Published Ahead of Print 3 April 2013. 2013, 87(11):6469. DOI: 10.1128/JVI.03456-12. J. Virol. 

Alessandro MarcelloHoppe, Jacomine Krijnse-Locker, Ralf Bartenschlager and Lisa Miorin, Inés Romero-Brey, Paolo Maiuri, Simone Sites and Trafficking of the Replicated RNATick-Borne Encephalitis Virus Replication Three-Dimensional Architecture of

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Three-Dimensional Architecture of Tick-Borne Encephalitis VirusReplication Sites and Trafficking of the Replicated RNA

Lisa Miorin,a Inés Romero-Brey,b Paolo Maiuri,a* Simone Hoppe,b,c Jacomine Krijnse-Locker,b,c Ralf Bartenschlager,b

Alessandro Marcelloa

Laboratory of Molecular Virology, The International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano, Trieste, Italya; Department of InfectiousDiseases, Molecular Virology, University of Heidelberg, Heidelberg, Germanyb; Electron Microscopy Core Facility, University of Heidelberg, Heidelberg, Germanyc

Flavivirus replication is accompanied by the rearrangement of cellular membranes that may facilitate viral genome replicationand protect viral components from host cell responses. The topological organization of viral replication sites and the fate of rep-licated viral RNA are not fully understood. We exploited electron microscopy to map the organization of tick-borne encephalitisvirus (TBEV) replication compartments in infected cells and in cells transfected with a replicon. Under both conditions, 80-nmvesicles were seen within the lumen of the endoplasmic reticulum (ER) that in infected cells also contained virions. By electrontomography, the vesicles appeared as invaginations of the ER membrane, displaying a pore that could enable release of newlysynthesized viral RNA into the cytoplasm. To track the fate of TBEV RNA, we took advantage of our recently developed methodof viral RNA fluorescent tagging for live-cell imaging combined with bleaching techniques. TBEV RNA was found outside virus-induced vesicles either associated to ER membranes or free to move within a defined area of juxtaposed ER cisternae. From ourresults, we propose a biologically relevant model of the possible topological organization of flavivirus replication compartmentscomposed of replication vesicles and a confined extravesicular space where replicated viral RNA is retained. Hence, TBEV modi-fies the ER membrane architecture to provide a protected environment for viral replication and for the maintenance of newlyreplicated RNA available for subsequent steps of the virus life cycle.

Tick-borne encephalitis virus (TBEV) is the etiological agent oftick-borne encephalitis, a potentially fatal infection of the cen-

tral nervous system occurring throughout wide areas in Europeand Asia (1–3). TBEV is the most medically important member ofthe mammalian tick-borne group of the genus Flavivirus withinthe family Flaviviridae (4). Flaviviruses are a large group of arbo-viruses that are responsible for severe diseases in humans andanimals. This virus group includes, in addition to TBEV, the den-gue virus (DENV), yellow fever virus (YFV), West Nile virus(WNV), and Japanese encephalitis virus (JEV). They have in com-mon an enveloped virus particle that contains a single-stranded,positive-sense RNA genome, a similar genomic organization, andcomparable replication strategies (5, 6). After entry, the incomingviral RNA is translated, giving rise to a polyprotein precursor thatis processed by cellular proteases and the viral protease NS2B/3 toobtain three structural and seven nonstructural proteins (NS).The RNA-dependent RNA polymerase (RdRp) residing in NS5synthesizes complementary negative-strand RNA from genomicRNA, with negative strands serving as the template for the synthe-sis of new positive-strand viral RNAs.

Like all positive-strand RNA viruses, flaviviruses replicate inthe cytoplasm in close association with virus-induced intracellularmembrane structures. It is generally accepted that the formationof these replication compartments (RC) provide an optimal mi-croenvironment for viral RNA replication by limiting diffusion ofviral/host proteins and viral RNA, thereby increasing the concen-tration of components required for RNA synthesis, and by pro-viding a scaffold for anchoring the replication complex (7). Inaddition, these virus-induced membranes may also shield double-stranded RNA (dsRNA) replication intermediates from host cell-intrinsic surveillance (8–10). Elegant electron tomography (ET)studies on DENV- and WNV-infected cells have recently providedthe first three-dimensional (3D) view of the architecture of flavi-

virus RCs (11, 12). In these studies, different virus-induced mem-brane structures appeared to be part of a highly organized networkof ER-derived rearranged membranes. Vesicle packets, containingdsRNA and proteins of the replication complex, have been de-scribed as the sites of virus replication and appear in ET as invagi-nations of the ER membrane bearing pore-like connections to thecytoplasm and possibly between themselves. Convoluted mem-branes (CM) that are specifically enriched in NS2B/3 have beenproposed as the putative sites of protein synthesis and proteolyticcleavage (11, 13–15).

However, although ET images from fixed cells can provide ahigh-resolution snapshot of the complex network of vesicles andinterconnections, they cannot address the dynamic exchange ofproteins and viral RNA throughout these compartments. In orderto provide a global picture of the spatiotemporal organization ofthe RCs, it is therefore important to integrate high-resolution im-aging approaches with innovative techniques that allow exploringin real time the dynamic interplay between viruses and their hosts.Engineered subgenomic replicons expressing fluorescently tagged

Received 18 December 2012 Accepted 22 March 2013

Published ahead of print 3 April 2013

Address correspondence to Alessandro Marcello, marcello@icgeb.org, or LisaMiorin, miorin@icgeb.org.

* Present address: Paolo Maiuri, Systems Cell Biology of Cell Polarity and CellDivision, Institut Curie, CNRS, UMR144, Paris, France.

L.M. and I.R.-B. contributed equally to this article.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.03456-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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nonstructural proteins (16–18) have been exploited to investigatethe distribution and dynamics of HCV replicase proteins. How-ever, the only available tool to explore flaviviral RNA traffickinghas been reported for TBEV (19). The method is based on TBEVreplicons containing multiple high-affinity binding sites for thephage MS2 core protein fused to an autofluorescent protein. Rep-licated viral RNA could then be visualized in living cells, thusproviding the first description of flavivirus RNA dynamics in liv-ing cells (19).

In this work, we took advantage of immunoelectron micros-copy (immuno-EM), thin-section transmission electron micros-copy (TEM), and high-resolution ET in combination with live-imaging approaches for TBEV RNA to identify replicationcompartments in infected and replicon-transfected cells. We pre-cisely mapped the spatial organization of virus-induced mem-branes and vesicles and studied the mobility of replicated viralRNA. We propose a model where replication vesicles and the ex-travesicular space form an optimized environment to support ef-ficient virus replication.

MATERIALS AND METHODSCells, viruses, and plasmids. Baby hamster kidney (BHK-21) and Africangreen monkey (Vero E6) cell lines were grown under standard conditionsin Dulbecco’s modified Eagle medium (DMEM) supplemented with 10%fetal bovine serum. BHK21-enhanced yellow fluorescent protein (EYFP)-MS2nls cells are a cell pool stably expressing EYFP-MS2nls and blasticidinS-deaminase, and they were cultured in the presence of 10 �g/ml blasti-cidin. Lentiviral particles for the transduction of the EYFP-MS2nls genewere prepared in 293T cells exactly as described previously (20) usingpWPI-BLR-EYFP-MS2nls and packaging constructs pCMVR8.91 andpMD.G (provided by Didier Trono). Working stocks of TBEV strain Neu-doerfl were propagated and titrated on Vero E6 cells. pWPI-BLR-EYFP-MS2nls was used to generate a cell line constitutively expressing theEYFP-MS2nls protein suitable for immuno-EM studies. The EYFP-MS2nls gene was isolated from pEYFP-MS2nls (21) by XbaI digestionand then inserted into the multiple cloning site (MCS) of the pWPI-BLR vector (provided by Volker Lohmann) via the SpeI restriction site.The construct pTNd/�ME_24�MS2, the replication-deficient TNd/�ME_24�MS2_GAA replicon carrying the GDD-to-GAA mutation inthe viral NS5 protein, and the pMS2-EYFP, pEYFP-MS2nls, andpCherry-MS2nls vectors used for visualization purposes were previ-ously described (19, 21).

RNA transcription and transfection. Subgenomic replicon RNAswere transcribed in vitro as described in detail elsewhere (19, 22). A total of4 � 106 cells were resuspended in 400 �l ice-cold phosphate-bufferedsaline (PBS) and mixed in a 0.4-cm gene-pulser cuvette with 10 �g of RNAto be electroporated with a Bio-Rad Gene Pulser apparatus at 0.25 kV witha capacitance of 960 �F (19). After electroporation, cells were washed incomplete growth medium without antibiotics and seeded in the samemedium.

Sample staining and imaging for indirect immunofluorescenceanalysis. Immunofluorescence (IF) analysis was performed 24 h uponinfection or transfection. Cells were washed with PBS, fixed with 4% para-formaldehyde (PFA) for 15 min, incubated for 5 min with 100 mM gly-cine, and permeabilized with 0.1% Triton X-100 for 5 min. Subsequently,the cells were incubated at 37°C for 30 min with PBS, 1% bovine serumalbumin (BSA), and 0.1% Tween 20 before incubation with antibodies.The coverslips were rinsed three times with PBS-0.1% Tween 20 (washingsolution) and incubated for 1 h with secondary antibodies. Donkey anti-bodies specific for rabbit or mouse immunoglobulin G and conjugated toAlexa Fluor 594 or Alexa Fluor 488 (Molecular Probes) were used for thisanalysis. Coverslips were finally washed three times with washing solutionand mounted on slides using Vectashield mounting medium (Vector Lab-oratories). For the detection of viral antigens, we used the following anti-

bodies: mouse monoclonal antibody detecting NS1 (provided by ConnieSchmaljohn), polyclonal rabbit anti-TBEV serum that can be used forboth structural and nonstructural protein detection (23) (provided byFranz X. Heinz), and polyclonal rabbit anti-prM serum (provided byFranz X. Heinz). The J2 mouse monoclonal anti-dsRNA antibody (Eng-lish and Scientific Consulting, Szirak, Hungary) was used to detect repli-cation complexes, whereas for the ER staining we used the mouse mono-clonal anti- protein disulfide isomerase (PDI; AB2792; Abcam) antibody.Fluorescent images of fixed cells were captured on a Zeiss LSM510 METAconfocal microscope with a 63� Plan-Apochromat oil objective having a1.4 numerical aperture (NA). The pinhole of the microscope was adjustedto get an optical slice of less than 1.0 �m for any wavelength acquired.

Epoxy embedding of cells for transmission electron microscopy.BHK-21 cells grown on 10-cm-diameter dishes were infected with TBEVat a multiplicity of infection (MOI) of 2 or transfected with in vitro tran-scribed TNd/�ME_24�MS2 replicon RNA. After 24 h, cells were washed3 times with prewarmed PBS and fixed for 30 min with 2.5% glutaralde-hyde (GA) in 50 mM Na-cacodylate buffer (pH 7.4) containing 1 M KCl,0.1 M MgCl2, 0.1 M CaCl2, and 2% sucrose. Cells were washed 5 times for5 min each with 50 mM Na-cacodylate buffer and postfixed on ice in thedark with 2% OsO4 in 50 mM Na-cacodylate buffer for 40 min. After thecells were washed overnight in distilled water, they were treated with 0.5%uranylacetate (UA; dissolved in water) for 30 min, rinsed thoroughly withwater, and dehydrated in a graded ethanol series at room temperature(40%, 50%, 60%, 70%, and 80%, 5 min each; then 95% and 100%, 20 mineach). Cells were immersed in 100% propylene oxide and immediatelyembedded in an Araldite-Epon mixture (Araldite 502/Embed 812 kit;Electron Microscopy Sciences). After polymerization at 60°C for 2 days,embedded cells were sectioned using a Leica Ultracut UCT ultrami-crotome and a 35° diamond knife (Diatome, Biel, Switzerland). Sectionswith a thickness of 60 nm were collected onto 100-mesh copper grids thathad been coated with Formvar (Plano, Wetzlar, Germany) and coatedwith carbon. Sections were counterstained with 2% lead citrate in H2O for2 min. Samples were analyzed by using a Biotwin CM120 Philips electronmicroscope (100 kV) equipped with a bottom-mounted 1K charge-cou-pled-device (CCD) camera (Keen View; SIS, Münster, Germany).

Immunolabeling of thawed cryosections. TBEV-infected and repli-con-transfected cells were fixed by adding an equal amount of 8% PFAand 0.2% GA in 0.2 M PHEM buffer (120 mM Pipes, 100 mM HEPES, 4mM MgCl2, 40 mM EGTA, pH 6.9) to the culture medium for 1 h at roomtemperature. Cells were then fixed for 1 h with 4% PFA and 0.1% GA in0.1 M PHEM at room temperature. The fixative was removed and the cellswere stored at 4°C in 4% PFA in 0.1 M PHEM until further processing.After extensive washing with 0.1 M PHEM, remaining aldehyde groupswere blocked with 30 mM glycine in 0.1 M PHEM. Cells were scraped offthe plate, embedded in 10% gelatin, and infiltrated in 2.3 M sucrose over-night at 4°C. Cell pellets were mounted onto sample holder pins, frozen,and stored in liquid nitrogen. Cryosections (60 nm) were prepared usinga Leica Ultracut UC6 microtome (Leica Microsystems, Wetzlar, Ger-many) and a diamond knife (Diatome, Biel, Switzerland). Sections werepicked up with a mixture of 2% methylcellulose and 2.3 M sucrose (1:1)and, after being thawed, were transferred to 100-mesh Formvar- and car-bon-coated grids. Labeling of thawed cryosections was performed essen-tially as described elsewhere (24). In brief, sections were molten by float-ing on 2% gelatin for 30 min at 37°C and incubated in 30 mM glycine inPBS for 10 min and then at 30 min at room temperature in blockingsolution (PBG; 0.8% [wt/vol] BSA [Sigma], 0.1% [wt/vol] fish skin gelatin[Sigma] in PBS). Sections were then incubated with primary antibodydiluted in blocking buffer, for 30 min at room temperature. After beingwashed 5 times, for 5 min each time, in blocking buffer, sections wereincubated with rabbit anti-mouse antibody followed by protein A coupledto 10-nm gold particles (Cell Microscopy Center, Utrecht, The Nether-lands) diluted in blocking solution. After being washed with PBS anddistilled water, grids were contrasted with a mix of 2% methylcelluloseand 3% UA (1:6) for 10 min on ice.

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Electron tomography. Sections of 250-nm thickness were collectedon palladium-copper slot grids (Science Services, Munich, Germany)coated with Formvar (Plano, Wetzlar, Germany). Protein A-gold (10 nm)was added to both sides of the sections as fiducial markers. Single- anddual-axis tilt series were acquired with an FEI Tecnai TF30 microscopeoperated at 300 kV and equipped with a 4k FEI Eagle camera (binningfactor 2, on the specimen level) over a �65° to 65° tilt range (increment of1°) and at an average defocus of �0.2 �m. Tomograms were recon-structed using the weighted back projection method implemented in theIMOD software package (version 3.11.5) (25). Rendering of the three-dimensional surface of the tomograms was performed by using theAMIRA visualization software package (version 5.4.2; Visage Imaging,Berlin, Germany). Models were generated from unfiltered and 2� binnedtomograms by manually masking areas of interest, thresholding, andsmoothing labels.

Microscopy and live imaging acquisition. BHK-21 cells were electro-porated with the TBEV replicons’ RNA and plated on glass-bottom plates(MatTek, Ashland, MA). For the visualization of the viral RNA, cells werecotransfected either with MS2-EYFPnls, expressing a hybrid protein com-posed by the core protein of the MS2 bacteriophage fused to EYFP and toa nuclear localization signal (NLS), or with the same construct without theNLS. At the appropriate time point posttransfection, cells were trans-ferred on a humidified and CO2-controlled on-stage incubator (PeConGmbH, Erbach, Germany) at 37°C in complete DMEM without phenolred for live cell imaging. For fluorescence recovery after photobleaching(FRAP) experiments, images of 512 by 512 pixels (29.25 by 29.25 �m) andoptical thickness of 1 �m were acquired using 1% or less of the power ofthe 514-nm laser line. EYFP was bleached at 514 nm (Argon laser; maxi-mum output of 500 of milliwatts) in a circle of 30 pixels of diameter, at fulllaser power, for 10 passages. For fluorescence loss in photobleaching(FLIP) measurements, images were acquired as described above, andbleaching was performed at every acquisition. Images were analyzed withImageJ (W. S. Rasband, ImageJ; National Institutes of Health, Bethesda,MA; http://rsb.info.nih.gov/ij/).

RESULTSSubcellular localization of TBEV proteins and dsRNA in in-fected cells. As a first step to study the organization of TBEVreplication sites, we characterized by immunofluorescence thesubcellular localization of proteins and dsRNA in virus-infectedBHK-21 cells. At 24 h postinfection (hpi), viral proteins detectedwith a polyclonal TBEV-specific antiserum (predominantly rec-ognizing the structural protein E and NS1) were localized in theperinuclear region and in discrete and irregularly shaped foci(Fig. 1A). As expected, these structures partially colocalized withNS1-containing cytoplasmic foci (Fig. 1A) as well as with thedsRNA replication intermediate (Fig. 1B), a well-accepted markerfor flaviviral replication vesicles (11, 12, 15, 26). A similar distri-bution pattern was observed with the prM-specific antibody thatalso showed partial colocalization with NS1 cytoplasmic foci(Fig. 1C). In addition, coimmunolocalization studies of viral pro-teins and the rER marker protein disulfide isomerase (PDI) sug-gested that ER-derived membranes provide the framework for themembranous TBEV replication compartments (Fig. 1D). Thesedata are in agreement with our previous observations for the MS2-tagged TBEV replicon where newly synthesized viral RNA associ-ates with viral proteins and dsRNA replication intermediates inrER-derived cytoplasmic compartments (8, 19).

Electron tomography analysis of TBEV-induced membranealterations. To reveal the three-dimensional organization of vi-rus-induced membrane alterations, we performed a detailed ETanalysis of TBEV-infected cells. At 24 hpi, BHK-21 cells werefixed, resin embedded, and sectioned as described in Materials and

Methods. Tomograms from 250-nm-thick sections were acquiredand reconstructed. As shown in Fig. 2, TBEV infection inducedmembrane alterations reminiscent of those previously describedfor mosquito-borne flaviviruses like DENV and WNV (11, 12, 27)and more recently also for Langat virus (LGTV), a naturally atten-uated tick-borne flavivirus (28). Virus-induced single-membranevesicles (Ve) with a diameter of 80 nm (�10.6 nm; n � 70) wereobserved in the lumen of a dilated rER and appeared to be part ofan elaborate reticulovesicular network of interconnected mem-branes (Fig. 2A and B). ER-derived cisternae filled with viral par-ticles in the proximity of virus-induced vesicles, as well as individ-ual virions trafficking throughout the secretory pathway, couldalso be detected (see Movie S1 in the supplemental material). Inaddition, during our studies, we frequently noticed virus particleswith a diameter of about 40 nm (Fig. 2A and C, yellow arrow-heads) and TBEV-induced vesicles sharing the same ER lumen(Fig. 2B; see also Movie S1 in the supplemental material). Thisfinding supports the notion that replication, occurring in vesicles,and assembly of new virions takes place in very close proximity(11).

In agreement with earlier reports (11, 12, 28, 29), detailed to-mographic analysis revealed pore-like structures (Fig. 2C; yellowarrowheads) connecting the lumen of the vesicles to the cytosol.These pore-like openings could be detected in approximately 50%of the vesicles included in these tomograms (n � 14) (Fig. 2D). Inaddition, we found that adjacent/neighboring vesicles were fre-quently tightly packed, but pore-like openings between them werenot observed (Fig. 2E). This observation differs from what hasbeen previously reported for WNV (12) or LGTV (28). Whetherthis depends on the virus or the cell type or the sample preparationmethod remains to be established.

Association of viral proteins with ER-derived membrane al-terations induced in infected cells. To further characterize theultrastructural modifications induced by TBEV infection and toallocate viral proteins to specific membrane alterations, we per-formed immuno-EM experiments on TBEV-infected BHK-21cells. Thawed cryosections labeled with antibodies against the ERmarker PDI clearly showed that virus-induced vesicles as well asnewly assembled immature virions (Vi; arrowheads) localize inthe lumen of a dilated and rearranged ER compartment (Fig. 3A),thus supporting our earlier immunofluorescence microscopystudies (Fig. 1D) (19). A monoclonal antibody to the NS1 protein(30) was used to mark TBEV-induced replication vesicles. Asshown in Fig. 3B and C, gold particles predominantly labeled80-nm virus-induced vesicles that were frequently located in theproximity of ER cisternae containing progeny virions (arrow-heads). However, NS1 could also be detected in the lumen of theER or within the Golgi compartment, consistent with its putativeroles in different steps of the viral life cycle (data not shown).TBEV-induced vesicles were also efficiently labeled with the rabbitpolyclonal anti-TBEV serum we previously used for immunoflu-orescence analysis of infected BHK-21 cells, which recognizesboth NS1 and the structural protein E (Fig. 3D). Attempts to labelreplication sites in immuno-EM with the J2 antibody againstdsRNA or with other antibodies raised against NS3 or NS5 wereunsuccessful. Viral particles trafficking toward the Golgi compart-ment were also labeled with the polyclonal anti-TBEV serum (Vi;arrowheads). In addition, antibodies against the structural proteinprM showed specific labeling of TBEV particles (Fig. 3E and F).These particles were either located in the lumen of the ER, fre-

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quently adjacent to virus-induced vesicles, or accumulated withindilated ER cisternae, arguing that they represent newly assembledprogeny virions.

Ultrastructural analysis of replicon-transfected BHK-21cells. The ultimate goal of this study was to address TBEV RNAtrafficking within virus-induced intracellular compartments byexploiting the MS2-based replicon system we previously estab-lished (19). In order to ascertain that transfection of MS2-taggedTBEV-derived subgenomic replicons induces the same character-istic membrane alterations observed upon virus infection, we con-ducted the analogous analysis as described above and comparedthe morphology of membranous structures by using semithicksections (250 nm) prepared from replicon-transfected or virus-infected cells. For this set of experiments, we used BHK21-EYFP-MS2nls cells transduced with lentiviral vectors expressing the nu-

clear variant of the EYFP-MS2 fusion protein. Previously, in orderto tag TBEV replicons, we always used a form of EYFP-tagged MS2that localized both in the nucleus and in the cytoplasm (8, 19, 31).However, we noticed that the accumulation of tagged TBEV RNAin the cytoplasm corresponded to a depletion of the MS2 nuclearstain. Therefore, in order to increase the signal-to-noise ratio inthe cytoplasm, we exploited an EYFP-MS2 protein with a nuclearlocalization signal (NLS) for exclusive nuclear localization in theabsence of TBEV replication. EYFP-MS2nls remains associatedwith the replicated TBEV RNA carrying the MS2 binding sites inthe cytoplasm against a dark background, offering a better tool forboth dynamic and ultrastructural studies. As shown in Fig. 4A,extensive colocalization of EYFP-MS2nls with dsRNA was ob-served in the ER-derived perinuclear compartment in cells trans-fected with the TNd/�ME_24�MS2 wild-type replicon, whereas

FIG 1 Localization of TBEV proteins and dsRNA in BHK-21-infected cells. BHK-21 cells were either mock infected (MOCK; right) or infected with TBEV at anMOI of 2 (TBEV; left). After 24 h, cells were fixed and processed for immunofluorescence as described in Materials and Methods. (A) Cells costained with theanti-TBEV antiserum (TBEV; Alexa Fluor 594, red) and with the monoclonal anti-NS1 antibody (NS1; Alexa Fluor 488, green); (B) cells costained with theanti-TBEV antiserum (TBEV; Alexa Fluor 594, red) and with the J2 monoclonal anti-dsRNA antibody (dsRNA; Alexa Fluor 488, green); (C) cells costained withthe anti-prM antiserum (prM; Alexa Fluor 594, red) and with the monoclonal anti-NS1 antibody (NS1; Alexa Fluor 488, green); (D) cells costained with theanti-TBEV antiserum (TBEV; Alexa Fluor 594, red) and with the anti-PDI monoclonal antibody (PDI; Alexa Fluor 488, green). The zoomed images shown in themiddle panels correspond to boxed regions in the left panels.

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control cells transfected with the replicon carrying an inactive NS5RdRp showed only nuclear localization of EYFP-MS2nls (Fig. 4B).Detailed tomographic analysis of cells fixed 24 h after transfectionrevealed rather small rER cisternae in comparison to infected cells.

These smaller ER tubules form a network of fragmented ER in theperinuclear region of transfected cells (Fig. 4C; see also Movies S3and S4 in the supplemental material). Nevertheless, vesicles wereobserved in the rER lumen, having an average diameter of 85 nm

FIG 2 Ultrastructural analysis of the membrane alterations induced by TBEV infection. (A) BHK-21 cells were infected with TBEV at an MOI of 2, fixed 24 hpi,and processed for ET as described in Materials and Methods. Left, tomographic slice of a dual-axis tomogram, shown in Movie S2 in the supplemental material.Upon infection, vesicles (Ve) and virions (yellow arrowheads) were observed in the lumen of the rough ER. Right, 3D surface reconstruction of the wholetomogram displaying the TBEV-induced vesicles (in light yellow) in the lumen of the ER (in light brown), as well as virions (in dark red). (B) Two examples (leftand right) of tomographic slices through the whole tomogram depicting connections between ER tubules (yellow arrows). (C) Left, tomographic slices of thesame dual-axis tomogram depicting several of these vesicles in the lumen of the ER in close proximity to newly assembled virions (yellow arrows), also observedin the Golgi apparatus (see Movie S1); right, 3D surface rendering of the same area displaying the TBEV-associated vesicles (in light yellow) sharing the ER lumenwith TBE virions (in dark red) and surrounded by ER membranes (in light brown). (D) Left, serial single slices depicting openings of several TBEV-induced vesicles tothe cytosol (green, yellow, and blue arrows); right, XZ view of the 3D reconstruction of the whole tomogram displaying these openings toward the cytosol (arrows withmatching colors). (E) Left, slices through the tomogram showing tight contacts of TBEV-induced vesicles with their neighboring vesicles (yellow arrows); right, 3Dreconstruction displaying these contacts between vesicles. Note that openings connecting vesicles were not observed in these tomograms.

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(�19.6 nm; n � 70). In addition, similar to what we found ininfected cells, most of the vesicles (75%; n � 35) detected in rep-licon-transfected cells had a pore-like opening toward the cytosol(Fig. 4D). Based on the similarity between replicon- and infec-tion-induced membranous structures and by analogy to denguevirus (11), we concluded that TBEV-induced vesicles play a role invirus replication.

Immuno-EM analysis of replicon-transfected BHK-21 cells.Membrane alterations observed upon transfection of MS2-taggedsubgenomic replicons were then further characterized by im-muno-EM (Fig. 5). As already suggested by our detailed three-dimensional analysis of replicon-induced ultrastructural changes(Fig. 4; see also Movies S3 and S4 in the supplemental material),PDI labeling clearly confirmed that TBEV-induced vesicles arepart of an intricate network of rearranged intracellular mem-branes that originate from the ER compartment (Fig. 5A). Thesevesicles were labeled with anti-NS1 antibodies, again supportingtheir role in virus replication (Fig. 5B). Clusters of vesicles, definedas vesicle packets (14, 15, 32), could be observed in close proximityto highly rearranged membranous structures most likely repre-senting convoluted membranes (CM), the putative sites for syn-thesis, and processing of the flavivirus polyprotein. As shown inFig. 5C and D, the TBEV-specific polyclonal antiserum labeledboth vesicles and CM (Fig. 5D, arrows). In addition, gold particlesfor the same antiserum were found also in the lumen of the ER,consistent with our previous findings on TBEV-infected cells.

Replicated viral RNA is not freely diffusible in the cytoplasm.The MS2-tagging method was then further exploited to study the

dynamic interchange of newly replicated TBEV RNA betweenMS2-defined perinuclear compartments and the cytoplasm. Tothis end, we took advantage of the fluorescence recovery afterphotobleaching (FRAP) technique. FRAP is a method for measur-ing the mobility of fluorescent particles in living cells (33, 34). Adefined portion of the system containing mobile fluorescent mol-ecules is exposed to a brief and intense focused laser beam, therebycausing irreversible photochemical bleaching of the fluorophorein that region. The subsequent kinetics of fluorescence recovery inthe bleached region, which results from transport of fluorescentproteins into the bleached area from nonirradiated regions of thecell, as well as transport of bleached fluorescent proteins out of thebleached area, provides a quantitative measure of the mobility ofthe protein of interest. If the fluorescent protein cannot move or isbound to an immobile substrate, the recovery of fluorescence willnot reach the prebleach values showing the so-called immobilefraction. In our case, the fluorescent protein is the MS2 phage coatprotein fused to EYFP. TBEV-replicated RNA carrying an array ofMS2 binding sites previously characterized in our laboratory isdetected by high-affinity specific interaction between the RNAstem-loops and the fluorescently labeled MS2 protein (19, 34).The viral RNA is continuously synthesized by the viral polymeraseand should be able to diffuse in the cytoplasm in complex withEYFP-MS2 unless bound to an immobile structure or limited in itsmovements by physical barriers. As shown in Fig. 6A and B andMovie S5 in the supplemental material, BHK-21 cells expressingthe TNd/�ME_24�MS2 replicon RNA and EYFP-MS2 were sub-jected to FRAP analysis at different time points. Full recovery of

FIG 3 Immunogold EM of TBEV-infected cells. BHK-21 cells were infected with TBEV at an MOI of 2 and fixed 24 hpi, and thawed cryosections were labeledwith antibodies against PDI (A), NS1 (B and C), and prM (E and F) and with the anti-TBEV serum (D) that predominantly recognizes the structural protein Eand the nonstructural protein NS1. Vesicles within the lumen of dilated ER cisternae (A) are specifically labeled with anti-NS1 (B and C) and anti-TBEVantibodies (D). Arrowheads in all panels highlight TBEV virions, specifically labeled with prM (E and F) and TBEV (D) antibodies, in the lumen of the ER ortrafficking toward the Golgi apparatus. Abbreviations: M, mitochondria; N, nucleus; ER, endoplasmic reticulum; Ve, vesicles; Vi, virions.

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FIG 4 Ultrastructural analysis of the membrane alterations induced by pTNd/�ME_24�MS2. (A) BHK21-EYFP-MS2nls cells were electroporated with theTNd/�ME_24�MS2 replicon RNA. Twenty-four hours postelectroporation (hpe), cells were fixed, permeabilized with Triton X-100, and incubated with anantibody against dsRNA that was then revealed by a secondary antibody conjugated with Alexa-594. The EYFP channel is shown in the left panel, dsRNA in themiddle panel, and the merge in the right panel. (B) BHK21-EYFP-MS2nls cells were electroporated with the TNd/�ME_24xMS2_GAA replicon RNA and treatedas described for panel A. (C) BHK21-EYFP-MS2nls cells were electroporated with TNd/�ME_24�MS2, fixed 24 hpe, and processed for ET as described inMaterials and Methods. Left, slice of a dual-axis tomogram showing the TBEV-induced vesicles (Ve) in the ER lumen; right, 3D reconstruction of the completetomogram. ER membranes are depicted in light brown and TBEV-induced vesicles in light yellow. This tomogram and its 3D membrane rendering are shown inMovie S4 in the supplemental material. (D) Left, serial single slices through the same tomogram displaying connections between adjacent ER tubules; right, 3Dreconstruction of the whole tomogram showing a network of interconnected ER tubules. Note that the ER is highly fragmented in these cells in comparison toinfected cells. (E) Left, serial single slices through the same tomogram displaying an opening toward the cytosol of a TBEV-induced vesicle (yellow arrows); right,3D surface model showing this opening that connects the interior of the vesicle with the cytosol.

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the signal within a few seconds was observed 6 h after electropo-ration, indicating free mobility of EYFP-MS2 at early time pointsof replicon amplification. In contrast, 12 h after transfection andat later time points, fluorescence recovery was severely impaired.Time points between 12 and 36 h showed very similar recoveryprofiles, with an initial recovery that quickly tails off and neverreaches prebleach values, indicative of the presence of a relevantimmobile fraction. The initial portion of the recovery curve rep-resents the unbound EYFP-MS2 that remains free to diffuse in thebleached area. At later time points, the amount of free EYFP-MS2is reduced, being sequestered by the increasing amount of repli-cated RNA (compare the red curve in Fig. 6A taken at 12 h post-electroporation [hpe] to the blue and green curves taken at 24 to36 hpe, respectively). These data suggest that the spatial con-straints are established early and maintained thereafter. These re-sults can be interpreted in different ways that were addressed asfollows.

The first possibility is that the replicated viral RNA is secludedinto compartments that are not accessible by the EYFP-MS2. Toaddress this assumption, we exploited the red-shifted variantCherry-MS2nls to mark viral RNA while the mobility of free greenfluorescent protein (GFP) was monitored by FRAP as previouslydescribed (21, 34–36). As shown in Fig. 6C, D, and E and Movie S6in the supplemental material, mobility of GFP within the com-partment did not differ significantly from the free mobility of theprotein in the cytoplasm. The second possibility is that the viralRNA is only slowly or not at all replicated, resulting in little or no

substrate available for free EYFP-MS2. However, bleaching didnot affect virus replication (data not shown), and we have earliershown that replicated viral RNA accumulates continuously up to72 h posttransfection (19, 31). Therefore, we can exclude that theRCs are inactive with respect to replication. The third possibility isthat a slow release of replicated viral RNA bound to bleachedEYFP-MS2 residing in the region of interest (ROI) results in anincrease of the immobile fraction in the FRAP experiment. Toaddress such a release of replicated, MS2-tagged, viral RNA fromthis region, we depleted fluorescent EYFP-MS2 from the cyto-plasm and measured loss of florescence at the compartment. Thisapproach is called fluorescence loss in photobleaching (FLIP) andis complementary to FRAP (37, 38). As shown in Fig. 6F andMovie S7 in the supplemental material, we chose a region forbleaching in the cytoplasm distant from the MS2-enriched peri-nuclear compartment and measured loss of fluorescence at a sitein the cytoplasm as well as within regions of EYFP-MS2 accumu-lation in order to assess RNA exchange. Both sites were chosenapproximately at the same distance from the bleaching area. Aftereach bleaching, the intensity of fluorescence was measured in ev-ery ROI. In principle, each mobile fluorescent molecule that dif-fuses at the bleaching site will be irreversibly bleached. Hence,regions of freely mobile proteins will be losing fluorescencequickly, whereas regions where the proteins are immobile or se-cluded into membrane compartments will resist bleaching. Thiskind of measurements clearly showed that fluorescent TBEVRNA-bound EYFP-MS2 (blue line) is depleted less efficiently than

FIG 5 Immunogold EM of replicon-induced membrane alteration. BHK21-EYFP-MS2nls cells were electroporated with TNd/�ME_24�MS2 and fixed 24 hafter, and thawed cryosections were labeled with antibodies against PDI (A) and NS1 (B) and with the anti-TBEV serum (C and D). All images showTBEV-induced vesicles (Ve) that are specifically labeled with anti-NS1 (B) and anti-TBEV (C and D) antibodies. (D) Higher-magnification image of membranealterations induced by replicon transfection corresponding to the boxed region in panel C. Vesicles (Ve) and convoluted membranes (CM) labeled withanti-TBEV antibodies are shown.

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freely diffusible EYFP-MS2 (green line). Importantly, as observedfor the FRAP experiments (Fig. 6A) where the immobile fractiondecreased at later time points, immobilization of trapped TBEVRNA within the MS2-defined compartment is not complete, since30% depletion is observed after 4 min of FLIP. This clearly pointstoward a certain degree of TBEV RNA exchange between the com-partments and the cytoplasm.

Free diffusion of viral RNA within interconnected regions ofvirus-induced membrane alterations. Finally, we wanted to in-vestigate the dynamic interchange of TBEV RNA within the areaenriched for the EYFP signal. To this end, we took again advantageof the EYFP-MS2nls reporter that, in the presence of replicatingTNd/�ME_24�MS2, accumulates efficiently in the cytoplasmagainst a dark background, greatly enhancing the signal-to-noiseratio (Fig. 4A). This strategy is also particularly useful when doing

dynamic studies, because it allows to selectively measure viralRNA kinetics without taking into account free diffusion of un-bound MS2 proteins in the cytoplasm. We designed a FLIP exper-iment where the bleached area is located within perinuclear re-gions of EYFP-MS2 accumulation (Fig. 7A, red circle, bottompanels). If the tagged viral RNAs were all interconnected, contin-uous bleaching would have resulted in depletion of fluorescencefrom the entire compartment. Conversely, as shown in Fig. 7A andMovie S8 in the supplemental material, depletion of fluorescencewas restricted to a portion of the compartment, defined as ROI_1in Fig. 7A, leaving the rest unaffected. This experiment indicatesthat the perinuclear region, where replication of TBEV is likely tooccur, is not just a continuous network of virus-induced mem-brane alterations but is rather composed of physically separatedsubcompartments. Within a subcompartment, the viral RNA is

FIG 6 TBEV-replicated RNA is not freely diffusible in the cytoplasm. (A) Analysis of TBEV RNA dynamics by FRAP time course. BHK-21 cells wereelectroporated with the TNd/�ME_24�MS2 replicon RNA together with a vector expressing EYFP-MS2. At the indicated time points postelectroporation, thefluorescence recovery of the EYFP-MS2 protein in the area of bleaching was analyzed. The graph shows values of fluorescence intensity normalized to theprebleach values and corrected for the loss of fluorescence due to the imaging procedure (34, 36, 55). Data represent the averages of acquisitions from at least 10cells � standard deviations. (B) Image sequence from a FRAP experiment performed in BHK-21 cells 14 h after electroporation (29.25 by 29.25 �m). The brightperinuclear region represents the subcellular compartment into which replicated viral RNA is clustered and where ROIs were drawn. Times were collected beforebleaching (prebleach, 0 s), immediately after the bleaching (bleach, 20 s), and at 400 s after the bleaching event (postbleach). A movie is also available assupporting information (see Movie S5 in the supplemental material). (C) Analysis of GFP mobility within the replication compartment. BHK-21 cells wereelectroporated with a vector expressing CherryMS2nls, to mark the RC, and with a GFP-expressing plasmid (pEGFP-N1), both in the presence (red line, GFP andTNd/�ME_24�MS2) and in the absence (green line, GFP) of the TBEV replicon RNA. After 24 h, GFP kinetics was investigated by FRAP. In the graph, therecovery curves of the GFP protein in the two different experimental conditions are compared. The values of fluorescence intensity are normalized to theprebleach values and corrected for the loss of fluorescence due to the imaging procedure as already described. Data represent the averages of acquisitions from10 cells � standard deviations. (D) Representative image of BHK-21 cells transfected with GFP and CherryMS2nls in the presence of the TBEV replicating RNA.A z-projection of 41 images 0.5 �m apart is shown. (E) Image sequence from the FRAP experiment described in panel C (36.56 by 7.14 �m). Top, prebleach stacksin both channels (0 s). The circle indicates the area of bleach chosen in the RC marked by CherryMS2nls. Middle, time point immediately after the bleaching event(0.5 s); bottom, postbleach stack (13 s). A video is also available as supporting information (see Movie S6 in the supplemental material). (F) BHK-21 cells wereelectroporated with the TNd/�ME_24�MS2 replicon RNA together with a vector expressing EYFP-MS2. After 24 h, viral RNA release from the RC wasmonitored by FLIP. (G) Selected images from a FLIP experiment are shown (36.56 by 36.56 �m). The region for bleaching (red circle in the bottom panels) waschosen in the cytoplasm away from the clustered TBEV RNA. Loss of fluorescence was then measured in the cytoplasm both within (blue circle, ROI_1) andoutside (green circle, ROI_2) the RC. In the top graph, the loss in fluorescence intensity within the three different ROIs (bleach; ROI_1 and ROI_2) is compared.Data are normalized as described for panel A and represent the averages of acquisitions from 10 cells � standard deviations. A video is also available as supportinginformation (see Movie S7 in the supplemental material).

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able to freely diffuse (compare decay of ROI_1 in Fig. 7A withROI_1 in Fig. 6F), indicating that most likely the RNA is not com-pletely associated to membranes.

To unambiguously identify the subcellular localization ofnewly synthesized viral genomes, we selectively labeled the MS2protein bound to TBEV RNA by using antibodies specific to thefluorescent tag. BHK-21 cells transduced with lentiviral vectorsexpressing EYFP-MS2nls were either mock electroporated or elec-troporated with the TNd/�ME_24�MS2 replicon RNA. Twenty-four hours posttransfection, cells were fixed and processed forimmuno-EM as described in Materials and Methods. As shown inFig. 7, immunogold-labeled TBEV RNA specifically localized inthe cytoplasm of TNd/�ME_24�MS2-transfected cells, while, asexpected, only nuclear MS2 labeling could be detected in mock-transfected cells (data not shown). Strikingly, detailed analysis ofGFP-labeled cryosections revealed that more than 50% of the goldparticles localized in the cytosol, frequently surrounding virus-induced vesicles (Fig. 7E). In addition, particles associated or inclose proximity to ER-derived membranes (� 35%), as well asadjacent to vesicle-surrounding membranes (� 9%), were alsoobserved. To the contrary, labeled TBEV RNA was never detectedwithin the lumen of the vesicles, indicating that either the EYFP-MS2nls protein is too big to translocate into the vesicle or that

actively replicating viral RNA is part of a huge protein/RNA com-plex that shields the RNA from the MS2 protein and/or anti-GFPantibodies.

DISCUSSION

Flaviviruses hijack cytoplasmic membranes in order to build func-tional sites for protein synthesis, processing, and RNA replication(7, 39–42). These sites, generally defined as replication compart-ments, are required to efficiently coordinate different steps of theviral life cycle and to protect replicating RNA from innate immu-nity surveillance (8–10). Over the last few years, several electronmicroscopy studies have revealed important information aboutthe ultrastructural organization of these sophisticated intracellu-lar membrane compartments (11, 12, 28). However, this infor-mation is rather static and does not provide insights into intra-cellular trafficking of proteins and viral RNA. In this work, wehave combined high-resolution ET and immuno-EM with live-cell imagining studies of TBEV RNA dynamics to provide thefirst comprehensive picture of the spatiotemporal organizationof the flaviviral RC.

Our detailed 3D ultrastructural analysis clearly shows thatTBEV infection triggers a remarkable alteration of intracellularmembranes (Fig. 2). These virus-induced membranes are derived

FIG 7 TBEV replication compartments are organized in discrete clusters. (A) BHK-21 cells were electroporated with the TNd/�ME_24�MS2 replicon RNA andwith the EYFP-MS2nls reporter. At 24 h after transfection, viral RNA trafficking within the RC was analyzed by FLIP. For this purpose, as shown in the bottomimages (29.25 by 29.25 �m), the area of bleaching (red circle) was located inside the compartment. Loss of fluorescence was then measured in two differentregions, one surrounding the bleaching area (blue region; ROI_1) and the other one more distant (green circle; ROI_2). The top panel compares the loss offluorescence curves of the three selected ROIs. Data are normalized as already described and represent the average of acquisitions from 10 cells � standarddeviation. A video is also available as supporting information (see Movie S8 in the supplemental material). (B) BHK21-EYFP-MS2nls cells were electroporatedwith TNd/�ME_24�MS2 and fixed 24 h after, and thawed cryosections were immunogold labeled with antibodies against GFP. Selected examples of the differentsituations are shown in panels C and D. (E) Quantification of GFP-labeled EM cryosections prepared as described in Fig. 7B. Counting was performed intriplicate, measuring the distribution of �100 gold particles for each grid. Data are plotted as percentages � SD.

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from the ER since they contain viral proteins and the ER-residentchaperone PDI (Fig. 1). PDI did not directly label the vesicles(Fig. 3A), indicating that this protein is not recruited but mostlikely excluded from virus-induced membranes, as observed forDENV-induced vesicles (11). Interestingly, we consistently ob-served packets of vesicles with a diameter of about 80 nm thatappear as invaginations of the ER within a highly organized net-work of interconnected membranes (Fig. 2A and B; see also Mov-ies S1 and S2 in the supplemental material). Attempts to labelvesicles with antibodies recognizing dsRNA were ambiguous; al-though we detected immunogold labeling within the vesicles, thesignal appeared to be unspecific (data not shown). Nevertheless,labeling of the vesicles with antibodies against the TBEV NS1 pro-tein (Fig. 3B, C, and D) supports the hypothesis that these struc-tures correspond to sites of virus replication. NS1 is an essentialgene and modulates early viral RNA replication (13, 43), possiblythrough its ability to regulate negative-strand synthesis of viralRNA (44) and/or by interacting with NS4B (45). NS1 is located inthe ER lumen and is associated with the outer side of the replica-tion vesicles through interaction with other transmembrane viralproteins. Localization of DENV NS1 at replication vesicles wasreported by Mackenzie et al. (13) and more recently by Welsch etal. (11). Finally, immunoprecipitation of dsRNA intermediateswas shown to contain NS1 in addition to NS5, confirming previ-ous cosedimentation data (46, 47). Therefore, NS1 can be consid-ered a bona fide marker for replication sites. The observation thatNS1 is associated to other compartments in addition to replica-tion vesicles may reflect its involvement in different steps of theviral life cycle, beyond RNA replication.

Approximately half of the vesicles showed a single pore-likeconnection to the cytoplasm. There might be several explanationsfor this observation. Besides technical problems that may not al-low clear detection of pores in all vesicles, there could be a dy-namic life cycle of virus-induced vesicles, from their formation,function in replication with a pore connection, and eventual latestep where the vesicles are not connected with the cytosol by thepore anymore. This pore likely allows recruitment of cellular co-factors to active sites of virus replication as well as for the release ofnewly synthesized viral RNA to be used for translation and assem-bly (Fig. 2D). However, although the vesicles were frequentlyclosely associated, we could never detect direct connections be-tween neighboring vesicles in the same packet, as described forKunjin virus and more recently also for the naturally attenuatedtick-borne LGTV (12, 28). This discrepancy might be due to dif-ferences in the sample preparation. Alternatively, it might impli-cate that the architecture of flaviviral RCs slightly changes basedon the specific virus and/or cell line used for the analysis.

We also often observed newly assembled virions sharing thesame ER lumen with putative replicative vesicles, suggesting thatreplication and assembly sites are located in very close proximity.Given the absence of pore-like openings directly connecting vesi-cles with the ER lumen, it is likely that, once synthesized, the viralRNA is released via the pore to the cytosolic side of these invagi-nations. The basic C protein would then interact with progenyRNA genomes to form a nucleocapsid that buds back to the lumenof the ER, acquiring the viral envelope. Thereafter, immature viralparticles would be either stored in clusters within dilated ER cis-ternae (Fig. 2A) or transported through the cellular secretorypathway (see Movie S1 in the supplemental material), where mat-

uration occurs, in order to be released to the extracellular milieu(48, 49).

In this study, we also compared ultrastructural alterations in-duced by virus infection with those associated with the replicationof MS2-tagged TBEV subgenomic replicons. As observed uponTBEV infection, dramatic reorganization of intracellular mem-branes with the appearance of single membrane vesicles could bedetected in the perinuclear region of the cell. Although ER tubuleshosting replicon-induced vesicles appeared fragmented andsmaller than those observed in virus-infected cells, vesicles weresimilar in size, and our detailed tomographic analysis revealed thatthey also originated from the ER and were connected to the cyto-plasm via pore-like channels. Interestingly, at variance with virus-infected cells, replicon-induced vesicles did not accumulate inpackets but were rather dispersed through the cytoplasm as one orfew invaginations of the ER membrane (Fig. 4; see also Movies S3and S4 in the supplemental material). We also observed a higherfraction of vesicles with pores in replicon-transfected cells. How-ever, the latter observation could be simply related to a betteraccessibility of dispersed vesicles during ET. These data suggestthat replicons, despite the induction of massive ER rearrange-ments, are less efficient at forming vesicles, which is consistentwith the lower level of viral replication previously reported (31, 50,51). The induction of membrane rearrangements could be due tothe altered expression or regulation of viral proteins resultingfrom the electroporation of large amounts of genomic RNA. Forexample, it has been shown that the NS4A protein of mosquito-borne KUNV and DENV viruses possesses the intrinsic capacity toinduce membrane rearrangements similar to those induced uponvirus infection (52, 53). A similar role for TBEV NS4A remains tobe clarified.

When we investigated the mobility of viral RNA by FRAP, wefound a consistent delay of the recovery of fluorescence withinperinuclear regions of EYFP-MS2 accumulation (Fig. 6A). In con-trast, GFP did not show significant differences of mobility withinthe cytosol compared to that within TBEV-induced replicationcompartments that are marked with cherry-MS2nls (Fig. 6C),suggesting that these compartments are open to the cytosol andallow exchange of proteins. Interestingly, recovery after 36 h didnot differ significantly from that at 12 h after replicon transfection.Although we cannot formally exclude that replication rates slowdown at late time points, thus masking an increase of recovery inthe FRAP curve due to an increased permeability, we can assumethat the mobility of viral RNA is similar at early and late timepoints. In other terms, at later time points there is not a massivedisruption or reorganization of these membranous compart-ments compatible with the liberation of viral RNA. Free accessi-bility of the fluorescent MS2 concomitant with a slow recoveryafter photobleaching within the compartment suggests an im-paired movement of the genomic RNA out of this area. This couldbe coupled with a low rate of viral RNA biogenesis and/or with theassociation of a fraction of the viral RNA to polysomes on ERmembranes. To address this hypothesis, we performed a FLIP ex-periment, and we directly measured trafficking of viral RNA be-tween cytosol and sites of virus replication (Fig. 6F). Strikingly, wecould observe a slow release of replicated RNA from the compart-ment.

Another important observation relates to the mobility oftagged viral RNA within MS2-defined regions of the cytoplasm.Previous FRAP studies performed with components of the HCV

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replication complex NS4B and NS5A showed that the internalarchitecture of the membranous web is relatively static, with lim-ited exchange of viral nonstructural proteins between neighboringfactories (17, 18, 54). In the present study, for the first time, wemonitored viral RNA dynamics within perinuclear regions of vi-rus replication. By using continuous bleaching of MS2 fluores-cence in the region where the RNA is clustered, we could observea fast depletion of fluorescence, consistent with a high mobility ofthe viral RNA (Fig. 7A; see Movie S8 in the supplemental mate-rial). Mobility within this area was indeed higher than mobilitybetween the compartment and the cytosol, confirming that aphysical impediment restricts viral RNA egress rather than an in-trinsic slow mobility of the viral RNP (compare the blue lines inFig. 6F and 7A). However, depletion of fluorescence was clearlyrestricted only to a discrete region in the cluster, leading us to theconclusion that these TBEV-induced compartments are parti-tioned with respect to viral RNA mobility. In agreement with thisfinding, specific labeling of the newly replicated RNA clearly re-vealed a high proportion of labeled MS2-tagged RNA genomeslocalized in the cytosolic space surrounding TBEV-induced mem-brane alterations (Fig. 7E). Therefore, we propose that upon syn-thesis within ER-derived vesicles, progeny genomes must be re-leased to a larger virus-induced extravesicular subcompartmentwhere RNA translation and virus assembly occur. This subcom-partment would be full of viral RNA that travels from replicationvesicles to the virion assembly sites and is connected to the cyto-plasm (Fig. 8). Such an extremely organized architecture wouldcertainly help in coordinating genomic RNA recruitment fortranslation and/or packaging.

In conclusion, we generated a high-resolution structure of thethree-dimensional organization of the TBEV-induced replicationcompartment. In addition, by exploiting the MS2-tagged repliconsystem, we could combine structural information with dynamicdata of newly replicated TBEV-RNA within functional intracellu-lar compartments, providing new insights into the spatiotemporalorganization of flavivirus replication compartments.

ACKNOWLEDGMENTS

We thank the Electron Microscopy Core Facility (EMCF) at the Univer-sity of Heidelberg and the EM Facility at the European Molecular BiologyLaboratory (EMBL; Heidelberg) for providing access to their equipment,expertise, and technical support. We also thank Anil Kumar for the experthelp in handling lentiviral vectors and for useful discussions.

L.M. benefited from a short-term EMBO fellowship (ASTF 217-2012).Work on flaviviruses in A.M.’s laboratory is supported by the Beneficien-tia Stiftung. R.B. is supported by the Deutsche Forschungsgemeinschaft(SFB 638, TPA5, TRR83, TP13).

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