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JVI Accepts, published online ahead of print on 3 January 2014
J. Virol. doi:10.1128/JVI.03006-13
Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Rice Stripe Tenuivirus NSvc2 Glycoproteins Targeted to Golgi

Body by N-Terminal Transmembrane Domain and Adjacent

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

Secretion Pathway

Min Yao

Xiaorong Tao 1 *

1 †

, Xiaofan Liu

1†

, Shuo Li

2†

, Yi Xu 3 †, Yijun Zhou

*, Xueping Zhou

3,4

* and

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

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

China;

Hangzhou 310029, P. R. China;

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

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

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

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

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18

*Corresponding

authors:

Xiaorong

Tao

(taoxiaorong@njau.edu.cn);

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

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21

† These authors contributed equally to this study.

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Running title: Requirements for Golgi targeting of RSV glycoproteins

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Word count: Abstract, 203
Main body of the text, 4988

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1

Xueping

Zhou

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Abstract

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

characteristics common to the glycoproteins found among Bunyaviridae. Within this

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

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

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

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

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

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

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

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

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

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

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

NSvc2 proteins demonstrates that the Golgi targeting signal comprises amino acids

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

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

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

cells.

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Importance

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

many characteristics in common with glycoprotein found among Bunyaviridae in

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

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

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

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

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

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

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

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

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

physiologically relevant mechanism in the maturation of RSV particle complex for

insect vector transmission.

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INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

Based on phylogenetic relationship and their genome organization and gene

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

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

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

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

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

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

processed into two proteins, Gn (the amino-terminal glycoprotein) and Gc (the
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carboxyl-terminal glycoprotein), which together form the surface spikes of the mature

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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nonenveloped teniuvirus encode glycoproteins.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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glycoproteins in plant cells.

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MATERIALS AND METHODS

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Plasmid constructs and organelle markers

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

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

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

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and NSvc2-C PCR fragments were digested with Kpn I and BamH I and inserted into
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p1300S-YFP using the same restriction sites to obtain p1300S-NSvc2-N-YFP and

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p1300S-NSvc2-C-YFP, respectively.

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

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

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

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

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

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

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p1300S-YFP using the same restriction sites.

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150

p1300S-NSvc2-N-46del-YFP

and

p1300S-NSvc2-N-63del-YFP.

NSvc2-N

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

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

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

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p1300S-SSNTMDNCTN-YFP,

p1300S-SSNTMDNCTNdel46-YFP

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

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

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

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

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

and

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

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

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

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of p1300S-YFP, respectively.

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

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

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

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

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

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

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

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and BamH I and cloned into p1300S-YFP.

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

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

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

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

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

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

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

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

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

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cloned into p1300S.

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

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

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

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Taiyun Wei (25).

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Plant material, transient expression and treatment

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

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

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

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containing various RSV constructs and organelle markers) were grown using

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

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

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

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benthamiana leaves. All agroinfiltrated plants were grown in growth chambers

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

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

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

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

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before fluorescence observation.

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Confocal laser scanning microscopy

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

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

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

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

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

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

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

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

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X4 software (Ottawa, Ontario, Canada).

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Western blot analysis

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

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

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

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

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

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

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

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

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

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

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solutions; Shenggong, Shanghai, China).

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For subcellular fractionations, the soluble and microsomal fractions were isolated

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

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

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

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

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visualized by Licor Odyssey scanner.

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RESULTS

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The NSvc2-N protein is targeted to the Golgi apparatus

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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2C-E).

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

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

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

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

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

co-infiltrated

the

Golgi

marker

construct

Man49-mCherry

(24)

with

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

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The NSvc2-C protein accumulates in the ER membrane

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

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

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

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

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

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

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leaves were characterized using confocal laser scanning microscopy. The green

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

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

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

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

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

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

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

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

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

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accumulation in the Golgi stacks, we checked the fluorescent signal of NSvc2-C-YFP
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at 24, 48 and 72 hpi. The NSvc2-C-YFP protein did not form any small bodies that

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

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

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

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To determine the localization and trafficking of the NSvc2 glycoproteins when

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

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

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

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

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

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

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

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

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

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

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

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

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

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membranes and Golgi bodies (Fig. 4B).

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

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benthamiana and the infiltrated leaves were examined using Zeiss confocal laser

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

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

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

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

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

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

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

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

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

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

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benthamiana

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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the Golgi stacks along the ER/actin network.

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

347

II complex

348

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

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

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

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

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

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

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

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

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heterodimer, to the Golgi apparatus.

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

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

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

361

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

362

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

363

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

364

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

365

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

366

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

367

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

368

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

369

370

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

371

active COP I

372

To investigate whether the Golgi targeting of viral glycoproteins also involves

373

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

374

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

375

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

376

labeled by Arf1-CFP.
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358

377

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

379

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

380

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

381

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

382

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

383

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

384

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

385

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

386

glycoproteins is also dependent on an active retrograde export route.

387

388

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

389

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

390

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

391

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

392

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

393

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

394

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

395

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

396

intracellular localization of this protein was determined by confocal fluorescence

397

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

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378

398

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

399

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

400

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

401

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

402

are required for entering into the Golgi.

404

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

405

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

406

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

407

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

408

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

409

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

410

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

411

resulting SSNTMDNCTNdel46-YFP construct also localized to the Golgi apparatus

412

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

413

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

414

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

415

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

416

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

417

NSvc2-N protein are required for Golgi targeting.

418

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403

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

420

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

421

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

422

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

423

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

424

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

425

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

426

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

427

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

428

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

429

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

430

domain

431

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

432

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

433

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

434

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

435

cytosolic tail.

NSvc2-C.

illustrated

Fig.

8S-U,

this

chimeric

436
437

DISCUSSION

438

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

439

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

440

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

441

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

442

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

443

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

444

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

445

glycoproteins to the Golgi apparatus was dependent on an active COP I or COP II
20

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419

446

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

447

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

448

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

449

cytosolic tail.

450

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

452

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

453

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

454

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

455

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

456

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

457

the formation of an enveloped spherical particle remains to be extensively

458

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

459

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

460

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

461
462

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

463

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

464

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

465

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

466

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

467

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

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451

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

469

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

470

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

471

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

472

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

473

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

474

RNP particle and subsequent vector transmission.

475
476

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

477

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

478

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

479

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

480

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

481

subcellular localization patterns in different systems. The NSvc2 glycoproteins target

482

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

483

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

484

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

485

may require glycoproteins which need to obtain glycosylation or similar modification

486

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

487

back into plant host may not require glycoproteins.

488
489

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

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468

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

491

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

492

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

493

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

494

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

495

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

496

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

497

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

498

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

499

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

500

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

501

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

502

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

503

over time.

504
505

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

506

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

507

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

508

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

509

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

510

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

511

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

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490

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

513

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

514

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

515

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

516

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

517

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

518

into Golgi.

519

520

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

521

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

522

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

523

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

524

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

525

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

526

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

527

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

528

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

529

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

530

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

531

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

532

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

533

the Golgi targeting motif of the RSV glycoprotein is more closely related to that of
24

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512

534

RVFV, instead of UUKV glycoprotein.

535

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

537

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

538

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

539

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

540

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

541

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

542

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

543

represents a physiologically relevant mechanism in the maturation of RSV particle

544

complex for insect vector transmission.

545
546

ACKNOWLEDGMENTS

547

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

548

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

549

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

550

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

551

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

552

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

553

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

554

comments on earlier version of this paper.

555
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536

556

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FIGURE LEGENDS:

683

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

684

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

685

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

686

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

687

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

688

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

689

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

690

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

691

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

692
693

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

694

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

695

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

696

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

697

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

698

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

699

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

700

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

701

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

702

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

703

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

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682

704

Scale bars, 20 μm.

705

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

707

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

708

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

709

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

710

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

711

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

712

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

713

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

714

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

715

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

716

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

717

of (F) and (G). Scale bars, 20 μm.

718
719

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

720

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

721

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

722

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

723

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

724

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

725

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

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706

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

727

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

728

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

729

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

730

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

731

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

732

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

733

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

734
735

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

736

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

737

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

738

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

739

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

740

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

741

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

742

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

743

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

744

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

745
746

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

747

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

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726

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

749

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

750

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

751

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

752

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

753

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

754

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

755

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

756

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

757

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

758
759

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

760

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

761

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

762

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

763

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

764

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

765

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

766

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

767

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

768

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

769

bars, 20 μm.

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748

770

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

772

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

773

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

774

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

775

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

776

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

777
778
779
780
781
782
783

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