JVI Accepts, published online ahead of print on 20 August 2014 J. Virol. doi:10.1128/JVI.01815-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

1

Trafficking of Bluetongue virus visualized by recovery of

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tetracysteine-tagged virion particles

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Junzheng Du§*, Bishnupriya Bhattacharya*, Theresa H. Ward and Polly Roy#

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Department of Pathogen and Molecular Biology, Faculty of Infectious and

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Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1 7HT, United Kingdom

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#

Corresponding author: [email protected]

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*

Du and Bhattacharya* equally contributed

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§

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Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary

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Research Institute, Chinese Academy of Agricultural Science, Lanzhou

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730046, China

Current Address: State Key Laboratory of Veterinary Etiological Biology, Key

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Running title: Bluetongue virus with TC-tagged VP2

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Abstract: 235 words

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Text: 5826 words

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35

Abstract

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Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae

37

family, is a double-capsid insect-borne virus enclosing a genome of 10

38

double-stranded RNA segments. As with other members of the family, BTV

39

virions are non-enveloped particles containing two architecturally complex

40

capsids. The two proteins of the outer capsid, VP2 and VP5, are involved in

41

BTV entry and the delivery of the transcriptionally active core in the cell

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cytoplasm. Although the importance of endocytic pathway in BTV entry has

43

been reported, a detailed analysis of entry and the role of each protein on virus

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trafficking have not been possible due to unavailability of a tagged virus. Here

45

for the first time we report on the successful manipulation of a segmented

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genome of a non-enveloped capsid virus by the introduction of tags that were

47

subsequently fluorescently visualized in infected cells. The genetically

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engineered fluorescent BTV particles were observed to enter live cells

49

immediately after virus adsorption. Further, we showed separation of VP2 from

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VP5 during virus entry and confirmed that while VP2 is shed from virions in

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early endosomes, virus particles still consisting of VP5 were trafficked

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sequentially from early to late endosomes. Since BTV infects both mammalian

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and insect cells, the generation of tagged viruses will allow visualization of

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further downstream trafficking of BTV in different host cells. In addition, the

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tagging technology also has potential for transferable application on other

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non-enveloped complex viruses.

57 58 59 60 2

61

Importance

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Live virus trafficking in host cells has been highly informative in understanding

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interactions between virus and host cells. Although insertion of fluorescent

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markers in viral genome have made it possible to study trafficking of enveloped

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viruses, the physical constraints of architecturally complex capsid viruses have

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led to practical limitations. In this study, we have successfully genetically

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engineered the segmented RNA genome of Bluetongue virus (BTV), a

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complex non-enveloped virus belonging to the Reoviridae family. The resulting

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fluorescent virus particles could be visualized in virus entry studies for both live

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and fixed cells. This is the first time a structurally complex capsid virus has

71

been successfully genetically manipulated to generate virus particles that

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could be visualized in infected cells.

73 74 75 76 77 78 79 80 81 82 83

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Introduction

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Bluetongue virus (BTV), the prototype Orbivirus within the Reoviridae family is

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a non-enveloped, architecturally complex virus. BTV has 26 distinct serotypes

87

and is endemic in most parts of the world, often resulting in high morbidity and

88

mortality

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double-stranded RNA (dsRNA) segments (S1 to S10) encodes 7 structural

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(VP1 to VP7) and 4 non-structural proteins (NS1 to NS4) (1, 2). In the virus

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particles, the structural proteins are organized in two capsids; an outer capsid

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of VP2 and VP5, an inner capsid or “core,” of VP7 and VP3 that encloses the

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viral transcription complex (VP1, VP4 and VP6) in addition to the viral genome

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(1, 3). Three-dimensional structural studies of virions by cryo-electron

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microscopy revealed that VP2 arranges as trimers on the virion surface

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protruding as spike-like structures from the surface of the virus particles (4, 5).

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VP2 alone is responsible for the viral haemagglutination activity, serotype

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specificity and for the attachment of virions to the host cell (5-9). The second

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outer capsid protein VP5, is also arranged in trimers, but is less exposed than

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VP2 and is globular in shape (5). Structurally, VP5 resembles the fusion

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proteins of enveloped viruses and consists of amphipathic Į-helical regions on

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its external surface that have been suggested to play an active role in the

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penetration of endosomal membranes to release BTV cores into the cytoplasm

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(5, 10, 11).

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Although studies have elucidated the role of cellular factors in BTV entry, it is

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still not clear how the virus particles are trafficked into cells. Compared to other

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viruses, such as polio and vaccinia (12-14), a major difficulty in previous

108

studies on BTV entry process and its interactions with various cellular

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components has been the lack of real time live-cell imaging. To this end,

in

ruminants.

The

BTV

viral

4

genome,

comprising

of

10

110

fluorescence visualization of single virus particles or viral proteins in fixed and

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live cells not only provides a valuable means to study interactions between

112

viral and cellular proteins during virus entry, but also during trafficking,

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assembly and release of the virus (15, 16). Since the BTV genome consists of

114

10 segmented dsRNA molecules each with 0.8 to 3.9kb approximate sizes, the

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capacity of each segment to accommodate foreign genes is limited (17).

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Hence, as an alternate strategy we used the biarsenical tetracysteine (TC)

117

technology that involves the use of small TC tags with a CCPGCC motif, which

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can be inserted into a protein without the risk of disrupting the overall structure

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of the targeted protein (18-20). The tagged proteins are specifically recognized

120

by membrane-permeable biarsenical dyes that fluoresce when bound to the

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cysteine pairs in the TC motif. In addition, the differential labeling of the tagged

122

proteins with two fluorescent biarsenical dyes, FlAsH (green) and ReAsH (red)

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(18, 19), makes it a powerful tool for the real-time visualization of nascent

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protein synthesis and trafficking in cells. To date, this technology has been

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used successfully in enveloped viruses (21-24), but not for any complex capsid

126

virus such as BTV.

127

Although the functions of VP2 and VP5 have been documented (5-9, 11,

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25-27), the detailed mechanism of both proteins’ role in virus trafficking has not

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been studied in depth. In addition, while a 7Å structure of VP2 has been

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reported (5), the lack of a VP2 structure at atomic level has made it difficult to

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design strategies for the insertion of foreign tags that will not disrupt the

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secondary structure of the protein. Thus, this study was designed to

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investigate whether biarsenical TC tagging technology could be utilized for

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BTV as a means for investigating the trafficking of virus particles during virus

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entry. In this study we have successfully utilized biochemical methods, 5

136

sequence comparison data and BTV reverse genetics system (28) to insert

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tags into VP2 that do not disrupt the structure-function relationship of the virus

138

particles. To our knowledge this is the first report on the successful tagging of a

139

structural protein for any non-enveloped viruses. This resulted in clarification of

140

the BTV entry pathway in mammalian cells and showed, for the first time, that

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the two outer capsid proteins are separated from each other during the early

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stages of virus entry. Our study not only provides the possibility for further

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investigation of VP2 protein trafficking in live cells infected with the mutant

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virus, but also opens up possibilities for tagging other BTV proteins and other

145

orbiviruses.

146 147 148 149

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Materials and Methods

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Cell lines, viruses and bacteria.

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BSR and HeLa cells were maintained as described (29). Wild-type BTV1

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(South African Strain) and TC-tagged BTV1 stocks were propagated and

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titered in BSR cells (28). Recombinant baculoviruses expressing S-tag

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BTV10-VP2 or His-tag BTV10-VP294 were propagated in Spodoptera

156

frugiperda (Sf9) cells as described (29).

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Antibodies and reagents.

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All antibodies against BTV proteins used were generated in our laboratory.

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Purified, recomibinant VP2, VP5 and NS2 expressed from baculoviruses were

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used to generate monospecific polyclonal antibody in rabbits as described

161

previously (30). Antibodies against EEA1 and CD63 were obtained from

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Abcam. Fluorescent-labeled secondary antibodies, Alexa Fluor 488, Alexa

163

Fluor 546, biarsenical dye FlAsH were obtained from Invitrogen. Ammonium

164

chloride, trypsin and dynasore were obtained from Sigma. Cells were treated

165

with 30μM of ammonium chloride or 80nM of dynasore for 30 minutes (mins)

166

prior to infection (31). In the dynasore treated cells, the drug was maintained in

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the media during the 30 mins of virus incubation.

168

Plasmids and site-directed mutagenesis.

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Site-directed mutagenesis were performed to insert TC tags in BTV1 S2

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sequence (32). Briefly, two complementary primers were used to insert the

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nucleotide sequence (TGTTGTCCCGGGTGTTGT) encoding the TC tags in

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pUCBTV1T7S2 (28) template. The following primers used for site-directed

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mutagenesis:

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VP2TC94_F,5ƍCGGTTGTTGAAAGTACGAGATGTTGTCCCGGGTGTTGTCA

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CAAGAGTTTCCATACGAA3ƍ;VP2TC94_R,5’TTCGTATGGAAACTCTTGTGA 7

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CAACACCC GGGACAACATCTCGTACTTTCAACAACCG3’;VP2TC352_F,5’

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CGATACTTTTAATTGTTGTCCCGGGTGTTGTACACGAGTGTGGTGGTCGA

178

AC3ƍ; VP2TC352_R, 5’ACAACACCCGGGACAACAATTAAAAGTATCGGAGG

179

CTG3ƍ; VP2TC420_F, 5ƍTTGACTTT GTCGCGGAACCTTGTTGTCCCGGGTG

180

TTGTGGGATTAAAATTGTTCATTG3ƍ;and VP2TC420_R, 5ƍCAATGAACAATT

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TTAATCCCACAACACCCGGGACAACAAGCGACAAAGTCAA3ƍ.

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Recovery of tagged viruses.

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The T7 BTV capped (BTV1S1, BTV1S3-S10) and uncapped transcripts

184

(BTV1S2, BTV1S2 with TC tag) were generated as described (28). The mutant

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BTV particles were recovered following the method described previously (33).

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Genomic dsRNA from cells infected with control or mutant BTV was analyzed

187

as described (28).

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Virus growth kinetics.

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Monolayers of BSR cells were synchronously infected with either wild-type or

190

mutant BTV1 viruses at a MOI of 1 and plaque assays at 0, 24 and 48 hours

191

(hrs) post infection (pi) were carried out as described previously (29). Western

192

Blot (WB) was undertaken to monitor the expression of BTV proteins VP2, VP5

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and NS2, while cellular protein tubulin was used as the loading control. Each

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blotting experiment was repeated three times and the amount of protein

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expression was quantitated by ImageJ. The mean and standard error of the

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virus titers and intensities of the protein bands were calculated (Sigma Plot

197

2000; Systat Software Inc.) and the p values were also determined by Excel

198

(Microsoft).

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Fluorescence and confocal microscopic analysis of TC-tagged proteins

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and viruses.

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Live and fixed cell analysis of tagged-virus trafficking was undertaken by 8

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synchronously infecting HeLa cells at a MOI of 50 and 10, respectively. BSR

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cells infected with BTV1-VP2TC1 or BTV1-VP2TC2 were processed for

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biarsenical labeling at different times pi with FlAsH according to the

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manufacturer’s recommendations. Both live and fixed HeLa cells were imaged

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by confocal microscopy on a Zeiss LSM 510. Cells infected for live cell imaging

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were, washed with Opti-MEM I Reduced-Serum Medium (Invitrogen) and

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stained with FlAsH solution (2μM) for 30 minutes (min) at 4°C. After washing

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the cells with BAL (2, 3-dimercapto-1-propanol), live images were captured

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every 16.7 seconds by confocal microscopy with 488 nm laser and appropriate

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fluorescein filters on a pre-warmed stage that was maintained at 37°C.

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Subsequently, the images compiled into a movie at 8 images per second in

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Image J and cartoon was created using Photoshop Element 2.0 software.

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Fixed cell analysis was undertaken as described (29). The images were

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obtained using LSM 510 image browser software and processed using

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Photoshop Element 2.0 software (Microsoft). Each set of fixed cell experiments

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were repeated at least three times to generate either localization or

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co-localization data that could be quantified. Co-localization was judged by the

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appearance of yellow spots formed by the merging of red and green signals

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generated by the florescent tags attached to the secondary antibodies. The

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mean and standard error of percentage of localization or co-localization were

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calculated (Sigma Plot 2000; Systat Software Inc.) and the p values were also

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determined by Excel (Microsoft).

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Recombinant expression of amino terminal fragment of VP2.

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For the production of a recombinant baculovirus expressing BTV10-VP294, 94

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amino acids from amino terminal end of BTV10 VP2 were inserted in the 9

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baculovirus expression vector pAcYM1 and a His-tag was introduced

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upstream of the start codon of VP2 (30). In addition for bacterial expression,

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the N terminal first 94 residues of BTV1 were inserted into pRSETA

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(Invitrogen). Each construct was verified by sequencing. Recombinant

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baculoviruses expressing His BTV10-VP294 was produced, plaque purified and

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propagated using standard baculovirus recombination procedures (34).

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His-tagged BTV1-VP294 was expressed in Escherichia coli BL21(E.coli) as

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described (35). The soluble and insoluble fractions from bacterial and insect

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cells were prepared and analyzed by Western Blot (36).

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Trypsin

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Spectrophotometry analysis.

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Purified virus particles and VP2 were incubated with increasing concentrations

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of trypsin (1ng, 10ng, 100ng) for 30mins at 37°C and resolved by SDS-12%

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PAGE. Bands representing 100 and 110 kDa sizes that were excised from

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trypsin digested purified VP2 were subjected to in-gel digestion with trypsin,

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and analyzed by a liquid chromatography-tandem mass spectrometer

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QToF-micro; Waters Corp., Milford, MA) as described (37).

digestion

of

purified

virus

244 245 246 247 248 249 250 251 252 10

particles,

VP2

and

Mass

253

Results

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Identification of putative exposed regions in VP2.

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VP2, the host attachment protein, is responsible for virus entry. Hence

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strategies were adopted to minimize the potential impediment to virus

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infectivity that may be caused by fusion of the TC tag to VP2. Since, in the

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absence of atomic structure, the localization of flexible loop-linker regions

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that could be utilized for the insertion of TC tag were still not clear, two

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strategies were adopted to identify such exposed loop regions in VP2. Firstly,

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analysis of VP2 amino acid sequences (ExPASy) of two different BTV

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serotypes (BTV1 and BTV10) revealed the presence of putative trypsin

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cleavage sites that were common between the two VP2 sequences (data not

264

shown). Since trypsin could only access sites that are exposed on the

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surface of the protein and not those present internally, it was hypothesized

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that these putative cleavage sites could be the exposed loop-linker regions in

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VP2 and thus could be potentially utilized for the insertion of TC tags.

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Consequently, purified virus particles (BTV1) and VP2 protein (of BTV10) were

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digested with increasing concentrations of trypsin for 30mins at 37°C to identify

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the presence of potential enzyme cleavage sites in VP2 (Fig. 1). Although

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digestion of purified virus particles (Fig. 1A, left) and VP2 protein (Fig. 1A, right)

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with 100ng of trypsin showed the presence of protein bands with smaller sizes

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on SDS-PAGE gel, the pattern of the digested products was different between

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virus particles and VP2 protein (compare Fig. 1A left with right). Briefly,

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digestion of purified virus particles yielded a very faint fragment of 110kDa size

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and two smaller sized fragments measuring 40kDa and 10kDa (Fig. 1A, left). In

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comparison, digestion of purified VP2 protein under the same conditions 11

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resulted in two fragments of 100kDa and 10kDa size products (Fig. 1A, right).

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The difference in the digestion pattern between purified virus particles versus

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VP2 alone can be attributed to VP2 in virion adopting conformations that are

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more susceptible to proteolysis. Furthermore, the smaller digested products of

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VP2 in both purified virus particles (Fig. 1A, left) and VP2 protein (Fig. 1A, right)

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were only detectable in higher concentrations of trypsin (100ng) and not in the

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lower concentrations of trypsin (10 or 1ng). Control virus particles incubated at

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37°C for 30 mins in the absence of trypsin did not show any breakdown

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products. Since the presence of the 10kDa protein band was noted in both the

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digested virus particle and purified VP2 protein, the larger fragment 100kDa

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product from digested VP2 (Fig. 1A, right) was excised from the SDS-PAGE

289

gel (indicated with an asterisk). Subsequently, after purification from

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SDS-PAGE gel, smaller peptide fragments were generated by trypsin

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digestion of the 100kDa purified product and the peptides were further

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analyzed by mass spectrometry. The undigested 110kDa purified protein,

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treated in the same way, served as control. Evaluation of the peptide

294

fragments generated from both the 100kDa digested product and 110kDa

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undigested VP2 by SwissProt based Mascot search confirmed that these

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peptides were BTV10 VP2 derived (Table. 1). Peptides generated from the

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100kDa fragment mapped perfectly to the BTV10 VP2 sequence after amino

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acid position number 94 suggesting that the 100kDa protein fragment lacked

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the first 94 amino acids (Fig. 2A), consistent with the 10kDa fragment released

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as a result of trypsin digestion of purified VP2. In comparison, peptides

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generated from the undigested full-length VP2 (110kDa) mapped the entire

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length of the protein (Fig. 2B).

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In order to analyze whether the 10 KDa VP2 fragments was an autonomous 12

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domain that could be expressed as a stable soluble product, the amino

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terminal fragments of two different BTV serotypes, BTV1 (Fig. 1B, upper) and

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BTV10 (Fig. 1B, lower) were expressed as His-tagged fusion proteins

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(BTV1-VP294 and BTV10-VP294) either in bacterial (E. coli, Fig. 1B, upper) or

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eukaryotic (Baculovirus, Fig. 1B, lower) expression systems. The E. coli and

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Sf9

310

respectively were lysed, fractionated into soluble and insoluble fractions and

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run on a SDS-PAGE gel. Western Blot analysis of the His-tagged products

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revealed the presence of the BTV1-VP294 and BTV10-VP294 fusion proteins in

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both soluble and insoluble fractions, in both expression systems (Fig. 1B,

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upper & lower). Thus, this data suggests the presence of an exposed loop after

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the first 94 residues of VP2 which separates autonomous folded domains as

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soluble fraction. Hence, insertion of a TC in this region of VP2 might allow the

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construction of a VP2 with preserved overall folding and biological activity.

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In a second, alternate, approach to identify the presence of exposed loop

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regions in VP2 the similarity of VP2 of BTV with that of African horse sickness

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virus, AHSV, a closely related orbivirus (38) was assessed. A database search

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of the available AHSV (4 of the 9 serotypes) and BTV (24 of the 26 serotypes)

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VP2 peptide sequences (data not shown) revealed that VP2 from AHSV

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serotypes are generally longer (ranging from 1051 to 1060 residues) than BTV

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serotypes (ranging from 950 to 962 residues). Further, a careful sequence

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alignment of BTV and AHSV VP2 proteins revealed the presence of two

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in-frame deletions between residues 352-371 and 420-452 in all BTV

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serotypes that were absent in AHSV (Fig. 3A). This led us to hypothesize that

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the two deletions might also be exposed loop/linker regions of the protein

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which could also be explored for the insertion of TC tags in VP2. Hence, based

cells

expressing

BTV1-VP294

and

13

BTV10-VP294 fusion

proteins

330

on these results it was predicted that three regions in VP2 could be potentially

331

exploited for the introduction of TC tags (Fig. 3A).

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Generation and characterization of recovered mutant viruses containing

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TC-tagged VP2.

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The TC tags were inserted in the coding region of BTV1 VP2 between amino

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acids at position 94-95, 352-353 and 420-421 (Fig. 3B). As described

336

previously (33), uncapped S2 T7 transcripts (BTV1-S2) were generated for all

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constructs (BTV1-S294, BTV1-S2352 and BTV1-S2420) to recover TC tagged

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mutant viruses. The reverse genetics system used transfection of BSR cells

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with 9T7-derived RNA transcripts (S1 and S3~9) together with either wild-type

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BTV1-S2 or each of the tagged S2 transcripts. As with wild-type, all mutant

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viruses were recovered successfully.

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Plaque assays were undertaken to investigate the plaque morphology of the

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newly generated BTV1-VP2TC1 (BTV1-S294), BTV1-VP2TC2 (BTV1-S2352)

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and BTV1-VP2TC3 (BTV1-S2420) tagged viruses. Although clear plaques were

345

visible 3 days post transfection for all three BTV1 VP2 tagged viruses and also

346

the control BTV1 wild-type (WT) virus recovered at the same time, the plaques

347

formed by BTV1-VP2TC3 were smaller than those of both the wild-type BTV1

348

and mutants BTV1-VP2TC1 and BTV1-VP2TC2 (Fig. 4A). This suggested that,

349

compared to BTV1-VP2TC1 and BTV1-VP2TC2, the TC tagged VP2 in

350

BTV1-VP2TC3 might have generated a mildly attenuated virus. To confirm the

351

replication of the recovered viruses, genomic dsRNA from cells infected with

352

independent

353

non-denaturing polyacrylamide gel (Fig. 4B). The results revealed that the 10

354

dsRNA segments synthesized by the three tagged viruses had dsRNA profiles

355

that were indistinguishable from that of WT BTV1. Subsequently, using forward

plaques

were

extracted,

14

purified

and

analyzed

on

a

356

and reverse primers flanking the full-length S2, cDNA from both WT and

357

tagged viruses was generated from viral dsRNA by RT-PCR and sequenced.

358

The data confirmed the presence of the TC tag sequence at the relevant

359

position in the S2 segment of each tagged virus (Fig. 4C).

360

The difference in the plaque morphology of BTV1-VP2TC3 to that of

361

BTV1-VP2TC1, BTV1-VP2TC2 and WT virus prompted us to investigate virus

362

infectivity and growth characteristics of the three newly generated viruses. For

363

this purpose, BSR cells were infected at MOI of 1 with BTV1-VP2TC1,

364

BTV1-VP2TC2 or BTV1-VP2TC3 for different times. Virus growth and the viral

365

protein expression kinetics of each recovered tagged virus were then

366

monitored by total plaque assay titer (Fig. 5A) and by WB (Fig. 5B),

367

respectively. Control cells were infected with BTV1 WT virus and treated

368

similarly to the tagged viruses. Monitoring the total plaque assay titers at 0, 24

369

and 48 hrs pi demonstrated that although all three tagged viruses showed a

370

similar overall growth profile to the WT (Fig. 5A), the total titer of

371

BTV1-VP2TC3 was significantly reduced (p 0.05) with that of WT BTV1 at all times analyzed (Fig. 5A). This indicates

375

that the insertion of a TC tag in VP2 after amino acid position 94 or 352 did not

376

significantly impede the function of VP2, whereas TC tagging after amino acid

377

position 420 caused some loss of function of VP2 such that growth of the

378

tagged virus was impaired. WBs were undertaken to analyze the production of

379

two viral structural (VP2 and VP5) and one non-structural proteins (NS2)

380

encoded by the virus at 0, 24 and 48 hrs pi in cells infected with the tagged and

381

WT BTV1 viruses (Fig. 5B). BSR cells infected with BTV1-VP2TC1 or 15

382

BTV1-VP2TC2 or WT BTV1 showed similar expression profiles for VP2, VP5

383

and NS2 at all times pi (Fig. 5B). In all blots the level of tubulin, used as a

384

loading control were equivalent. Further, when the production of VP5, VP2 and

385

NS2 was quantified and normalized to that of tubulin production, the virus

386

proteins produced by BTV1-VP2TC3 at 24 (Fig. 5C) and 48 (Fig. 5D) hrs pi

387

was statistically significantly different (p 0.05) in the expression of

389

VP5, VP2 and NS2 in cells infected with either BTV1-VP2TC1 or

390

BTV1-VP12TC2 with that of WT. Since BTV1-VP2TC1 and BTV1-VP2TC2

391

have similar growth curves and protein production to WT BTV1, these two

392

tagged viruses were utilized for virus entry studies.

393

Internalization of tagged viruses in cells.

394

Live cell imaging was undertaken to assess whether the fluorescent labelling of

395

the tagged viruses with biarsenical dye FlAsH generated sufficient signal for

396

investigating trafficking of tagged viruses during entry into the host cells (Fig. 6).

397

For this purpose HeLa cells infected with BTV1-VP2TC2 were stained with the

398

fluorescent biarsenical dye FlAsH, which binds specifically to tetracysteine

399

tags, and live cells were imaged by confocal microscopy immediately after

400

infection (Fig. 6A). Control containing uninfected cells stained with FlAsH did

401

not show any fluorescent signal for VP2 (Fig. 6B). When the movement of the

402

fluorescently labelled TC tagged BTV1-VP2TC2 was monitored over time (Fig.

403

6C), the movement of labelled virus particle (indicated by white arrow) from its

404

initial position at zero time pi (yellow arrow) confirmed that the movement of

405

tagged BTV1-VP2TC2 can be tracked over time in an infected cell (Fig. 6C and

406

Movie. S1). It has been shown previously that BTV enters cells through the

407

clathrin-mediated endocytic pathway (11). Cellular dynamin is known to 16

408

mediate the pinching of the clathrin-coated pits to form the coated vesicles (39,

409

40) and it has been reported that inhibition of dynamin with dynasore (31)

410

impedes this process. Hence, further experiments were undertaken to confirm

411

whether entry of the tagged viruses is also influenced by dynamin (Fig. 7A, B).

412

For this purpose BTV1-VP2TC1 or BTV1-VP2TC2 were adsorbed on both

413

mock-treated and dynasore-treated HeLa cells for 30 mins at 4°C. The cells

414

were washed and either processed for zero time pi or incubated at 37°C for 30

415

mins. At both times the cells were fixed with 4% paraformaldehyde, stained with

416

FlAsH and visualized by confocal imaging. While analysis of control untreated

417

cell at zero time pi showed presence of majority of the tagged BTV1-VP2TC1

418

(94% ± 3.1) or BTV1-VP2TC2 (95% ± 2.9) on the plasma membrane (Fig. 7A,

419

upper panel) of infected cells, at the later time point (30 mins) pi both

420

BTV1-VP2TC1 (84% ± 1.9) and BTV1-VP2TC2 (80% ± 3.2) were observed

421

within the cellular cytoplasm (Fig. 7A, middle panel). In comparison,

422

dynasore-treated cells analyzed at 0 (93% ± 3.3 for BTV1-VP2TC1 and 94% ±

423

3.1 for BTV1-VP2TC2) and 30 mins (84% ± 2.3 for BTV1-VP2TC1 and 84% ±

424

0.6 for BTV1-VP2TC2) pi demonstrated the presence of majority of tagged

425

virus particles on the plasma membrane (Fig. 7A, lower panel). To further

426

confirm whether dynamin is required for BTV entry, HeLa cells infected with

427

BTV1-VP2TC1 or BTV1-VP2TC2 were monitored for the expression of one of

428

the BTV nonstructural protein, NS2 at 16 hrs pi in the presence (Fig. 7B, upper

429

panel) and absence (Fig. 7B, lower panel) of dynasore. Although similar to Fig.

430

7A the presence of dynasore did not completely abolish the expression of NS2

431

(Fig. 7B, upper panel), however, a clear decrease in NS2 was observed

432

between cells that were treated with dynasore from the untreated control cells

433

(Fig. 7B, lower panel). While dynasore dependent decrease in NS2 expression 17

434

was greater for cells infected with BTV1-VP2TC1 (94% ± 1) than

435

BTV1-VP2TC2 (90.5% ± 2.8), a similar decrease of NS2 expression in

436

dynasore treated WT BTV1 infected cells (95.7% ± 0.3) showed that tagged

437

viruses and WT BTV1 behaved similarly. Further, statistical analysis also

438

confirmed that the difference in NS2 expression in cells treated with dynasore

439

and infected with either BTV1-VP2TC1 or BTV1-VP2TC2 was statistically

440

insignificant (p > 0.05) to that of treated cells infected with WT. This data is

441

consistent with an earlier study which has also noted the role for dynamin in

442

BTV entry (41) and confirmed that dynamin might play a functionally important

443

role during tagged BTV entry.

444

The canonical view of BTV entry is that the outer capsid (VP2 and VP5) is shed

445

in an early endosome to release a fusion competent core which then fuses with

446

the endocytic membrane and formally enters the cytoplasm (5, 8, 11, 26, 27).

447

However, whether VP2 and VP5, which are juxtaposed on the surface of the

448

virus particle remain together during this period is unknown. To investigate this

449

using tagged virions, cells were infected with BTV1-VP2TC1 or BTV1-VP2TC2

450

and the localization of VP2 and VP5 (Fig. 7C) were assessed at 2, 5 and 15

451

mins pi, following fixation with 4% paraformaldehyde and immunostaining.

452

FlAsH was used to label tagged viruses VP2 in BTV1-VP2TC1- and

453

BTV1-VP2TC2 infected cells. The second outer capsid protein VP5 was also

454

immunolabeled with a polyclonal VP5 antibody. Although co-localization

455

(yellow) of VP2 and VP5 by confocal microscopy was observed at 2 mins

456

(95.8% ± 4.2 for BTV1-VP2TC1 and 96.7% ± 3.3 for BTV1-VP2TC2) and at 5

457

mins pi (90.5% ± 4.8 for BTV1-VP2TC1 and 87.9% ± 2.4 for BTV1-VP2TC2)

458

(Fig. 7C, upper and middle panel), from 15 mins pi onwards VP2 and VP5 were

459

seen as separate entities (Fig. 7C, lower panel) in the majority of the infected 18

460

cells (72.9% ± 1.5 for BTV1-VP2TC1 and 72.7% ± 2.03 for BTV1-VP2TC2).

461

The separation of VP2 and VP5 after virus internalization suggests that the two

462

proteins disengage early in virus entry.

463

Our previous studies showed that BTV entry is pH-dependent (25) and that

464

VP5 has pH-dependent fusogenic activity (27). Further, the VP5 structure has

465

certain features analogous to the fusion proteins of some enveloped virus

466

proteins, in particular influenza HA (5). To confirm biochemically that the TC

467

tagged virus particles behave similarly to the WT virus, the effect of acidic pH

468

in BTV entry was explored by treating the cells with ammonium chloride (Fig.

469

7D), a lysosomotropic weak base that immediately raises the pH of

470

intracellular acidic vesicles. The possible effects of ammonium chloride

471

induced cytotoxicity assessed by cell viability assays did not show toxicity

472

(data not shown). HeLa cells were exposed to 30mM of ammonium chloride

473

prior to BTV infection, and BTV replication was examined at 24 hrs pi by

474

determining the virus titers. A decrease of almost two and a half logs in virus

475

titer by plaque assay was observed in BTV1-VP2TC1, BTV1-VP2TC2 and WT

476

BTV1 infected cells pre-treated with ammonium chloride (Fig. 7D) confirmed

477

that tagged virus entry was similar to that of the WT. Western blotting of

478

infected cells also indicated that ammonium chloride had a strong inhibitory

479

effect on BTV replication (data not shown). The accumulating results suggest

480

that TC tagged viruses behave in a similar manner to WT BTV1.

481

Segregation of VP2 and VP5 in endocytic pathways.

482

As VP2 appeared to segregate from VP5 quite early during virus infection,

483

further investigations were undertaken to identify the cellular compartments

484

that might be involved in BTV virus entry. Since BTV particles enter cells by

485

clathrin-mediated endocytosis (11), both early (EEA1) and late (CD63) 19

486

endosome markers were used to ascertain the distribution of tagged VP2

487

during entry into mammalian cells (Fig. 8). HeLa cells infected at 10 MOI with

488

either BTV1-VP2TC1 or BTV1-VP2TC2 were incubated at 4°C for 1hr to

489

synchronize virus infection followed by incubation at 37°C for 5, 15 or 30 min

490

and processed for FlAsH labeling of the tagged VP2. Confocal analysis of

491

tagged VP2 in cells infected with BTV1-VP2TC1 (Fig. 8A, B left panel) or

492

BTV1-VP2TC2 (Fig. 8A, B right panel) demonstrated that for both the tagged

493

viruses VP2 co-localized with EEA1 (Fig. 8A) but not CD63 (Fig. 8B). Further,

494

quantification of co-localization for VP2 and EEA1 showed that both

495

BTV1-VP2TC1 and BTV1-VP2TC2 were localized with EEA1 by 5 mins pi

496

(89.2% ± 0.8 for BTV1-VP2TC1 and 88.8% ± 2.3 for BTV1-VP2TC2). On

497

further incubation, co-localization of VP2 and EEA1 were maintained at both

498

15 (92.8% ± 3.73 for BTV1-VP2TC1 and 89.2% ± 0.4 for BTV1-VP2TC2) and

499

30 mins (93.9% ± 3.1 for BTV1-VP2TC1 and 94.4% ± 2.8 for BTV1-VP2TC2)

500

pi. Since VP2 and VP5 co-localization studies revealed that VP2 and VP5

501

segregates from each other 15 mins pi, the retention of VP2 in the EEA1

502

labeled early endosomal compartments indicated that VP2 had been shed and

503

that virus particles containing an outer layer of only VP5 might have trafficked

504

to the CD63 labelled late endosomal compartments.

505

Trafficking of VP5 during virus entry.

506

The role of VP5 in BTV entry has been elucidated by two independent studies

507

which disagree on the precise site of fusion. While one study undertaken in

508

BTV10 infected HeLa cells established that virus entered through receptor

509

mediated

510

demonstrated that BTV1 particles entered BHK cells through clathrin

endocytosis

and

early endosomes (11), a

20

second

study

511

independent macropinocytosis and that late endosomes played a crucial role

512

in this process (41). Since our data showed that VP2 is retained on the early

513

endosomes, the role of VP5 in BTV trafficking was investigated further. As the

514

early endosome based trafficking of the two tagged viruses BTV1-VP2TC1 and

515

BTV1-VP2TC2 was similar to WT BTV1, only VP2TC1 was used to analyze

516

the relationship of VP5 with early and late endosomal compartments in BTV

517

entry (Fig. 9). HeLa cells infected at 10 MOI with BTV1-VP2TC1 were

518

incubated at 4°C for 1hr to synchronize virus infection followed by incubation at

519

37°C for 5, 15 or 30 min, fixed and further processed for immunolabeling of

520

both VP5 and EEA1 or CD63 compartments by their respective antibodies.

521

Confocal analysis of VP5 in cells infected with BTV1-VP2TC1 (Fig. 9A)

522

demonstrated that co-localization of VP5 and EEA1 labelled compartments

523

was only observed up to 15 mins pi (Fig. 9A, middle column). In comparison,

524

when the cells fluorescently labelled for VP5 and CD63 were analyzed (Fig.

525

9B), no co-localization was observed at 5 mins pi for the tagged virus (Fig. 9B,

526

left). At 15 mins pi some co-localization between VP5 and CD63 was apparent

527

(Fig. 9B, middle column). In comparison VP5 was entirely co-localized with

528

CD63 by 30 mins pi (Fig. 9B, extreme right). Further, quantitation of

529

co-localization of VP5 and EEA1 established that with an increase in times pi

530

there was a decrease in co-localization between VP5 and EEA1 (Fig. 9C).

531

comparison, when the cells fluorescently labelled for VP5 and CD63 were

532

analyzed (Fig. 9B), almost negligible co-localization (about 2%) was observed

533

at 5 mins pi for the tagged virus (Fig. 9B, left). Subsequently, a sequential

534

increase in co-localization of VP5 and CD63 was of observed at 15 (67%) and

535

30 mins pi (87%) (Fig. 9C). These results therefore demonstrate show that, 21

In

536

following infection of cells by BTV, VP2 is lost in an early endosomal

537

compartment while BTV particles containing an outer layer of VP5 traffic from

538

the early to the late endosomes.

539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 22

562

Discussion

563

Viruses have developed different strategies to hijack intrinsic host cellular

564

pathways for entry and to deliver their genomes to specific cellular locations for

565

replication. With enveloped viruses, fusion of viral and cellular membranes

566

prompts release of capsid or genome into the cytoplasm. For non-enveloped

567

viruses the ability of the outer capsid proteins to disrupt cellular membranes or

568

to form pores in them results in the delivery of the inner capsid or viral genome

569

into the cytosol.

570

Fluorescent labeling of virus particles and cellular structures have made it

571

possible to monitor live virus trafficking in infected cells (15, 16, 42).Whereas

572

enveloped viruses have been successfully labeled with genetically encoded

573

fluorescent proteins (21-24, 43), the structural constraints of non-enveloped

574

virus capsid structures have made the insertion of tags difficult. An alternate

575

strategy involving nonspecific labeling of non-enveloped virus capsid

576

structures with fluorescent dyes has helped to elucidate the entry pathway of

577

viruses with naked capsids. To this end, live cell imaging of cells infected with

578

fluorescent labeled poliovirus have revealed that after internalization through a

579

clathrin-, caveolin-, and flotillin-independent, but actin- and tyrosine

580

kinase-dependent pathway, the virus releases its RNA rapidly from vesicles

581

located very close to the plasma membrane and does not require endocytic

582

acidification or microtubule-dependent transport (14). In this study, however,

583

for the first time we have successfully identified exposed loop regions in the

584

BTV outer capsid protein VP2 and have generated replication competent

585

tagged viruses via reverse genetics (28, 33). Since, to date, the functional role

586

of VP2 in BTV entry has been limited to studies based mainly on recombinant

587

protein expression (9), the tagging of VP2 and its successful incorporation in 23

588

the BTV particles have provided valuable insights into its role during virus entry

589

and trafficking.

590

Among the different viral entry pathways that have been reported for the

591

internalization of virus particles, receptor mediated endocytic pathways

592

regulated by clathrin-coated pits and cellular proteins such as the AP-2

593

complex and the GTPase dynamin, have been held accountable for the

594

majority of viral entrance mechanisms in cells. We have previously reported

595

that entry and infection of HeLa cells by BTV10 occurs via clathrin-mediated

596

endocytosis and that the early but not the late endosomes play an essential

597

role in the early stages of BTV entry (11). However, Gold et al (41) found that

598

clathrin-mediated entry is not the major entry route used by BTV1 to enter

599

BHK-21 cells and that the virus particles are directly delivered to the late

600

endosomes through a pathway that share certain common factors with

601

macropinocytosis. Since it is well established that dynasore, an inhibitor of

602

dynamin2 modulates clathrin mediated entry, we used this drug to study BTV

603

entry. Our data showed that although dynasore did not completely block virus

604

entry, it was able to block the entry of majority of the virus particles. Dynamin 2

605

is also involved in several exocytic traffic steps (44) including exit from the

606

Golgi (45). As BTV uses a non-lytic exocytic pathway for virus release (46),

607

prolonged incubation with dynasore was not carried out and the study was

608

limited to first 30 mins of virus infection.

609

trafficking revealed that VP2 and VP5, the two outer surface proteins, separate

610

from each other by 15 mins pi. The involvement of endosomal vesicles was

611

also confirmed by infecting cells pretreated with ammonium chloride, a

612

chemical that raises the pH of the endosomes. This further re-established the

613

importance of acidic pH in BTV replication (25, 27). When the BTV entry 24

Further investigation of BTV

614

pathway was investigated for the involvement of early and late endosomes,

615

fluorescently labeled VP2 localized to the early and not the late endosomes. In

616

comparison, although fluorescent labeling of antibody mediated detection of

617

VP5 in infected cells demonstrated its distribution to both early and endosomal

618

compartments, the co-localization of VP5 with EEA1 was observed to occur at

619

earlier times pi than that of its co-localization with CD63. Together, these

620

results suggest that, after the early removal of VP2, the virus particles retain an

621

outer layer of VP5 and traffic from the early to the late endosomes for the

622

putative pH induced structural modification of VP5. This facilitates pore

623

formation in the endosomal membranes leading to release of transcriptionally

624

active cores into the cytoplasm. In addition, while it was difficult to infer from

625

the live cell data whether the moving BTV particles entered the cells, analysis

626

of BTV entry with the same tagged viruses in fixed cells clearly showed that

627

BTV particles enter cells within first 5 minutes of infection.

628

current results have demonstrated a slightly different mechanism to the two

629

published studies on BTV entry, an increasing number of viruses have also

630

been revealed to use more than one pathway to enter cells (47-52). Since BTV

631

is able to infect a wide variety of tissue culture cells such as BHK-21, Vero,

632

Hela, and C6/36 cell lines as well as other cell types (11, 36, 53), it is possible

633

that BTV might utilize different entry mechanisms to initiate infection in the

634

different cells that it infects. In addition, serotype of BTV might also play a role

635

in determining the pathway for viral entry.

636

Interestingly, studies with untagged rotaviruses have revealed that virus strain

637

determines the choice of endocytic entry pathway into MA104 gut cells and

638

that VP4, the outer spike protein of rotavirus, is responsible for this

639

phenomenon (52). While bovine rotavirus UK strain enters cells through a 25

Although our

640

clathrin-mediated endocytic process, the rhesus rotavirus strain uses a poorly

641

defined endocytic pathway that is clathrin- and caveolin-independent (47, 50).

642

Recently, an in-depth study on rotavirus entry has further shown that although

643

both bovine and rhesus strains reach maturing endosomes to establish virus

644

infection, unlike the rhesus strain, bovine rotavirus has to traffic to late

645

endosomes (54). This requirement for the late endosomes was also shared by

646

other rotavirus strains of human and porcine origin. In another study using

647

mammalian reoviruses that were bound to fluorescent dyes in vitro, it was

648

shown that virus particles and infectious subvirion particles (ISVPs) were both

649

internalized by clathrin-mediated endocytosis in Madin–Darby canine kidney

650

cells (55). However, virions were trafficked to both early and late endosomes,

651

while ISVPs escaped the endocytic pathway from a location before early

652

endosomes (55).

653

In this study it was possible for us to identify accessible loop-linker regions in

654

VP2 that could accommodate the insertion of a tag in a replication-competent

655

viral genome allowing visualization of virus trafficking. The creation of tagged

656

viruses also provide a valuable tool for studying BTV pathogenesis, including

657

virus entry pathways, uncoating and capsid synthesis. With biarsenical

658

labeling, we also report for the first time that VP2 and VP5 segregate from

659

each other very early during BTV entry. Using the tagged virus this study has

660

paved the way for detailed analysis of interaction with intracellular markers in

661

real time by live cell imaging.

662 663 664 665 26

666

Acknowledgments

667

This work was partly funded by the Wellcome Trust Senior Investigator award

668

(UK), and the US National Institutes of Health (AI094386). We are grateful to

669

Dr. Kit-Yi Leung (William Harvey Research Institute, Barts and the London

670

School of Medicine and Dentistry, London EC1M 6BQ, United Kingdom) for

671

assistance with the Mass Spectrophotometry analysis.

672

27

673

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Celma CC, Roy P. 2011. Interaction of calpactin light chain

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trafficking of nonenveloped bluetongue virus. J Virol 85:4783-4791.

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Characterization of rotavirus cell entry. J. Virol 78:2310-2318.

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Sánchez-San Martín C, López T, Arias CF, López S. 2004.

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Rust MJ, Lakadamyali M, Zhang F, Zhuang X. 2004 Assembly of

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endocytic machinery around individual influenza viruses during viral

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entry. Nat Struct Mol Biol 11:567-573.

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Rojek JM, Sanchez AB, Nguyen NT, de la Torre JC, Kunz S. 2008.

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Different mechanisms of cell entry by human-pathogenic Old World and

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New World arenaviruses. J Virol 82:7677-7687.

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Gutiérrez M, Isa P, Sánchez-San Martín C, Pérez-Vargas J, Espinosa R,

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Arias CF, López S. 2010. Different rotavirus strains enter MA104 cells

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through different endocytic pathways: the role of clathrin-mediated

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endocytosis. J Virol 84:9161-9169.

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pathways for cell entry. J Virol 86:12665-12675.

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Schulz WL, Haj AK, Schiff LA. 2012. Reovirus uses multiple endocytic

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Díaz-Salinas MA, Romero P, Espinosa R, Hoshino Y, López S, Arias

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CF. 2013. The spike protein VP4 defines the endocytic pathway used

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by rotavirus to enter MA104 cells. J Virol 87:1658-1663.

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Tan B, Nason E, Staeuber N, Jiang W, Monastryrskaya K, Roy P. 2001.

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RGD tripeptide of bluetongue virus VP7 protein is responsible for core

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attachment to Culicoides cells. JVirol 75:3937-3947.

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Díaz-Salinas MA, Silva-Ayala D, López S, Arias CF. 2014. Rotaviruses 33

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reach

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mannose-6-phosphate receptor and the activity of cathepsin proteases

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to enter the cell. J Virol 88:4389-4402.

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late

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cation-dependent

Boulant S, Stanifer M, Kural C, Cureton DK, Massol R, Nibert ML,

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Kirchhausen T. 2013. Similar uptake but different trafficking and escape

834

routes of reovirus virions and infectious subvirion particles imaged in

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polarized Madin-Darby canine kidney cells. Mol Biol Cell 24:1196-1207.

836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 34

855

Figure Legends

856

Fig. 1 Identification of putative exposed regions in VP2 by biochemical

857

and sequence analysis. (A) Digestion of purified BTV1 intact virus (left) and

858

recombinant BTV10 VP2 protein with increasing concentration of trypsin (1ng

859

to 100ng). Control consists of undigested BTV1 and BTV10 VP2. The digested

860

products were run on SDS-PAGE and stained with coomasie blue stain.

861

Concentration of trypsin, molecular weight and virus proteins are indicated on

862

the top, left and right, respectively. The arrows indicate digested products and

863

the asterisk indicates products that were sent for Mass Spectrometry analysis.

864

(B) Expression of 10kDa BTV1 (top panel) and BTV10 (lower) VP2 as soluble

865

and insoluble fractions in E.coli and Sf9, respectively. The lysates were

866

fractionated, run on SDS-PAGE and analyzed by Western blotting. Molecular

867

masses and virus proteins are indicated on left and right, respectively.

868

Fig. 2 Peptide mapping. (A) Peptide fragments generated by digestion of 100

869

KDa fragment of BTV-10 VP2 was mapped against the amino acids sequence

870

of full length BTV-10 VP2. (B) Control consisted of mapping peptides generated

871

from digestion of whole length VP2 against BTV-10 VP2. The peptides have

872

been highlighted in grey.

873

Fig. 3 Schematic demonstration of insertion of tags in VP2. (A) Schematic

874

representation depicting deletions in amino acid sequence of BTV1 VP2 (lower)

875

that is present in AHSV4 (upper). The numbers designate amino acid positions

876

in VP2 sequence. Arrow indicates the position of amino acid number 94. (B)

877

Insertion of TC tag in BTV1 VP2 sequence. Position of the inserted tags has

878

been indicated.

879

Fig.4 Recovery of tagged BTV particles. (A) Plaque morphology of the

880

WT-BTV1 and TC tagged virus. (B) Genomic dsRNA from BSR cells infected 35

881

with BTV1 WT virus (lane 1) or tagged BTV1-VP2TC1 (lane 2) or

882

BTV1-VP2TC2 (lane 3) or BTV1-VP2TC3 (lane 4) was purified and analyzed

883

on a non-denaturing polyacrylamide gel. (C) Sequence electropherograms of

884

segment 2 RT-PCR products from TC-tagged virus. The position of TC tags are

885

indicated on top of each panel.

886

Fig. 5 Characterization of recovered mutant viruses containing

887

TC-tagged VP2. (A) The total titer at different times pi for either mutant or WT

888

viruses in BSR was determined, expressed as PFU/ml, and plotted on a

889

logarithmic scale. Asterisk indicates that the decrease in titers of

890

BTV1-VP2TC3 at 24 and 48 hrs pi is statistically significant to WT BTV-1

891

(p

Trafficking of bluetongue virus visualized by recovery of tetracysteine-tagged virion particles.

Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a double-capsid insect-borne virus enclosing a genome of 10 doubl...
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