycoproteins results from traffic 349

out of the ER to the Golgi apparatus via ERES. To address this question, we 350

co-infiltrated an ERES-marker, CFP-Sec24 (30), with NSvc2-N-YFP or NSvc2-YFP 351

proteins into N. benthamiana leaf cells. As shown in Fig. 6A-C and G-I, the 352

NSvc2-N-YFP or NSvc2-YFP bodies co-localized with CFP-Sec24 fluorescence at 353

the ERES. These results suggest that NSvc2-N is able to redirect NSvc2-C from the 354

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ER to the ERES, from where they subsequently co-migrate, most likely as a 355

heterodimer, to the Golgi apparatus. 356

357

The COP II complex is responsible for anterograde traffic out of the ER to the Golgi 358

apparatus (31). To test whether COPII vesicles are involved in ER-to-Golgi transport 359

of RSV NSvc2 glycoproteins, wild-type Sar1 or its dominant-negative mutant (H74L) 360

(32) was co-infiltrated with NSvc2-N-YFP or NSvc2-YFP together with the Golgi 361

marker Man49-mCherry into N. benthamiana. As shown in Fig. 6D-F and J-L, upon 362

co-expression of NSvc2-N-YFP or NSvc2-YFP with Sar1 (H74L), the florescence of 363

NSvc2-N-YFP or NSvc2-YFP, as well as of the Golgi bodies, was retrieved back to 364

the ER network, while co-expression with wild-type Sar1 did not cause the 365

NSvc2-N-YFP or NSvc2-YFP bodies to redistribute back to the ER (data not shown). 366

These results suggest that the accumulation of the RSV glycoproteins at the ERES and 367

in the Golgi bodies is dependent on a functional anterograde secretion pathway. 368

369

The accumulation of the NSvc2 glycoproteins at the Golgi bodies depends on 370

active COP I 371

To investigate whether the Golgi targeting of viral glycoproteins also involves 372

retrograde traffic, we co-infiltrated Arf1 tagged with CFP, a COP I vesicle marker 373

(33), with NSvc2-N-YFP or NSvc2-YFP in N. benthamiana. As shown in Fig. 7A-C 374

and G-I, the NSvc2-N-YFP or NSvc2-YFP bodies co-localized with COP I vesicles 375

labeled by Arf1-CFP. 376

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377

To determine the dependency of the ER-to-Golgi transport of RSV NSvc2 378

glycoproteins on active COP I, wild-type Arf1 or Arf1 (T31N), a dominant-negative 379

mutant of COP I (33, 34), was co-infiltrated with NSvc2-N-YFP or NSvc2-YFP along 380

with the Golgi marker Man49-mCherry into N. benthamiana. We found that 381

NSvc2-N-YFP or NSvc2-YFP as well as Man49-mCherry labeled Golgi bodies 382

redistributed back to the ER membrane in the presence of the dominant-negative Arf1 383

(T31N) (Fig. 7D-E and J-L). However, the co-expression of wild-type Arf1 has no 384

such effect (data not shown). These data demonstrate that the Golgi targeting of RSV 385

glycoproteins is also dependent on an active retrograde export route. 386

387

The Golgi targeting signal resides in a region of NSvc2-N encompassing a 388

transmembrane domain and the 24 adjacent amino acids of the cytosolic tail 389

Both the NSvc2-N-YFP and NSvc2-YFP expressed in N. benthamiana localized to 390

the Golgi complex, indicating that the Golgi retention signal resides in the N-terminus 391

of the NSvc2 protein. To map the domain responsible for the Golgi targeting of RSV 392

NSvc2-N, a truncated NSvc2-N del46-YFP protein, where 46 amino acids at the 393

C-terminal end of NSvc2-N within the cytosolic tail were deleted and fused with YFP 394

(Fig. 1), was constructed and transiently expressed in N. benthamiana. The 395

intracellular localization of this protein was determined by confocal fluorescence 396

analysis after 48 hpi. As illustrated in Fig. 8A-C, the truncated NSvc2-N del46-YFP 397

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protein was still capable of targeting to the Golgi complex. Subsequently, 63 amino 398

acids of the C-terminal end of the NSvc2-N protein within the cytosolic tail were 399

deleted (Fig. 1). This truncated NSvc2-N del63-YFP protein was no longer targeted to 400

the Golgi apparatus (Fig. 8D-F), suggesting that the amino acids in the cytosolic tail 401

are required for entering into the Golgi. 402

403

To determine the minimum region required for Golgi targeting, the predicted 404

transmembrane domain (amino acids 269-291) and the entire cytosolic domain (amino 405

acids 292-361) of NSvc2-N were fused with its signal peptide sequence (amino acids 406

1-23) (Fig. 1). When this chimeric SSNTMDNCTN-YFP construct was expressed in N. 407

benthamiana leaf cells, we found that it accumulated in the Golgi apparatus (Fig. 408

8G-I). Subsequently, the transmembrane domain and the 24 adjacent amino acids 409

(CTdel46, amino acids 292-315) were fused with its signal peptide (Fig. 1). The 410

resulting SSNTMDNCTNdel46-YFP construct also localized to the Golgi apparatus 411

(Fig. 8J-L). Lastly, the transmembrane domain and the 7 adjacent amino acids 412

(CTdel63, amino acids 292-298) were fused with its signal peptide (Fig. 1). As shown 413

in Fig. 8M-O, this SSNTMDNCTNdel63-YFP construct was incapable of targeting to 414

the Golgi complex. These analyses suggest that both the transmembrane domain 415

(amino acids 269-291) and the 24 adjacent amino acids in the cytosolic tail of the 416

NSvc2-N protein are required for Golgi targeting. 417

418

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To substantiate the observation that the Golgi retention signal is located within the 419

TMD and CT domains of NSvc2-N, the transmembrane domain (amino acids 269-291) 420

and the entire cytosolic domain (amino acids 292-361) of NSvc2-N were swapped 421

with those of NSvc2-C (Fig. 1). The resulting NSvc2-C(TMDNCTN)-YFP construct 422

was co-expressed with mCherry-HDEL and Man49-mCherry separately in N. 423

benthamiana. As shown in Fig. 8P-R, the chimeric NSvc2-C(TMDNCTN)-YFP 424

construct was capable of targeting to the Golgi apparatus, suggesting that the 425

transmembrane domain and the cytosolic domain of NSvc2-N was sufficient to direct 426

NSvc2-C-YFP to the Golgi complex (Fig. 8P-R). To analyze the requirement for the 427

Golgi targeting signal further, the transmembrane domain and the 24 adjacent amino 428

acids in the cytosolic domain of NSvc2-N were swapped with the corresponding 429

domain of NSvc2-C. As illustrated in Fig. 8S-U, this chimeric 430

NSvc2-C(TMDNCTNdel46)-YFP protein was also capable of localizing to the Golgi 431

apparatus. Taken together, these data suggest that the ER-to-Golgi targeting signal 432

resides in the C-terminal region (amino acids 269-315) of NSvc2-N, encompassing 433

the 23-amino-acids transmembrane domain and 24 adjacent amino acids in the 434

cytosolic tail. 435

436

DISCUSSION 437

In this study, using N. benthamiana as a model system we demonstrated here for the 438

first time that the glycoproteins from an unenveloped Tenuivirus could target into 439

Golgi bodies in plant cells. The RSV NSvc2-N glycoprotein alone targeted to the 440

Golgi apparatus, while the NSvc2-C glycoprotein accumulated in the ER membrane 441

in the absence of NSvc2-N. Upon co-expression, NSvc2-N was able to redirect 442

NSvc2-C from the ER to the Golgi apparatus. Using the Sar1 or Arf1 443

dominant-negative mutants, we demonstrated that the targeting of NSvc2 444

glycoproteins to the Golgi apparatus was dependent on an active COP I or COP II 445

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secretion pathway. We further revealed that the Golgi targeting signal mapped to a 446

region of the NSvc2-N protein (amino acids 269-315) encompassing the 447

23-amino-acids transmembrane domain (TMD) and the adjacent 24 amino acids of the 448

cytosolic tail. 449

450

The targeting of viral glycoproteins to the Golgi apparatus plays a pivotal role in the 451

formation of enveloped spherical particles for the viruses (animal- and plant-infecting) 452

in the Bunyaviridae family (15, 17, 35-41). Although RSV particle adopt long 453

filamentous morphology (20, 21), the subcellular targeting to the Golgi apparatus 454

seems to be a conserved mechanism between the unenveloped Rice stripe tenuivirus 455

and the enveloped viruses in Bunyaviridae. Why RSV glycoproteins do not facilitate 456

the formation of an enveloped spherical particle remains to be extensively 457

investigated in the future. It is interesting to note that despite the common 458

glycoprotein characteristics shared by RSV and viruses in the Bunyaviridae, all of the 459

viruses in the Bunyaviridae have larger size of glycoproteins than are found in RSV. 460

461

For TSWV, the type member of Tospovirus which is the only genus containing 462

plant-infecting viruses in the family Bunyaviridae, the glycoproteins forming the 463

surface spikes of the mature viral particle play an important role in insect transmission 464

(42). The key step where the virus enters the insect midgut cells is mediated by these 465

glycoproteins (42). RSV particles must also enter the midgut cells of L. striatellus to 466

complete their circulative-propagative transmission. The RSV-encoded glycoproteins 467

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were predicted to have a similar role in vector transmission. Although the NSvc2 468

protein was not detected in the filamentous RSV particle, this protein may function as 469

a bridge between the virus particle and recognition sites on the insect cell, as is seen, 470

for example, with helper component-proteinase (Hc-Pro) of potyvirus (43). The 471

targeting of RSV NSvc2 proteins to the Golgi apparatus could be an essential process 472

for glycoprotein modification and maturation, allowing the attachment of the RSV 473

RNP particle and subsequent vector transmission. 474

475

Zhao et al. (2012) reported that all of the RSV NSvc2 glycoproteins, including 476

NSvc2-N, NSvc2-C and the full-length NSvc2 localized exclusively to the ER 477

membrane in Sf9 insect cells (21). Our findings on the Golgi targeting of NSvc2 478

glycoproteins in N. benthamiana cells were different from those reported by Zhao et 479

al. (2012) in Sf9 insect cells. The RSV NSvc2 glycoproteins may have different 480

subcellular localization patterns in different systems. The NSvc2 glycoproteins target 481

to the Golgi apparatus in plant cells, while they were arrested in the ER membrane in 482

insect cells. These two different findings together lead to an interesting new concept 483

that acquisition of RSV viral particle from plant host by L. striatellus insect vector 484

may require glycoproteins which need to obtain glycosylation or similar modification 485

in the Golgi apparatus whereas transmission of RSV viral particle from insect vector 486

back into plant host may not require glycoproteins. 487

488

The leaf Golgi complex functions as a motile system that acquires products from a 489

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relatively stationary ER system (28, 31). The glycoproteins of TSWV have shown to 490

target into Golgi body using a tobacco protoplast system (44). However, the 491

movement of the viral glycoproteins in the plant cell has not been shown previously. 492

We demonstrated in this study that the targeted NSvc2 glycoproteins moved together 493

with the Golgi stacks along the ER/actin network in N. benthamiana epidermal cells. 494

The movement of the NSvc2-N glycoprotein together with the Golgi stacks in the N. 495

benthamiana epidermal cells gives rise to an interesting hypothesis that the NSvc2-N 496

could be acting as a mobile system for picking up NSvc2-C from the ER and transport 497

it into the Golgi stacks. This hypothesis is consistent with the finding that the 498

NSvc2-N protein accumulated in the Golgi stacks as early as 24 hpi, whereas the 499

NSvc2-C protein alone remained consistently localized in the ER. NSvc2-C only 500

began to accumulate in the Golgi apparatus at 48 hpi in the presence of NSvc2-N. The 501

constant movement of NSvc2-N will continue to pick up NSvc2-C in the Golgi stacks 502

over time. 503

504

RSV NSvc2-N was able to facilitate NSvc2-C transport from the ER to the Golgi 505

apparatus. Export of proteins from the ER in plant cells has been suggested to occur 506

through different routes (45-48). For ER-to-Golgi transport, a widely accepted 507

pathway is based on the sequential action of COP II and COP I complexes (27). Our 508

results showed that RSV NSvc2-N and the NSvc2-N::NSvc2-C complex migrate to 509

the Golgi apparatus via the ERES and that Golgi targeting was strictly dependent on a 510

functional anterograde traffic out of the ER to the Golgi or a retrograde transport route 511

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from the Golgi apparatus, as over-expression of Sar1 (H74L) and Arf1 (T31N) 512

aborted NSvc2-N as well as NSvc2-N::NSvc2-C complex trafficking to the Golgi. In 513

the mammalian system, it has been demonstrated that the COPII coat recognizes and 514

selects export cargo into ERES vesicles (49). Our finding that the targeting of NSvc2 515

protein into Golgi via ERES suggests that COPII machineries, such as Sar1 or 516

Sec23-Sec24 complex, may be involved in selecting NSvc2 glycoproteins to target 517

into Golgi. 518

519

For viruses in the Bunyaviridae family, intracellular maturation and budding in the 520

Golgi complex is mediated by the targeting and accumulation of the viral 521

glycoproteins in this cellular compartment (17, 18, 35, 38-40). Previous work has 522

shown that the Golgi targeting signal of the TSWV and BUNV glycoproteins resides 523

in the transmembrane domain of the Gn protein, allowing for sufficient ER-exit and 524

transport to the Golgi (35, 36). However, the Golgi localization signal of RVFV was 525

mapped to a 48-amino-acid region of Gn containing the transmembrane domain and 526

the adjacent 28 amino acids of the cytosolic tail (17). Although UUKV is also a 527

phlebovirus, the Golgi localization signal for the UUKV glycoproteins resides in the 528

cytosolic tail of Gn (15, 50). In this study, we have mapped the Golgi targeting signal 529

of RSV to a region encompassing the transmembrane domain and the 24 adjacent 530

amino acids of the cytosolic tail of the N-terminus of NSvc2. Although the 531

tenuiviruses has very close relationship to the phleboviruses, our finding support that 532

the Golgi targeting motif of the RSV glycoprotein is more closely related to that of 533

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RVFV, instead of UUKV glycoprotein. 534

535

In summary, our results presented here reveal that Rice stripe tenuivirus glycoproteins 536

were able to target into Golgi apparatus in plant cells. Targeting of RSV glycoproteins 537

into Golgi apparatus is mediated by the N-Terminal transmembrane domain and the 538

adjacent cytosolic 24 amino-acids of NSvc2 in a COP I- and COP II-dependent 539

manner. In light of the evidence from viruses in Bunyavidae that targeting into Golgi 540

apparatus is important for the viral particle assembly and vector transmission, we 541

propose that targeting of RSV glycoproteins into Golgi apparatus in plant cells 542

represents a physiologically relevant mechanism in the maturation of RSV particle 543

complex for insect vector transmission. 544

545

ACKNOWLEDGMENTS 546

This work was financially supported by the Program for New Century Excellent 547

Talents in the University (NCET-12-0888), the National Natural Science Foundation 548

of China (31222045, 31171813 and 31170142), the Special Fund for Agro-scientific 549

Research in the Public Interest (201303021 and 201003031) and the National Program 550

on Key Basic Research Project of China (973 Program, 2014CB138400). We would 551

like to thank Professor Taiyun Wei for kindly providing the Sar1 (H74L) 552

dominant-negative mutant. We also thank three anonymous referees for their valuable 553

comments on earlier version of this paper. 554

555

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675

676

677

678

679

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FIGURE LEGENDS: 682

FIG 1 Schematic diagrams of the viral constructs used for expression analysis (the 683

glycoprotein constructs are aligned below the precursor). Predicted cleavage sites 684

(scissor symbols) and amino acid positions are indicated. SS, TMD and CT refer to 685

the signal sequence, the transmembrane domain and the cytosolic tail, respectively. 686

SSN and SSC refer to the SS of NSvc2-N and NSvc2-C, respectively. TMDN and 687

TMDC refer to the TMD of NSvc2-N and NSvc2-C, respectively. CTN and CTC refer 688

to the CT of NSvc2-N and NSvc2-C, respectively. An intron of the potato ST-LS1 689

was inserted at the nucleotide position of 1182 on NSvc2. In all constructs, the YFP 690

fluorophore was fused in frame at the site of the stop codon. 691

692

FIG 2 Subcellular localization of the NSvc2-N protein in Nicotiana benthamiana leaf 693

epidermal cells. (A) Immunoblot analysis of NSvc2-N-YFP fusion proteins expressed 694

by agroinfiltration in N. benthamiana leaves. The blots were probed using anti-YFP. 695

Empty vector (EV) was used as a negative control. Ponseau S was used as a loading 696

control. (B) Subcellular fractionation analysis of NSvc2-N-YFP fusion protein. The 697

soluble (S30) and microsomal (P30) fractions were isolated from agroinfiltrated 698

leaves of N. benthamiana. The membrane blots were probed using anti-YFP. (C-E) 699

The co-localization of the NSvc2-N-YFP (C) with the ER labeled by mCherry-HDEL 700

at 36 hpi (D). (E) Merged image of (C) and (D). (F-H) The co-localization of the 701

NSvc2-N-YFP (F) with the Golgi apparatus labeled by Man49-mCherry at 36 hpi (G). 702

The merged image illustrates the NSvc2-N protein targeted to the Golgi apparatus (H). 703

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Scale bars, 20 μm. 704

705

FIG 3 Subcellular localization of the NSvc2-C protein in Nicotiana benthamiana leaf 706

epidermal cells. (A) Western blot analysis of NSvc2-C-YFP fusion proteins expressed 707

by agroinfiltration in N. benthamiana leaves. The blots were probed using anti-YFP. 708

Ponseau S was used as a loading control. Empty vector (EV) was used as a negative 709

control. (B) Subcellular distribution of NSvc2-C-YFP protein by fractionation 710

analysis. The soluble (S30) and microsomal (P30) fractions were isolated from 711

agroinfiltrated leaves of N. benthamiana. The membrane blots were probed using 712

anti-YFP. (C-E) The co-localization of the NSvc2-C-YFP (C) with the ER labeled by 713

mCherry-HDEL at 36 hpi (D). The merged image shows that NSvc2-C-YFP align 714

well with the ER membrane (E). (F-H) The co-localization of the NSvc2-C-YFP (F) 715

with the Golgi apparatus labeled by Man49-mCherry at 36 hpi (G). (H) Merged image 716

of (F) and (G). Scale bars, 20 μm. 717

718

FIG 4 Subcellular localization of NSvc2-YFP in Nicotiana benthamiana leaf 719

epidermal cells. (A) Immunoblot analysis of NSvc2-YFP fusion proteins (NSvc2-N 720

and NSvc2-C-YFP glycoproteins were processed from its common glycoprotein 721

precursor NSvc2-YFP) expressed by agroinfiltration in N. benthamiana leaves. The 722

membrane blots were probed using anti-YFP. Ponseau S was used as a loading control. 723

Empty vector (EV) was used as a negative control. (B) Subcellular distribution 724

analysis of NSvc2-YFP protein by fractionation. The soluble (S30) and microsomal 725

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(P30) fractions were isolated from agroinfiltrated leaves of N. benthamiana. The 726

membrane blots were probed using anti-YFP. (C-E) Co-expression of the NSvc2-YFP 727

(NSvc2-C-YFP was processed from this glycoprotein precursor) (C) with 728

mCherry-HDEL (D) at 48 hpi. (E) Merged image of (C) and (D). (F-H) The 729

co-localization of the NSvc2-YFP (NSvc2-C-YFP was processed from the 730

glycoprotein precursor) (F) with the Golgi apparatus labeled by Man49-mCherry (G) 731

at 48 hpi. The merged image shows the NSvc2-C protein targeted to the Golgi 732

apparatus in the presence of NSvc2-N (H). Scale bars, 20 μm. 733

734

FIG 5 NSvc2 glycoproteins trafficking together with the Golgi stacks along the ER 735

track in Nicotiana benthamiana leaf epidermal cells. (A-C) Time-lapse confocal 736

images showing the movement of NSvc2-N-YFP (A) and the Golgi apparatus (B) 737

labeled by Man49-mCherry at the times indicated. The position of the tracked signal 738

is marked with an arrow. (C) Merged image of (A) and (B). (D-F) Time-lapse 739

confocal images showing the movement of NSvc2-YFP (NSvc2-N and NSvc2-C-YFP 740

were processed from the glycoprotein precursor NSvc2-YFP) (D) and the Golgi 741

apparatus (E) at the times indicated. The position of the tracked signal is marked with 742

an arrow. The merged images demonstrate that the NSvc2 proteins move together 743

with the Golgi apparatus along the ER track (F). Scale bars, 20 μm. 744

745

FIG 6 ER-to-Golgi targeting of RSV NSvc2 glycoproteins depends on a functional 746

COP II complex. (A-C) Confocal images of Nicotiana benthamiana epidermal cells 747

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co-expressing NSvc2-N-YFP (A) and the COP II marker CFP-Sec24 at 36 hpi (B). (C) 748

Merged image of (A) and (B). The arrows mark co-localization of NSvc2-N-YFP 749

bodies with the ERES labeled with CFP-Sec24. (D-F) Co-expression of the 750

dominant-negative mutant Sar1 (H74L) causes the redistribution of NSvc2-N-YFP (D) 751

as well as the Golgi apparatus (E) back to the ER. (F) Merged image of (D) and (E). 752

(G-I) Cells co-expressing NSvc2-N and NSvc2-C-YFP (from their common precursor 753

NSvc2- YFP) (G) and the ERES labeled with CFP-Sec24 at 48 hpi (H). (I) Merged 754

image of (G) and (I). (J-L) Co-expression of the dominant-negative mutant Sar1 755

(H74L) inhibits the transport of NSvc2-N and NSvc2-C-YFP (co-expressed from their 756

common precursor NSvc2-YFP) to the Golgi complex. Scale bars, 20 μm. 757

758

FIG 7 ER-to-Golgi targeting of RSV NSvc2 glycoproteins depends on an active COP 759

I complex. (A-C) Confocal images of Nicotiana benthamiana epidermal cells 760

co-expressing NSvc2-N-YFP (A) and the COP I marker labeled with Arf1-CFP at 36 761

hpi (B). (C) Merged image of (A) and (B). (D-F) Co-expression of the 762

dominant-negative mutant Arf1 (T31N) led to the retention of NSvc2-N-YFP (D) as 763

well as the Golgi apparatus (E) in the ER at 48 hpi. (F) Merged image of (D) and (E). 764

(G-I) Cells co-expressing NSvc2-YFP (G) and the COP I marker labeled with 765

Arf1-CFP (H). (I) Merged image of (G) and (H). (J-L) Co-expression of the 766

dominant-negative Arf1 (T31N) blocks transport of NSvc2-N and NSvc2-C-YFP 767

(co-expressed from their common precursor NSvc2-YFP) to the Golgi complex. Scale 768

bars, 20 μm. 769

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770

FIG 8 Golgi targeting signal analysis of truncated and chimeric NSvc2-N proteins. 771

(A-U) Confocal images of Nicotiana benthamiana epidermal cells co-expressing 772

Man49-mCherry with the truncated or chimeric proteins NSvc2-N del46-YFP (A-C), 773

NSvc2-N del63-YFP (D-F), SSNTMDNCTN-YFP (G-I), SSnTMDN-CTNdel46-YFP 774

(J-L), SSNTMDNCTNdel63-YFP (M-O), NSvc2-C(TMDNCTN)-YFP (P-R), and 775

NSvc2-C(TMDNCTNdel46)-YFP (S-U), respectively, at 48 hpi. Scale bars, 20 μm. 776

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XT800 JVI.03006 13.full

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