1 three dimensional architecture of tick-borne encephalitis virus
TRANSCRIPT
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Three Dimensional Architecture of Tick-borne Encephalitis Virus Replication Sites and 1
Trafficking of the Replicated RNA 2
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Lisa Miorin1,5,*, Inés Romero-Brey2,5, Paolo Maiuri1,3, Simone Hoppe2,4, Jacomine Krijnse-4
Locker2,4, Ralf Bartenschlager2, Alessandro Marcello1,* 5 6 1Laboratory of Molecular Virology, The International Center for Genetic Engineering and 7
Biotechnology (ICGEB), Padriciano, Trieste, 34149 – Italy 8
9 2Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer 10
Feld 345, D-69120, Heidelberg, Germany 11
12 3Current address: Systems Cell Biology of Cell polarity and Cell division, Institut Curie, CNRS, 13
UMR144, 26 rue d'Ulm, 75005 Paris, France 14
15 4Electron Microscopy Core Facility, University of Heidelberg, Im Neuenheimer Feld 267, D-69120, 16
Heidelberg, Germany 17
18 5These authors contributed equally to this work 19
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*Corresponding authors: [email protected]; [email protected] 21
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Running title: Spatio-temporal organization of TBEV replication 24
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Word count: 26
Abstract: 235 27
Main text: 6389 28
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Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.03456-12 JVI Accepts, published online ahead of print on 3 April 2013
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Abstract 30
Flavivirus replication is accompanied by the rearrangement of cellular membranes that may 31
facilitate viral genome replication and protect viral components from host cell responses. The 32
topological organization of viral replication sites and the fate of replicated viral RNA are not fully 33
understood. We exploited electron microscopy to map the organization of tick-borne encephalitis 34
virus (TBEV) replication compartments in infected cells and in cells transfected with a replicon. 35
Under both conditions 80 nm vesicles were seen within the lumen of the endoplasmic reticulum 36
(ER) that in infected cells also contained virions. By electron tomography the vesicles appeared as 37
invaginations of the ER membrane displaying a pore that could enable release of newly synthesized 38
viral RNA into the cytoplasm. To track the fate of TBEV RNA we took advantage of our recently 39
developed method of viral RNA fluorescent tagging for live cell imaging combined with bleaching 40
techniques. TBEV RNA was found outside virus-induced vesicles either associated to ER 41
membranes or free to move within a defined area of juxtaposed ER cisternae. From our results, we 42
propose a biologically relevant model of the possible topological organization of flavivirus 43
replication compartments composed of replication vesicles and a confined extra-vesicular space 44
where replicated viral RNA is retained. Hence, TBEV modifies the ER membrane architecture to 45
provide a protected environment for viral replication and for the maintenance of newly replicated 46
RNA available for subsequent steps of the virus life cycle. 47
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Introduction 49
Tick-borne encephalitis virus (TBEV) is the etiological agent of tick-borne encephalitis, a 50
potentially fatal infection of the central nervous system occurring throughout wide areas in Europe 51
and Asia (1-3). TBEV is the most medically important member of the mammalian tick-borne group 52
of the genus Flavivirus within the family Flaviviridae (4). Flaviviruses are a large group of 53
arboviruses that are responsible for severe diseases in humans and animals. This virus group 54
includes, in addition to TBEV, the dengue virus (DENV), yellow fever virus (YFV), West Nile 55
virus (WNV) and Japanese encephalitis virus (JEV). They have in common an enveloped virus 56
particle that contains a single-stranded, positive sense RNA genome, a similar genomic 57
organization and comparable replication strategies (5, 6). After entry, the incoming viral RNA is 58
translated giving rise to a polyprotein precursor that is processed by cellular proteases and the viral 59
protease NS2B/3 to obtain three structural and seven non-structural proteins (NS). The RNA-60
dependent RNA polymerase (RdRp) residing in NS5 synthesizes complementary negative strand 61
RNA from genomic RNA with negative strands serving as template for the synthesis of new 62
positive strand viral RNAs. 63
Like all positive strand RNA viruses, flaviviruses replicate in the cytoplasm in close association 64
with virus-induced intracellular membrane structures. It is generally accepted that the formation of 65
these replication compartments (RC) provide an optimal microenvironment for viral RNA 66
replication by limiting diffusion of viral/host proteins and viral RNA, thereby increasing the 67
concentration of components required for RNA synthesis, and by providing a scaffold for anchoring 68
the replication complex (7). In addition, these virus-induced membranes may also shield double 69
strand (ds)RNA replication intermediates from host cell-intrinsic surveillance (8-10). Elegant 70
electron tomography (ET) studies on DENV- and WNV-infected cells have recently provided the 71
first three-dimensional view of the architecture of flavivirus RCs (11, 12). In these studies different 72
virus-induced membrane structures appeared to be part of a highly organized network of ER-73
derived rearranged membranes. Vesicle packets, containing dsRNA and proteins of the replication 74
complex, have been described as the sites of virus replication and appear in ET as invaginations of 75
the ER membrane bearing pore-like connections to the cytoplasm and possibly between themselves. 76
Convoluted membranes (CM) that are specifically enriched in NS2B/3 have been proposed as the 77
putative sites of protein synthesis and proteolytic cleavage (11, 13-15). 78
However, although ET images from fixed cells can provide a high-resolution snapshot of the 79
complex network of vesicles and interconnections, they cannot address the dynamic exchange of 80
proteins and viral RNA throughout these compartments. In order to provide a global picture of the 81
spatio-temporal organization of the RCs it is therefore important to integrate high-resolution 82
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imaging approaches with innovative techniques that allow exploring in real time the dynamic 83
interplay between viruses and their host. Engineered subgenomic replicons expressing fluorescently 84
tagged nonstructural proteins (16-18) have been exploited to investigate the distribution and 85
dynamics of HCV replicase proteins. However, the only available tool to explore flaviviral RNA 86
trafficking has been reported for TBEV (19). The method is based on TBEV replicons containing 87
multiple high-affinity binding sites for the phage MS2 core protein fused to an autofluorescent 88
protein. Replicated viral RNA could then be visualized in living cells thus providing the first 89
description of flavivirus RNA dynamics in living cells (19). 90
In this work we took advantage of immuno-EM, thin-section TEM and high-resolution ET in 91
combination with live imaging approaches for TBEV RNA to identify replication compartments in 92
infected and replicon-transfected cells. We precisely mapped the spatial organization of virus-93
induced membranes and vesicles and studied the mobility of replicated viral RNA. We propose a 94
model where replication vesicles and the extra-vesicular space form an optimized environment to 95
support efficient virus replication. 96
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Materials and Methods 99
Cells, viruses and plasmids. Baby hamster kidney (BHK-21) and African green-monkey (Vero 100
E6) cell lines were grown under standard conditions in Dulbecco’s modified Eagle medium 101
supplemented with 10% foetal bovine serum. BHK21-EYFP-MS2nls cells are a cell pool stably 102
expressing EYFP-MS2nls and blasticidin S-deaminase, and they were cultured in the presence of 10 103
μg/ml blasticidin. Lentiviral particles for the transduction of the EYFP-MS2nls gene were prepared 104
in 293T-cells exactly as described previously (20) using pWPI-BLR-EYFP-MS2nls and packaging 105
constructs pCMVR8.91 and pMD.G (provided by Didier Trono). Working stocks of TBEV strain 106
Neudoerfl were propagated and titrated on Vero E6 cells. pWPI-BLR-EYFP-MS2nls was used to 107
generate a cell line constitutively expressing the EYFP-MS2nls protein suitable for immuno-EM 108
studies. The EYFP-MS2nls gene was isolated from pEYFP-MS2nls (21) by XbaI digestion and then 109
inserted into the multiple cloning site (MCS) of the pWPI-BLR vector (provided by Volker 110
Lohmann) via the SpeI restriction site. The construct pTNd/ΔME_24×MS2, the replication-111
deficient TNd/ΔME_24×MS2_GAA replicon carrying the GDD to GAA mutation in the viral NS5 112
protein, the pMS2-EYFP, pEYFP-MS2nls and pCherry-MS2nls vectors used for visualization 113
purposes were previously described (19, 21). 114
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RNA transcription and transfection. Subgenomic replicon RNAs were transcribed in vitro as 116
described in detail elsewhere (19, 22). 4×106 cells were resuspended in 400μl ice-cold PBS and 117
mixed in a 0.4 cm gene-pulser cuvette with 10 μg of RNA to be electroporated with a Bio-Rad 118
Gene Pulser apparatus at 0,25 kV with a capacitance of 960 μF (19). After electroporation, cells 119
were washed in complete growth medium without antibiotics and seeded in the same medium. 120
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Samples staining and imaging for indirect immunofluorescence analysis. IF analysis was 122
performed 24h upon infection or transfection. Cells were washed with PBS, fixed with 4% PFA for 123
15 min, incubated 5 min with 100mM glycine and permeabilized with 0.1% Triton X-100 for 5 min. 124
Subsequently the cells were incubated at 37°C for 30 min with PBS, 1% bovine serum albumine 125
(BSA) and 0,1% Tween 20 before incubation with antibodies. The coverslips were rinsed three 126
times with PBS 0,1% tween 20 (washing solution) and incubated for 1h with secondary antibodies. 127
Donkey antibodies specific for rabbit or mouse immunoglobulin G and conjugated to Alexa Fluor 128
594 or Alexa Fluor 488 (Molecular Probes) were used for this analysis. Coverslips were finally 129
washed three times with washing solution and mounted on slides using Vectashield mounting 130
medium (Vector Laboratories). For the detection of viral antigens we used the following antibodies: 131
mouse monoclonal antibody detecting NS1 (provided by Connie Schmaljohn); polyclonal rabbit 132
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anti-TBEV serum that can be used for both structural and non-structural protein detection (23) 133
(provided by Franz X. Heinz); polyclonal rabbit anti-prM serum (provided by Franz X. Heinz). The 134
J2 mouse monoclonal anti-dsRNA antibody (English and Scientific Consulting, Szirak, Hungary) 135
was used to detect replication complexes, whereas for the ER staining we used the mouse 136
monoclonal anti-PDI (AB2792, Abcam) antibody. Fluorescent images of fixed cells were captured 137
on Zeiss LSM510 META confocal microscope with a 63× NA 1.4 Plan-Apochromat oil objective. 138
The pinhole of the microscope was adjusted to get an optical slice of less than 1.0µm for any 139
wavelength acquired. 140
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Epoxy embedding of cells for transmission electron microscopy. BHK-21 cells grown on 10 cm 142
diameter dishes were infected with TBEV at MOI = 2 or transfected with in vitro transcribed 143
TNd/ΔME_24xMS2 replicon RNA. After 24 h cells were washed 3 times with pre-warmed PBS 144
and fixed for 30 min with 2.5% glutaraldehyde (GA) in 50 mM Na-cacodylate buffer [pH 7.4] 145
containing 1 M KCl, 0.1 M MgCl2, 0.1 M CaCl2 and 2% sucrose. Cells were washed 5 times for 5 146
min each with 50 mM Na-cacodylate buffer and post-fixed on ice in the dark with 2% OsO4 in 50 147
mM Na-cacodylate buffer for 40 min. After washing the cells overnight in distilled water they were 148
treated with 0.5% uranylacetate (UA, dissolved in water) for 30 min, rinsed thoroughly with water 149
and dehydrated in a graded ethanol series at room temperature (40%, 50%, 60%, 70% and 80%, 5 150
min each; then 95% and 100%, 20 min each). Cells were immersed in 100% propylene oxide and 151
immediately embedded in an Araldite-epon mixture (Araldite 502/Embed 812 kit, Electron 152
Microscopy Sciences). After polymerization at 60°C for 2 days embedded cells were sectioned 153
using a Leica Ultracut UCT ultramicrotome and a 35° diamond knife (Diatome, Biel, Switzerland). 154
Sections with a thickness of 60 nm were collected onto 100 mesh copper grids that had been coated 155
with Formvar (Plano, Wetzlar, Germany) and coated with carbon. Sections were counter-stained 156
with 2% lead citrate in H2O for 2 min. Samples were analyzed by using a Biotwin CM120 Philips 157
electron microscope (100 kV) equipped with a bottom-mounted 1K CCD camera (Keen View, SIS, 158
Münster, Germany). 159
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Immunolabeling of thawed cryo-sections. TBEV infected and replicon transfected cells were 161
fixed by adding an equal amount of 8% PFA and 0.2% GA in 0.2 M PHEM buffer (120 mM Pipes, 162
100 mM Hepes, 4 mM MgCl2, 40 mM EGTA, pH 6.9) to the culture medium for 1 h at room 163
temperature. Cells were then fixed for 1 h with 4% PFA and 0.1% GA in 0.1 M PHEM at room 164
temperature. The fixative was removed and the cells were stored at 4 °C in 4% PFA in 0.1 M 165
PHEM until further processing. After extensive washing with 0.1 M PHEM remaining aldehyde 166
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groups were blocked with 30 mM glycine in 0.1 M PHEM. Cells were scraped off the plate, 167
embedded in 10% gelatine and infiltrated in 2.3M sucrose overnight at 4 °C. Cell pellets were 168
mounted onto sample holder pins, frozen and stored in liquid nitrogen. 60nm cryo-sections were 169
prepared using a Leica Ultracut UC6 microtome (Leica Microsystems, Wetzlar, Germany) and a 170
diamond knife (Diatome, Biel, Switzerland). Sections were picked up with a mixture of 2% 171
methylcellulose and 2.3 M sucrose (1:1) and after thawing transferred to 100 mesh formvar and 172
carbon-coated grids. Labeling of thawed cryosections was performed essentially as described 173
elsewhere (24). In brief, sections were molten by floating on 2% gelatine for 30 min at 37 °C, 174
incubated in 30 mM glycine in PBS for 10 min and then 30 min at room temperature in blocking 175
solution (PBG: (0.8% [w/v] BSA [Sigma], 0.1% [w/v] fish skin gelatin [Sigma] in PBS). Sections 176
were then incubated with primary antibody diluted in blocking buffer, for 30 min at room 177
temperature. After 5 times washing for each 5 min in blocking buffer, sections were incubated with 178
rabbit anti-mouse followed by protein A coupled to 10 nm gold (Cell Microscopy Center, Utrecht, 179
The Netherlands) diluted in blocking solution. After washing with PBS and distilled water, grids 180
were contrasted with a mix of 2% methylcellulose and 3% UA (1:6) for 10 min on ice. 181
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Electron tomography. Sections of 250 nm thickness were collected on palladium-copper slot grids 183
(Science Services, Munich, Germany) coated with Formvar (Plano, Wetzlar, Germany). Protein A-184
gold (10 nm) was added to both sides of the sections as fiducial markers. Single and dual axis tilt 185
series were acquired with a FEI TECNAI TF30 microscope operated at 300 kV and equipped with a 186
4k FEI Eagle camera (binning factor 2, on the specimen level) over a -65° to 65° tilt range 187
(increment 1°) and at an average defocus of -0.2 µm. Tomograms were reconstructed using the 188
weighted back-projection method implemented in the IMOD software package (version 3.11.5) 189
(25). Rendering of the 3D surface of the tomograms was performed by using the AMIRA 190
Visualization software Package (version 5.4.2, Visage Imaging, Berlin, Germany). Models were 191
generated from unfiltered and 2x binned tomograms by manually masking areas of interest, 192
thresholding and smoothing labels. 193
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Microscopy and live imaging acquisition. BHK-21 cells were electroporated with the TBEV 195
replicons’ RNA and plated on glass-bottom plates (MatTek, Ashland, MA, USA). For the 196
visualization of the viral RNA cells were co-transfected either with MS2-EYFPnls, expressing a 197
hybrid protein composed by the core protein of the MS2 bacteriophage fused to EYFP and to a 198
nuclear localization signal (nls), or with the same construct without the nls. At the appropriate time 199
point post-transfection, cells were transferred on a humidified and CO2-controlled on-stage 200
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incubator (PeCon GmbH, Erbach, Germany) at 37°C in complete DMEM medium without phenol 201
red for live cell imaging. For FRAP experiments images of 512×512 pixels (29,25×29,25 µm) and 202
optical thickness of 1 µm were acquired using 1% or less of the power of the 514 nm laser line. 203
EYFP was bleached at 514 nm (Argon laser, maximum output 500 MilliWatt) in a circle of 30 204
pixels of diameter, at full laser power, for 10 passages. For FLIP measurements images were 205
acquired as described above and bleaching was performed at every acquisition. Images were 206
analyzed with ImageJ (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, Maryland, 207
USA, http://rsb.info.nih.gov/ij/). 208
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Results 212
Subcellular localization of TBEV proteins and dsRNA in infected cells 213
As a first step to study the organization of TBEV replication sites we characterized by 214
immunofluorescence the sub-cellular localization of proteins and dsRNA in virus-infected BHK-21 215
cells. At 24 hours post-infection (hpi), viral proteins detected with a polyclonal TBEV-specific 216
antiserum (predominantly recognizing the structural protein E and NS1) were localized in the 217
perinuclear region and in discrete and irregularly shaped foci (Fig. 1A). As expected, these 218
structures partially colocalized with NS1-containing cytoplasmic foci (Fig. 1A) as well as with the 219
dsRNA replication intermediate (Fig. 1B), a well-accepted marker for flaviviral replication vesicles 220
(11, 12, 15, 26). A similar distribution pattern was observed with the prM-specific antibody that 221
also showed partial co-localization with NS1 cytoplasmic foci (Fig. 1C). In addition, co-222
immunolocalization studies of viral proteins and the rER marker protein disulphide isomerase (PDI) 223
suggested that ER-derived membranes provide the framework for the membranous TBEV 224
replication compartments (Fig. 1D). These data are in agreement with our previous observations for 225
the MS2-tagged TBEV replicon where newly synthesized viral RNA associates with viral proteins 226
and dsRNA replication intermediates in rER-derived cytoplasmic compartments (8, 19). 227
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Electron tomography analysis of TBEV-induced membrane alterations 229
To reveal the three-dimensional organization of virus-induced membrane alterations we performed 230
a detailed ET analysis of TBEV-infected cells. At 24 hpi, BHK-21 cells were fixed, resin-embedded 231
and sectioned as described in Materials and Methods. Tomograms from 250 nm thick sections were 232
acquired and reconstructed. As shown in Fig. 2, TBEV infection induced membrane alterations 233
reminiscent of those previously described for mosquito-borne flaviviruses like DENV and WNV 234
(11, 12, 27) and more recently also for Langat virus (LGTV), a naturally attenuated tick-borne 235
flavivirus (28). Virus-induced single-membrane vesicles (Ve) with a diameter of 80 nm (± 10.6 nm; 236
n = 70) were observed in the lumen of a dilated rER, and appeared to be part of an elaborate 237
reticulovesicular network of interconnected membranes (Fig. 2A and B). ER-derived cisternae filled 238
with viral particles in the proximity of virus-induced vesicles, as well as individual virions 239
trafficking throughout the secretory pathway could also be detected (Movie S1). In addition, during 240
our studies, we frequently noticed virus particles with a diameter of about 40 nm (Fig. 2A and C, 241
yellow arrowheads) and TBEV-induced vesicles sharing the same ER lumen (Fig. 2B and Movie 242
S1). This finding supports the notion that replication, occurring in vesicles, and assembly of new 243
virions takes place in very close proximity (11). 244
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In agreement with earlier reports (11, 12, 28, 29), detailed tomographic analysis revealed pore-like 245
structures (Fig. 2C, yellow arrowheads) connecting the lumen of the vesicles to the cytosol. These 246
pore-like openings could be detected in approximately 50% of the vesicles included in these 247
tomograms (n=14) (Fig. 2D). In addition, we found that adjacent/neighboring vesicles were 248
frequently tightly packed, but pore-like openings between them were not observed (Fig. 2E). This 249
observation differs from what has been previously reported for WNV (12) or LGTV (28). Whether 250
this depends on the virus or the cell-type or the sample preparation method remains to be 251
established. 252
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Association of viral proteins with ER-derived membrane alterations induced in infected cells 254
To further characterize the ultrastructural modifications induced by TBEV infection and to allocate 255
viral proteins to specific membrane alterations we performed immuno-EM experiments on TBEV-256
infected BHK-21 cells. Thawed cryosections labeled with antibodies against the ER marker PDI 257
clearly showed that virus-induced vesicles (Ve) as well as newly assembled immature virions (Vi, 258
arrowheads) localize in the lumen of a dilated and rearranged ER compartment (Fig. 3A), thus 259
supporting our earlier immunofluorescence microscopy studies (Fig. 1D; (19)). A monoclonal 260
antibody to the NS1 protein (30) was used to mark TBEV-induced replication vesicles. As shown in 261
Fig. 3B and 3C, gold particles predominantly labeled 80 nm virus-induced vesicles that were 262
frequently located in the proximity of ER cisternae containing progeny virions (Vi, arrowheads). 263
However, NS1 could also be detected in the lumen of the ER or within the Golgi compartment 264
consistent with its putative roles in different steps of the viral life cycle (data not shown). TBEV-265
induced vesicles were also efficiently labeled with the rabbit polyclonal anti-TBEV serum we 266
previously used for immunofluorescence analysis of infected BHK-21 cells, which recognizes both 267
NS1 and the structural protein E (Fig. 3D). Attempts to label replication sites in immuno-EM with 268
the J2 antibody against dsRNA or with other antibodies raised against NS3 or NS5 were 269
unsuccessful. Viral particles trafficking towards the Golgi compartment were also labeled with the 270
polyclonal anti-TBEV serum (Vi, arrowheads). In addition, antibodies against the structural protein 271
prM showed specific labeling of TBEV particles (Fig. 3E and F). These particles were either 272
located in the lumen of the ER, frequently adjacent to virus-induced vesicles, or accumulated within 273
dilated ER cisternae, arguing that they represent newly assembled progeny virions. 274
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Ultrastructural analysis of replicon-transfected BHK-21 cells 276
The ultimate goal of this study was to address TBEV RNA trafficking within virus-induced 277
intracellular compartments by exploiting the MS2-based replicon system we previously established 278
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(19). In order to ascertain that transfection of MS2-tagged TBEV-derived subgenomic replicons 279
induces the same characteristic membrane alterations observed upon virus infection, we conducted 280
the analogous analysis as described above and compared the morphology of membranous structures 281
by using semi-thick sections (250 nm) prepared from replicon-transfected or virus-infected cells. 282
For this set of experiments we used BHK21-EYFP-MS2nls cells transduced with lentiviral vectors 283
expressing the nuclear variant of the EYFP-MS2 fusion protein. Previously, in order to tag TBEV 284
replicons, we always used a form of EYFP-tagged MS2 that localized both in the nucleus and in the 285
cytoplasm (8, 19, 31). However, we noticed that the accumulation of tagged TBEV RNA in the 286
cytoplasm corresponded to a depletion of the MS2 nuclear stain. Therefore, in order to increase the 287
signal to noise ratio in the cytoplasm, we exploited an EYFP-MS2 protein with a nuclear 288
localization signal (nls) for exclusive nuclear localization in the absence of TBEV replication. 289
EYFP-MS2nls remains associated with the replicated TBEV RNA carrying the MS2 binding sites 290
in the cytoplasm against a dark background offering a better tool for both dynamic and 291
ultrastructural studies. As shown in Fig. 4A, extensive co-localization of EYFP-MS2nls with 292
dsRNA was observed in the ER-derived perinuclear compartment in cells transfected with the 293
TNd/ΔME_24×MS2 wild type replicon whereas control cells transfected with the replicon carrying 294
an inactive NS5 RdRp showed only nuclear localization of EYFP-MS2nls (Fig. 4B). Detailed 295
tomographic analysis of cells fixed 24 h after transfection revealed rather small rER cisternae in 296
comparison to infected cells. These smaller ER tubules form a network of fragmented ER in the 297
perinuclear region of transfected cells (Movie S3, Fig. 4C and Movie S4). Nevertheless, vesicles 298
were observed in the rER lumen, having an average diameter of 85 nm (± 19.6 nm; n = 70). In 299
addition, similar to what we found in infected cells, most of the vesicles (75%, n=35) detected in 300
replicon-transfected cells had a pore-like opening towards the cytosol (Fig. 4D). Based on the 301
similarity between replicon- and infection-induced membranous structures and by analogy to 302
dengue virus (11) we concluded that TBEV-induced vesicles play a role in virus replication. 303
304
Immuno-EM analysis of replicon-transfected BHK-21 cells 305
Membrane alterations observed upon transfection of MS2-tagged subgenomic replicons were then 306
further characterized by immuno-EM (Fig. 5). As already suggested by our detailed three-307
dimensional analysis of replicon-induced ultrastructural changes (Fig. 4, Movie S3 and Movie S4), 308
PDI labeling clearly confirmed that TBEV-induced vesicles are part of an intricate network of 309
rearranged intracellular membranes that originate from the ER compartment (Fig. 5A). These 310
vesicles were labeled with anti-NS1 antibodies, again supporting their role in virus replication (Fig. 311
5B). Clusters of vesicles, defined as vesicle packets (14, 15, 32), could be observed in close 312
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proximity to highly rearranged membranous structures most likely representing convoluted 313
membranes (CM), the putative sites for synthesis and processing of the flavivirus polyprotein. As 314
shown in Fig. 5C and D the TBEV-specific polyclonal antiserum labeled both vesicles and CM 315
(Fig. 5D, arrows). In addition, gold particles for the same antiserum were found also in the lumen of 316
the ER consistent with our previous findings on TBEV-infected cells. 317
318
Replicated viral RNA is not freely diffusible in the cytoplasm 319
The MS2-tagging method was then further exploited to study the dynamic interchange of newly 320
replicated TBEV RNA between MS2-defined perinuclear compartments and the cytoplasm. To this 321
end we took advantage of the fluorescence recovery after photobleaching (FRAP) technique. FRAP 322
is a method for measuring the mobility of fluorescent particles in living cells (33, 34). A defined 323
portion of the system containing mobile fluorescent molecules is exposed to a brief and intense 324
focused laser beam, thereby causing irreversible photochemical bleaching of the fluorophore in that 325
region. The subsequent kinetics of fluorescence recovery in the bleached region, which results from 326
transport of fluorescent proteins into the bleached area from non-irradiated regions of the cell, as 327
well as transport of bleached fluorescent proteins out of the bleached area, provides a quantitative 328
measure of the mobility of the protein of interest. If the fluorescent protein cannot move, or is 329
bound to an immobile substrate, the recovery of fluorescence will not reach the pre-bleach values 330
showing the so-called immobile fraction. In our case the fluorescent protein is the MS2 phage coat 331
protein fused to EYFP. TBEV replicated RNA carrying an array of MS2 binding sites previously 332
characterized in our laboratory is detected by high affinity specific interaction between the RNA 333
stem-loops and the fluorescently labeled MS2 protein (19, 34). The viral RNA is continuously 334
synthesized by the viral polymerase and should be able to diffuse in the cytoplasm in complex with 335
EYFP-MS2 unless bound to an immobile structure or limited in its movements by physical barriers. 336
As shown in Fig. 6A, 6B and Movie S5, BHK-21 cells expressing the TNd/ΔME_24×MS2 replicon 337
RNA and MS2-EYFP were subjected to FRAP analysis at different time points. Full recovery of the 338
signal within few seconds was observed 6 hours after electroporation indicating free mobility of 339
MS2-EYFP at early time points of replicon amplification. In contrast, 12 hours after transfection 340
and at later time points, fluorescence recovery was severely impaired. Time points between 12 and 341
36 hours showed very similar recovery profiles with an initial recovery that quickly tails off and 342
never reaches pre-bleach values indicative of the presence of a relevant immobile fraction. The 343
initial portion of the recovery curve represents the unbound EYFP-MS2 that remains free to diffuse 344
in the bleached area. At later time points the amount of free EYFP-MS2 is reduced being 345
sequestered by the increasing amount of replicated RNA (compare the red curve in Fig. 6A taken at 346
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12 hpe to the blue and green curves taken at 24-36 hpe, respectively). These data suggest that the 347
spatial constraints are established early and maintained thereafter. These results can be interpreted 348
in different ways that were addressed as follows: 349
The first possibility is that the replicated viral RNA is secluded into compartments that are not 350
accessible by the MS2-EYFP. To address this assumption we exploited the red-shifted variant 351
Cherry-MS2nls to mark viral RNA while the mobility of free GFP was monitored by FRAP as 352
previously described (21, 34-36). As shown in Fig. 6C, 6D, 6E and Movie S6, mobility of GFP 353
within the compartment did not differ significantly from the free mobility of the protein in the 354
cytoplasm. The second possibility is that the viral RNA is only slowly or not at all replicated, 355
resulting in little or no substrate available for free MS2-EYFP. However, bleaching did not affect 356
virus replication (data not shown) and we have earlier shown that replicated viral RNA accumulates 357
continuously up to 72 hours post-transfection (19, 31). Therefore we can exclude that the RCs are 358
inactive with respect to replication. The third possibility is that, a slow release of replicated viral 359
RNA bound to bleached MS2-EYFP residing in the region of interest (ROI) results in an increase of 360
the immobile fraction in the FRAP experiment. To address such a release of replicated, MS2-361
tagged, viral RNA from this region we depleted fluorescent MS2-EYFP from the cytoplasm and 362
measured loss of florescence at the compartment. This approach is called fluorescence loss in 363
photobleaching (FLIP) and is complementary to FRAP (37, 38). As shown in Fig. 6F and Movie 364
S7, we chose a region for bleaching in the cytoplasm distant from the MS2-enriched perinuclear 365
compartment and measured loss of fluorescence at a site in the cytoplasm as well as within regions 366
of MS2-EYFP accumulation in order to assess RNA exchange. Both sites were chosen 367
approximately at the same distance from the bleaching area. After each bleaching the intensity of 368
fluorescence was measured in every ROI. In principle each mobile fluorescent molecule that 369
diffuses at the bleaching site will be irreversibly bleached. Hence, regions of freely mobile proteins 370
will be loosing fluorescence quickly, whereas regions where the proteins are immobile or secluded 371
into membrane compartments will resist bleaching. This kind of measurements clearly showed that 372
fluorescent TBEV RNA-bound MS2-EYFP (blue line) is depleted less efficiently than freely 373
diffusible MS2-EYFP (green line). Importantly, as observed for the FRAP experiments (Fig. 6A) 374
where the immobile fraction decreased at later time points, immobilization of trapped TBEV RNA 375
within the MS2-defined compartment is not complete since 30% depletion is observed after 4 376
minutes of FLIP. This clearly points towards a certain degree of TBEV RNA exchange between the 377
compartments and the cytoplasm. 378
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Free diffusion of viral RNA within interconnected regions of virus-induced membrane 380
alterations 381
Finally, we wanted to investigate the dynamic interchange of TBEV RNA within the area enriched 382
for the EYFP signal. To this end we took again advantage of the EYFP-MS2nls reporter that, in the 383
presence of replicating TNd/ΔME_24×MS2, accumulates efficiently in the cytoplasm against a dark 384
background, greatly enhancing the signal-to-noise ratio (Fig. 4A). This strategy is also particularly 385
useful when doing dynamic studies because it allows to selectively measure viral RNA kinetics 386
without taking into account free diffusion of unbound MS2 proteins in the cytoplasm. We designed 387
a FLIP experiment where the bleached area is located within perinuclear regions of MS2-EYFP 388
accumulation (Fig. 7A, red circle, bottom panels). If the tagged viral RNAs were all interconnected, 389
continuous bleaching would have resulted in depletion of fluorescence from the entire 390
compartment. Conversely, as shown in Fig. 7A and Movie S8, depletion of fluorescence was 391
restricted to a portion of the compartment, defined as ROI_1 in Fig. 7A, leaving the rest unaffected. 392
This experiment indicates that the perinuclear region, where replication of TBEV is likely to occur, 393
is not just a continuous network of virus-induced membrane alterations, but is rather composed of 394
physically separated sub-compartments. Within a sub-compartment the viral RNA is able to freely 395
diffuse (compare decay of ROI_1 in Fig. 7A with ROI_1 in Fig. 6F) indicating that most likely the 396
RNA is not completely associated to membranes. 397
To unambiguously identify the subcellular localization of newly synthesized viral genomes we 398
selectively labeled the MS2 protein bound to TBEV RNA by using antibodies specific to the 399
fluorescent tag. BHK-21 cells transduced with lentiviral vectors expressing EYFP-MS2nls were 400
either mock electroporated or electroporated with the TNd/ΔME_24×MS2 replicon RNA. At 24 h 401
upon transfection, cells were fixed and processed for immuno-EM as described in Material and 402
Methods. As shown in Fig. 7, immunogold-labeled TBEV RNA specifically localized in the 403
cytoplasm of TNd/ΔME_24×MS2 transfected cells, while, as expected only nuclear MS2 labeling 404
could be detected in mock-transfected cells (data not shown). Strikingly, detailed analysis of GFP-405
labeled cryosections revealed that more than 50% of the gold particles localized in the cytosol, 406
frequently surrounding virus-induced vesicles (Fig. 7E). In addition, particles associated or in close 407
proximity to ER-derived membranes (~ 35%), as well as adjacent to vesicle-surrounding 408
membranes (~ 9%) were also observed. To the contrary, labeled TBEV RNA was never detected 409
within the lumen of the vesicles, indicating that either the EYFP-MS2nls protein is too big to 410
translocate into the vesicle, or that actively replicating viral RNA is part of a huge protein/RNA 411
complex that shields the RNA from the MS2 protein and/or anti-GFP antibodies. 412
413
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Discussion 415
Flaviviruses hijack cytoplasmic membranes in order to build functional sites for protein synthesis, 416
processing and RNA replication (7, 39-42). These sites, generally defined as replication 417
compartments, are required to efficiently coordinate different steps of the viral life cycle and to 418
protect replicating RNA from innate immunity surveillance (8-10). Over the last few years, several 419
electron microscopy studies have revealed important information about the ultrastructural 420
organization of these sophisticated intracellular membrane compartments (11, 12, 28). However, 421
this information is rather static and does not provide insights into intracellular trafficking of 422
proteins and viral RNA. In this work we have combined high-resolution ET and immuno-EM with 423
live-cell imagining studies of TBEV RNA dynamics to provide the first comprehensive picture of 424
the spatio-temporal organization of the flaviviral RC. 425
Our detailed 3D ultrastructural analysis clearly shows that TBEV infection triggers a remarkable 426
alteration of intracellular membranes (Fig. 2). These virus-induced membranes are derived from the 427
ER since they contain viral proteins and the ER-resident chaperone PDI (Fig. 1). PDI did not 428
directly label the vesicles (Fig. 3A), indicating that this protein is not recruited, but most likely 429
excluded from virus-induced membranes as observed for DENV-induced vesicles (11). 430
Interestingly, we consistently observed packets of vesicles with a diameter of about 80 nm that 431
appear as invaginations of the ER within a highly organized network of interconnected membranes 432
(Fig. 2A and B and Movie S1 and S2). Attempts to label vesicles with antibodies recognizing 433
dsRNA were ambiguous; although we detected immuno-gold labeling within the vesicles, the signal 434
appeared to be unspecific (data not shown). Nevertheless, labeling of the vesicles with antibodies 435
against the TBEV NS1 protein (Fig. 3B, 3C and 3D) supports the hypothesis that these structures 436
correspond to sites of virus replication. NS1 is an essential gene and modulates early viral RNA 437
replication (13, 43), possibly through its ability to regulate negative-strand synthesis of viral RNA 438
(44) and/or by interacting with NS4B (45). NS1 is located in the ER lumen and is associated with 439
the outer side of the replication vesicles through interaction with other transmembrane viral 440
proteins. Localization of DENV NS1 at replication vesicles was reported by Mackenzie (13) and 441
more recently by Welsch (11). Finally, immunoprecipitation of dsRNA intermediates was shown to 442
contain NS1 in addition to NS5 confirming previous co-sedimentation data (46, 47). Therefore, 443
NS1 can be considered a bona-fide marker for replication sites. The observation that NS1 is 444
associated to other compartments in addition to replication vesicles may reflect its involvement in 445
different steps of the viral life cycle, beyond RNA replication. 446
Approximately half of the vesicles showed a single pore-like connection to the cytoplasm. There 447
might be several explanations for this observation. Besides technical problems that may not allow 448
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clear detection of pores in all vesicles, there could be a dynamic life-cycle of virus-induced vesicles, 449
from their formation, function in replication with a pore connection and eventual late step where the 450
vesicles are not connected with the cytosol by the pore anymore. This pore likely allows 451
recruitment of cellular cofactors to active sites of virus replication as well as for the release of 452
newly synthesized viral RNA to be used for translation and assembly (Fig. 2D). However, although 453
the vesicles were frequently closely associated, we could never detect direct connections between 454
neighboring vesicles in the same packet as described for Kunjin virus, and more recently also for 455
the naturally attenuated tick-borne LGTV (12, 28). This discrepancy might be due to differences in 456
the sample preparation. Alternatively, it might implicate that the architecture of flaviviral RCs 457
slightly changes based on the specific virus and/or cell line used for the analysis. 458
We also often observed newly assembled virions sharing the same ER lumen with putative 459
replicative vesicles, suggesting that replication and assembly sites are located in very close 460
proximity. Given the absence of pore-like openings directly connecting vesicles with the ER lumen, 461
it is likely that, once synthesized, the viral RNA is released via the pore to the cytosolic side of 462
these invaginations. The basic C protein would then interact with progeny RNA genomes to form a 463
nucleocapsid that buds back to the lumen of the ER acquiring the viral envelope. Thereafter, 464
immature viral particles would be either stored in clusters within dilated ER cisternae (Fig. 2A) or 465
transported through the cellular secretory pathway (Movie S1), where maturation occurs, in order to 466
be released to the extracellular milieu (48, 49). 467
In this study we also compared ultrastructural alterations induced by virus infection with those 468
associated with the replication of MS2-tagged TBEV subgenomic replicons. As observed upon 469
TBEV infection, dramatic reorganization of intracellular membranes with the appearance of single 470
membrane vesicles could be detected in the perinuclear region of the cell. Although ER tubules 471
hosting replicon-induced vesicles appeared fragmented and smaller than those observed in virus-472
infected cells, vesicles were similar in size and our detailed tomographic analysis revealed that they 473
also originated from the ER and were connected to the cytoplasm via pore-like channels. 474
Interestingly, at variance with virus-infected cells, replicon-induced vesicles did not accumulate in 475
packets but were rather dispersed through the cytoplasm as one or few invaginations of the ER 476
membrane (Fig. 4, Movie S3 and S4). We also observed a higher fraction of vesicles with pores in 477
replicon-transfected cells. However, the latter observation could be simply related to a better 478
accessibility of dispersed vesicles during ET. These data suggest that replicons, despite the 479
induction of massive ER rearrangements, are less efficient at forming vesicles which is consistent 480
with the lower level of viral replication previously reported (31, 50, 51). The induction of 481
membrane rearrangements could be due to the altered expression or regulation of viral proteins 482
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resulting from the electroporation of large amounts of genomic RNA. For example, it has been 483
shown that the NS4A protein of mosquito-borne KUNV and DENV viruses possesses the intrinsic 484
capacity to induce membrane rearrangements similar to those induced upon virus infection (52, 53). 485
A similar role for TBEV NS4A remains to be clarified. 486
When we investigated the mobility of viral RNA by FRAP we found a consistent delay of the 487
recovery of fluorescence within perinuclear regions of MS2-EYFP accumulation (Fig. 6A). In 488
contrast, GFP did not show significant differences of mobility within the cytosol as compared to 489
that within TBEV induced replication compartments that are marked with cherry-MS2nls (Figure 490
6C), suggesting that these compartments are open to the cytosol and allow exchange of proteins. 491
Interestingly, recovery after 36 hours did not differ significantly from that at 12 hours after replicon 492
transfection. Although we cannot formally exclude that replication rates slow down at late time 493
points, thus masking an increase of recovery in the FRAP curve due to an increased permeability, 494
we can assume that the mobility of viral RNA is similar at early and late time points. In other terms, 495
at later time points there is not a massive disruption or reorganization of these membranous 496
compartments compatible with the liberation of viral RNA. Free accessibility of the fluorescent 497
MS2 concomitant with a slow recovery after photobleaching within the compartment suggests an 498
impaired movement of the genomic RNA out of this area. This could be coupled with a slow rate of 499
viral RNA biogenesis and/or with the association of a fraction of the viral RNA to polysomes on ER 500
membranes. To address this hypothesis we performed a FLIP experiment and we directly measured 501
trafficking of viral RNA between cytosol and sites of virus replication (Fig. 6F). Strikingly, we 502
could observe a slow release of replicated RNA from the compartment. 503
Another important observation relates to the mobility of tagged viral RNA within MS2-defined 504
regions of the cytoplasm. Previous FRAP studies performed with components of the HCV 505
replication complex NS4B and NS5A showed that the internal architecture of the membranous web 506
is relatively static, with limited exchange of viral nonstructural proteins between neighboring 507
factories (17, 18, 54). In the present study, for the first time, we monitored viral RNA dynamics 508
within perinuclear regions of virus replication. By using continuous bleaching of MS2 fluorescence 509
in the region where the RNA is clustered, we could observe a fast depletion of fluorescence, 510
consistent with a high mobility of the viral RNA (Fig. 7A and Movie S8). Mobility within this area 511
was indeed higher than mobility between the compartment and the cytosol, confirming that a 512
physical impediment restricts viral RNA egress rather than an intrinsic slow mobility of the viral 513
RNP (compare the blue line in Fig. 6F and 7A). However, depletion of fluorescence was clearly 514
restricted only to a discrete region in the cluster leading us to the conclusion that these TBEV-515
induced compartments are partitioned with respect to viral RNA mobility. In agreement with this 516
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finding, specific labeling of the newly replicated RNA clearly revealed a high proportion of labeled 517
MS2-tagged RNA genomes localized in the cytosolic space surrounding TBEV-induced membrane 518
alterations (Fig. 7E). Therefore we propose that upon synthesis within ER-derived vesicles, progeny 519
genomes must be released to a larger virus-induced extra-vesicular sub-compartment where RNA 520
translation and virus assembly occur. This subcompartment would be full of viral RNA that travels 521
from replication vesicles to the virion assembly sites and is connected to the cytoplasm (Fig. 8). 522
Such extremely organized architecture would certainly help in coordinating genomic RNA 523
recruitment for translation and/or packaging. 524
In conclusion, we generated a high-resolution structure of the three-dimensional organization of 525
TBEV-induced replication compartment. In addition, by exploiting the MS2-tagged replicon 526
system, we could combine structural information with dynamic data of newly replicated TBEV-527
RNA within functional intracellular compartments providing new insights into the spatio-temporal 528
organization of flavivirus replication compartments. 529
530
Acknowledgments 531
We thank the Electron Microscopy Core Facility (EMCF) at University of Heidelberg, and the EM 532
Facility at European Molecular Biology Laboratory (EMBL, Heidelberg) for providing access to 533
their equipment, expertise, and technical support. We also thank Anil Kumar for the expert help in 534
handling lentiviral vectors and for useful discussions. LM benefited of a short-term EMBO 535
Fellowship (ASTF 217 – 2012). Work on Flaviviruses in AM’s laboratory is supported by the 536
Beneficientia Stiftung. R.B. is supported by the Deutsche Forschungsgemeinschaft (SFB 638, TP 537
A5 and TRR83, TP13). 538
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Figures Legends 540
Figure 1. Localization of TBEV proteins and dsRNA in BHK-21 infected cells. BHK-21 cells 541
were either mock infected (MOCK, right panels) or infected with TBEV at MOI = 2 (TBEV, left 542
panels). After 24h, cells were fixed and processed for immunofluorescence as described in materials 543
and methods. (A) Cells co-stained with the anti-TBEV antiserum (TBEV; Alexa Fluor 594, red) and 544
with the monoclonal anti-NS1 antibody (NS1, Alexa Fluor 488, green). (B) Cells co-stained with 545
the anti-TBEV antiserum (TBEV; Alexa Fluor 594, red) and with the J2 monoclonal anti-dsRNA 546
antibody (dsRNA; Alexa Fluor 488, green). (C) Cells co-stained with the anti-prM antiserum (prM; 547
Alexa Fluor 594, red) and with the monoclonal anti-NS1 antibody (NS1; Alexa Fluor 488, green). 548
(D) Cells co-stained with the anti-TBEV antiserum (TBEV; Alexa Fluor 594, red) and with the anti-549
PDI monoclonal antibody (PDI; Alexa Fluor 488, green). The zoomed images shown in the middle 550
panels correspond to boxed regions in the left panels. 551
552
Figure 2. Ultrastructural analysis of the membrane alterations induced by TBEV infection. 553
(A) BHK-21 cells were infected with TBEV at MOI = 2, fixed 24 hpi and processed for ET as 554
described in materials and methods. Left: tomographic slice of a dual axis tomogram, shown in 555
Movie S2. Upon infection vesicles (Ve), as well as virions (yellow arrows) were observed in the 556
lumen of the rough ER. Right: 3D surface reconstruction of the whole tomogram displaying the 557
TBEV-induced vesicles (in light yellow) in the lumen of the ER (in light brown), as well as virions 558
(in dark red). (B) Two examples (left and right, respectively) of tomographic slices through the 559
whole tomogram depicting connections between ER tubules (yellow arrows). (C) Left: tomographic 560
slices of the same dual axis tomogram depicting several of these vesicles in the lumen of the ER in 561
close proximity to newly assembled virions (yellow arrows), also observed in the Golgi apparatus 562
(Movie S1). Right: 3D surface rendering of the same area displaying the TBEV-associated vesicles 563
(in light yellow) sharing the ER lumen with TBE-virions (in dark red) and surrounded by ER 564
membranes (in light brown). (D) Left: serial single slices depicting openings of several TBEV-565
induced vesicles to the cytosol (green, yellow and blue arrows). Right: XZ view of the 3D 566
reconstruction of the whole tomogram displaying these openings towards the cytosol (arrows with 567
matching colors). (E) Left: slices through the tomogram showing tight contacts of TBEV-induced 568
vesicles with their neighboring vesicles (yellow arrows). Right: 3D reconstruction displaying these 569
contacts between vesicles. Note that openings connecting vesicles were not observed in these 570
tomograms. 571
572
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Figure 3. Immunogold EM of TBEV infected cells. BHK-21 cells were infected with TBEV at 573
MOI = 2, fixed 24 hpi, and thawed cryosections were labeled with antibodies against PDI (A), NS1 574
(B and C), prM (E and F), and with the anti-TBEV serum (D) that predominantly recognizes the 575
structural protein E and the nonstructural protein NS1. Vesicles within the lumen of dilated ER 576
cisternae (A) are specifically labeled with anti-NS1 (B and C) and anti-TBEV antibodies (D). 577
Arrowheads in all panels highlight TBEV virions, specifically labeled with prM (E and F) and 578
TBEV (D) antibodies, in the lumen of the ER or trafficking toward the Golgi apparatus. 579
Abbreviations: M, mitocondria; N, nucleus; ER, endoplasmic reticulum; Ve, vesicles; Vi, virions. 580
581
Figure 4. Ultrastructural analysis of the membrane alterations induced by 582
pTNd/∆ME_24×MS2. 583
(A) BHK21-EYFP-MS2nls cells were electroporated with the TNd/ΔME_24xMS2 replicon RNA. 584
24 hours post electroporation (hpe) cells were fixed, permeabilized with Triton X-100 and 585
incubated with an antibody against dsRNA that was then revealed by a secondary antibody 586
conjugated with Alexa-594. The EYFP channel is shown in the left panel, dsRNA in the middle 587
panel and the merge in the right panel. (B) BHK21-EYFP-MS2nls cells were electroporated with 588
the TNd/ΔME_24xMS2_GAA replicon RNA and treated as in (A). 589
(C) BHK21-EYFP-MS2nls cells were electroporated with TNd/∆ME_24×MS2, fixed 24 hpe and 590
processed for ET as described in materials and methods. Left: slice of a dual axis tomogram 591
showing the TBEV-induced vesicles (Ve) in the ER lumen. Right: 3D reconstruction of the 592
complete tomogram. ER membranes are depicted in light brown and TBEV-induced vesicles in 593
light yellow. This tomogram and its 3D membrane rendering are shown in Movie S4. (D) Left: 594
serial single slices through the same tomogram displaying connections between adjacent ER 595
tubules. Right: 3D reconstruction of the whole tomogram showing a network of interconnected ER 596
tubules. Note that the ER is highly fragmented in these cells in comparison to infected cells. (E) 597
Left: serial single slices through the same tomogram displaying an opening towards the cytosol of a 598
TBEV-induced vesicle (yellow arrows). Right: 3D surface model showing this opening that 599
connects the interior of the vesicle with the cytosol. 600
601
Figure 5. Immunogold EM of replicon-induced membrane alteration. BHK21-EYFP-MS2nls 602
cells were electroporated with TNd/∆ME_24×MS2, fixed 24 h after, and thawed cryosections were 603
labeled with antibodies against PDI (A), NS1 (B), and with the anti-TBEV serum (C and D). All 604
images show TBEV-induced vesicles (Ve) that are specifically labeled with anti-NS1 (B) and anti-605
TBEV antibodies (C and D). (D) Higher magnification image of membrane alterations induced by 606
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replicon transfection corresponding to the boxed region in panel (C). Vesicles (Ve) and convoluted 607
membranes (CM) labeled with anti-TBEV antibodies are shown. 608
609
Figura 6. TBEV replicated RNA is not freely diffusible in the cytoplasm. (A) Analysis of 610
TBEV RNA dynamics by FRAP time course. BHK-21 cells were electroporated with the 611
TNd/ΔME_24×MS2 replicon RNA together with a vector expressing MS2-EYFP. At the indicated 612
time-points post-electroporation the fluorescence recovery of the MS2-EYFP protein in the area of 613
bleaching was analyzed. The graph shows values of fluorescence intensity normalized to the pre-614
bleach values and corrected for the loss of fluorescence due to the imaging procedure (34, 36, 55). 615
Data represent the average of acquisitions from at least 10 cells ± standard deviation. (B) Image 616
sequence from a FRAP experiment performed in BHK-21 cells 14 hours upon electroporation 617
(29,25 × 29,25 μm). The bright perinuclear region represents the subcellular compartment into 618
which replicated viral RNA is clustered and where ROIs were drawn. Times were collected before 619
bleaching (pre-bleach, 0 sec.), immediately after the bleaching (bleach, 20 sec.) and at 400 sec. after 620
the bleaching event (post-bleach). A movie is also available as supporting information (Movie S5). 621
(C) Analysis of GFP mobility within the replication compartment. BHK-21 cells were 622
electroporated with a vector expressing CherryMS2nls, to mark the RC, and with a GFP expressing 623
plasmid (pEGFP-N1) both in the presence (red line, GFP + TNd/ΔME_24×MS2) and in the absence 624
(green line, GFP) of the TBEV replicon RNA. After 24 hours GFP kinetics was investigated by 625
FRAP. In the graph the recovery curves of the GFP protein in the two different experimental 626
conditions are compared. The values of fluorescence intensity are normalized to the pre-bleach 627
values and corrected for the loss of fluorescence due to the imaging procedure as already described. 628
Data represent the average of acquisitions from 10 cells ± standard deviation. (D) Representative 629
image of BHK-21 cells transfected with GFP and CherryMS2nls in the presence of the TBEV 630
replicating RNA. A z-projection of 41 images 0,5 µm apart is shown. (E) Image sequence from the 631
FRAP experiment described in (C) (36,56 × 7,14 μm). Top, pre-bleach stacks in both channels (0 632
sec.). The circle indicates the area of bleach chosen in the RC marked by CherryMS2nls. Middle, 633
time point immediately after the bleaching event (0,5 sec.); bottom, post-bleach stack (13 sec.). A 634
video is also available as supporting information (Movie S6). (F) BHK-21 cells were electroporated 635
with the TNd/ΔME_24×MS2 replicon RNA together with a vector expressing MS2-EYFP. After 24 636
hours viral RNA release from the RC was monitored by FLIP. (G) selected images from a FLIP 637
experiment are shown (36,56 × 36,56 μm). The region for bleaching (red circle in the bottom 638
panels) was chosen in the cytoplasm away from the clustered TBEV RNA. Loss of fluorescence 639
was then measured in the cytoplasm both within (blue circle, ROI_1) and outside (green circle, 640
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ROI_2) the RC. In the top graph the loss in fluorescence intensity within the three different ROIs 641
(Bleach; ROI_1 and ROI_2) is compared. Data are normalized as described in (A) and represent the 642
average of acquisitions from 10 cells ± standard deviation. A video is also available as supporting 643
information (Movie S7). 644
645
Figure 7. TBEV replication compartments are organized in discrete clusters. (A) BHK-21 cells 646
were electroporated with the TNd/ΔME_24×MS2 replicon RNA and with the EYFP-MS2nls 647
reporter. At 24 hours upon transfection viral RNA trafficking within the RC was analyzed by FLIP. 648
For this purpose, as shown in the bottom images (29,25 × 29,25 μm), the area of bleaching (red 649
circle) was located inside the compartment. Loss of fluorescence was then measured in two 650
different regions, one surrounding the bleaching area (blue region; ROI_1) and the other one more 651
distant (green circle; ROI_2). The top panel compares the loss of fluorescence curves of the three 652
selected ROIs. Data are normalized as already described and represent the average of acquisitions 653
from 10 cells ± standard deviation. A video is also available as supporting information (Movie S8). 654
(B) BHK21-EYFP-MS2nls cells were electroporated with TNd/∆ME_24×MS2, fixed 24 h after, 655
and thawed cryosections were immunogold-labeled with antibodies against GFP. Selected examples 656
of the different situations are shown in (C) and (D). 657
(E) Quantification of GFP-labeled EM cryosections prepared as described in Fig. 7B. Counting was 658
performed in triplicate measuring the distribution of >100 gold particles for each grid. Data are 659
plotted as percentage ± SD. 660
661
Figure 8. Model of TBEV-induced membrane alterations. Left: slice of the dual axis tomogram 662
shown in Fig. 4B. Right: two-dimension schematic model of the possible organization of TBEV 663
replication compartments. TBEV replication leads to the formation of a highly organized network 664
of interconnected and juxtaposed ER membranes in the perinuclear region of the cells. This 665
compartment is composed of vesicles (Ve, in light yellow) connected to the cytoplasm via pore-like 666
channels, of dilated ER cisternae (in brown), and of a cytoplasmic extra-vesicular space (in light 667
blue). Progeny viral RNAs are synthesized within the lumen of these ER-invaginations and are then 668
extruded through the pore into the cytoplasmic extra-vesicular space. Once released into this area 669
viral RNAs are available for downstream assembly into new viral particles that bud back into the 670
lumen of ER cisternae of infected cells (Fig. 2). Alternatively these RNAs may be engaged in 671
further rounds of translation and replication. 672
673
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