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
2
tetracysteine-tagged virion particles
3 4 5 6 7
Junzheng Du§*, Bishnupriya Bhattacharya*, Theresa H. Ward and Polly Roy#
8 9
Department of Pathogen and Molecular Biology, Faculty of Infectious and
10
Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1 7HT, United Kingdom
11 12 13 14 15
#
Corresponding author:
[email protected] 16 17 18
*
Du and Bhattacharya* equally contributed
19 20 21
§
22
Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary
23
Research Institute, Chinese Academy of Agricultural Science, Lanzhou
24
730046, China
Current Address: State Key Laboratory of Veterinary Etiological Biology, Key
25 26 27
Running title: Bluetongue virus with TC-tagged VP2
28 29
Abstract: 235 words
30
Text: 5826 words
31 32 33 34 1
35
Abstract
36
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
42
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
44
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
46
genome of a non-enveloped capsid virus by the introduction of tags that were
47
subsequently fluorescently visualized in infected cells. The genetically
48
engineered fluorescent BTV particles were observed to enter live cells
49
immediately after virus adsorption. Further, we showed separation of VP2 from
50
VP5 during virus entry and confirmed that while VP2 is shed from virions in
51
early endosomes, virus particles still consisting of VP5 were trafficked
52
sequentially from early to late endosomes. Since BTV infects both mammalian
53
and insect cells, the generation of tagged viruses will allow visualization of
54
further downstream trafficking of BTV in different host cells. In addition, the
55
tagging technology also has potential for transferable application on other
56
non-enveloped complex viruses.
57 58 59 60 2
61
Importance
62
Live virus trafficking in host cells has been highly informative in understanding
63
interactions between virus and host cells. Although insertion of fluorescent
64
markers in viral genome have made it possible to study trafficking of enveloped
65
viruses, the physical constraints of architecturally complex capsid viruses have
66
led to practical limitations. In this study, we have successfully genetically
67
engineered the segmented RNA genome of Bluetongue virus (BTV), a
68
complex non-enveloped virus belonging to the Reoviridae family. The resulting
69
fluorescent virus particles could be visualized in virus entry studies for both live
70
and fixed cells. This is the first time a structurally complex capsid virus has
71
been successfully genetically manipulated to generate virus particles that
72
could be visualized in infected cells.
73 74 75 76 77 78 79 80 81 82 83
3
84
Introduction
85
Bluetongue virus (BTV), the prototype Orbivirus within the Reoviridae family is
86
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
89
double-stranded RNA (dsRNA) segments (S1 to S10) encodes 7 structural
90
(VP1 to VP7) and 4 non-structural proteins (NS1 to NS4) (1, 2). In the virus
91
particles, the structural proteins are organized in two capsids; an outer capsid
92
of VP2 and VP5, an inner capsid or “core,” of VP7 and VP3 that encloses the
93
viral transcription complex (VP1, VP4 and VP6) in addition to the viral genome
94
(1, 3). Three-dimensional structural studies of virions by cryo-electron
95
microscopy revealed that VP2 arranges as trimers on the virion surface
96
protruding as spike-like structures from the surface of the virus particles (4, 5).
97
VP2 alone is responsible for the viral haemagglutination activity, serotype
98
specificity and for the attachment of virions to the host cell (5-9). The second
99
outer capsid protein VP5, is also arranged in trimers, but is less exposed than
100
VP2 and is globular in shape (5). Structurally, VP5 resembles the fusion
101
proteins of enveloped viruses and consists of amphipathic Į-helical regions on
102
its external surface that have been suggested to play an active role in the
103
penetration of endosomal membranes to release BTV cores into the cytoplasm
104
(5, 10, 11).
105
Although studies have elucidated the role of cellular factors in BTV entry, it is
106
still not clear how the virus particles are trafficked into cells. Compared to other
107
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
109
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
111
live cells not only provides a valuable means to study interactions between
112
viral and cellular proteins during virus entry, but also during trafficking,
113
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
115
capacity of each segment to accommodate foreign genes is limited (17).
116
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
118
can be inserted into a protein without the risk of disrupting the overall structure
119
of the targeted protein (18-20). The tagged proteins are specifically recognized
120
by membrane-permeable biarsenical dyes that fluoresce when bound to the
121
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)
123
(18, 19), makes it a powerful tool for the real-time visualization of nascent
124
protein synthesis and trafficking in cells. To date, this technology has been
125
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,
128
25-27), the detailed mechanism of both proteins’ role in virus trafficking has not
129
been studied in depth. In addition, while a 7Å structure of VP2 has been
130
reported (5), the lack of a VP2 structure at atomic level has made it difficult to
131
design strategies for the insertion of foreign tags that will not disrupt the
132
secondary structure of the protein. Thus, this study was designed to
133
investigate whether biarsenical TC tagging technology could be utilized for
134
BTV as a means for investigating the trafficking of virus particles during virus
135
entry. In this study we have successfully utilized biochemical methods, 5
136
sequence comparison data and BTV reverse genetics system (28) to insert
137
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
141
the two outer capsid proteins are separated from each other during the early
142
stages of virus entry. Our study not only provides the possibility for further
143
investigation of VP2 protein trafficking in live cells infected with the mutant
144
virus, but also opens up possibilities for tagging other BTV proteins and other
145
orbiviruses.
146 147 148 149
6
150
Materials and Methods
151
Cell lines, viruses and bacteria.
152
BSR and HeLa cells were maintained as described (29). Wild-type BTV1
153
(South African Strain) and TC-tagged BTV1 stocks were propagated and
154
titered in BSR cells (28). Recombinant baculoviruses expressing S-tag
155
BTV10-VP2 or His-tag BTV10-VP294 were propagated in Spodoptera
156
frugiperda (Sf9) cells as described (29).
157
Antibodies and reagents.
158
All antibodies against BTV proteins used were generated in our laboratory.
159
Purified, recomibinant VP2, VP5 and NS2 expressed from baculoviruses were
160
used to generate monospecific polyclonal antibody in rabbits as described
161
previously (30). Antibodies against EEA1 and CD63 were obtained from
162
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
167
the media during the 30 mins of virus incubation.
168
Plasmids and site-directed mutagenesis.
169
Site-directed mutagenesis were performed to insert TC tags in BTV1 S2
170
sequence (32). Briefly, two complementary primers were used to insert the
171
nucleotide sequence (TGTTGTCCCGGGTGTTGT) encoding the TC tags in
172
pUCBTV1T7S2 (28) template. The following primers used for site-directed
173
mutagenesis:
174
VP2TC94_F,5ƍCGGTTGTTGAAAGTACGAGATGTTGTCCCGGGTGTTGTCA
175
CAAGAGTTTCCATACGAA3ƍ;VP2TC94_R,5’TTCGTATGGAAACTCTTGTGA 7
176
CAACACCC GGGACAACATCTCGTACTTTCAACAACCG3’;VP2TC352_F,5’
177
CGATACTTTTAATTGTTGTCCCGGGTGTTGTACACGAGTGTGGTGGTCGA
178
AC3ƍ; VP2TC352_R, 5’ACAACACCCGGGACAACAATTAAAAGTATCGGAGG
179
CTG3ƍ; VP2TC420_F, 5ƍTTGACTTT GTCGCGGAACCTTGTTGTCCCGGGTG
180
TTGTGGGATTAAAATTGTTCATTG3ƍ;and VP2TC420_R, 5ƍCAATGAACAATT
181
TTAATCCCACAACACCCGGGACAACAAGCGACAAAGTCAA3ƍ.
182
Recovery of tagged viruses.
183
The T7 BTV capped (BTV1S1, BTV1S3-S10) and uncapped transcripts
184
(BTV1S2, BTV1S2 with TC tag) were generated as described (28). The mutant
185
BTV particles were recovered following the method described previously (33).
186
Genomic dsRNA from cells infected with control or mutant BTV was analyzed
187
as described (28).
188
Virus growth kinetics.
189
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
193
and NS2, while cellular protein tubulin was used as the loading control. Each
194
blotting experiment was repeated three times and the amount of protein
195
expression was quantitated by ImageJ. The mean and standard error of the
196
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).
199
Fluorescence and confocal microscopic analysis of TC-tagged proteins
200
and viruses.
201
Live and fixed cell analysis of tagged-virus trafficking was undertaken by 8
202
synchronously infecting HeLa cells at a MOI of 50 and 10, respectively. BSR
203
cells infected with BTV1-VP2TC1 or BTV1-VP2TC2 were processed for
204
biarsenical labeling at different times pi with FlAsH according to the
205
manufacturer’s recommendations. Both live and fixed HeLa cells were imaged
206
by confocal microscopy on a Zeiss LSM 510. Cells infected for live cell imaging
207
were, washed with Opti-MEM I Reduced-Serum Medium (Invitrogen) and
208
stained with FlAsH solution (2μM) for 30 minutes (min) at 4°C. After washing
209
the cells with BAL (2, 3-dimercapto-1-propanol), live images were captured
210
every 16.7 seconds by confocal microscopy with 488 nm laser and appropriate
211
fluorescein filters on a pre-warmed stage that was maintained at 37°C.
212
Subsequently, the images compiled into a movie at 8 images per second in
213
Image J and cartoon was created using Photoshop Element 2.0 software.
214
Fixed cell analysis was undertaken as described (29). The images were
215
obtained using LSM 510 image browser software and processed using
216
Photoshop Element 2.0 software (Microsoft). Each set of fixed cell experiments
217
were repeated at least three times to generate either localization or
218
co-localization data that could be quantified. Co-localization was judged by the
219
appearance of yellow spots formed by the merging of red and green signals
220
generated by the florescent tags attached to the secondary antibodies. The
221
mean and standard error of percentage of localization or co-localization were
222
calculated (Sigma Plot 2000; Systat Software Inc.) and the p values were also
223
determined by Excel (Microsoft).
224
Recombinant expression of amino terminal fragment of VP2.
225
For the production of a recombinant baculovirus expressing BTV10-VP294, 94
226
amino acids from amino terminal end of BTV10 VP2 were inserted in the 9
227
baculovirus expression vector pAcYM1 and a His-tag was introduced
228
upstream of the start codon of VP2 (30). In addition for bacterial expression,
229
the N terminal first 94 residues of BTV1 were inserted into pRSETA
230
(Invitrogen). Each construct was verified by sequencing. Recombinant
231
baculoviruses expressing His BTV10-VP294 was produced, plaque purified and
232
propagated using standard baculovirus recombination procedures (34).
233
His-tagged BTV1-VP294 was expressed in Escherichia coli BL21(E.coli) as
234
described (35). The soluble and insoluble fractions from bacterial and insect
235
cells were prepared and analyzed by Western Blot (36).
236
Trypsin
237
Spectrophotometry analysis.
238
Purified virus particles and VP2 were incubated with increasing concentrations
239
of trypsin (1ng, 10ng, 100ng) for 30mins at 37°C and resolved by SDS-12%
240
PAGE. Bands representing 100 and 110 kDa sizes that were excised from
241
trypsin digested purified VP2 were subjected to in-gel digestion with trypsin,
242
and analyzed by a liquid chromatography-tandem mass spectrometer
243
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
254
Identification of putative exposed regions in VP2.
255
VP2, the host attachment protein, is responsible for virus entry. Hence
256
strategies were adopted to minimize the potential impediment to virus
257
infectivity that may be caused by fusion of the TC tag to VP2. Since, in the
258
absence of atomic structure, the localization of flexible loop-linker regions
259
that could be utilized for the insertion of TC tag were still not clear, two
260
strategies were adopted to identify such exposed loop regions in VP2. Firstly,
261
analysis of VP2 amino acid sequences (ExPASy) of two different BTV
262
serotypes (BTV1 and BTV10) revealed the presence of putative trypsin
263
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
265
surface of the protein and not those present internally, it was hypothesized
266
that these putative cleavage sites could be the exposed loop-linker regions in
267
VP2 and thus could be potentially utilized for the insertion of TC tags.
268
Consequently, purified virus particles (BTV1) and VP2 protein (of BTV10) were
269
digested with increasing concentrations of trypsin for 30mins at 37°C to identify
270
the presence of potential enzyme cleavage sites in VP2 (Fig. 1). Although
271
digestion of purified virus particles (Fig. 1A, left) and VP2 protein (Fig. 1A, right)
272
with 100ng of trypsin showed the presence of protein bands with smaller sizes
273
on SDS-PAGE gel, the pattern of the digested products was different between
274
virus particles and VP2 protein (compare Fig. 1A left with right). Briefly,
275
digestion of purified virus particles yielded a very faint fragment of 110kDa size
276
and two smaller sized fragments measuring 40kDa and 10kDa (Fig. 1A, left). In
277
comparison, digestion of purified VP2 protein under the same conditions 11
278
resulted in two fragments of 100kDa and 10kDa size products (Fig. 1A, right).
279
The difference in the digestion pattern between purified virus particles versus
280
VP2 alone can be attributed to VP2 in virion adopting conformations that are
281
more susceptible to proteolysis. Furthermore, the smaller digested products of
282
VP2 in both purified virus particles (Fig. 1A, left) and VP2 protein (Fig. 1A, right)
283
were only detectable in higher concentrations of trypsin (100ng) and not in the
284
lower concentrations of trypsin (10 or 1ng). Control virus particles incubated at
285
37°C for 30 mins in the absence of trypsin did not show any breakdown
286
products. Since the presence of the 10kDa protein band was noted in both the
287
digested virus particle and purified VP2 protein, the larger fragment 100kDa
288
product from digested VP2 (Fig. 1A, right) was excised from the SDS-PAGE
289
gel (indicated with an asterisk). Subsequently, after purification from
290
SDS-PAGE gel, smaller peptide fragments were generated by trypsin
291
digestion of the 100kDa purified product and the peptides were further
292
analyzed by mass spectrometry. The undigested 110kDa purified protein,
293
treated in the same way, served as control. Evaluation of the peptide
294
fragments generated from both the 100kDa digested product and 110kDa
295
undigested VP2 by SwissProt based Mascot search confirmed that these
296
peptides were BTV10 VP2 derived (Table. 1). Peptides generated from the
297
100kDa fragment mapped perfectly to the BTV10 VP2 sequence after amino
298
acid position number 94 suggesting that the 100kDa protein fragment lacked
299
the first 94 amino acids (Fig. 2A), consistent with the 10kDa fragment released
300
as a result of trypsin digestion of purified VP2. In comparison, peptides
301
generated from the undigested full-length VP2 (110kDa) mapped the entire
302
length of the protein (Fig. 2B).
303
In order to analyze whether the 10 KDa VP2 fragments was an autonomous 12
304
domain that could be expressed as a stable soluble product, the amino
305
terminal fragments of two different BTV serotypes, BTV1 (Fig. 1B, upper) and
306
BTV10 (Fig. 1B, lower) were expressed as His-tagged fusion proteins
307
(BTV1-VP294 and BTV10-VP294) either in bacterial (E. coli, Fig. 1B, upper) or
308
eukaryotic (Baculovirus, Fig. 1B, lower) expression systems. The E. coli and
309
Sf9
310
respectively were lysed, fractionated into soluble and insoluble fractions and
311
run on a SDS-PAGE gel. Western Blot analysis of the His-tagged products
312
revealed the presence of the BTV1-VP294 and BTV10-VP294 fusion proteins in
313
both soluble and insoluble fractions, in both expression systems (Fig. 1B,
314
upper & lower). Thus, this data suggests the presence of an exposed loop after
315
the first 94 residues of VP2 which separates autonomous folded domains as
316
soluble fraction. Hence, insertion of a TC in this region of VP2 might allow the
317
construction of a VP2 with preserved overall folding and biological activity.
318
In a second, alternate, approach to identify the presence of exposed loop
319
regions in VP2 the similarity of VP2 of BTV with that of African horse sickness
320
virus, AHSV, a closely related orbivirus (38) was assessed. A database search
321
of the available AHSV (4 of the 9 serotypes) and BTV (24 of the 26 serotypes)
322
VP2 peptide sequences (data not shown) revealed that VP2 from AHSV
323
serotypes are generally longer (ranging from 1051 to 1060 residues) than BTV
324
serotypes (ranging from 950 to 962 residues). Further, a careful sequence
325
alignment of BTV and AHSV VP2 proteins revealed the presence of two
326
in-frame deletions between residues 352-371 and 420-452 in all BTV
327
serotypes that were absent in AHSV (Fig. 3A). This led us to hypothesize that
328
the two deletions might also be exposed loop/linker regions of the protein
329
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).
332
Generation and characterization of recovered mutant viruses containing
333
TC-tagged VP2.
334
The TC tags were inserted in the coding region of BTV1 VP2 between amino
335
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
337
constructs (BTV1-S294, BTV1-S2352 and BTV1-S2420) to recover TC tagged
338
mutant viruses. The reverse genetics system used transfection of BSR cells
339
with 9T7-derived RNA transcripts (S1 and S3~9) together with either wild-type
340
BTV1-S2 or each of the tagged S2 transcripts. As with wild-type, all mutant
341
viruses were recovered successfully.
342
Plaque assays were undertaken to investigate the plaque morphology of the
343
newly generated BTV1-VP2TC1 (BTV1-S294), BTV1-VP2TC2 (BTV1-S2352)
344
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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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